Patent Grant
10544191
The present invention relates to methods and compositions for generating macrocyclic peptides from genetically encoded, ribosomally produced polypeptide precursors. The invention also relates to nucleic acid molecules, polypeptides, and methods for generating macrocyclic peptides and combinatorial libraries of macrocyclic peptides.
Peptides molecules represent valuable tools for investigating biological systems, studying the binding and activity properties of biomolecules (e.g., enzymes, cell receptors, antibodies, kinases), exploring the etiopathological causes of diseases, and for validating pharmacological targets. Peptides are also attractive ligands for targeting protein-protein interactions and modulating the function of biological molecules such as enzymes and nucleic acids. The synthesis of combinatorial libraries of small peptides followed by screening of these chemical libraries in biological assays can enable the identification of compounds that exhibit a variety of biological and pharmacological properties. Bioactive peptides identified in this manner can constitute valuable lead compounds or facilitate the development of lead compounds towards the discovery of new drugs.
While many peptides exhibit interesting biological activity, linear peptides do not generally represent suitable pharmacological agents as they are generally only poorly adsorbed, do not cross biological membranes readily, and are prone to proteolytic degradation. In addition, linear peptides fail to bind proteins that recognize discontinuous epitopes. The use of molecular constraints to restrict the conformational freedom of the molecule backbone can be used to overcome these limitations. In many cases, conformationally constrained peptides exhibit enhanced enzymatic stability (Fairlie, Tyndall et al. 2000; Wang, Liao et al. 2005), membrane permeability (Walensky, Kung et al. 2004; Rezai, Bock et al. 2006; Rezai, Yu et al. 2006), and protein binding affinity (Tang, Yuan et al. 1999; Dias, Fasan et al. 2006) and selectivity (Henchey, Porter et al. 2010), compared to their linear counterparts. Constraints that lock-in the active conformation of a peptide molecule can result in increased affinity due to the reduced conformational entropy loss upon binding to the receptor. Many bioactive and therapeutically relevant peptides isolated from natural sources occur indeed in cyclized form or contain intramolecular bridges that reduce the conformational flexibility of these molecules (e.g., immunosuppressant cyclosporin A, antitumor dolastatin 3 and diazonamide A, anti-HIV luzopeptin E2, and the antimicrobial vancomycin). Since macrocyclic peptides constitute promising molecular scaffolds for the development of bioactive compounds and therapeutic agents (Katsara, Tselios et al. 2006; Driggers, Hale et al. 2008; Obrecht, Robinson et al. 2009; Mars ault and Peterson 2011), methods for generating macrocyclic peptides and combinatorial libraries thereof, are of high synthetic value and practical utility, in particular in the context of drug discovery.
While cyclic peptides can be prepared synthetically via a variety of known methods (White and Yudin 2011), the possibility to generate macrocyclic peptides starting from genetically encoded polypeptide precursors offers several advantages (Frost, Smith et al. 2013; Smith, Frost et al. 2013). Among these, there are: (a) the high combinatorial potential inherent to the ribosomal synthesis of genetically encoded polypeptides, which can enable the production of very large collections of peptide sequences (108-1010 members or higher) in a cost- and time-effective manner; (b) the possibility to link these peptide libraries to powerful, high-throughput screening platforms such as phage display, mRNA display, or yeast display, in order to identify peptide ligands with the desired property (e.g., high binding affinity toward a target protein); (c) the ease by which these chemical libraries can be deconvoluted in order to identify the library members of interest (i.e., via sequencing of the peptide-encoding DNA or RNA sequence).
Various methods have been developed for producing biological libraries of conformationally constrained peptides (Frost, Smith et al. 2013; Smith, Frost et al. 2013). For example, libraries of disulfide-constrained cyclic peptides have been prepared using phage display and fusing randomized polypeptide sequences flanked by two cysteines to a phage particle as described, e.g., in U.S. Pat. No. 7,235,626. Disulfide bridges are however potentially reactive and this chemical linkage is unstable under reducing conditions or in a reductive environment such as the intracellular milieu. Alternatively, ribosomally produced peptides have also been constrained through the use of cysteine- or amine-reactive cross-linking agents (Millward, Takahashi et al. 2005; Seebeck and Szostak 2006; Heinis, Rutherford et al. 2009; Schlippe, Hartman et al. 2012). A drawback of these methods is the risk of producing multiple undesired products via reaction of the cross-linking agents with multiple sites within the randomized peptide sequence or the carrier protein in a display system. In addition, these methods do not allow for the formation of macrocyclic peptides inside the polypeptide-producing cell host. Other methods have been described that are useful for preparing head-to-tail cyclic peptides by using natural (i.e., naturally occurring) or engineered (i.e., non-naturally occurring, artificial or synthetic) split inteins, as described in U.S. Pat. Nos. 7,354,756, 7,252,952 and 7,105,341. An advantage of these strategies is the possibility to couple the intracellular formation of cyclic peptide libraries with an cell-based reporter or selection system, which can facilitate the identification of functional peptide ligands (Horswill, Savinov et al. 2004; Cheng, Naumann et al. 2007; Naumann, Tavassoli et al. 2008; Young, Young et al. 2011). However, the peptide cyclization efficiency was found to be highly dependent on the peptide sequence (Scott, Abel-Santos et al. 2001). In addition, only head-to-tail cyclic peptides can be obtained through these strategies, which limits the extent of structural diversity of the ligand libraries generated through these methods. Finally, methods have also been reported for generating cyclic peptides through the enzymatic modification of linear peptide precursors (Hamamoto, Sisido et al. 2011; Touati, Angelini et al. 2011). However, the need for exogenous reagents and/or enzyme catalysts for mediating peptide cyclization and, in some cases, moderate cyclization efficiency limit the scope and utility of these approaches toward the generation and screening of cyclic peptide libraries.
Efficient and versatile methods for generating macrocyclic peptides from ribosomally produced polypeptides would thus be highly desirable in the art. The methods and compositions described herein provide a solution to this need, enabling the ribosomal synthesis of cyclic peptides in vitro (i.e., in a cell-free system) and in vivo (i.e., inside a cell or on a surface of a cell) and in various ‘configurations’, namely in the form of macrocyclic peptides, lariat-shaped peptides, or as cyclic peptides fused to a N-terminus or C-terminus of a protein of interest, such as a carrier protein of a display system.
Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.
A method is provided for making a macrocyclic peptide, the method comprising:
a. providing an artificial nucleic acid molecule encoding for a polypeptide of structure:
(AA)m-Z-(AA)n-Cys-(AA)p (I)
or
(AA)m-Cys-(AA)n-Z-(AA)p (II)
or
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p (V)
In certain embodiments, FG1 and FG2 can be either the same group among those groups listed above or two different groups among those groups listed above.
In one embodiment of the method, Z is an amino acid of structure:
wherein FG1 is a functional group selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO2C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO2; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane, unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide; where each R and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group;
wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups.
In another embodiment of the method, Z is an amino acid of structure (IV) and Y is a linker group selected from the group consisting of C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, and C5-C24 aryloxy groups.
In another embodiment of the method, Y is a linker group selected from the group consisting of —CH2—C6H4—, —CH2—C6H4—O—, —CH2—C6H4—NH—, —(CH2)4—, —(CH2)4NH—, —(CH2)4NHC(O)—, and —(CH2)4NHC(O)O—.
In another embodiment of the method, the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, Nε-((2-bromoethoxy)carbonyl)-lysine, Nε-((2-chloroethoxy)carbonyl)-lysine, Nε-(buta-2,3-dienoyl)-lysine, Nε-acryl-lysine, Nε-crotonyl-lysine, Nε-(2-fluoro-acetyl)-lysine, and Nε-(2-chloro-acetyl)-lysine.
In another embodiment of the method, Z2 is an amino acid of structure:
wherein each of FG1 and FG2 is a functional group independently selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO2C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO2, —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R and R′ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group;
wherein Y2, Y3, and L are linker groups selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups.
In certain embodiments, FG1 and FG2 can be either the same group among those groups listed above or two different groups among those groups listed above.
In another embodiment of the method, Z2 is an amino acid of structure (VI) and Y2 is a linker group selected from the group consisting of C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, and C5-C24 aryloxy groups.
In another embodiment of the method, Y is a linker group selected from the group consisting of —CH2—C6H4—, —CH2—C6H4—O—, —CH2—C6H4—NH—, —CH2—C6H4—OCH2—, —(CH2)4NH—, —(CH2)4NHC(O)—, —(CH2)4NHC(O)O—, —(CH2)4NHC(O)OCH2—,
In another embodiment of the method, the amino acid Z2 is selected from the group consisting of 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, Nε-4(1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, Nε-4(1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, Nε-((2,3-dibromopropoxy)carbonyl)-lysine, and Nε-((2,3-dichloropropoxy)carbonyl)-lysine.
In another embodiment of the method, the codon encoding for Z or Z2 is an amber stop codon TAG, an ochre stop codon TAA, an opal stop codon TGA, or a four base codon.
In another embodiment of the method, the expression system comprises:
an aminoacyl-tRNA synthetase polypeptide or an engineered variant thereof that is at least 90% identical to SEQ ID NO:77, 78, 79, or 80; and
a transfer RNA molecule encoded by a polynucleotide that is at least 90% identical to SEQ ID NO:101, 105, 109, 113, or 117.
In another embodiment of the method,
(a) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises an amino acid substitution at a position selected from the group consisting of position: X32, X63, X65, X70, X107, X108, X109, X155, X158, X159, X160, X161, X162, X163, X164, X167, and X286 of SEQ ID NO:77,
(b) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises an amino acid substitution at a position selected from the group consisting of position: X302, X305, X306, X309, X346, X348, X364, X384, X401, X405, and X417 of SEQ ID NO:78,
(c) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises an amino acid substitution at a position selected from the group consisting of position: X76, X266, X270, X271, X273, X274, X313, X315, and X349 of SEQ ID NO:79, or
(d) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises an amino acid substitution at a position selected from the group consisting of position: X37, X182, X183, X186, and X265 of SEQ. ID NO. 204.
In another embodiment of the method,
(a) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises at least one of the features selected from the group consisting of: X32 is Tyr, Leu, Ala, Gly, Thr, His, Glu, Val, or Gln; X65 is Leu, His, Tyr, Val, Ser, Thr, Gly, or Glu; X67 is Ala or Gly; X70 is His, Ala, Cys, or Ser; X107 is Glu, Pro, Asn, or Thr; X108 is Phe, Trp, Ala, Ser, Arg, Gly, Tyr, His, Trp, or Glu; X109 is Gln, Met, Asp, Lys, Glu, Pro, His, Gly, Met, or Leu; X155 is Gln, Glu, or Gly; X158 is Asp, Gly, Glu, Ala, Pro, Thr, Ser, or Val; X159 is Ile, Cys, Pro, Leu, Ser, Trp, His, or Ala; X160 is His or Gln; X161 is Tyr or Gly; X162 is Leu, Arg, Ala, Gln, Gly, Lys, Ser, Glu, Tyr, or His; X163 is Gly or Asp; X164 is Val or Ala; X167 is Ala or Val; X286 is Asp or Arg;
(b) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises at least one of the features selected from the group consisting of: X302 is Ala or Thr; X305 is Leu or Met; X306 is Tyr, Ala, Met, Ile, Leu, Thr, Gly; X309 is Leu, Ala, Pro, Ser, or Arg; X346 is Asn, Ala, Ser, or Val; X348 is Cys, Ala, Thr, Leu, Lys, Met, or Trp; X364 is Thr or Lys; X384 is Tyr or Phe; X405 is Ile or Arg; X401 is Val or Leu; and X417 is Trp, Thr, or Leu;
(c) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises at least one of the features selected from the group consisting of: X76 is Asp or Gly; X266 is Leu, Val, or Met; X270 is Leu or Ile; X271 is Tyr, Phe, Leu, Met, or Ala; X274 is Leu, Ala, Met, or Gly; X313 is Cys, Phe, Ala, Val, or Ile; X315 is Met or Phe; and X349 is Tyr, Phe, or Trp; or
(d) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises at least one of the features selected from the group consisting of: X37 is Tyr, Ile, Gly, Val, Leu, Thr, or Ser; X182 is Asp, Gly, Ser, or Thr; X183 is Phe, Met, Tyr, or Ala; X186 is Leu, Ala, Met, or Val; and X265 is Asp or Arg.
In another embodiment of the method, the expression system comprises:
an aminoacyl-tRNA synthetase selected from the group consisting of SEQ ID NOs. 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100; and
a transfer RNA molecule encoded by a polynucleotide selected from the group consisting of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120.
In another embodiment of the method, the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), or (V) comprise(s):
In another embodiment of the method, the polypeptide comprised within the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), and (V), is a polypeptide selected from the group of polypeptides consisting of SEQ ID NOs 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, and 158.
In another embodiment of the method, the intein polypeptide comprised within the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), or (V), is a selected from the group consisting of a naturally occurring intein, an engineered variant of a naturally occurring intein, a fusion of the N-terminal and C-terminal fragments of a naturally occurring split intein and a fusion of the N-terminal and C-terminal fragments of an engineered split intein.
In another embodiment of the method, the intein is selected from the group consisting of Mxe GyrA (SEQ ID NO:1), eDnaB (SEQ ID NO:2), Hsp-NRC1 CDC21 (SEQ ID NO:3), Ceu ClpP (SEQ ID NO:4), Tag Pol-1 (SEQ ID NO:5), Tfu Pol-1 (SEQ ID NO:6), Tko Pol-1 (SEQ ID NO:7), Psp-GBD Pol (SEQ ID NO:8), Tag Pol-2 (SEQ ID NO:9), Thy Pol-1 (SEQ ID NO:10), Tko Pol-2 (SEQ ID NO:11), Tli Pol-1 (SEQ ID NO:12), Tma Pol (SEQ ID NO:13), Tsp-GE8 Pol-1 (SEQ ID NO:14), Tthi Pol (SEQ ID NO:15), Tag Pol-3 (SEQ ID NO:16), Tfu Pol-2 (SEQ ID NO:17), Thy Pol-2 (SEQ ID NO:18), Tli Pol-2 (SEQ ID NO:19), Tsp-GE8 Pol-2 (SEQ ID NO:20), Pab Pol-II (SEQ ID NO:21), Mtu-CDC1551 DnaB (SEQ ID NO:22), Mtu-H37Rv DnaB (SEQ ID NO:23), Rma DnaB (SEQ ID NO:24), Ter DnaE-1 (SEQ ID NO:25), Ssp GyrB (SEQ ID NO:26), Mfl GyrA (SEQ ID NO:27), Mgo GyrA (SEQ ID NO:28), Mkas GyrA (SEQ ID NO:29), Mle-TN GyrA (SEQ ID NO:30), Mma GyrA (SEQ ID NO:31), Ssp DnaX (SEQ ID NO:32), Pab Lon (SEQ ID NO:33), Mja PEP (SEQ ID NO:34), Afu-FRR0163 PRP8 (SEQ ID NO:35), Ani-FGSCA4 PRP8 (SEQ ID NO:36), Cne-A PRP8 (SEQ ID NO:37), Hca PRP8 (SEQ ID NO:38), Pch PRP8 (SEQ ID NO:39), Pex PRP8 (SEQ ID NO:40), Pvu PRP8 (SEQ ID NO:41), Mtu-H37Rv RecA (SEQ ID NO:42), Mtu-So93 RecA (SEQ ID NO:43), Mfl RecA (SEQ ID NO:44), Mle-TN RecA (SEQ ID NO:45), Nsp-PCC7120 RIR1 (SEQ ID NO:46), Ter RIR1-1 (SEQ ID NO:47), Pab RIR1-1 (SEQ ID NO:48), Pfu RIR1-1 (SEQ ID NO:49), Chy RIR1 (SEQ ID NO:50), Mth RIR1 (SEQ ID NO:51), Pab RIR1-3 (SEQ ID NO:52), Pfu RIR1-2 (SEQ ID NO:53), Ter RIR1-2 (SEQ ID NO:54), Ter RIR1-4 (SEQ ID NO:55), CIV RIR1 (SEQ ID NO:56), Ctr VMA (SEQ ID NO:57), Sce VMA (SEQ ID NO:58), Tac-ATCC25905 VMA (SEQ ID NO:59), Ssp DnaB (SEQ ID NO:60),
engineered variant(s) thereof, and
engineered variant(s) thereof wherein the N-terminal cysteine or serine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than cysteine or serine, or wherein the C-terminal asparagine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than asparagine.
In another embodiment of the method, the intein is a fusion product of a split intein selected from the group consisting of Ssp DnaE (SEQ ID NO:61-SEQ ID NO:62), Neq Pol (SEQ ID NO:63-SEQ ID NO:64), Asp DnaE (SEQ ID NO:65-SEQ ID NO:66), Npu-PCC73102 DnaE (SEQ ID NO:67-SEQ ID NO:68), Nsp-PCC7120 DnaE (SEQ ID NO:69-SEQ ID NO:70), Oli DnaE (SEQ ID NO:71-SEQ ID NO:72), Ssp-PCC7002 DnaE (SEQ ID NO:73-SEQ ID NO:74), Tvu DnaE (SEQ ID NO:75-SEQ ID NO:76),
engineered variant(s) thereof, and
engineered variant(s) thereof wherein the N-terminal cysteine or serine residue of the split intein N-domain of the engineered variant is mutated to any of the natural (or naturally occurring) amino acid residues other than cysteine or serine, or wherein the C-terminal asparagine residue of the split intein C-domain of the engineered variant is mutated to any of the natural (or naturally occurring) amino acid residues other than asparagine.
In another embodiment of the method,
the N-terminal tail polypeptide, (AA)m, of the precursor polypeptide of formula (I), (II), or (V) comprises the C-domain of a split intein, and
the C-terminal tail polypeptide, (AA)p, comprises the corresponding N-domain of the split intein.
In another embodiment of the method, the split intein C-domain is selected from the group consisting of Ssp DnaE-c (SEQ ID NO:62), Neq Pol-c (SEQ ID NO:64), Asp DnaE-c (SEQ ID NO:66), Npu-PCC73102 DnaE-c (SEQ ID NO:68), Nsp-PCC7120 DnaE-c (SEQ ID NO:70), Oli DnaE-c (SEQ ID NO:72), Ssp-PCC7002 DnaE-c (SEQ ID NO:74), Tvu DnaE-c (SEQ ID NO:76), and engineered variant(s) thereof; and the split intein N-domain is selected from the group consisting of Ssp DnaE-n (SEQ ID NO:61), Neq Pol-n (SEQ ID NO:63), Asp DnaE-n (SEQ ID NO:65), Npu-PCC73102 DnaE-n (SEQ ID NO:67), Nsp-PCC7120 DnaE-n (SEQ ID NO:69), Oli DnaE-n (SEQ ID NO:71), Ssp-PCC7002 DnaE-n (SEQ ID NO:73), Tvu DnaE-n (SEQ ID NO:75), and engineered variant(s) thereof.
In another embodiment of the method, the expression system is selected from the group consisting of a prokaryotic cell, an eukaryotic cell, and a cell-free expression system.
In another embodiment of the method, the prokaryotic cell is Escherichia coli.
In another embodiment of the method, the eukaryotic cell is a yeast, a mammalian, an insect or a plant cell.
In another embodiment of the method, any of polypeptides (AA)n, (AA)o, (AA)m, or (AA)p, is fully or partially genetically randomized so that a plurality of macrocyclic peptides is obtained upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG1 in Z or between the cysteine (Cys) residues and the side-chain functional groups FG1 and FG2 in Z2.
In another embodiment of the method, the method comprises fully or partially randomizing any of polypeptides (AA)n, (AA)o, (AA)m, or (AA)p, wherein, upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG1 in Z or between the cysteine (Cys) residues and the side-chain functional groups FG1 and FG2 in Z2, a plurality of macrocyclic peptides is produced.
Artificial, engineered and recombinant nucleic acid molecules and peptide sequences (or amino acid sequences) for use in this method are also provided.
A recombinant host cell is provided comprising a polypeptide of structure:
(AA)m-Z-(AA)n-Cys-(AA)p (I)
or
(AA)m-Cys-(AA)n-Z-(AA)p (II)
or
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p (V)
In certain embodiments, FG1 and FG2 can be either the same group among those groups listed above or two different groups among those groups listed above.
In one embodiment of the cell, the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, Nε-((2-bromoethoxy)carbonyl)-lysine, Nε-((2-chloroethoxy)carbonyl)-lysine, Nε-(buta-2,3-dienoyl)-lysine, Nε-acryl-lysine, Nε-crotonyl-lysine, Nε-(2-fluoro-acetyl)-lysine, and Nε-(2-chloro-acetyl)-lysine.
In another embodiment of the cell, the amino acid Z2 is selected from the group consisting of 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, Nε-(((1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, Nε-(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, Nε-((2,3-dibromopropoxy)carbonyl)-lysine, and Nε-((2,3-dichloropropoxy)carbonyl)-lysine.
In another embodiment of the cell, the polypeptide comprised within the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), and (V), is a polypeptide selected from the group of polypeptides consisting of SEQ ID NOs 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, and 158.
In another embodiment of the cell, the cell comprises a macrocyclic peptide produced by a thioether bond-forming reaction between the cysteine (Cys) residue and the FG1 functional group in the amino acid Z or between the cysteine (Cys) residues and the FG1 and FG2 functional groups in the amino acid Z2.
In another embodiment of the cell, the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, in the precursor polypeptides of formula (I), formula (II), or formula (V) comprise(s) an intein selected from the group consisting of a naturally occurring intein, an engineered variant of a naturally occurring intein, a fusion of the N-terminal and C-terminal fragments of a naturally occurring split intein and a fusion of the N-terminal and C-terminal fragments of an engineered split intein.
In another embodiment of the cell, the intein is selected from the group consisting of Mxe GyrA (SEQ ID NO:1), eDnaB (SEQ ID NO:2), Hsp-NRC1 CDC21 (SEQ ID NO:3), Ceu ClpP (SEQ ID NO:4), Tag Pol-1 (SEQ ID NO:5), Tfu Pol-1 (SEQ ID NO:6), Tko Pol-1 (SEQ ID NO:7), Psp-GBD Pol (SEQ ID NO:8), Tag Pol-2 (SEQ ID NO:9), Thy Pol-1 (SEQ ID NO:10), Tko Pol-2 (SEQ ID NO:11), Tli Pol-1 (SEQ ID NO:12), Tma Pol (SEQ ID NO:13), Tsp-GE8 Pol-1 (SEQ ID NO:14), Tthi Pol (SEQ ID NO:15), Tag Pol-3 (SEQ ID NO:16), Tfu Pol-2 (SEQ ID NO:17), Thy Pol-2 (SEQ ID NO:18), Tli Pol-2 (SEQ ID NO:19), Tsp-GE8 Pol-2 (SEQ ID NO:20), Pab Pol-II (SEQ ID NO:21), Mtu-CDC1551 DnaB (SEQ ID NO:22), Mtu-H37Rv DnaB (SEQ ID NO:23), Rma DnaB (SEQ ID NO:24), Ter DnaE-1 (SEQ ID NO:25), Ssp GyrB (SEQ ID NO:26), Mfl GyrA (SEQ ID NO:27), Mgo GyrA (SEQ ID NO:28), Mkas GyrA (SEQ ID NO:29), Mle-TN GyrA (SEQ ID NO:30), Mma GyrA (SEQ ID NO:31), Ssp DnaX (SEQ ID NO:32), Pab Lon (SEQ ID NO:33), Mja PEP (SEQ ID NO:34), Afu-FRR0163 PRP8 (SEQ ID NO:35), Ani-FGSCA4 PRP8 (SEQ ID NO:36), Cne-A PRP8 (SEQ ID NO:37), Hca PRP8 (SEQ ID NO:38), Pch PRP8 (SEQ ID NO:39), Pex PRP8 (SEQ ID NO:40), Pvu PRP8 (SEQ ID NO:41), Mtu-H37Rv RecA (SEQ ID NO:42), Mtu-So93 RecA (SEQ ID NO:43), Mfl RecA (SEQ ID NO:44), Mle-TN RecA (SEQ ID NO:45), Nsp-PCC7120 RIR1 (SEQ ID NO:46), Ter RIR1-1 (SEQ ID NO:47), Pab RIR1-1 (SEQ ID NO:48), Pfu RIR1-1 (SEQ ID NO:49), Chy RIR1 (SEQ ID NO:50), Mth RIR1 (SEQ ID NO:51), Pab RIR1-3 (SEQ ID NO:52), Pfu RIR1-2 (SEQ ID NO:53), Ter RIR1-2 (SEQ ID NO:54), Ter RIR1-4 (SEQ ID NO:55), CIV RIR1 (SEQ ID NO:56), Ctr VMA (SEQ ID NO:57), Sce VMA (SEQ ID NO:58), Tac-ATCC25905 VMA (SEQ ID NO:59), Ssp DnaB (SEQ ID NO:60),
engineered variant(s) thereof, and
engineered variant(s) thereof wherein the N-terminal cysteine or serine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than cysteine or serine, or wherein the C-terminal asparagine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than asparagine
In another embodiment of the cell, the intein is a fusion product of a split intein selected from the group consisting of Ssp DnaE (SEQ ID NO:61-SEQ ID NO:62), Neq Pol (SEQ ID NO:63-SEQ ID NO:64), Asp DnaE (SEQ ID NO:65-SEQ ID NO:66), Npu-PCC73102 DnaE (SEQ ID NO:67-SEQ ID NO:68), Nsp-PCC7120 DnaE (SEQ ID NO:69-SEQ ID NO:70), Oli DnaE (SEQ ID NO:71-SEQ ID NO:72), Ssp-PCC7002 DnaE (SEQ ID NO:73-SEQ ID NO:74), Tvu DnaE (SEQ ID NO:75-SEQ ID NO:76),
engineered variant(s) thereof,
engineered variant(s) thereof, wherein the N-terminal cysteine or serine residue of the split intein N-domain of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than cysteine or serine, or wherein the C-terminal asparagine residue of the split intein C-domain of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than asparagine.
In another embodiment of the cell, the cell comprises a macrocyclic peptide produced by a thioether bond-forming reaction between the cysteine (Cys) residue and the FG1 functional group in the amino acid Z or between the cysteine (Cys) residues and the FG1 and FG2 functional groups in the amino acid Z2, and an intein-catalyzed N-terminal splicing, C-terminal splicing, or self-splicing reaction.
In another embodiment of the cell, the N-terminal tail polypeptide, (AA)m, comprises the C-domain of a naturally occurring split intein, or of an engineered variant thereof, and the C-terminal tail polypeptide, (AA)p, comprises the N-domain of said split intein.
In another embodiment of the cell, the split intein C-domain is selected from the group consisting of Ssp DnaE-c (SEQ ID NO:62), Neq Pol-c (SEQ ID NO:64), Asp DnaE-c (SEQ ID NO:66), Npu-PCC73102 DnaE-c (SEQ ID NO:68), Nsp-PCC7120 DnaE-c (SEQ ID NO:70), Oli DnaE-c (SEQ ID NO:72), Ssp-PCC7002 DnaE-c (SEQ ID NO:74), Tvu DnaE-c (SEQ ID NO:76), and engineered variant(s) thereof; and the split intein N-domain is selected from the group consisting of Ssp DnaE-n (SEQ ID NO:61), Neq Pol-n (SEQ ID NO:63), Asp DnaE-n (SEQ ID NO:65), Npu-PCC73102 DnaE-n (SEQ ID NO:67), Nsp-PCC7120 DnaE-n (SEQ ID NO:69), Ohi DnaE-n (SEQ ID NO:71), Ssp-PCC7002 DnaE-n (SEQ ID NO:73), Tvu DnaE-n (SEQ ID NO:75), and engineered variant(s) thereof.
In another embodiment of the cell, the cell comprises a polycyclic peptide produced by a thioether bond-forming reaction between the cysteine (Cys) residue and the FG1 functional group in the amino acid Z or between the cysteine (Cys) residues and the FG1 and FG2 functional groups in the amino acid Z2, and a split intein-catalyzed trans-splicing reaction.
Embodiments are described herein with reference to the accompanying drawings, in which similar reference characters denote similar elements throughout the several views. It is to be understood that in some instances, various aspects of the embodiments may be shown exaggerated or enlarged to facilitate an understanding of the invention.
For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections set forth below.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
The singular forms “a,” “an,” and “the” used herein include plural referents unless the content clearly dictates otherwise.
The term “plurality” refers to two or more referents unless the content clearly dictates otherwise. The term “at least one” refers to one or more referents.
The term “functional group” as used herein refers to a contiguous group of atoms that, together, may undergo a chemical reaction under certain reaction conditions. Examples of functional groups are, among many others, —OH, —NH2, —SH, —(C═O)—, —N3, —C≡CH.
The term “aliphatic” or “aliphatic group” as used herein means a straight or branched C1-15 hydrocarbon chain that is completely saturated or that contains at least one unit of unsaturation, or a monocyclic C3-3 hydrocarbon, or bicyclic C8-12 hydrocarbon that is completely saturated or that contains at least one unit of unsaturation, but which is not aromatic (also referred to herein as “cycloalkyl”). For example, suitable aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl, alkynyl groups or hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkynyl)alkyl. The alkyl, alkenyl, or alkynyl group may be linear, branched, or cyclic and may contain up to 15, up to 8, or up to 5 carbon atoms. Alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, pentyl, and cyclopentyl groups. Alkenyl groups include, but are not limited to, propenyl, butenyl, and pentenyl groups. Alkynyl groups include, but are not limited to, propynyl, butynyl, and pentynyl groups.
The term “aryl” and “aryl group” as used herein refers to an aromatic substituent containing a single aromatic or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such as linked through a methylene or an ethylene moiety). An aryl group may contain from 5 to 24 carbon atoms, 5 to 18 carbon atoms, or 5 to 14 carbon atoms.
The terms “heteroatom” means nitrogen, oxygen, or sulphur, and includes, but is not limited to, any oxidized forms of nitrogen and sulfur, and the quaternized form of any basic nitrogen. Heteroatom further includes, but is not limited to, Se, Si, or P.
The term “heteroaryl” as used herein refer to an aryl group in which at least one carbon atom is replaced with a heteroatom. In various embodiments, a heteroaryl group is a 5- to 18-membered, a 5- to 14-membered, or a 5- to 10-membered aromatic ring system containing at least one heteroatom selected from the group consisting of oxygen, sulphur, and nitrogen atoms. Heteroaryl groups include, but are not limited to, pyridyl, pyrrolyl, furyl, thienyl, indolyl, isoindolyl, indolizinyl, imidazolyl, pyridonyl, pyrimidyl, pyrazinyl, oxazolyl, thiazolyl, purinyl, quinolinyl, isoquinolinyl, benzofuranyl, and benzoxazolyl groups.
A heterocyclic group may be any monocyclic or polycyclic ring system which contains at least one heteroatom and may be unsaturated or partially or fully saturated. The term “heterocyclic” thus includes, but is not limited to, heteroaryl groups as defined above as well as non-aromatic heterocyclic groups. In various embodiments, a heterocyclic group is a 3- to 18-membered, a 3- to 14-membered, or a 3- to 10-membered, ring system containing at least one heteroatom selected from the group consisting of oxygen, sulphur, and nitrogen atoms. Heterocyclic groups include, but are not limited to, the specific heteroaryl groups listed above as well as pyranyl, piperidinyl, pyrrolidinyl, dioaxanyl, piperazinyl, morpholinyl, thiomorpholinyl, morpholinosulfonyl, tetrahydroisoquinolinyl, and tetrahydrofuranyl groups.
A halogen atom may be a fluorine, chlorine, bromine, or iodine atom.
By “optionally substituted”, it is intended that in the any of the chemical groups listed above (e.g., alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, heterocyclic, triazolyl groups), at least one of the hydrogen atoms is optionally replaced with an atom or chemical group other than hydrogen. Specific examples of such substituents include, but are not limited to, halogen atoms, hydroxyl (—OH), sulfhydryl (—SH), substituted sulfhydryl, carbonyl carboxy (—COOH), amino (—NH2), nitro (—NO2), sulfo (˜SO2—OH), cyano (—C≡N), thiocyanato (—S—C≡N), phosphono (—P(O)OH2), alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, heterocyclic, alkylthiol, alkyloxy, alkylamino, arylthiol, aryloxy, or arylamino groups. Where “optionally substituted” modifies a series of groups separated by commas (e.g., “optionally substituted A, B, or C”; or “A, B, or C optionally substituted with”), it is intended that each of the groups (e.g., A, B, or C) is optionally substituted.
The term “heteroatom-containing aliphatic” as used herein refer to an aliphatic moiety where at least one carbon atom is replaced with a heteroatom, e.g., oxygen, nitrogen, sulphur, selenium, phosphorus, or silicon, and typically oxygen, nitrogen, or sulphur.
The terms “alkyl” and “alkyl group” as used herein refer to a linear, branched, or cyclic saturated hydrocarbon typically containing 1 to 24 carbon atoms, or 1 to 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl and the like.
The term “heteroatom-containing alkyl” as used herein refers to an alkyl moiety where at least one carbon atom is replaced with a heteroatom, e.g., oxygen, nitrogen, sulphur, phosphorus, or silicon, and typically oxygen, nitrogen, or sulphur.
The terms “alkenyl” and “alkenyl group” as used herein refer to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, or of 2 to 12 carbon atoms, containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like.
The term “heteroatom-containing alkenyl” as used herein refer to an alkenyl moiety where at least one carbon atom is replaced with a heteroatom.
The terms “alkynyl” and “alkynyl group” as used herein refer to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, or of 2 to 12 carbon atoms, containing at least one triple bond, such as ethynyl, n-propynyl, and the like.
The term “heteroatom-containing alkynyl” as used herein refer to an alkynyl moiety where at least one carbon atom is replaced with a heteroatom.
The term “heteroatom-containing aryl” as used herein refer to an aryl moiety where at least one carbon atom is replaced with a heteroatom.
The terms “alkoxy” and “alkoxy group” as used herein refer to an aliphatic group or a heteroatom-containing aliphatic group bound through a single, terminal ether linkage. In various embodiments, aryl alkoxy groups contain 1 to 24 carbon atoms, or contain 1 to 14 carbon atoms.
The terms “aryloxy” and “aryloxy group” as used herein refer to an aryl group or a heteroatom-containing aryl group bound through a single, terminal ether linkage. In various embodiments, aryloxy groups contain 5 to 24 carbon atoms, or contain 5 to 14 carbon atoms.
The term “substituent” refers to a contiguous group of atoms. Examples of “substituents” include, but are not limited to: alkoxy, aryloxy, alkyl, heteroatom-containing alkyl, alkenyl, heteroatom-containing alkenyl, alkynyl, heteroatom-containing alkynyl, aryl, heteroatom-containing aryl, alkoxy, heteroatom-containing alkoxy, aryloxy, heteroatom-containing aryloxy, halo, hydroxyl (—OH), sulfhydryl (—SH), substituted sulfhydryl, carbonyl (—CO—), thiocarbonyl, (—CS—), carboxy (—COOH), amino (—NH2), substituted amino, nitro (—NO2), nitroso (—NO), sulfo (˜SO2—OH), cyano (—C≡N), cyanato (—O—C≡N), thiocyanato (—S—C≡N), formyl (—CO—H), thioformyl (—CS—H), phosphono (—P(O)OH2), substituted phosphono, and phospho (—PO2).
The term “contact” as used herein with reference to interactions of chemical units indicates that the chemical units are at a distance that allows short range non-covalent interactions (such as Van der Waals forces, hydrogen bonding, hydrophobic interactions, electrostatic interactions, dipole-dipole interactions) to dominate the interaction of the chemical units. For example, when a protein is ‘contacted’ with a chemical species, the protein is allowed to interact with the chemical species so that a reaction between the protein and the chemical species can occur.
The term “bioorthogonal” as used herein with reference to a reaction, reagent, or functional group, indicates that such reaction, reagent, or functional group does not exhibit significant or detectable reactivity towards biological molecules such as those present in a bacterial, yeast or mammalian cell. The biological molecules can be, e.g., proteins, nucleic acids, fatty acids, or cellular metabolites.
In general, the term “mutant” or “variant” as used herein with reference to a molecule such as polynucleotide or polypeptide, indicates that such molecule has been mutated from the molecule as it exists in nature. In particular, the term “mutate” and “mutation” as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, but are not limited to, any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, or gene. A mutation can occur in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues. A mutation in a polynucleotide includes, but is not limited to, mutations arising within a protein-encoding region of a gene as well as mutations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A mutation in a coding polynucleotide such as a gene can be “silent”, i.e., not reflected in an amino acid alteration upon expression, leading to a “sequence-conservative” variant of the gene. A mutation in a polypeptide includes, but is not limited to, mutation in the polypeptide sequence and mutation resulting in a modified amino acid. Non-limiting examples of a modified amino acid include, but are not limited to, a glycosylated amino acid, a sulfated amino acid, a prenylated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like.
The term “engineer” refers to any manipulation of a molecule that result in a detectable change in the molecule, wherein the manipulation includes, but is not limited to, inserting a polynucleotide and/or polypeptide heterologous to the cell and mutating a polynucleotide and/or polypeptide native to the cell.
The term “nucleic acid molecule” as used herein refers to any chain of at least two nucleotides bonded in sequence. For example, a nucleic acid molecule can be a DNA or a RNA.
The term “peptide”, “polypeptide”, and “protein” as used herein refers to any chain of at least two amino acids bonded in sequence, regardless of length or post-translational modification.
The term “peptide-containing molecule” as used herein refers to a molecule that contains at least two amino acids.
The term “non-natural” and “unnatural” as used herein means being directly or indirectly made or caused to be made through human action. Thus, a “non-natural amino acid” is an amino acid that has been produced through human manipulation and does not occur in nature. The term “non-canonical amino acid” is equivalent in meaning to the terms “non-natural amino acid” or “unnatural amino acid”.
The term “cyclic” and “macrocyclic” as used herein means having constituent atoms forming a ring. Thus, a “macrocyclic peptide” is a peptide molecule that contains at least one ring formed by atoms comprised in the molecule. As such, the term “macrocyclic peptide” comprises peptides that contain at least two rings separated from each other via a polypeptide sequence (also referred to herein as “polycyclic peptides”) and peptides that contain at least two rings fused to each other (also referred to herein as “polycyclic peptides”). The term “macrocyclic peptide” also comprises peptides that contain two rings fused to each other (referred to herein also as “bicyclic peptides”).
The terms “cyclization” or “macrocyclization” as used herein refer to a process or reaction whereby a cyclic molecule is formed or is made to be formed.
The term “peptidic backbone” as used herein refers to a sequence of atoms corresponding to the main backbone of a natural protein.
The term “precursor polypeptide” or “polypeptide precursor” as used herein refers to a polypeptide that is capable of undergoing macrocyclization according to the methods disclosed herein.
The term “ribosomal polypeptide”, “ribosomally produced polypeptide” or “ribosomally derived polypeptide” as used herein refers to a polypeptide that is produced by action of a ribosome, and specifically, by the ribosomal translation of a messenger RNA encoding for such polypeptide. The ribosome can be a naturally occurring ribosome, e.g., a ribosome derived from an archea, procaryotic or eukaryotic organism, or an engineered (i.e., non-naturally occurring, artificial or synthetic) variant of a naturally occurring ribosome.
The term “intein” and “intein domain” as used herein refers to a naturally occurring or artificially constructed polypeptide sequence embedded within a precursor protein that can catalyze a splicing reaction during post-translational processing of the protein. The NEB Intein Registry (neb.com/neb/inteins.html) provides a list of known inteins.
The term “split intein” as used herein refers to an intein that has at least two separate components not fused to one another.
The term “splicing” as used herein refers to the process involving the cleavage of the main backbone of an intein-containing polypeptide by virtue of a reaction or process catalyzed by an intein or portions of an intein. “N-terminal splicing” refers to the cleavage of a polypeptide chain fused to the N-terminus of an intein, such reaction typically involving the scission of the thioester (or ester) bond formed via intein-catalyzed N→S (or N→O acyl) transfer, by action of a nucleophilic functional group or a chemical species containing a nucleophilic functional group. “C-terminal splicing” refers to the cleavage of a polypeptide chain fused to the C-terminus of an intein. “Self-splicing” as used herein refers to the process involving the cleavage of an intein from a polypeptide, within which the intein is embedded. “Trans-splicing” as used herein refers to a self-splicing process involving split inteins.
The term “affinity tag” as used herein refers to a polypeptide that is able to bind reversibly or irreversibly to an organic molecule, a metal ion, a protein, or a nucleic acid molecule.
The terms “vector” and “vector construct” as used herein refer to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can be readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include, but are not limited to, pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. The terms “express” and “expression” refer to allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
The term “fused” as used herein means being connected through at least one covalent bond. The term “bound” as used herein means being connected through non-covalent interactions. Examples of non-covalent interactions are van der Waals, hydrogen bond, electrostatic, and hydrophobic interactions. Thus, a “DNA-binding peptide” refers to a peptide capable of connecting to a DNA molecule via non-covalent interactions. The term “tethered” as used herein means being connected through non-covalent interactions or through covalent bonds. Thus, a “polypeptide tethered to a solid support” refers to a polypeptide that is connected to a solid support (e.g., surface, resin bead) either via non-covalent interactions or through covalent bonds.
Methods and compositions are provided for making artificial macrocyclic peptides from genetically encoded, ribosomally produced artificial polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue carrying a thiol-reactive functional group (referred to as FG1); and (b) a cysteine residue that is positioned either upstream or downstream of the non-canonical amino acid in the polypeptide sequence. These methods are based on the ability of the FG1-bearing amino acid and cysteine residue to react with each other after ribosomal synthesis of the polypeptide, so that a macrocyclic peptide carrying a side-chain-to-side-chain covalent (thioether) linkage is formed. Schematic representations of these embodiments are provided in
Methods and compositions are also provided for making macrocyclic peptides from genetically encoded, ribosomally produced, intein-fused polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue with a thiol-reactive functional group (referred to as FG1); (b) a cysteine residue positioned upstream or downstream of the non-canonical amino acid within the polypeptide sequence; and (c) an intein protein positioned upstream or downstream of the non-canonical amino acid or of the cysteine residue within the polypeptide sequence. These methods exploit the ability of this non-canonical amino acid and cysteine residue to react with each other after ribosomal synthesis of the precursor polypeptide, so that a macrocyclic peptide carrying a side-chain-to-side-chain covalent (thioether) linkage is formed. These methods also exploit the ability of the intein to undergo N-terminal splicing, C-terminal splicing, or self-splicing, so that the macrocyclic peptide is released upon intein splicing. Schematic representations of these embodiments are provided in
Methods and compositions are also provided for making artificial macrocyclic peptides from genetically encoded, ribosomally produced, split intein-fused polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue with a thiol-reactive functional group (referred to as FG1); (b) a cysteine residue positioned upstream or downstream of the non-canonical amino acid within the polypeptide sequence; and (c) a split intein domain positioned upstream or downstream of the non-canonical amino acid or the cysteine residue within the polypeptide sequence. These methods exploit the ability of this non-canonical amino acid and cysteine residue to react with each other after ribosomal synthesis of the precursor polypeptide, so that a macrocyclic peptide carrying a side-chain-to-side-chain covalent (thioether) linkage is formed. These methods also exploit the ability of the split intein to undergo trans-splicing, so that the bicyclic peptide is released upon split intein trans-splicing. Schematic representations of these embodiments are provided in
Methods and compositions are also provided for making artificial macrocyclic peptides from genetically encoded, ribosomally produced, split intein-fused polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue with two thiol-reactive functional groups (referred to as FG1 and FG2); (b) two cysteine residues positioned upstream and downstream of the non-canonical amino acid within the polypeptide sequence. These methods are based on the ability of the FG1/FG2-bearing amino acid to react with the two cysteine residues after ribosomal synthesis of the polypeptide, so that a bicyclic peptide carrying two side-chain-to-side-chain covalent (thioether) linkages is formed. Schematic representations of these embodiments are provided in
Artificial, engineered and recombinant nucleic acid molecules and peptide sequences (or amino acid sequences) for use in these methods are also provided.
In some embodiments, a method is provided for making an artificial macrocyclic peptide, the method comprising:
a. providing a nucleic acid molecule encoding for a polypeptide of structure:
(AA)m-Z-(AA)n-Cys-(AA)p (I)
or
(AA)m-Cys-(AA)n-Z-(AA)p (II)
In other embodiments, a method is provided for making an artificial macrocyclic peptide, the method comprising:
a. providing a nucleic acid molecule encoding for a polypeptide of structure:
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p (V)
According to the method, (AA)m is a N-terminal sequence comprising at least one amino acid, where AA corresponds to a generic amino acid residue and m corresponds to the number of amino acid residues composing such sequence. (AA)m is also referred to as “N-terminal tail”. (AA)p is a C-terminal sequence that has 0 or at least one amino acid, where AA corresponds to a generic amino acid residue and p corresponds to the number of amino acid residues composing such sequence. (AA)p is also referred to as “C-terminal tail”. (AA)n (and (AA)o, when present) is a peptide sequence of variable length (also referred to as “target peptide sequence”), where AA corresponds to a generic amino acid residue and n corresponds to the number of amino acid residues composing such peptide sequence. Cys is a cysteine amino acid residue. Z is an amino acid that carries a side-chain functional group FG1, which can react with the side-chain sulfhydryl group (—SH) of the cysteine residue to form a stable thioether bond.
As disclosed herein, the ability of an artificial polypeptide of formula (I) or (II) (also referred herein to as “precursor polypeptide”) to produce a macrocyclic peptide is conferred by the ability of the nucleophilic sulfhydryl group carried by the cysteine residue to react intramolecularly with the electrophilic functional group FG1 carried by the amino acid Z, thereby forming a covalent, inter-side-chain thioether bond. Depending on the nature of FG1, this reaction proceeds via a thiol-mediated nucleophilic substitution reaction, a thiol-mediated Michael-type addition reaction, or a radical thiol-ene or thiol-yne reaction. Whereas the electrophilic functional group FG1 in the precursor polypeptide could in principle react intermolecularly with free cysteine or other thiol-containing molecules contained in the expression system (e.g., glutathione), it was discovered by the inventors that appropriate functional groups FG1 can be found so that the desired intramolecular thioether-bond forming reaction occurs exclusively or preferentially over the undesired intermolecular side-reactions. This result can be achieved because of the spatial proximity between the nucleophilic cysteine residue and the electrophilic Z amino acid, resulting in an increased effective concentration of the reacting species (i.e., —SH and FG1 groups, respectively) in the intramolecular settings as compared to the intermolecular settings, which in turn favors the intramolecular peptide cyclization reaction over undesired intermolecular reactions. Similar considerations can be made in the context of certain embodiments, wherein a precursor polypeptide of formula (V) along with a bifunctional cysteine-reactive amino acid capable of forming thioether bonds with two cysteine residues within the polypeptide (residue Z2) is used.
A first advantage of the methods described herein is that they provide a highly versatile approach for the preparation of structurally diverse artificial macrocyclic peptides. Indeed, they offer multiple opportunities toward the structural and functional diversification of these compounds, e.g., through variation of the length and composition of the target peptide sequence ((AA)n), variation of the structure of the amino acid Z, variation of the position of the amino acid Z relative to the cysteine residue (e.g., precursor polypeptide (I) versus (II)), variation of the length and composition of the N-terminal tail ((AA)m), and variation of the length and composition of the C-terminal tail ((AA)p). Further structural diversification can be achieved by combining multiple Z/Cys pairs within the same precursor polypeptide or by using bifunctional cysteine-reactive amino acids (Z2) in order to obtain polycyclic and bicyclic peptides. Accordingly, and because of the genetically encoded and ribosomal nature of the precursor polypeptides, the methods and compositions described herein can be used to produce vast libraries of structurally and functionally diverse macrocyclic peptides, which can be screened to identify compounds that can modulate, inhibit or promote interactions between biomolecules (e.g., enzymes, proteins, nucleic acids) for a variety of applications, including drug discovery.
A second advantage of the methods disclosed herein is that they produce peptide molecules whose conformational flexibility is restrained by virtue of at least one intramolecular thioether linkage. As illustrated in Example 8, this feature can confer these molecules with advantageous properties such as, for example, enhanced binding affinity, increased stability against proteolysis, and/or more favorable membrane-crossing properties, as compared to linear peptides or peptides lacking the intramolecular thioether linkage. In addition, the thioether linkage is redox and chemically stable in biological milieu, including the intracellular environment.
A third advantage of the methods disclosed herein is they allow for the preparation of macrocyclic peptides from genetically encoded, ribosomally produced polypeptides. Accordingly, these macrocyclic peptides can be produced as fused to a genetically encoded affinity tag, DNA-binding protein/peptide, protein-binding protein/peptide, fluorescent protein, or enzyme, which can be achieved via the introduction of one or more of these elements within the N-terminal tail and/or within the C-terminal tail of the precursor polypeptide. On one hand, these tags/proteins/enzymes can be useful to facilitate the purification and/or immobilization of the macrocyclic peptides for functional screening as demonstrated in Examples 4, 5 and 8. On the other hand, very large libraries of macrocyclic peptides can be rapidly and cost-effectively produced utilizing precursor polypeptides in which the target peptide sequence ((AA)n), N-terminal tail ((AA)m), and/or C-terminal tail ((AA)m), is partially or fully randomized genetically. These features of the method can allow one to produce macrocyclic peptides as fused to a carrier protein of a display system such as phage display, mRNA display, ribosome display, yeast display, and the like. So, for example, the methods described herein allow one to generate combinatorial libraries of macrocyclic peptides that are fused to the pIII protein of M13 bacteriophage. These phage-displayed macrocyclic peptide libraries can be then ‘panned’ against a target biomolecule of interest according to procedures well known in the art (Lane and Stephen 1993; Giebel, Cass et al. 1995; Sidhu, Lowman et al. 2000) in order to identify macrocyclic peptide binders or inhibitors of such biomolecule.
A fourth advantage of the methods described herein is that they also enable the production of macrocyclic peptides inside a cell-based expression host such as a bacterial, yeast, insect, or mammalian cell. Intracellular production of the macrocyclic peptide can then be coupled to an (intra)cellular reporter system, phenotypic screen, or selection system, in order to identify a macrocyclic peptide capable of inhibiting or activating a certain cellular process, biomolecule, or enzymatic reaction linked to the reporter output, phenotype, or cell survival, respectively.
A fifth advantage of the methods disclosed herein is that the production of the macrocyclic peptides can be carried out under physiological conditions (e.g., in aqueous buffer, neutral pH, physiological temperature) and in complex biological media (e.g., inside a cell, in cell lysate) and in the presence of biological molecules (proteins, nucleic acids, cell metabolites) and biological material. One implication of this is that the production of macrocyclic peptides according to the methods disclosed herein can be coupled to one of the several techniques known in the art for the display and high-throughput screening of biological peptide libraries.
Because of the aforementioned advantageous features, the methods described herein can be useful to greatly accelerate and facilitate the discovery of bioactive peptide-based compounds as potential drug molecules and chemical probes or the identification of lead structures for the development of new chemical probes and drugs.
In some embodiments, Z is an amino acid of structure:
wherein FG1 is a functional group selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″), —SO2C(R′)═C(R′)(R″), —C(O)C(R′)═C(R′)(R″), —C(R′)═C(R′)C(O)OR′, —C(R′)═C(R′)C(O)N(R′)(R″), —C(R′)═C(R′)—CN, —C(R′)═C(R′)—NO2, —C≡C—C(O)OR′, —C≡C—C(O)N(R′)(R″), unsubstituted or substituted oxirane, unsubstituted or substituted aziridine, 1,2-oxathiolane 2,2-dioxide, 4-fluoro-1,2-oxathiolane 2,2-dioxide, and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; and
wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups.
In some embodiments, Z is an amino acid of structure (IV) wherein Y is a linker group selected from the group consisting of C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, C5-C24 aryloxy groups.
In some embodiments, Z is an amino acid of structure (IV) wherein Y is a linker group selected from —CH2—C6H4—, —CH2—C6H4—O—, —CH2—C6H4—NH—, —(CH2)4—, —(CH2)4NH—, —(CH2)4NHC(O)—, and —(CH2)4NHC(O)O—.
In specific embodiments, the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, Nε-((2-bromoethoxy)carbonyl)-lysine, Nε-((2-chloroethoxy)carbonyl)-lysine, Nε-(buta-2,3-dienoyl)-lysine, Nε-acryl-lysine, Nε-crotonyl-lysine, Nε-(2-fluoro-acetyl)-lysine, and Nε-(2-chloro-acetyl)-lysine.
In some embodiments, Z2 is an amino acid of structure:
wherein FG1 and FG2 are a functional group independently selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″), —SO2C(R′)═C(R′)(R″), —C(O)C(R′)═C(R′)(R″), —C(R′)═C(R′)C(O)OR′, —C(R′)═C(R′)C(O)N(R′)(R″), —C(R′)═C(R′)—CN, —C(R′)═C(R′)—NO2, —C≡C—C(O)OR′, —C≡C—C(O)N(R′)(R″), unsubstituted or substituted oxirane, unsubstituted or substituted aziridine, 1,2-oxathiolane 2,2-dioxide, 4-fluoro-1,2-oxathiolane 2,2-dioxide, and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R and R′ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; and
wherein Y2, Y3, and L are linker groups selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, aryloxy groups.
In some embodiments, Z2 is an amino acid of structure (VI) wherein Y2 is a linker group selected from the group consisting of C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, C5-C24 aryloxy groups.
In some embodiments, Z2 is an amino acid of structure (VI) wherein Y2 is a linker group selected from the group consisting of —CH2—C6H4—, —CH2—C6H4—O—, —CH2—C6H4—NH—, —CH2—C6H4—OCH2—, —(CH2)4NH—, —(CH2)4NHC(O)—, —(CH2)4NHC(O)O—, —(CH2)4NHC(O)OCH2—,
In specific embodiments, the amino acid Z2 is selected from the group consisting of of 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, Nε-4(1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, Nε-4(1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, Nε-((2,3-dibromopropoxy)carbonyl)-lysine, and Nε-((2,3-dichloropropoxy)carbonyl)-lysine.
Artificial nucleic acid molecules for use according to the methods provided herein include, but are not limited to, those that encode for a polypeptide of general formula (I), (II), or (V) as defined above. The codon encoding for the amino acid Z (or Z2) in these polypeptides can be one of the 61 sense codons of the standard genetic code, a stop codon (TAG, TAA, TGA), or a four-base frameshift codon (e.g., TAGA, AGGT, CGGG, GGGT, CTCT). In some embodiments, the codon encoding for the amino acid Z (or Z2) within the nucleotide sequence encoding for the precursor polypeptide of formula (I), (II) or (V) is an amber stop codon (TAG), an ochre stop codon (TAA), an opal stop codon (TGA), or a four-base frameshift codon (see Example 2). In other embodiments, the codon encoding for Z (or Z2) in the nucleotide sequence encoding for these precursor polypeptides is the amber stop codon, TAG, or the 4-base codon, TAGA.
The non-canonical amino acid Z (or Z2) can be introduced into the precursor polypeptide through direct incorporation during ribosomal synthesis of the precursor polypeptide, or generated post-translationally through enzymatic or chemical modification of the precursor polypeptide, or by a combination of these procedures. In some embodiments, the amino acid Z (or Z2) is introduced into the precursor polypeptide during ribosomal synthesis of the precursor polypeptide via either stop codon suppression or four-base frameshift codon suppression. In other embodiments, the amino acid Z (or Z2) is introduced into the precursor polypeptide during ribosomal synthesis of the precursor polypeptide via amber (TAG) stop codon suppression or via 4-base TAGA codon suppression.
Several methods are known in the art for introducing a non-canonical amino acid into a recombinant or in vitro translated artificial polypeptide, any of which can be applied for preparing artificial precursor polypeptides suitable for the methods disclosed herein. These art-known methods include, but are not limited to, methods for suppression of a stop codon or of a four-based frameshift codon with a non-canonical amino acid using engineered (i.e., non-naturally occurring, artificial or synthetic) tRNA/aminoacyl-tRNA synthetase (AARS) pairs (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011; Lang and Chin 2014). Examples of tRNA/aminoacyl-tRNA synthetase (AARS) pairs used for this purpose include, but are not limited to, engineered variants of Methanococcus jannaschii AARS/tRNA pairs (e.g., TyrRS/tRNATyr), of Saccharomyces cerevisiae AARS/tRNA pairs (e.g., AspRS/tRNAAsp, GlnRS/tRNAGln, TyrRS/tRNATyr, and PheRS/tRNAPhe), of Escherichia coli AARS/tRNA pairs (e.g., TyrRS/tRNATyr, LeuRS/tRNALue), of Methanosarcina mazei AARS/tRNA pairs (PylRS/tRNAPyl), and of Methanosarcina mazei AARS/tRNA pairs (PylRS/tRNAPyl) (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011; Lang and Chin 2014). Alternatively, natural or engineered four-codon suppressor tRNAs and their cognate aminoacyl-tRNA synthetases can be used for the same purpose (Anderson, Wu et al. 2004; Rodriguez, Lester et al. 2006; Neumann, Slusarczyk et al. 2010; Neumann, Wang et al. 2010). Alternatively, a non-canonical amino acid can be incorporated into a polypeptide using chemically (Dedkova, Fahmi et al. 2003) or enzymatically (Bessho, Hodgson et al. 2002; Hartman, Josephson et al. 2006) aminoacylated tRNA molecules and using a cell-free protein expression system in the presence of the aminoacylated tRNA molecules (Kourouklis, Murakami et al. 2005; Murakami, Ohta et al. 2006). Alternatively, a non-canonical amino acid can be incorporated into a polypeptide by exploiting the promiscuity of wild-type aminoacyl-tRNA synthetase enzymes using a cell-free protein expression system, in which one or more natural amino acids are replaced with structural analogs(Josephson, Hartman et al. 2005; Hartman, Josephson et al. 2007). Any of these methods can be used to introduce an unnatural amino acid of the type (III), (IV), (VI) or (VII) into the precursor polypeptide for the purpose of generating macrocyclic peptides according to the methods disclosed herein.
In some embodiments, the non-canonical amino acid Z (or Z2) is incorporated into the precursor polypeptide via stop codon or four-base codon suppression methods using an engineered AARS/tRNA pair derived from Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS) and its cognate tRNA (MjtRNATyr), an engineered AARS/tRNA pair derived from Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPy1RS) and its cognate tRNA (tRNAPyl), an engineered AARS/tRNA pair derived from Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS) and its cognate tRNA (tRNAPyl), or an engineered AARS/tRNA pair derived from Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS) and its cognate tRNA (EctRNATyr).
In the characterization of the aminoacyl-tRNA synthetase enzymes disclosed herein, these enzymes can be described in reference to the amino acid sequence of a naturally occurring aminoacyl-tRNA synthetase or another engineered aminoacyl-tRNA synthetase. As such, the amino acid residue is determined in the aminoacyl-tRNA synthetase enzyme beginning from the first amino acid after the initial methionine (M) residue (i.e., the first amino acid after the initial methionine M represents residue position 1). It will be understood that the initiating methionine residue may be removed by biological processing machinery such as in a host cell or in vitro translation system, to generate a mature protein lacking the initiating methionine residue. The amino acid residue position at which a particular amino acid or amino acid change is present is sometimes described herein as “Xn”, or “position n”, where n refers to the residue position.
In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of Methanococcus jannaschii tRNATyr as encoded by a nucleotide of sequence SEQ ID NO: 101, 102, 103, or 104; and an engineered variant of Methanococcus jannaschii tyrosyl-tRNA synthetase (SEQ ID NO: 77), said variant comprising an amino acid change at at least one of the following amino acid positions of SEQ ID NO:77: X32, X63, X65, X70, X107, X108, X109, X155, X158, X159, X160, X161, X162, X163, X164, X167, and X286.
In other embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide consists of a Methanococcus jannaschii tRNATyr variant selected from the group of tRNA molecules encoded by the nucleotide sequence of SEQ ID NOs: 101, 102, 103, and 104; and a Methanococcus jannaschii tyrosyl-tRNA synthetase variant selected from the group of polypeptides of SEQ ID NOs: 77, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90.
In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of Methanosarcina species tRNAPyl or Desulfitobacterium hafniense tRNAPyl as encoded by a nucleotide of sequence SEQ ID NO: 105, 106, 107, 108, 109, 110, 111, or 112; and an engineered variant of Methanosarcina mazei pyrrolysyl-tRNA synthetase (SEQ ID NO: 78), said variant comprising an amino acid change at least one of the following amino acid positions of SEQ ID NO:78: X302, X305, X306, X309, X346, X348, X364, X384, X401, X405, and X417.
In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of Methanosarcina species tRNAPyl or Desulfitobacterium hafniense tRNAPyl as encoded by a nucleotide of sequence SEQ ID NO: 105, 106, 107, 108, 109, 110, 111, or 112; and an engineered variant of Methanosarcina barkeri pyrrolysyl-tRNA synthetase (SEQ ID NO: 79), said variant comprising an amino acid change at at least one of the following amino acid positions of SEQ ID NO: 79: X76, X266, X270, X271, X273, X274, X313, X315, and X349.
In other embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide consists of a tRNAPyl variant selected from the group of tRNA molecules encoded by the nucleotide sequence of SEQ ID NO: 105, 106, 107, 108, 109, 110, 111, and 112; and a pyrrolysyl-tRNA synthetase variant selected from the group of polypeptides of SEQ ID NOs: 78, 79, 91, 92, 93, 94, 95, and 96.
In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of Escherichia coli tRNATyr or Bacillus stearothermophilus tRNATyr as encoded by a nucleotide of sequence SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, or 120; and an engineered variant of Escherichia coli tyrosyl-tRNA synthetase (SEQ ID NO: 80), said variant comprising an amino acid change at least one of the following amino acid positions of SEQ ID NO: 80: X37, X182, X183, X186, and X265.
In other embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide consists of a tRNATyr variant selected from the group of tRNA molecules encoded by the nucleotide sequence of SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, and 120; and a E. coli tyrosyl-tRNA synthetase variant selected from the group of polypeptides of SEQ ID NOs: 80, 97, 98, 99, and 100.
In some embodiments, the aminoacyl-tRNA synthetase used for incorporating the amino acid Z (or Z2) into the precursor polypeptide can have additionally at least one amino acid residue differences at positions not specified by an X above as compared to the sequence SEQ ID NO: 77, 78, 79, or 80. In some embodiments, the differences can be 1-2, 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-75, 1-100, 1-150, or 1-200 amino acid residue differences at other positions not defined by X above.
In some embodiments, the suppressor tRNA molecule used for incorporating the amino acid Z (or Z2) into the precursor polypeptide can have additionally at least one nucleotide difference as compared to the sequence encoded by the gene of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. In some embodiments, the differences can be 1-2, 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, or 1-60 nucleotide differences as compared to the sequences encoded by these genes.
In another embodiment of the method, the engineered variant of Methanococcus jannaschii tyrosyl-tRNA synthetase (SEQ ID NO: 77) comprises at least one of the features selected from the group consisting of: X32 is Tyr, Leu, Ala, Gly, Thr, His, Glu, Val, or Gln; X65 is Leu, His, Tyr, Val, Ser, Thr, Gly, or Glu; X67 is Ala or Gly; X70 is His, Ala, Cys, or Ser; X107 is Glu, Pro, Asn, or Thr; X108 is Phe, Trp, Ala, Ser, Arg, Gly, Tyr, His, Trp, or Glu; X109 is Gln, Met, Asp, Lys, Glu, Pro, His, Gly, Met, or Leu; X155 is Gln, Glu, or Gly; X158 is Asp, Gly, Glu, Ala, Pro, Thr, Ser, or Val; X159 is Ile, Cys, Pro, Leu, Ser, Trp, His, or Ala; X160 is His or Gln; X161 is Tyr or Gly; X162 is Leu, Arg, Ala, Gln, Gly, Lys, Ser, Glu, Tyr, or His; X163 is Gly or Asp; X164 is Val or Ala; X167 is Ala or Val; X286 is Asp or Arg.
In another embodiment of the method, the engineered variant of Methanosarcina mazei pyrrolysyl-tRNA synthetase (SEQ ID NO: 78) comprises at least one of the features selected from the group consisting of: X302 is Ala or Thr; X305 is Leu or Met; X306 is Tyr, Ala, Met, Ile, Leu, Thr, Gly; X309 is Leu, Ala, Pro, Ser, or Arg; X346 is Asn, Ala, Ser, or Val; X348 is Cys, Ala, Thr, Leu, Lys, Met, or Trp; X364 is Thr or Lys; X384 is Tyr or Phe; X405 is Ile or Arg; X401 is Val or Leu; X417 is Trp, Thr or Leu.
In another embodiment of the method, the engineered variant of Methanosarcina barkeri pyrrolysyl-tRNA synthetase (SEQ ID NO: 79) comprises at least one of the features selected from the group consisting of: X76 is Asp or Gly; X266 is Leu, Val, or Met; X270 is Leu or Ile; X271 is Tyr, Phe, Leu, Met, or Ala; X274 is Leu, Ala, Met, or Gly; X313 is Cys, Phe, Ala, Val, or Ile; X315 is Met or Phe; X349 is Tyr, Phe, or Trp.
In another embodiment of the method, the engineered variant of Escherichia coli tyrosyl-tRNA synthetase (SEQ ID NO: 80) comprises at least one of the features selected from the group consisting of: X37 is Tyr, Ile, Gly, Val, Leu, Thr, or Ser; X182 is Asp, Gly, Ser, or Thr; X183 is Phe, Met, Tyr, or Ala; X186 is Leu, Ala, Met, or Val; X265 is Asp or Arg.
An aspect of the methods disclosed herein is the identification and selection of a suitable aminoacyl-tRNA synthetase for incorporating an amino acid Z (or Z2) as defined above, into the artificial precursor polypeptide. Various methods are known in the art to evaluate and quantify the relative efficiency of a given wild-type or engineered aminoacyl-tRNA synthetase to incorporate a non-canonical amino acid into a protein (Young, Young et al. 2011). Any of these methods can be used to guide the identification and choice of a suitable aminoacyl-tRNA synthetase for incorporating a desired amino acid Z (or Z2) into the precursor polypeptide. For example, such efficiency can be measured via a fluorescence assay based on the expression of a reporter fluorescent protein (e.g., green fluorescent protein), whose encoding gene has been modified to contain a codon to be suppressed (e.g., amber stop codon). Expression of the reporter fluorescent protein is then induced in a suitable expression system (e.g., an E. coli or yeast cell) in the presence of the aminoacyl-tRNA synthetase to be tested, a cognate suppressor tRNA (e.g., amber stop codon suppressor tRNA), and the desired non-canonical amino acid. Under these conditions, the relative amount of the expressed (i.e., ribosomally produced) fluorescent protein is linked to the relative efficiency of the aminoacyl-tRNA synthetase to charge the cognate suppressor tRNA with the non-canonical amino acid, which can thus be quantified via fluorimetric means. A demonstration of how this procedure can be applied for selecting an aminoacyl-tRNA synthetase/suppressor tRNA pair for incorporating a desired amino acid Z (or Z2) into the precursor polypeptide is provided in Example 3.
If necessary, the ability of a given aminoacyl-tRNA synthetase/suppressor tRNA pair to incorporate a target non-canonical amino acid into a protein can be improved by means of rational design or directed evolution. While the fluorescence-based method described above can be used to screen several hundreds of engineered aminoacyl-tRNA synthetase variants and/or suppressor tRNA variants for this purpose, higher throughput procedures are also known in the art, which are, for example, based on selection systems (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011). One such system involves introducing a library of mutated aminoacyl-tRNA synthetases and/or of mutated suppressor tRNAs into a suitable cell-based expression host (e.g., E. coli or yeast cells), whose survival under a suitable selective medium or growth conditions is dependent upon the functionality of the aminoacyl-tRNA synthetase/suppressor tRNA pair. This can be achieved, for example, by introducing a stop codon or four-base codon that is to be suppressed, into a gene encoding for a protein or enzyme essential for survival of the cell, such as a protein or enzyme conferring resistance to an antibiotic. In this case, the ability of the aminoacyl-tRNA synthetase/suppressor tRNA pair to incorporate the desired non-canonical amino acid into the selection marker protein is linked to the survival of the host, thereby enabling the rapid isolation of suitable aminoacyl-tRNA synthetase/suppressor tRNA pair(s) for the incorporation of a particular non-canonical amino acid from very large engineered libraries. The selectivity of these aminoacyl-tRNA synthetase/suppressor tRNA pair toward the desired non-canonical amino acid over the twenty natural amino acids can be further improved by iterative rounds of positive and negative selection as described in (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011). Procedures such as those described above can be thus applied to generate and isolate an engineered aminoacyl-tRNA synthetase/suppressor tRNA pair suitable for incorporation of the amino acid Z as defined above, into the precursor polypeptide.
Engineered aminoacyl-tRNA synthetase/tRNA pairs for the incorporation of the amino acid Z (or Z2) into the precursor polypeptide can be prepared via mutagenesis of the polynucleotide encoding for the aminoacyl-tRNA synthetase enzymes of SEQ ID NOs: 77, 78, 79, 80, or an engineered variant thereof; and via mutagenesis of the tRNA-encoding polynucleotides of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or an engineered variant thereof. Many mutagenesis methods are known in the art and these include, but are not limited to, site-directed mutagenesis, site-saturation mutagenesis, random mutagenesis, cassette-mutagenesis, DNA shuffling, homologous recombination, non-homologous recombination, site-directed recombination, and the like. Detailed description of art-known mutagenesis methods can be found, among other sources, in U.S. Pat. Nos. 5,605,793; 5,830,721; 5,834,252; WO 95/22625; WO 96/33207; WO 97/20078; WO 97/35966; WO 98/27230; WO 98/42832; WO 99/29902; WO 98/41653; WO 98/41622; WO 98/42727; WO 00/18906; WO 00/04190; WO 00/42561; WO 00/42560; WO 01/23401; WO 01/64864.
As described above, the engineered aminoacyl-tRNA synthetases and cognate suppressor tRNA obtained from mutagenesis of SEQ ID NO:77 to 80, and from mutagenesis of SEQ ID NO:101 to 120, can be screened for identifying aminoacyl-tRNA synthetase/suppressor tRNA pairs being able, or having improved ability as compared to the corresponding wild-type enzyme/tRNA molecule, to incorporate the amino acid Z (or Z2) into the precursor polypeptide.
In some embodiments, the engineered aminoacyl-tRNA synthetase used in the method comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or more identical to the sequence SEQ ID NOs: 77, 78, 79, or 80.
In some embodiments, the engineered suppressor tRNA used in the method is encoded by a polynucleotide comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99% or more identical to the sequence SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120.
The target peptide sequence, (AA)n, in the precursor polypeptide of formula (I), (II) and (V) and the second target peptide sequence, (AA)o, in the precursor polypeptide of formula (V), can be a polypeptide comprising 1 to 1,000 amino acid residues. In some embodiments, (AA)m (and (AA)o) consists of a polypeptide comprising 1 to 50 amino acid residues and, in other embodiments, (AA)n (and (AA)o) consists of a polypeptide comprising 1 to 20 amino acid residues.
The N-terminal tail, (AA)m, in the precursor polypeptide of formula (I), (II), and (V) can be a polypeptide comprising 1 to 10,000 amino acid residues. In some embodiments, (AA)m consists of a polypeptide comprising 1 to 1,000 amino acid residues and, in other embodiments, (AA)m consists of a polypeptide comprising 1 to 600 amino acid residues.
The C-terminal tail, (AA)p, in the precursor polypeptide of formula (I), (II), and (V) may not be present, and when present, it can be a polypeptide comprising 1 to 10,000 amino acid residues. When present, (AA)m consists, in some embodiments, of a polypeptide comprising 1 to 1,000 amino acid residues and, in other embodiments, (AA)m consists of a polypeptide comprising 1 to 600 amino acid residues.
The N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), and (V) can comprise a polypeptide affinity tag, a DNA-binding polypeptide, a protein-binding polypeptide, an enzyme, a fluorescent protein, an intein protein, or a combination of these polypeptides.
Introduction of a polypeptide affinity tag within the N-terminal tail and/or C-terminal tail of the precursor polypeptide results in macrocyclic peptides fused to such polypeptide affinity tag. Such affinity tags can be useful for isolating, purifying, and/or immobilizing onto a solid support the macrocyclic peptides generated according to the methods disclosed herein. Accordingly, in some embodiments, the N-terminal tail, C-terminal tail, or both, of the precursor polypeptides comprise at least one polypeptide affinity tags selected from the group consisting of a polyarginine tag (e.g., RRRRR) (SEQ ID NO:121), a polyhistidine tag (e.g., HHHHHH) (SEQ ID NO:122), an Avi-Tag (SGLNDIFEAQKIEWHELEL) (SEQ ID NO:123), a FLAG tag (DYKDDDDK) (SEQ ID NO:124), a Strep-tag II (WSHPQFEK) (SEQ ID NO:125), a c-myc tag (EQKLISEEDL) (SEQ ID NO:126), a S tag (KETAAAKFERQHMDS) (SEQ ID NO:127), a calmodulin-binding peptide (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO:128), a streptavidin-binding peptide (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO:129), a chitin-binding domain (SEQ ID NO:130), a glutathione S-transferase (GST; SEQ ID NO:131), a maltose-binding protein (MBP; SEQ ID NO:132), streptavidin (SEQ ID NO:133), and engineered variants thereof. These aspects are illustrated in Example 2.
The N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), and (V) can comprise a reporter protein or enzyme. This approach will result in the formation of macrocyclic peptides fused to a reporter protein or enzyme, which can be useful to facilitate the functional screening of said macrocyclic peptides. Accordingly, in some embodiments, the N-terminal tail, (AA)m and/or the C-terminal tail, (AA)p, in the precursor polypeptides of formula (I), (II), and (V) comprise at least one polypeptide selected from the group consisting of green fluorescent protein (SEQ ID NO:134), luciferase (SEQ ID NO:135), alkaline phosphatase (SEQ ID NO:136), and engineered variants thereof.
The N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), or (V) can comprise a protein or enzyme that is part of a display system such as, for example, a phage display (e.g., M13, T7, or lambda phage display), a yeast display, a bacterial display, a DNA display, a plasmid display, a CIS display, a ribosome display, or a mRNA display system. As mentioned above, this approach can be useful for generating large libraries of macrocyclic peptides which are physically linked to, or compartmentalized with the polynucleotide sequence that encodes for the corresponding precursor polypeptides. In turn, this approach can be useful toward isolating functional macrocyclic peptides that are able to bind, inhibit or activate a certain target biomolecule (e.g., protein, enzyme, DNA or RNA molecule) or target biomolecular interaction.
Accordingly, in some embodiments, the N-terminal tail, (AA)m, comprises a polypeptide selected from the group consisting of M13 phage coat protein pVI (SEQ ID NO:137), T7 phage protein 10A (SEQ ID NO:138), T7 phage protein 10B (SEQ ID NO:139), E. coli NlpA (SEQ ID NO:140), E. coli OmpC (SEQ ID NO:141), E. coli FadL (SEQ ID NO:142), E. coli Lpp-OmpA (SEQ ID NO:143), E. coli PgsA (SEQ ID NO:144), E. coli EaeA (SEQ ID NO:145), S. cerevisiae Aga2p (SEQ ID NO:146), S. cerevisiae Flo 1p (SEQ ID NO:147), human NF-κB p50 protein (SEQ ID NO:148), M13 phage coat protein pIII leader sequence (SEQ ID NO:149), M13 phage coat protein pVIII leader sequence (SEQ ID NO:150), M13 phage protein pVI (SEQ ID NO:151), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), and engineered variants thereof.
In other embodiments, the C-terminal tail, (AA)p, comprises a polypeptide selected from the group consisting of M13 phage coat protein pIII (SEQ ID NO:154), M13 phage coat protein pVIII (SEQ ID NO:155), RepA protein (SED ID NO: 156), S. cerevisiae Aga1p (SEQ ID NO:157), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), P2A protein (SED ID NO: 158), and engineered variants thereof.
In other embodiments, the C-terminal tail, (AA)p, comprises a molecule selected from the group consisting of puromycin, puromycin analog, a puromycin-DNA conjugate, and a puromycin-RNA conjugate.
The N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), or (V) can comprise an intein protein. Inteins are polypeptides that are found as in-frame insertions in various natural proteins and can undergo a self-catalyzed intramolecular rearrangement leading to self-excision (self-splicing) of the intein and ligation of the flanking polypeptides together. The mechanism of intein splicing is well known (Xu and Perler 1996; Paulus 2000) and it involves the formation of a (thio)ester bond at the junction between the intein and the polypeptide fused the N-terminus of the intein (commonly referred to as “N-extein”) by action of a catalytic cysteine or serine residue at the first position of the intein sequence. This reversible N(backbone)→S(side-chain) or a N(backbone)→O(side-chain) acyl transfer is followed by a trans(thio)esterification step whereby the N-extein acyl unit is transferred to the side-chain thiol/hydroxyl group of a cysteine, serine, or threonine residue at the first position of the polypeptide fused the C-terminus of the intein (“C-extein”). The last step of the intein self-splicing process involves cleavage of the peptide bond connecting the intein to the C-extein via an intramolecular transamidation reaction by action of a conserved catalytic asparagine residue at the C-terminal position of the intein sequence (Paulus 2000).
Knowledge of the splicing mechanism of intein has enabled the preparation of engineered inteins with altered splicing behavior (Perler 2005; Xu and Evans 2005; Elleuche and Poggeler 2010). For example, it is known that removal of the conserved asparagine residue at the C-terminus of the intein sequence can result in an engineered intein protein capable of only N-terminal splicing (i.e., cleavage of the peptide bond between the N-extein and the intein), which can occurs spontaneously (i.e., via hydrolysis of N-terminal (thio)ester bond) or upon incubation with a thiol reagent (e.g., thiophenol, benzylmercaptan, dithiothreitol, sodium 2-sulfanylethanesulfonate), depending on the nature of the intein and of the C-terminal amino acid(s) in the N-extein sequence. Similarly, removal of the conserved cysteine or serine residue at the N-terminus of the intein sequence can result in an engineered intein protein capable of only C-terminal splicing (i.e., cleavage of the peptide bond between the intein and C-extein), which can occurs spontaneously or promoted via a change in pH or temperature, depending on the nature of the intein and of the N-terminal amino acid(s) in the C-extein sequence. Furthermore, certain intein proteins occur as split inteins, having an N-domain and C-domain. Upon association of the N-domain with the C-domain, split inteins acquires the ability to self-splice according to a mechanism analogous to single-polypeptide intein proteins (Mootz 2009). As for the latter, the N-terminal cysteine or serine residue and C-terminal asparagine residue can be mutated, resulting in altered splicing behavior as described above (Perler 2005; Xu and Evans 2005; Mootz 2009; Elleuche and Poggeler 2010).
According to the methods described herein, introduction of a natural or engineered intein protein within the N-terminal tail, (AA)m, or C-terminal tail, (AA)p, of the precursor polypeptide of formula (I), (II), or (V) results in the formation of a macrocyclic peptide that is fused to either the C-terminus or the N-terminus, respectively, of such natural or engineered intein. This aspect enables one to control and modulate the release of the macrocyclic peptide from the intein-fused polypeptide based on the self-splicing and altered splicing behavior of natural and engineered intein proteins as summarized above. This aspect can be useful to facilitate the isolation and characterization of the macrocyclic peptide from a complex mixture such as, for example, the lysate of a cell expressing the precursor polypeptide or a cell-free translation system. This aspect can also be useful to facilitate the accumulation, and if desired, control the formation of a target macrocyclic peptide, prepared according the methods described herein, inside a cell-based expression host. In turn, this capability can facilitate the functional screening of in vivo (i.e., in-cell) produced macrocyclic peptide libraries, prepared according the methods disclosed herein, using an intracellular reporter system or a selection system as described above. These aspects are illustrated by Examples 4-8.
Nucleotide sequences encoding for intein proteins that can be used can be derived from naturally occurring inteins and engineered variants thereof. A rather comprehensive list of such inteins is provided by the Intein Registry (neb.com/neb/inteins.html). Inteins that can be used include, but are not limited to, any of the naturally occurring inteins from organisms belonging to the Eucarya, Eubacteria, and Archea. Among these, for example, inteins of the GyrA group (e.g., Mxe GyrA, Mfl GyrA, Mgo GyrA, Mkas GyrA, Mle-TN GyrA, Mma GyrA), DnaB group (e.g., Ssp DnaB, Mtu-CDC1551 DnaB, Mtu-H37Rv DnaB, Rma DnaB), RecA group (e.g., Mtu-H37Rv RecA, Mtu-So93 RecA), RIR1 group (e.g., Mth RIR1, Chy RIR1, Pfu RIR1-2, Ter RIR1-2, Pab RIR1-3), and Vma group (e.g., Sce Vma, Ctr Vma), intein Mxe GyrA (SEQ ID NO:1) and the engineered ‘mini Ssp DnaB (‘eDnaB’, SEQ ID NO:2) can be used.
Intein proteins suitable in the methods described herein include, but are not limited to, engineered variants of natural inteins (or genetic fusion of split inteins), which have been modified by mutagenesis in order, for example, to prevent or minimize splicing at the N-terminal or C-terminal end of the intein. Examples of these modifications include, but are not limited to, mutation of the conserved cysteine or serine residue at the N-terminus of the intein (e.g., via substitution to an alanine) with the purpose, for example, of preventing cleavage at the N-terminus of the intein. Examples of these modifications include, but are not limited to, mutation of the conserved asparagine residue at the C-terminus of the intein (e.g., via substitution to an alanine) with the purpose, for example, of preventing cleavage at the C-terminus of the C-terminus of the intein. Examples of these modifications are provided in Example 2. Intein variants useful for the methods disclosed herein also include, but are not limited to, engineered inteins whose internal endonuclease domain, which is not essential for the splicing mechanism, is removed. For example, a variant of Ssp DnaB (‘eDnaB’, SEQ ID NO:2) lacking the internal endonuclease domain is used for the preparation of the precursor polypeptides. Inteins to be comprised in the precursor polypeptide can also be engineered with the purpose, for example, of altering the splicing properties of the intein in order to increase or reduce the splicing efficiency or in order to make the intein-catalyzed splicing process dependent upon variation of certain parameters such as pH or temperature.
Accordingly, in some embodiments, the N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), and (V) comprise an intein protein, or an engineered variant thereof. In some embodiments, the N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), and (V) comprise an intein protein selected from the group consisting of Mxe GyrA (SEQ ID NO:1), eDnaB (SEQ ID NO:2), Hsp-NRC1 CDC21 (SEQ ID NO:3), Ceu ClpP (SEQ ID NO:4), Tag Pol-1 (SEQ ID NO:5), Tfu Pol-1 (SEQ ID NO:6), Tko Pol-1 (SEQ ID NO:7), Psp-GBD Pol (SEQ ID NO:8), Tag Pol-2 (SEQ ID NO:9), Thy Pol-1 (SEQ ID NO:10), Tko Pol-2 (SEQ ID NO:11), Tli Pol-1 (SEQ ID NO:12), Tma Pol (SEQ ID NO:13), Tsp-GE8 Pol-1 (SEQ ID NO:14), Tthi Pol (SEQ ID NO:15), Tag Pol-3 (SEQ ID NO:16), Tfu Pol-2 (SEQ ID NO:17), Thy Pol-2 (SEQ ID NO:18), Tli Pol-2 (SEQ ID NO:19), Tsp-GE8 Pol-2 (SEQ ID NO:20), Pab Pol-II (SEQ ID NO:21), Mtu-CDC1551 DnaB (SEQ ID NO:22), Mtu-H37Rv DnaB (SEQ ID NO:23), Rma DnaB (SEQ ID NO:24), Ter DnaE-1 (SEQ ID NO:25), Ssp GyrB (SEQ ID NO:26), Mfl GyrA (SEQ ID NO:27), Mgo GyrA (SEQ ID NO:28), Mkas GyrA (SEQ ID NO:29), Mle-TN GyrA (SEQ ID NO:30), Mma GyrA (SEQ ID NO:31), Ssp DnaX (SEQ ID NO:32), Pab Lon (SEQ ID NO:33), Mja PEP (SEQ ID NO:34), Afu-FRR0163 PRP8 (SEQ ID NO:35), Ani-FGSCA4 PRP8 (SEQ ID NO:36), Cne-A PRP8 (SEQ ID NO:37), Hca PRP8 (SEQ ID NO:38), Pch PRP8 (SEQ ID NO:39), Pex PRP8 (SEQ ID NO:40), Pvu PRP8 (SEQ ID NO:41), Mtu-H37Rv RecA (SEQ ID NO:42), Mtu-So93 RecA (SEQ ID NO:43), Mfl RecA (SEQ ID NO:44), Mle-TN RecA (SEQ ID NO:45), Nsp-PCC7120 RIR1 (SEQ ID NO:46), Ter RIR1-1 (SEQ ID NO:47), Pab RIR1-1 (SEQ ID NO:48), Pfu RIR1-1 (SEQ ID NO:49), Chy RIR1 (SEQ ID NO:50), Mth RIR1 (SEQ ID NO:51), Pab RIR1-3 (SEQ ID NO:52), Pfu RIR1-2 (SEQ ID NO:53), Ter RIR1-2 (SEQ ID NO:54), Ter RIR1-4 (SEQ ID NO:55), CIV RIR1 (SEQ ID NO:56), Ctr VMA (SEQ ID NO:57), Sce VMA (SEQ ID NO:58), Tac-ATCC25905 VMA (SEQ ID NO:59), Ssp DnaB (SEQ ID NO:60), engineered variants thereof, and engineered variants thereof wherein the N-terminal cysteine or serine residue of the engineered variant is mutated to any of the natural amino acid residues other than cysteine or serine, or wherein the C-terminal asparagine residue of the engineered variant is mutated to any of the natural amino acid residues other than asparagine.
In some embodiments, the N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), and (V) comprise the N-domain, C-domain, or both the N-domain and C-domain of a split intein, or an engineered variant thereof. In some embodiments, the N-terminal tail, (AA)m, the C-terminal tail, (AA)p, or both, in the precursor polypeptides of formula (I), (II), and (V) comprise the N-domain, C-domain, or both the N-domain and C-domain of a split intein selected from the group consisting of Ssp DnaE (SEQ ID NO:61-SEQ ID NO:62), Neq Pol (SEQ ID NO:63-SEQ ID NO:64), Asp DnaE (SEQ ID NO:65-SEQ ID NO:66), Npu-PCC73102 DnaE (SEQ ID NO:67-SEQ ID NO:68), Nsp-PCC7120 DnaE (SEQ ID NO:69-SEQ ID NO:70), Oli DnaE (SEQ ID NO:71-SEQ ID NO:72), Ssp-PCC7002 DnaE (SEQ ID NO:73-SEQ ID NO:74), Tvu DnaE (SEQ ID NO:75-SEQ ID NO:76), engineered variants thereof, and engineered variants wherein the N-terminal cysteine or serine residue of the split intein N-domain of the engineered variant is mutated to any of the natural amino acid residues other than cysteine or serine, or wherein the C-terminal asparagine residue of the split intein C-domain of the engineered variant is mutated to any of the natural amino acid residues other than asparagine.
In some embodiments, the N-terminal tail, (AA)m, in the precursor polypeptides of formula (I), (II), and (V) comprises the C-domain of a split intein and the C-terminal tail, (AA)p, of said precursor polypeptides comprises the corresponding N-domain of the split intein. In some embodiments, the N-terminal tail, (AA)m, in the precursor polypeptides of formula (I), (II), and (V) comprises the C-domain of a split intein selected from the group consisting of Ssp DnaE-c (SEQ ID NO:62), Neq Pol-c (SEQ ID NO:64), Asp DnaE-c (SEQ ID NO:66), Npu-PCC73102 DnaE-c (SEQ ID NO:68), Nsp-PCC7120 DnaE-c (SEQ ID NO:70), Oli DnaE-c (SEQ ID NO:72), Ssp-PCC7002 DnaE-c (SEQ ID NO:74), Tvu DnaE-c (SEQ ID NO:76), and engineered variants thereof; and the C-terminal tail, (AA)p, comprises the corresponding N-domain of the split intein selected from the group consisting of Ssp DnaE-n (SEQ ID NO:61), Neq Pol-n (SEQ ID NO:63), Asp DnaE-n (SEQ ID NO:65), Npu-PCC73102 DnaE-n (SEQ ID NO:67), Nsp-PCC7120 DnaE-n (SEQ ID NO:69), Oli DnaE-n (SEQ ID NO:71), Ssp-PCC7002 DnaE-n (SEQ ID NO:73), Tvu DnaE-n (SEQ ID NO:75), and engineered variants thereof.
In another aspect, polynucleotide molecules are provided encoding for precursor polypeptides of formula (I), (II), and (V) as defined above. Polynucleotide molecules are provided for encoding for the aminoacyl-tRNA synthetases and cognate tRNA molecules for the ribosomal incorporation of the amino acid Z into the precursor polypeptides of formula (I) and (II) and for the ribosomal incorporation of the amino acid Z2 into the precursor polypeptides of formula (V). Polynucleotide molecules are provided encoding for polypeptide sequences that can be introduced within the N-terminal tail ((AA)m) or C-terminal tail ((AA)p) of the precursor polypeptides of formula (I), (II) and (V), such as peptide and protein affinity tags, reporter proteins and enzymes, carrier proteins of a display system, and intein proteins, as described above. Since the correspondence of all the possible three-base codons to the various amino acids is known, providing the amino acid sequence of the polypeptide provides also a description of all the polynucleotide molecules encoding for such polypeptide. Thus, a person skilled in the art will be able, given a certain polypeptide sequence, to generate any number of different polynucleotides encoding for the same polypeptide. In some embodiments, the codons are selected to fit the host cell in which the polypeptide is being expressed. For example, codons used in bacteria can be used to express the polypeptide in a bacterial host. The polynucleotides may be linked to one or more regulatory sequences controlling the expression of the polypeptide-encoding gene to form a recombinant polynucleotide capable of expressing the polypeptide.
Numerous methods for making nucleic acids encoding for polypeptides having a predetermined or randomized sequence are known to those skilled in the art. For example, oligonucleotide primers having a predetermined or randomized sequence can be prepared chemically by solid phase synthesis using commercially available equipments and reagents. Polynucleotide molecules can then be synthesized and amplified using a polymerase chain reaction, digested via endonucleases, ligated together, and cloned into a vector according to standard molecular biology protocols known in the art (e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Harbor Press, 2001). These methods, in combination with the mutagenesis methods mentioned above, can be used to generate polynucleotide molecules that encode for the aforementioned polypeptides as well as suitable vectors for the expression of these polypeptides in a host expression system.
The precursor polypeptides can be produced by introducing said polynucleotides into an expression vector, by introducing the resulting vectors into an expression host, and by inducing the expression of the encoded precursor polypeptides in the presence of the amino acid Z (or Z2) and, whenever necessary, also in the presence of a suitable stop codon or frameshift codon suppression system for mediating the incorporation of the amino acid Z (or Z2) into the precursor polypeptides.
Nucleic acid molecules can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include, but are not limited to, chromosomal, nonchromosomal, artificial and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated viruses, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used. A large number of expression vectors and expression hosts are known in the art, and many of these are commercially available. A person skilled in the art will be able to select suitable expression vectors for a particular application, e.g., the type of expression host (e.g., in vitro systems, prokaryotic cells such as bacterial cells, and eukaryotic cells such as yeast, insect, or mammalian cells) and the expression conditions selected.
Expression hosts that may be used for the preparation of the precursor polypeptides and macrocyclic peptides include, but are not limited to, any systems that support the transcription, translation, and/or replication of a nucleic acid. In some embodiments, the expression host system is a cell. Host cells for use in expressing the polypeptides encoded by the expression vector of this disclosure are well known in the art and include, but are not limited to, bacterial cells (e.g., Escherichia coli, Streptomyces); fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris); insect cells; plant cells; and animal cells, such as mammalian cells and human cells. These systems also include, but are not limited to, lysates of prokaryotic cells (e.g., bacterial cells) and lysates of eukaryotic cells (e.g., yeast, insect, or mammalian cells). These systems also include, but are not limited to, in vitro transcription/translation systems, many of which are commercially available. The choice of the expression vector and host system depends on the type of application intended for the methods disclosed herein and a person skilled in the art will be able to select a suitable expression host based on known features and application of the different expression hosts. As an example, when it is desired to evaluate the interaction between the macrocyclic peptide(s) generated via the methods disclosed herein with a bacterial, yeast, or a human cell component, a bacterial, yeast, or a human expression host, respectively, can be used. In some embodiments, the expression host system is a cell.
In some embodiments, the formation of the macrocyclic peptides from the biosynthetic polypeptides as defined above is carried out within the cell-based expression host that produces the precursor polypeptides, so that the macrocyclic peptides are produced within this cell-based expression host. This method comprises providing a nucleic acid encoding for the precursor polypeptide, introducing the nucleic acid into the cell-based expression host, inducing the expression of the precursor polypeptide, allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG1 group of the amino acid Z (or between the cysteines and the FG1 and FG2 groups of the amino acid Z2), thereby producing the macrocyclic peptide inside the cell-based expression host. These aspects are illustrated in Examples 4 through 8.
In some embodiments, the formation of the macrocyclic peptides from the biosynthetic polypeptides as defined above is carried out on the surface of a cell or on a viral particle, so that the macrocyclic peptides are produced as tethered to a cell or a viral particle, respectively. This method comprises providing a nucleic acid encoding for the precursor polypeptide, wherein the N- or C-terminal tail comprises a polypeptide component of the cell membrane (e.g., S. cerevisiae membrane protein Aga2p) or of the viral particle (e.g., M13 phage pIII protein), introducing the nucleic acid into the expression host, inducing the expression of the precursor polypeptide, allowing for the precursor polypeptide to be integrated into the cell membrane or viral particle, and allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG1 group of the amino acid Z (or between the cysteines and the FG1 and FG2 groups of the amino acid Z2), thereby producing the macrocyclic peptide as tethered to the membrane of the cell or to the viral particle.
In some embodiments, the formation of the macrocyclic peptides from the biosynthetic polypeptides as defined above is carried out within a cell-free expression system, so that the macrocyclic peptides are produced within this cell-free expression system. This method comprises providing a nucleic acid encoding for the precursor polypeptide, introducing the nucleic acid into the cell-free expression host, inducing the expression of the precursor polypeptide, allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG1 group of the amino acid Z (or between the cysteines and the FG1 and FG2 groups of the amino acid Z2), thereby producing the macrocyclic peptide within the cell-free expression host.
A method is also provided for making a library of macrocyclic peptides via cyclization of a plurality of precursor polypeptides of formula (I) or (II) that contain an heterogeneous peptide target sequence (AA)n, or an heterogeneous N-terminal tail (AA)m, or an heterogeneous C-terminal tail (AA)p, or a combination of these. This method comprises: (a) constructing a plurality of nucleic acid molecules encoding for a plurality of precursor polypeptides, said precursor polypeptides having an heterogeneous peptide target sequence (AA)n, or an heterogeneous N-terminal tail (AA)m, or an heterogeneous C-terminal tail (AA)p, or a combination of these; (b) introducing each of the plurality of said nucleic acid molecules into an expression vector, and introducing the resulting vectors into an expression host; (c) expressing the plurality of precursor polypeptides; (d) allowing for the precursor polypeptides to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG1 group of the amino acid Z, thereby producing a plurality of macrocyclic peptides.
A method is also provided for making a library of macrocyclic peptides via cyclization of a plurality of precursor polypeptides of formula (V) that contain an heterogeneous peptide target sequence (AA)n, or an heterogeneous second peptide target sequence (AA)o, or an heterogeneous N-terminal tail (AA)m, or an heterogeneous C-terminal tail (AA)p, or a combination of these. This method comprises: (a) constructing a plurality of nucleic acid molecules encoding for a plurality of precursor polypeptides, said precursor polypeptides having an heterogeneous peptide target sequence (AA)m, or an heterogeneous second peptide target sequence (AA)o, or an heterogeneous N-terminal tail (AA)m, or an heterogeneous C-terminal tail (AA)p, or a combination of these; (b) introducing each of the plurality of said nucleic acid molecules into an expression vector, and introducing the resulting vectors into an expression host; (c) expressing the plurality of precursor polypeptides; (d) allowing for the precursor polypeptides to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteines and the FG1 and FG2 group2 of the amino acid Z2, thereby producing a plurality of macrocyclic peptides.
In specific embodiments, each of the plurality of macrocyclic peptides prepared as described above is tethered to a cell component, to a cell membrane component, to a bacteriophage, to a viral particle, or to a DNA molecule, via a polypeptide comprised within the N-terminal tail or within the C-terminal tail of said macrocyclic peptide molecule.
Several methods of making polynucleotides encoding for heterogeneous peptide sequences are known in the art. These include, among many others, methods for site-directed mutagenesis (Botstein, D.; Shortie, D. Science (New York, N.Y., 1985, 229, 1193; Smith, M. Annual review of genetics, 1985, 19, 423; Dale, S. J.; Felix, I. R. Methods in molecular biology (Clifton, N.J., 1996, 57, 55; Ling, M. M.; Robinson, B. H. Analytical biochemistry, 1997, 254, 157), oligonucleotide-directed mutagenesis (Zoller, M. J. Current opinion in biotechnology, 1992, 3, 348; Zoller, M. J.; Smith, M. Methods Enzymol, 1983, 100, 468; Zoller, M. J.; Smith, M. Methods Enzymol, 1987, 154, 329), mutagenesis by total gene synthesis and cassette mutagenesis (Nambiar, K. P.; Stackhouse, J.; Stauffer, D. M.; Kennedy, W. P.; Eldredge, J. K.; Benner, S. A. Science (New York, N.Y., 1984, 223, 1299; Grundstrom, T.; Zenke, W. M.; Wintzerith, M.; Matthes, H. W.; Staub, A.; Chambon, P. Nucleic acids research, 1985, 13, 3305; Wells, J. A.; Vasser, M.; Powers, D. B. Gene, 1985, 34, 315), and the like. Additional methods are described in the following U.S. patents, PCT publications, and EPO publications: U.S. Pat. No. 5,605,793 “Methods for In vitro Recombination”, U.S. Pat. No. 5,830,721 “DNA Mutagenesis by Random Fragmentation and Reassembly”, WO 95/22625 “Mutagenesis by Random Fragmentation and Reassembly”, WO 96/33207 “End Complementary Polymerase Chain Reaction”, EP 752008 “DNA Mutagenesis by Random Fragmentation and Reassembly”, WO 98/27230 “Methods and Compositions for Polypeptide Engineering”, WO 00/00632, “Methods for Generating Highly Diverse Libraries”, WO 98/42832 “Recombination of Polynucleotide Sequences Using Random or Defined Primers”, WO 99/29902 “Method for Creating Polynucleotide and Polypeptide Sequences”. Any of these methods or modifications thereof can be utilized for generating nucleotide molecules that encode for precursor polypeptides of formula (I), (II), or (V) containing an heterogeneous peptide target sequence (AA)n, an heterogeneous second peptide target sequence (AA)o, an heterogeneous N-terminal tail (AA)m, an heterogeneous C-terminal tail (AA)p, or a combination of these.
The compounds provided herein may contain one or more chiral centers. Accordingly, the compounds are intended to include, but not be limited to, racemic mixtures, diastereomers, enantiomers, and mixture enriched in at least one stereoisomer or a plurality of stereoisomers. When a group of substituents is disclosed herein, all the individual members of that group and all subgroups, including any isomers, enantiomers, and diastereomers are intended to be included in the disclosure. Additionally, all isotopic forms of the compounds disclosed herein are intended to be included in the disclosure. For example, it is understood that any one or more hydrogens in a molecule disclosed herein can be replaced with deuterium or tritium.
The terms and expression that are employed herein are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described and portions thereof, but it is recognized that various modifications are possible within the scope of the subject matter claimed herein. Thus, it should be understood that although various embodiments and optional features have been disclosed herein, modification and variation of the concepts herein disclosed may be resorted to those skilled in the art, and that such modifications and variations are considered to be encompassed by the appended claims.
Unless otherwise indicated, the disclosure is not limited to specific molecular structures, substituents, synthetic methods, reaction conditions, or the like, as such may vary. It is to be understood that the embodiments are not limited to particular compositions or biological systems, which can, of course, vary.
A skilled artisan will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the methods and compositions disclosed herein. All art-known functional equivalents of any such materials and methods are intended to be included in the methods and compositions disclosed herein.
The following examples are offered by way of illustration and not by way of limitation.
This example demonstrates the preparation of various cysteine-reactive unnatural amino acids, i.e., various Z and Z2 amino acids, which can be used for preparation of macrocyclic peptide molecules according to the general methods illustrated in
The unnatural amino acid 4-(2-bromoethoxy)-phenylalanine (1, p-2beF) was prepared according to the synthetic route provide in Scheme 1 of
To a reaction flask containing N-tert-butoxycarbonyl-tyrosine (2 g, 7.1 mmol) and potassium carbonate (2.94 g, 21.3 mmol) in dry DMF (20 mL) dibromoethane (1.83 mL, 21.3 mmol) was added dropwise over 20 min. The reaction mixture was stirred at room temperature for 18 h after which the reaction mixture was filtered, diluted with 60 mL of water, acidified with acetic acid to pH 4 and extracted with 2×100 mL of EtOAc. Organic layers were combined and dried over sodium sulfate. The solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc acid as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding N-Boc-4-(2-bromoethoxy)-phenylalanine as an off-white powder (2.3 g, 84%). 1H NMR (400 MHz, CD3OD) δ 1.39 (s, 9H), 2.8-3.05 (m, 2H), 3.3 (t, 2H), 3.51 (t, 2H), 4.37 (t, 2H), 6.69 (d, 2H), 7.02 (d, 2H); 13C NMR (125 MHz, CD3OD) δ 28.73, 29.49, 37.92, 56.82, 65.77, 80.69, 116.27, 128.84, 131.32, 157.39, 157.77, 173.414. MS (ESI) calculated for C14H19NO5 M+: Mk 387.07, found 387.17. Purified N-Boc-4-(2-bromoethoxy)-phenylalanine was treated with 20 mL of 30% TFA/DCM to remove the N-terminal protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure, crude residue dissolved 2× in 10 mL of HOAc followed by solvent evaporation yielding the final product 1 as an off-white solid in quantitative yield (1.7 g). 1H NMR (400 MHz, CD3OD) δ 3.05-3.25 (m, 2H), 3.58 (t, 2H), 4.28 (t, 1H), 4.51 (t, 2H), 6.77 (d, 2H), 7.09 (d, 2H); 13C NMR (125 MHz, CD3OD) δ 29.1, 36.9, 55.35, 66.92, 116.92, 125.54, 131.59, 158.41, 169.93. MS (ESI) calculated for C11H14BrNO3 [M+H]+: m/z 288.02, found 288.51.
To a solution of Nα-tert-butoxycarbonyl-lysine (1 g, 4.06 mmol) and NaOH (162.4 mg, 4.06 mmol, 1 eq) dissolved in 20 mL of water 2-bromoethylchloroformate (0.435 mL, 4.06 mmol, 1 eq) and, separately, an additional equivalent of NaOH were added simultaneously dropwise over 30 min. The reaction mixture was stirred at room temperature for 18 h. Upon acidification with HOAc, the aqueous phase was extracted with EtOAc (3×80 mL). The combined organic phases were dried over sodium sulfate, solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding N-Boc-Nε-((2-bromoethoxy)carbonyl)-lysine as an off-white powder (1.1 g, 68%). 1H NMR (400 MHz, CD3OD) δ 1.43 (s, 9H), 1.5 (m, 2H), 1.65 (m, 2H), 1.79 (m, 2H), 3.09 (t, 2H), 3.54 (t, 2H), 4.05 (t, 1H), 4.29 (t, 2H); 13C NMR (125 MHz, CD3OD) δ 24.09, 28.78, 30.39, 30.47, 32.434, 41.44, 54.82, 65.51, 80.51, 158.15, 158.44, 176.24; MS (ESI) calculated for C14H19NO5 [M+H]+: m/z 397.1, found 397.47. Purified N-Boc-Nζ-((2-bromoethoxy)carbonyl)-lysine was treated with 20 mL of 30% TFA/DCM to remove the N-terminal protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure, crude residue dissolved 2× in 10 mL of acetic acid followed by solvent evaporation yielding the final product 2 as an off-white solid in quantitative yield (0.82 g). 1H NMR (400 MHz, CD3OD) δ 1.45 (m, 2H), 1.64 (m, 2H), 1.76 (m, 2H), 2.95 (t, 2H), 3.6 (t, 2H), 3.85 (t, 1H), 4.22 (t, 2H); 13C NMR (100 MHz, CD3OD) δ 20.74, 23.16, 30.36, 31.16, 41.21, 53.86, 65.54, 158.52, 175.21; MS (ESI) calculated for C11H14BrNO3 [M+H]+: m/z 297.04, found 297.7.
Solution of 4-acetylphenylalanine (0.5 g, 2.415 mmol), prepared as reported previously (Frost, Vitali et al. 2013), in methanol was placed in an ice bath followed by addition of triethylamine (0.51 mL, 3.63 mmol, 1.5 eq) and dropwise addition of di-tert-butyl dicarbonate (0.665 mL, 2.9 mmol, 1.2 eq) over 30 min. The reaction was left at room temperature for additional 3 h after which the solvent was removed in vacuo. The residue was redissolved in EtOAc and extracted with acidified water (pH 4). Organic phase was dried over sodium sulfate, solvent removed under reduced pressure and the crude yellow oil purified using flash column chromatography with 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions of interest were combined yielding N-Boc-4-acetylphenylalanine as a yellow powder (0.665 g, 90%) which was dissolved in MeOH, placed in an ice bath and treated with NaBH4 (0.164 g, 4.34 mmol, 2 eq) for 3 h. Following aqueous workup, the crude product was dissolved in DCM, placed in an ice bath and PBr3 (1 M solution in DCM) was added in portions (5.2 mL, 5.2 mmol, 2.4 eq) over 2 h. The reaction was warmed to reach room temperature and left stirring overnight. After workup, the aqueous layer was lyophilized and used as crude product 3 (0.382 g, 65%). 1H NMR (400 MHz, CD3OD) 1.99 (d, 3H), 2.8-3.2 (m, 2H), 4.31 (t, 1H), 4.78 (q, 1H), 7.18 (d, 2H), 7.27 (d, 2H); MS (ESI) calculated for C11H14BrNO2 [M+H]+: m/z 272.03, found 272.53.
To a solution of Nα-tert-butoxycarbonyl-lysine 1 (1 g, 4.06 mmol) and NaOH (162.4 mg, 4.06 mmol, 1 eq) dissolved in 20 mL of water 2-chloroethylchloroformate (0.419 mL, 4.06 mmol, 1 eq) and, separately, an additional equivalent of NaOH were added simultaneously dropwise over 30 min. The reaction mixture was stirred at room temperature for 10-12 h. Upon acidification with HOAc, the aqueous phase was extracted with EtOAc (3×80 mL). The combined organic phases were dried over sodium sulfate, solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding off-white powder as product (1.04 g, 75%). Purified product was treated with 20 mL of 30% TFA/DCM to remove the N-terminal Boc-protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure, yielding the final product 4 as off-white solid in quantitative yield (0.75 g). 1H NMR (400 MHz, CD3OD) δ 1.45 (m, 2H), 1.64 (m, 2H), 1.76 (m, 2H), 2.95 (t, 2H), 3.6 (t, 2H), 3.85 (t, 1H), 4.22 (t, 2H).
3-butynoic acid was prepared by oxidation of 3-butyn-1-ol. About 20 mL of water was added to a 150 mL single neck RBF followed by 65% HNO3 (45 μL, 0.66 mmol, 0.05 eq), Na2Cr2O7 (40 mg, 0.132 mmol, 0.01 eq) and NaIO4 (6.22 g, 29 mmol, 2.2 eq) and stirred vigorously on an ice bath. After 15 min 1 mL of 3-butyn-1-ol (1 eq, 13.2 mmol) dissolved in 5 mL of cold water was added dropwise over 30 min The reaction was left stirring overnight followed by product extraction with diethyl ether. Solvent was evaporated to yield off-white/yellow solid (g, %). 1H NMR (400 MHz, CDCl3) δ 3.35 (d, 2H), 2.22 (t, 1H). 3-butynoic acid (0.436 g, 5.2 mmol, 1 eq) was dissolved in dry DCM and 1.5 eq of 2-chloro-1-methylpyridinium iodide was added (2.2 g). The reaction was stirred for 1 h at room temperature followed by dropwise addition of Nα-tert-butoxycarbonyl-lysine (1.4 g, 5.72 mmol, 1.1 eq) and triethylamine (1.2 mL, 7.8 mmol, 1.5 eq). The reaction was monitored by TLC and upon completion (4-5 h) extracted with water. Organic layer was evaporated and the crude product was purified using flash column chromatography with 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions containing the desired product were pooled together and the solvent was removed under reduced pressure giving the desired product in 55% yield. 1H NMR (400 MHz, CD3OD) δ 1.4 (s, 9H), 1.5 (m, 2H), 1.62 (m, 2H), 1.81 (m, 2H), 3.13 (t, 2H), 4.51 (m, 3H), 5.8 (m, 1H). The final Boc-deprotection was achieved using 20 mL of 30% TFA/DCM for 30 min followed by solvent removal resulting in product 5 (g). 1H NMR (400 MHz, CD3OD) δ 1.48 (m, 2H), 1.63 (m, 2H), 1.82 (m, 2H), 3.12 (t, 2H), 4.21 (t, 1H), 4.51 (d, 2H), 5.8 (m, 1H).
To a reaction flask containing Nα-tert-butoxycarbonyl-tyrosine (2 g, 7.1 mmol) and potassium carbonate (2.94 g, 21.3 mmol, 2 eq) in dry DMF (20 mL) 1,2,3-tribromopropane (0.915 mL, 7.82 mmol, 1.1 eq) was added dropwise over 20 min. The reaction mixture was stirred at room temperature for 8 h after which the reaction mixture was filtered, diluted with 60 mL of water, acidified with acetic acid to pH 4 and extracted with 2×100 mL of EtOAc. Organic layers were combined and dried over sodium sulfate. The solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc acid as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding off-white powder as product (g, %). 1H NMR (400 MHz, CD3OD) δ 1.41 (s, 9H), 2.81-3.07 (m, 2H), 3.6-3.81 (m, 2H), 4.21-4.43 (m, 3H), 4.61-4.72 (m, 1H), 6.71 (d, 2H), 7.04 (d, 2H). Purified product was treated with 20 mL of 30% TFA/DCM to remove the N-terminal protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure yielding the final product 6 as an off-white solid in quantitative yield (g). 1H NMR (400 MHz, CD3OD) δ 2.81-3.07 (m, 2H), 3.6-3.81 (m, 2H), 4.12 (t, 1H), 4.21-4.43 (m, 2H), 4.61-4.72 (m, 1H), 6.71 (d, 2H), 7.04 (d, 2H).
This example demonstrates procedures for the construction of polynucleotide molecules for the expression of precursor polypeptides of the type (I), (II), or (V) according to the methods described herein.
To illustrate the various embodiments, a series of a plasmid-based vectors were prepared that encode for precursor polypeptides in different formats (Table 1) according to the macrocyclization methods schematically described in
A second series of constructs (Entries 10-12, Table 1) were prepared for the expression of precursor polypeptides of general formula (II), in which (i) the N-terminal tail, (AA)m, consists of a short (2 to 6 amino acid-long) polypeptide; (ii) the target peptide sequence, (AA)n, consists of a 3 to 7-amino acid long polypeptide; and (iii) the C-terminal tail, (AA)p, consists of the N198A variant of Mxe GyrA intein (SEQ ID NO:1) followed by a polyhistidine tag. In these constructs, an amber stop codon was used to enable the introduction of the desired, cysteine-reactive unnatural amino acid Z, downstream of the peptide target sequence via amber stop codon suppression. These constructs were designed to probe the functionality of the general methods described in
A third series of constructs (Entries 16-20, Table 1) were prepared for the expression of precursor polypeptides of general formula (I), in which (i) the N-terminal tail, (AA)m, contains the C-domain of Synechocystis sp. DnaE split intein (SEQ ID NO:62); (ii) the C-terminal tail, (AA)p, contains the N-domain of Synechocystis sp. DnaE split intein (SEQ ID NO:61); and (iii) a streptavidin-binding HPQ motif (Naumann, Savinov et al. 2005) is included within (Entry 18-20, Table 1) or downstream of the target peptide sequence (AA)n (Entries 16-17, Table 1). In these constructs, an amber stop codon was used to enable the introduction of the desired, cysteine-reactive unnatural amino acid Z, upstream of the peptide target sequence. Furthermore, these constructs contain a CBD (cellulose binding domain) affinity tag fused to the C-terminal end of the split intein N-domain. These constructs were designed to probe the functionality of the general methods described in
An additional construct (Entry 21, Table 1) was prepared for the expression of a precursor polypeptide which carries two Cys/Z pairs comprising two different target peptide sequences (HPQF (SEQ ID NO:185) and NTSK (SEQ ID NO:186)) and being separated from each other by an intervening polypeptide sequence (ENLYFQS (SEQ ID NO:187)). This construct is instrumental for demonstrating the possibility to generate polycyclic peptides using the methods disclosed herein.
Finally, a construct (Entry 22, Table 1) was prepared for the expression of a precursor polypeptide which carries a bifunctional cysteine-reactive amino acid (Z2) and two cysteine residues. This construct is instrumental for demonstrating the possibility to generate polycyclic peptides according to the general methods described in
aGYrAN190A corresponds to the N190A variant of Mycobacterium xenopi GyrA (SEQ. ID NO: 1), CBD corresponds to the Chitin Binding Domain (CBD) of Bacillus circulans chitinase A1 (SEQ of ID NO: 130), DnaEN and DnaEC correspond to the N-domain and C-domain, respectively, Synechocystis sp. DnaE split intein (SEQ ID NOS: 61 and 62). The reactive amino acid residues are involved in peptide macrocyclization (i.e., Cys and Z residues; Cys and Z2 residues) are highlighted in bold.
Cloning and plasmid construction. The plasmid vector pET22b(+) (Novagen) was used as cloning vector to prepare the plasmids for the expression of the precursor polypeptides of Entries 1-15 and 21-22 in Table 1. Briefly, synthetic oligonucleotides (Integrated DNA Technologies) were used for the PCR amplification of a gene encoding for N-terminal peptide and peptide target sequence fused to GyrAN198A intein using a previously described GyrA-containing vector (pBP_MG6) (Smith, Vitali et al. 2011) as template. The resulting PCR product (ca. 0.6 Kbp) was digested with Nde I and Xho I and cloned into pET22b(+) to provide the plasmids for the expression of the precursor polypeptides of Entries 1-15 and 21-22 in Table 1. The cloning process placed the polypeptide-encoding gene under the control of an IPTG-inducible T7 promoter and introduced a poly-histidine tag at the C-terminus of the intein. Plasmids for the expression of the polypeptide constructs of Entries 16 through 20 of Table 1 were prepared in a similar manner but using pBAD plasmid (Life Technologies) as the cloning and expression vector. The genes encoding for DnaEN and DnaEc were amplified from Addgene plasmids pSFBAD09 and pJJDuet30. The sequences of the plasmid constructs were confirmed by DNA sequencing.
This example illustrates how a suitable tRNA/aminoacyl-tRNA synthetase pair can be identified for the purpose of incorporating a desired cysteine-reactive, unnatural amino acid into a precursor polypeptide of general formula (I), (II), or (V) according to the methods disclosed herein. In particular, this example describes the identification of tRNA/aminoacyl-tRNA synthetase pairs for the incorporation of the unnatural amino acid 4-(2-bromoethoxy)-phenylalanine (p-2beF), Nε-((2-bromoethoxy)carbonyl)-lysine (2becK), 4-(1-bromoethyl)-phenylalanine (p-1beF), Nε-((2-chloroethoxy)carbonyl)-lysine (2cecK), Nε-(buta-2,3-dienoyl)-lysine (bdnK), and O-(2,3-dibromoethyl)-tyrosine (OdbpY), which were synthesized as described in Example 1.
A high-throughput fluorescence-based screen was applied to identify viable tRNA/aminoacyl-tRNA synthetase (AARS) pairs for the ribosomal incorporation of the unnatural amino acid p-2beF, 2becK, p-1beF, 2cecK, bdnK, or OdbpY, in response to an amber stop codon. In this assay, E. coli cells are co-transformed with two plasmids with compatible origins of replications and selection markers; one plasmid directs the expression of the tRNA/AARS pair to be tested, whereas the second plasmid contains a gene encoding for a variant of Yellow Fluorescence Protein (YFP), in which an amber stop codon (TAG) is introduced at the second position of the polypeptide sequence following the initial Met residue (called YFP(TAG)). The ability of the tRNA/AARS pair to suppress the amber stop codon with the unnatural amino acid of interest can be thus determined and quantified based on the relative expression of YFP as determined by fluorescence. Using this assay, a panel of engineered aminoacyl-tRNA synthetase (AARS) variants derived from M. jannaschii tyrosyl-tRNA synthetase (SEQ ID NO:77), M. barkeri pyrrolysyl-tRNA synthetase (SEQ ID NO:79), or M. mazei pyrrolysyl-tRNA synthetase (SEQ ID NO:78) in combination with their cognate amber stop codon suppressor tRNA (i.e., MjtRNACUATyr (SEQ ID NO:101) for Mj AARSs and Mm/MbtRNACUAPyl (SEQ ID NO:105) for the Mm and Mb AARSs) were tested for their ability to incorporate the target amino acids p-2beF, 2becK, p-1beF, 2cecK, bdnK, or OdbpY into the reporter YFP(TAG) protein. In a representative experiment, this panel of AARS enzymes included the known engineered AARSs Mj-pAcF-RS (SEQ ID NO:81), Mj-pAmF-RS (SEQ ID NO:87), Mb-CrtK-RS (SEQ ID NO:93), and Mm-pXF-RS (SEQ ID NO:91) (Young, Young et al. 2011)) as well as the newly engineered Mj-OpgY2-RS (SEQ ID NO:85). The latter, which is derived from Mj-OpgY-RS (SEQ ID NO:84) (Deiters and Schultz 2005), carries an Ala32G mutation that was designed to facilitate the recognition of 0-substituted tyrosine derivatives such as p-2beF and OdbpY based on the available crystal structure of the parent enzyme Mj-TyrRS (SEQ ID NO:77) (Kobayashi, Nureki et al. 2003). As illustrated by the representative data in
Using an analogous procedure, it was established that the Mj-pAcF-RS/MjtRNACUATyr pair can enable efficient amber stop codon suppression with p-1beF; the Mb-CrtK-RS/Mm/MbtRNACUAPyl pair can enable efficient amber stop codon suppression with 2cecK or bdnK; and the Mj-pOgY2-RS/MjtRNACUATyr pair can enable efficient amber stop codon suppression with OdbpY. These results provide an exemplary demonstration of viable procedures that can be used to identify suitable AARS/tRNA pairs for the ribosomal incorporation of cysteine-reactive unnatural amino acid into a polypeptide for the purpose of producing macrocyclic peptide according to methods disclosed herein and as illustrated in the following Examples.
YFP expression assay. Competent BL21(DE3) E. coli were cotransformed with a pEVOL-based plasmid (Smith, Vitali et al. 2011) for the expression of the desired AARS/tRNA pair and a pET22-YFP(TAG) plasmid for the expression of the reporter YFP protein. After overnight growth at 37° C. in LB medium supplemented with chloramphenicol (25 μg/mL) and ampicillin (50 μg/mL), cell cultures were used to inoculate 96-well plates containing 0.9 mL of minimal (M9) media (25 μg/mL chloramphenicol, 50 μg/mL ampicillin, 1% glycerol) per well. At OD600=0.6, protein expression was induced with 0.05% L-arabinose and 1 mM IPTG. Test wells were supplemented with the desired unnatural amino acid (e.g., 4-(2-bromoethoxy)-phenylalanine (p-2beF) at 2 to 5 mM, whereas no amino acid was added to the negative control wells. Cultures were grown overnight at 27° C. and then diluted 1:100 with phosphate buffer (50 mM KPi (pH 7.5), 150 mM NaCl) into microtiter plates. Fluorescence intensity was measured using a Tecan Infinite 1000 multi-well plate reader (λexc: 514 nm; λem: 527 nm).
This example demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (I) and containing the cysteine-reactive unnatural amino acid p-2beF. In particular, this example demonstrates certain embodiments as schematically described in
For these experiments, the precursor polypeptides corresponding to Entries 1 through 9 in Table 1 were expressed in BL21(DE3) E. coli cells containing a second, pEVOL-based plasmid for the co-expression of Mj-pOgY2-RS and MjtRNACUATyr. As described in Example 3, this AARS/tRNA pair was established to allow for the efficient ribosomal incorporation of p-2beF into a polypeptide in response to an amber stop codon. According to our strategy (
As summarized in
GyrA intein contains a Cys at its N-terminal end which is crucial for mediating protein splicing in the context of the application of the present methods for producing peptide macrocycles inside the cells (see Example 5). Since this residue is partially buried within the active site (Klabunde, Sharma et al. 1998), we did not expect it to readily react with p-2beF side chain. Notably, quantitative splicing of the GyrA moiety upon treatment of all the aforementioned contructs with benzyl mercaptan indicated that no reaction occurred between p-2beF and the catalytic Cys at the intein I+1 site (see representative results in
Protein Expression and Purification.
The protein constructs were expressed using BL21(DE3) E. coli cells co-transformed with a pET22-based vector for the expression of the precursor polypeptide and a pEVOL-based vector for the expression of the Mj-pOgY2-RS/MjtRNACUATyr pair. Cultures of these cells were grown overnight in LB media (50 mg/L ampicillin; 25 mg/L chloramphenicol) and used to inoculate 0.2 L of minimal (M9) media containing the same concentration of antibiotics, 1% glycerol, and 1 mM p-2BeF. At OD600=0.6, IPTG (1 mM) and L-arabinose (0.05%) was added to the culture media to induce protein expression. Cultures were grown for 14 h at 27° C. and then harvested by centrifugation. Cell pellets were resuspended in 50 mM Tris, 300 mM NaCl, 20 mM imidazole buffer (pH 7.5) and cells were lysed by sonication. The cell lysate was loaded on a Ni-NTA affinity column and proteins were eluted with 50 mM Tris, 150 mM NaCl, 300 mM imidazole buffer (pH 7.5). Fractions were combined and concentrated followed by buffer exchange with potassium phosphate buffer (50 mM, 150 mM NaCl, pH 7.5). The identity of the isolated proteins was confirmed using MALDI-TOF MS and LC-MS.
Intein Splicing and MS Analysis.
Aliquots of the purified proteins (200 μM) were incubated with 15 mM benzylmercaptan, 20 mM TCEP in 50 mM phosphate buffer (pH 8). The identity of the target macrocycles was confirmed using MALDI-TOF MS and LC-MS analysis. LC-MS analyses were performed on Thermo Scientific LTQ Velos ESI/ion-trap mass spectrometer coupled to an Accela U-HPLC. Macrocycles were analyzed using Thermo Scientific HyPurity C4 column (particle size 5 μm, 100×2.1 mm i.d.) and a linear gradient 5% to 95% ACN (with 0.1% formic acid) in water (with 0.1% formic acid) over 9 mM. MALDI-TOF spectra were acquired on the Bruker Autoflex III mass spectrometer.
This example further demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (I) and containing the cysteine-reactive unnatural amino acid p-2beF. In particular, this example provides a demonstration of the functionality of the methods described herein for the production of macrocyclic peptide within living bacterial cells.
For these studies, the constructs corresponding to Entries 13 through 15 of Table 1 were utilized. The corresponding precursor polypeptides were expressed in BL21(DE3) E. coli cells in the presence of the Mj-pOgY2-RS/MjtRNACUATyr to achieve the site-selective incorporation of the unnatural amino acid p-2beF into these proteins via amber stop codon suppression. These constructs were designed to contain an Asp residue in the position preceding the GyrA intein moiety in order to favor premature N-terminal splicing of this intein during expression (
Altogether, these results further demonstrate the versatility of the methods described herein for enabling the ribosomal synthesis of macrocyclic peptides of varying length and compositions. In addition, they demonstrate the possibility to apply these methods to enable the production of macrocyclic peptides in vivo, i.e., inside a living cell. Finally, they demonstrate that these in vivo produced macrocyclic peptides can be functional, that is capable of specifically bind to a target biomolecule (i.e., streptavidin).
Isolation and Analysis of HPQ-Containing Macrocyclic Peptides.
Protein expression was performed as described above (Example 5). After centrifugation, cells were resuspended in 50 mM Tris, 300 mM NaCl and 20 mM imidazole (pH 7.5) and lysed by sonication. Cell lysates were incubated with streptavidin-coated beads for 1 hour under gentle shaking on ice. Beads were washed two times with the same buffer followed by incubation with acetonitrile:H2O (70:30 v/v) for one minute to release any streptavidin-bound peptides. Eluates were lyophilized and the identity of the peptides evaluated using MALDI-TOF MS and LC-MS as described above (Example 5).
This example further demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (I). In particular, this example demonstrates how different cysteine-reactive unnatural amino acids of general structure (III) can be used for the purpose of generating macrocyclic peptides starting from ribosomally produced polypeptide precursors according to the methods described herein.
As described in Example 3, orthogonal AARS/tRNA pairs could be readily identified to achieve the specific incorporation of the unnatural amino acids 2becK, 2cecK, p-1beF, or bdnK into a precursor polypeptide of choice. Each of these amino acids contains an electrophilic side-chain functionality (i.e., alkylbromide group in 2becK and p-1beF; alkylchloride group in 2cecK; allenamide group in bdnK), which was expected to react chemoselectively with a neighboring cysteine residue within the precursor polypeptide sequence according to the general methods provided herein. To test the ability of 2becK and 2cecK to mediate peptide macrocyclization, the constructs corresponding to Entries 1 through 9 of Table 1 were expressed in E. coli as described above (Example 5) using the appropriate AARS/tRNA pairs (Example 3) for the incorporation of either 2becK or 2cecK as the cysteine-reactive residue (Z residue, Table 1). To establish the occurrence of the desired macrocyclization reaction, these proteins were purified by Ni-affinity chromatography and then reacted with benzyl mercaptan to splice the GyrA intein and release the macrocyclic peptide. Detection and quantification of the cyclic product was carried by LC-MS and MS/MS analysis as described in Example 4. These analyses revealed the occurrence of the desired macrocyclic peptide product in each case, as shown by the representative LC-MS extracted-ion chromatograms and MS/MS spectra in
To further investigate the generality of the methods presented herein, two additional amino acids, p-1beF and bdnK, were synthesized (Example 1) and tested here for their ability to induce peptide macrocyclization upon reaction with a proximal cysteine in the precursor polypeptide. p-1beF contains a benzylic, secondary alkyl bromide group, thus enabling the formation of more compact peptide ring structures as compared to those generated using p-2beF-mediated cysteine alkylation. On the other hand, bdnK was designed to contain an allenamide group, which is known to react chemoselectively with cysteine via a Michael addition reaction (Abbas, Xing et al. 2014). Using the appropriate AARS/tRNA pair as determined in Example 3, p-1beF was incorporated into the construct 12mer-Z4C (Entry 4, Table 1) to give 12mer-Z4C(p-1beF), whereas bdnK was incorporated into the construct 12mer-Z6C (Entry 6, Table 1) to give 12mer-Z6C(bdnK). After expression in E. coli and purification via Ni-affinity chromatography, these proteins were made react with benzyl mercaptan to splice the GyrA intein and release the macrocyclic peptide. The desired macrocyclic peptide product could be observed in each case (
This example demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (II). As such, this example demonstrates certain embodiments as schematically described in
For these studies, the constructs corresponding to Entries 10 through 12 of Table 1 were used. Three different cysteine-reactive amino acids, p-2beF, 2becK, and 2cecK, were tested as the Z residue in these constructs. The corresponding p-2beF-, 2becK-, or 2cecK-containing precursor polypeptides were expressed in BL21(DE3) E. coli cells using the appropriate AARS/tRNA pair as determined in Example 3 (Mj-pOgY2-RS/MjtRNACUATyr pair for the p-2beF-containing proteins and Mb-CrtK-RS/Mm/MbtRNACUAPyl for the 2becK and 2cecK-containing proteins). In these constructs, the reactive Cys is located upstream of the unnatural amino acid, and specifically at position Z-4, Z-6 and Z-8. Analysis of the p-2beF-containing proteins according to the procedure described above (Example 4) revealed the occurrence of the desired cyclic peptide as the largely predominant product (95-99%) for all of the constructs tested (
This example demonstrates certain embodiments as schematically described in
For these studies, the constructs corresponding to Entries 16 through 20 of Table 1 were utilized. The corresponding precursor polypeptides were expressed in BL21(DE3) E. coli cells in the presence of the Mj-pOgY2-RS/MjtRNACUATyr for incorporation of the unnatural amino acid p-2beY into these proteins via amber stop codon suppression, as described above (Example 5). These constructs were designed to comprise the C-domain and N-domain of split intein DnaE within the N-terminal tail and the C-terminal tail, respectively, of the precursor polypeptide. According to our strategy (
Notably, the desired bicyclic peptide was isolated as the largely predominant product in each case (70-95%), as determined by LC-MS (
The increased conformational rigidity imposed by the intra-side-chain thioether bridge is expected to improve the functional and/or stability properties of these bicyclic peptides as compared to the head-to-tail cyclized peptide counterpart. To investigate this aspect, the streptavidin-binding affinity of the bicyclic peptides obtained via cyclization of the cStrep3(S)-Z3C(p-2beF) and cStrep3(C)-Z8C(p-2beF) constructs was measured through an in-solution inhibition assay and compared with that of a ‘monocyclic’ counterpart (cyclo[S(OpgY)TNCHPQFANA] (SEQ ID NO:189) where OpgY is O-propargyl-tyrosine). In this assay (
Preparation and Isolation of Bicyclic Macrocycles.
Protein expression of constructs 16-20 was performed as described in the previous Examples with the difference that cells were incubated for additional 3 hours at 37° C. after overnight growth. Cells were harvested, lysed and the cell lysate treated as described above to isolate and analyze the streptavidin-bound peptides by LC-MS. To analyze the amount of protein splicing occurred in vivo, the same cell lysate samples were incubated with chitin beads for 1h on ice. Beads were washed two times with buffer followed by incubation with acetonitrile:H2O (70:30 v/v) for one minute to release any chitin-bound protein. Eluates were analyzed by LC-MS.
This example demonstrates the feasibility of generating polycyclic peptides using the methods provided herein. In particular, it demonstrates the formation and isolation of polycyclic peptides obtained via the post-translational cyclization of precursor polypeptides containing multiple Z/Cys pairs. It also demonstrates the formation and isolation of polycyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (V). In particular, this example demonstrates certain embodiments as schematically described in
For these studies, the constructs corresponding to Entries 21 and 22 of Table 1 were utilized. In Strep6_Z4C7C4Z, a Z/Cys pair encompassing a four-amino acid target peptide sequence (HPQF (SEQ ID NO:185)) is followed by a second Cys/Z pair encompassing a different target peptide sequence (NTSK) after a spacer sequence (ENLYFQS). To demonstrate the possibility to obtain polycyclic peptides in this manner, the corresponding precursor polypeptide was expressed in BL21(DE3) E. coli cells in the presence of the Mj-pOgY2-RS/MjtRNACUATyr to achieve the site-selective incorporation of the unnatural amino acid p-2beF in correspondence of the two Z residues. Although two possible bicyclic products could be generated via p-2beF-mediated cysteine alkylation, the structure-reactivity studies described in
The successful formation of cyclic peptides via the ribosomal incorporation of cysteine-reactive amino acids into precursor polypeptides as illustrated by the previous Examples suggested that macrocyclic peptide with a polycyclic architecture could also be obtained through the use of amino acids containing more than one cysteine-reactive functional group in their side chain, i.e., using amino acids with the general formula (VI) or (VII). To illustrate this aspect, one such amino acid, ObdpY, was designed and synthesized according to Scheme 6 of
A sample of the methods and cells that are described herein is set forth in the following numbered paragraphs:
1. A method for making a macrocyclic peptide, the method comprising:
a. providing an artificial nucleic acid molecule encoding for a polypeptide of structure:
(AA)m-Z-(AA)n-Cys-(AA)p (I)
or
(AA)m-Cys-(AA)n-Z-(AA)p (II)
or
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p (V)
2. The method of paragraph 1 wherein Z is an amino acid of structure:
wherein FG1 is a functional group selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO2C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO2; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane, unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide; where each R and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group;
wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups.
3. The method of paragraph 2 wherein Z is an amino acid of structure (IV) and Y is a linker group selected from the group consisting of C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, and C5-C24 aryloxy groups.
4. The method of paragraph 3 wherein Y is a linker group selected from the group consisting of —CH2—C6H4—, —CH2—C6H4—O—, —CH2—C6H4—NH—, —(CH2)4—, —(CH2)4NH—, —(CH2)4NHC(O)—, and —(CH2)4NHC(O)O—.
5. The method of paragraph 1 wherein the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, Nε-((2-bromoethoxy)carbonyl)-lysine, Nε-((2-chloroethoxy)carbonyl)-lysine, Nε-(buta-2,3-dienoyl)-lysine, Nε-acryl-lysine, Nε-crotonyl-lysine, Nε-(2-fluoro-acetyl)-lysine, and Nε-(2-chloro-acetyl)-lysine.
6. The method of paragraph 1 wherein Z2 is an amino acid of structure:
wherein each of FG1 and FG2 is a functional group independently selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO2C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO2, —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R and R′ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group;
wherein Y2, Y3, and L are linker groups selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups.
7. The method of paragraph 6 wherein Z2 is an amino acid of structure (VI) and Y2 is a linker group selected from the group consisting of C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, and C5-C24 aryloxy groups.
8. The method of paragraph 7 wherein Y is a linker group selected from the group consisting of —CH2—C6H4—, —CH2—C6H4—O—, —CH2—C6H4—NH—, —CH2—C6H4—OCH2—, —(CH2)4NH—, —(CH2)4NHC(O)—, —(CH2)4NHC(O)O—, —(CH2)4NHC(O)OCH2—,
9. The method of paragraph 1 wherein the amino acid Z2 is selected from the group consisting of 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, Nε-4(1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, Nε-(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, Nε-((2,3-dibromopropoxy)carbonyl)-lysine, and Nε-((2,3-dichloropropoxy)carbonyl)-lysine.
10. The method of paragraph 1 wherein the codon encoding for Z or Z2 is an amber stop codon TAG, an ochre stop codon TAA, an opal stop codon TGA, or a four base codon.
11. The method of paragraph 1, wherein the expression system comprises:
an aminoacyl-tRNA synthetase polypeptide or an engineered variant thereof that is at least 90% identical to SEQ ID NO:77, 78, 79, or 80; and
a transfer RNA molecule encoded by a polynucleotide that is at least 90% identical to SEQ ID NO:101, 105, 109, 113, or 117.
12. The method of paragraph 11, wherein:
(a) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises an amino acid substitution at a position selected from the group consisting of position: X32, X63, X65, X70, X107, X108, X109, X155, X158, X159, X160, X161, X162, X163, X164, X167, and X286 of SEQ ID NO:77,
(b) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises an amino acid substitution at a position selected from the group consisting of position: X302, X305, X306, X309, X346, X348, X364, X384, X401, X405, and X417 of SEQ ID NO:78,
(c) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises an amino acid substitution at a position selected from the group consisting of position: X76, X266, X270, X271, X273, X274, X313, X315, and X349 of SEQ ID NO:79, or
(d) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises an amino acid substitution at a position selected from the group consisting of position: X37, X182, X183, X186, and X265 of SEQ. ID NO. 204.
13. The method of paragraph 12, wherein:
(a) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises at least one of the features selected from the group consisting of: X32 is Tyr, Leu, Ala, Gly, Thr, His, Glu, Val, or Gln; X65 is Leu, His, Tyr, Val, Ser, Thr, Gly, or Glu; X67 is Ala or Gly; X70 is His, Ala, Cys, or Ser; X107 is Glu, Pro, Asn, or Thr; X108 is Phe, Trp, Ala, Ser, Arg, Gly, Tyr, His, Trp, or Glu; X109 is Gln, Met, Asp, Lys, Glu, Pro, His, Gly, Met, or Leu; X155 is Gln, Glu, or Gly; X158 is Asp, Gly, Glu, Ala, Pro, Thr, Ser, or Val; X159 is Ile, Cys, Pro, Leu, Ser, Trp, His, or Ala; X160 is His or Gln; X161 is Tyr or Gly; X162 is Leu, Arg, Ala, Gln, Gly, Lys, Ser, Glu, Tyr, or His; X163 is Gly or Asp; X164 is Val or Ala; X167 is Ala or Val; X286 is Asp or Arg;
(b) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises at least one of the features selected from the group consisting of: X302 is Ala or Thr; X305 is Leu or Met; X306 is Tyr, Ala, Met, Ile, Leu, Thr, Gly; X309 is Leu, Ala, Pro, Ser, or Arg; X346 is Asn, Ala, Ser, or Val; X348 is Cys, Ala, Thr, Leu, Lys, Met, or Trp; X364 is Thr or Lys; X384 is Tyr or Phe; X405 is Ile or Arg; X401 is Val or Leu; and X417 is Trp, Thr, or Leu;
(c) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises at least one of the features selected from the group consisting of: X76 is Asp or Gly; X266 is Leu, Val, or Met; X270 is Leu or Ile; X271 is Tyr, Phe, Leu, Met, or Ala; X274 is Leu, Ala, Met, or Gly; X313 is Cys, Phe, Ala, Val, or Ile; X315 is Met or Phe; and X349 is Tyr, Phe, or Trp; or
(d) the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises at least one of the features selected from the group consisting of: X37 is Tyr, Ile, Gly, Val, Leu, Thr, or Ser; X182 is Asp, Gly, Ser, or Thr; X183 is Phe, Met, Tyr, or Ala; X186 is Leu, Ala, Met, or Val; and X265 is Asp or Arg.
14. The method of paragraph 1, wherein the expression system comprises:
an aminoacyl-tRNA synthetase selected from the group consisting of SEQ ID NOs. 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100; and
a transfer RNA molecule encoded by a polynucleotide selected from the group consisting of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120.
15. The method of paragraph 1, wherein the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), or (V) comprise(s):
16. The method of paragraph 15, wherein the polypeptide comprised within the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), and (V), is a polypeptide selected from the group of polypeptides consisting of SEQ ID NOs 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, and 158.
17. The method of paragraph 15, wherein the intein polypeptide comprised within the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), or (V), is a selected from the group consisting of a naturally occurring intein, an engineered variant of a naturally occurring intein, a fusion of the N-terminal and C-terminal fragments of a naturally occurring split intein and a fusion of the N-terminal and C-terminal fragments of an engineered split intein.
18. The method of paragraph 17, wherein the intein is selected from the group consisting of Mxe GyrA (SEQ ID NO:1), eDnaB (SEQ ID NO:2), Hsp-NRC1 CDC21 (SEQ ID NO:3), Ceu ClpP (SEQ ID NO:4), Tag Pol-1 (SEQ ID NO:5), Tfu Pol-1 (SEQ ID NO:6), Tko Pol-1 (SEQ ID NO:7), Psp-GBD Pol (SEQ ID NO:8), Tag Pol-2 (SEQ ID NO:9), Thy Pol-1 (SEQ ID NO:10), Tko Pol-2 (SEQ ID NO:11), Tli Pol-1 (SEQ ID NO:12), Tma Pol (SEQ ID NO:13), Tsp-GE8 Pol-1 (SEQ ID NO:14), Tthi Pol (SEQ ID NO:15), Tag Pol-3 (SEQ ID NO:16), Tfu Pol-2 (SEQ ID NO:17), Thy Pol-2 (SEQ ID NO:18), Tli Pol-2 (SEQ ID NO:19), Tsp-GE8 Pol-2 (SEQ ID NO:20), Pab Pol-II (SEQ ID NO:21), Mtu-CDC1551 DnaB (SEQ ID NO:22), Mtu-H37Rv DnaB (SEQ ID NO:23), Rma DnaB (SEQ ID NO:24), Ter DnaE-1 (SEQ ID NO:25), Ssp GyrB (SEQ ID NO:26), Mfl GyrA (SEQ ID NO:27), Mgo GyrA (SEQ ID NO:28), Mkas GyrA (SEQ ID NO:29), Mle-TN GyrA (SEQ ID NO:30), Mma GyrA (SEQ ID NO:31), Ssp DnaX (SEQ ID NO:32), Pab Lon (SEQ ID NO:33), Mja PEP (SEQ ID NO:34), Afu-FRR0163 PRP8 (SEQ ID NO:35), Ani-FGSCA4 PRP8 (SEQ ID NO:36), Cne-A PRP8 (SEQ ID NO:37), Hca PRP8 (SEQ ID NO:38), Pch PRP8 (SEQ ID NO:39), Pex PRP8 (SEQ ID NO:40), Pvu PRP8 (SEQ ID NO:41), Mtu-H37Rv RecA (SEQ ID NO:42), Mtu-So93 RecA (SEQ ID NO:43), Mfl RecA (SEQ ID NO:44), Mle-TN RecA (SEQ ID NO:45), Nsp-PCC7120 RIR1 (SEQ ID NO:46), Ter RIR1-1 (SEQ ID NO:47), Pab RIR1-1 (SEQ ID NO:48), Pfu RIR1-1 (SEQ ID NO:49), Chy RIR1 (SEQ ID NO:50), Mth RIR1 (SEQ ID NO:51), Pab RIR1-3 (SEQ ID NO:52), Pfu RIR1-2 (SEQ ID NO:53), Ter RIR1-2 (SEQ ID NO:54), Ter RIR1-4 (SEQ ID NO:55), CIV RIR1 (SEQ ID NO:56), Ctr VMA (SEQ ID NO:57), Sce VMA (SEQ ID NO:58), Tac-ATCC25905 VMA (SEQ ID NO:59), Ssp DnaB (SEQ ID NO:60),
engineered variant(s) thereof, and
engineered variant(s) thereof wherein the N-terminal cysteine or serine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than cysteine or serine, or wherein the C-terminal asparagine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than asparagine.
19. The method of paragraph 17, wherein the intein is a fusion product of a split intein selected from the group consisting of Ssp DnaE (SEQ ID NO:61-SEQ ID NO:62), Neq Pol (SEQ ID NO:63-SEQ ID NO:64), Asp DnaE (SEQ ID NO:65-SEQ ID NO:66), Npu-PCC73102 DnaE (SEQ ID NO:67-SEQ ID NO:68), Nsp-PCC7120 DnaE (SEQ ID NO:69-SEQ ID NO:70), Oli DnaE (SEQ ID NO:71-SEQ ID NO:72), Ssp-PCC7002 DnaE (SEQ ID NO:73-SEQ ID NO:74), Tvu DnaE (SEQ ID NO:75-SEQ ID NO:76),
engineered variant(s) thereof, and
engineered variant(s) thereof wherein the N-terminal cysteine or serine residue of the split intein N-domain of the engineered variant is mutated to any of the natural (or naturally occurring) amino acid residues other than cysteine or serine, or wherein the C-terminal asparagine residue of the split intein C-domain of the engineered variant is mutated to any of the natural (or naturally occurring) amino acid residues other than asparagine.
20. The method of paragraph 1, wherein
the N-terminal tail polypeptide, (AA)m, of the precursor polypeptide of formula (I), (II), or (V) comprises the C-domain of a split intein, and
the C-terminal tail polypeptide, (AA)p, comprises the corresponding N-domain of the split intein.
21. The method of paragraph 20, wherein the split intein C-domain is selected from the group consisting of Ssp DnaE-c (SEQ ID NO:62), Neq Pol-c (SEQ ID NO:64), Asp DnaE-c (SEQ ID NO:66), Npu-PCC73102 DnaE-c (SEQ ID NO:68), Nsp-PCC7120 DnaE-c (SEQ ID NO:70), Oli DnaE-c (SEQ ID NO:72), Ssp-PCC7002 DnaE-c (SEQ ID NO:74), Tvu DnaE-c (SEQ ID NO:76), and engineered variant(s) thereof; and the split intein N-domain is selected from the group consisting of Ssp DnaE-n (SEQ ID NO:61), Neq Pol-n (SEQ ID NO:63), Asp DnaE-n (SEQ ID NO:65), Npu-PCC73102 DnaE-n (SEQ ID NO:67), Nsp-PCC7120 DnaE-n (SEQ ID NO:69), Oli DnaE-n (SEQ ID NO:71), Ssp-PCC7002 DnaE-n (SEQ ID NO:73), Tvu DnaE-n (SEQ ID NO:75), and engineered variant(s) thereof.
22. The method of paragraph 1 wherein the expression system is selected from the group consisting of a prokaryotic cell, an eukaryotic cell, and a cell-free expression system.
23. The method of paragraph 22 wherein the prokaryotic cell is Escherichia coli.
24. The method of paragraph 22 wherein the eukaryotic cell is a yeast, a mammalian, an insect or a plant cell.
25. The method of paragraph 1 wherein any of polypeptides (AA)n, (AA)o, (AA)m, or (AA)p, is fully or partially genetically randomized so that a plurality of macrocyclic peptides is obtained upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG1 in Z or between the cysteine (Cys) residues and the side-chain functional groups FG1 and FG2 in Z2.
26. The method of paragraph 1 comprising:
fully or partially randomizing any of polypeptides (AA)n, (AA)o, (AA)m, or (AA)p, wherein, upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG1 in Z or between the cysteine (Cys) residues and the side-chain functional groups FG1 and FG2 in Z2, a plurality of macrocyclic peptides is produced.
27. A recombinant host cell comprising a polypeptide of structure:
(AA)m-Z-(AA)n-Cys-(AA)p (I)
or
(AA)m-Cys-(AA)n-Z-(AA)p (II)
or
(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p (V)
wherein:
28. The cell of paragraph 27, wherein the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, Nε-((2-bromoethoxy)carbonyl)-lysine, Nε-((2-chloroethoxy)carbonyl)-lysine, Nε-(buta-2,3-dienoyl)-lysine, Nε-acryl-lysine, Nε-crotonyl-lysine, Nε-(2-fluoro-acetyl)-lysine, and Nε-(2-chloro-acetyl)-lysine.
29. The cell of paragraph 27 wherein the amino acid Z2 is selected from the group consisting of 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, Nε-4(1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, Nε-(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, Nε-((2,3-dibromopropoxy)carbonyl)-lysine, and Nε-((2,3-dichloropropoxy)carbonyl)-lysine.
30. The cell of paragraph 27, wherein the polypeptide comprised within the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, of the precursor polypeptides of formula (I), (II), and (V), is a polypeptide selected from the group of polypeptides consisting of SEQ ID NOs 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, and 158.
31. The cell of paragraph 30, wherein the cell comprises a macrocyclic peptide produced by:
a thioether bond-forming reaction between the cysteine (Cys) residue and the FG1 functional group in the amino acid Z or between the cysteine (Cys) residues and the FG1 and FG2 functional groups in the amino acid Z2.
32. The cell of paragraph 27, wherein the N-terminal tail polypeptide, (AA)m, or the C-terminal tail polypeptide, (AA)p, or both, in the precursor polypeptides of formula (I), formula (II), or formula (V) comprise(s) an intein selected from the group consisting of a naturally occurring intein, an engineered variant of a naturally occurring intein, a fusion of the N-terminal and C-terminal fragments of a naturally occurring split intein and a fusion of the N-terminal and C-terminal fragments of an engineered split intein.
33. The cell of paragraph 32, wherein the intein is selected from the group consisting of Mxe GyrA (SEQ ID NO:1), eDnaB (SEQ ID NO:2), Hsp-NRC1 CDC21 (SEQ ID NO:3), Ceu C1pP (SEQ ID NO:4), Tag Pol-1 (SEQ ID NO:5), Tfu Pol-1 (SEQ ID NO:6), Tko Pol-1 (SEQ ID NO:7), Psp-GBD Pol (SEQ ID NO:8), Tag Pol-2 (SEQ ID NO:9), Thy Pol-1 (SEQ ID NO:10), Tko Pol-2 (SEQ ID NO:11), Tli Pol-1 (SEQ ID NO:12), Tma Pol (SEQ ID NO:13), Tsp-GE8 Pol-1 (SEQ ID NO:14), Tthi Pol (SEQ ID NO:15), Tag Pol-3 (SEQ ID NO:16), Tfu Pol-2 (SEQ ID NO:17), Thy Pol-2 (SEQ ID NO:18), Tli Pol-2 (SEQ ID NO:19), Tsp-GE8 Pol-2 (SEQ ID NO:20), Pab Pol-II (SEQ ID NO:21), Mtu-CDC1551 DnaB (SEQ ID NO:22), Mtu-H37Rv DnaB (SEQ ID NO:23), Rma DnaB (SEQ ID NO:24), Ter DnaE-1 (SEQ ID NO:25), Ssp GyrB (SEQ ID NO:26), Mfl GyrA (SEQ ID NO:27), Mgo GyrA (SEQ ID NO:28), Mkas GyrA (SEQ ID NO:29), Mle-TN GyrA (SEQ ID NO:30), Mma GyrA (SEQ ID NO:31), Ssp DnaX (SEQ ID NO:32), Pab Lon (SEQ ID NO:33), Mja PEP (SEQ ID NO:34), Afu-FRR0163 PRP8 (SEQ ID NO:35), Ani-FGSCA4 PRP8 (SEQ ID NO:36), Cne-A PRP8 (SEQ ID NO:37), Hca PRP8 (SEQ ID NO:38), Pch PRP8 (SEQ ID NO:39), Pex PRP8 (SEQ ID NO:40), Pvu PRP8 (SEQ ID NO:41), Mtu-H37Rv RecA (SEQ ID NO:42), Mtu-So93 RecA (SEQ ID NO:43), Mfl RecA (SEQ ID NO:44), Mle-TN RecA (SEQ ID NO:45), Nsp-PCC7120 RIR1 (SEQ ID NO:46), Ter RIR1-1 (SEQ ID NO:47), Pab RIR1-1 (SEQ ID NO:48), Pfu RIR1-1 (SEQ ID NO:49), Chy RIR1 (SEQ ID NO:50), Mth RIR1 (SEQ ID NO:51), Pab RIR1-3 (SEQ ID NO:52), Pfu RIR1-2 (SEQ ID NO:53), Ter RIR1-2 (SEQ ID NO:54), Ter RIR1-4 (SEQ ID NO:55), CIV RIR1 (SEQ ID NO:56), Ctr VMA (SEQ ID NO:57), Sce VMA (SEQ ID NO:58), Tac-ATCC25905 VMA (SEQ ID NO:59), Ssp DnaB (SEQ ID NO:60),
engineered variant(s) thereof, and
engineered variant(s) thereof wherein the N-terminal cysteine or serine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than cysteine or serine, or wherein the C-terminal asparagine residue of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than asparagine.
34. The cell of paragraph 32, wherein the intein is a fusion product of a split intein selected from the group consisting of Ssp DnaE (SEQ ID NO:61-SEQ ID NO:62), Neq Pol (SEQ ID NO:63-SEQ ID NO:64), Asp DnaE (SEQ ID NO:65-SEQ ID NO:66), Npu-PCC73102 DnaE (SEQ ID NO:67-SEQ ID NO:68), Nsp-PCC7120 DnaE (SEQ ID NO:69-SEQ ID NO:70), Oli DnaE (SEQ ID NO:71-SEQ ID NO:72), Ssp-PCC7002 DnaE (SEQ ID NO:73-SEQ ID NO:74), Tvu DnaE (SEQ ID NO:75-SEQ ID NO:76),
engineered variant(s) thereof,
engineered variant(s) thereof, wherein the N-terminal cysteine or serine residue of the split intein N-domain of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than cysteine or serine, or wherein the C-terminal asparagine residue of the split intein C-domain of the engineered variant is mutated to any natural (or naturally occurring) amino acid residue other than asparagine.
35. The cell of paragraph 32, wherein the cell comprises a macrocyclic peptide produced by:
a thioether bond-forming reaction between the cysteine (Cys) residue and the FG1 functional group in the amino acid Z or between the cysteine (Cys) residues and the FG1 and FG2 functional groups in the amino acid Z2, and
an intein-catalyzed N-terminal splicing, C-terminal splicing, or self-splicing reaction.
36. The cell of paragraph 27, wherein:
the N-terminal tail polypeptide, (AA)m, comprises the C-domain of a naturally occurring split intein, or of an engineered variant thereof, and
the C-terminal tail polypeptide, (AA)p, comprises the N-domain of said split intein.
37. The cell of paragraph 36, wherein the split intein C-domain is selected from the group consisting of Ssp DnaE-c (SEQ ID NO:62), Neq Pol-c (SEQ ID NO:64), Asp DnaE-c (SEQ ID NO:66), Npu-PCC73102 DnaE-c (SEQ ID NO:68), Nsp-PCC7120 DnaE-c (SEQ ID NO:70), Oli DnaE-c (SEQ ID NO:72), Ssp-PCC7002 DnaE-c (SEQ ID NO:74), Tvu DnaE-c (SEQ ID NO:76), and engineered variant(s) thereof; and the split intein N-domain is selected from the group consisting of Ssp DnaE-n (SEQ ID NO:61), Neq Pol-n (SEQ ID NO:63), Asp DnaE-n (SEQ ID NO:65), Npu-PCC73102 DnaE-n (SEQ ID NO:67), Nsp-PCC7120 DnaE-n (SEQ ID NO:69), Oli DnaE-n (SEQ ID NO:71), Ssp-PCC7002 DnaE-n (SEQ ID NO:73), Tvu DnaE-n (SEQ ID NO:75), and engineered variant(s) thereof.
38. The cell of paragraph 36, wherein the cell comprises a polycyclic peptide produced by:
a thioether bond-forming reaction between the cysteine (Cys) residue and the FG1 functional group in the amino acid Z or between the cysteine (Cys) residues and the FG1 and FG2 functional groups in the amino acid Z2, and
a split intein-catalyzed trans-splicing reaction.
It will be appreciated that variants of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.
While embodiments of the present disclosure have been particularly shown and described with reference to certain examples and features, it will be understood by one skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the present disclosure as defined by claims that can be supported by the written description and drawings. Further, where exemplary embodiments are described with reference to a certain number of elements it will be understood that the exemplary embodiments can be practiced utilizing either less than or more than the certain number of elements.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
This application is a national stage application under 35 U.S.C. § 371 of PCT Application No. PCT/US14/72016, filed Dec. 23, 2014, which claims priority to and the benefit of U.S. provisional patent application Ser. No. 61/920,181, entitled Methods and Compositions for Ribosomal Synthesis of Macrocyclic Peptides, filed Dec. 23, 2013, which is incorporated herein by reference in its entirety.
This invention was made with government support under grant no. R21CA187502 awarded by the National Institutes of Health and grant no. CHE-1112342 awarded by the National Science Foundation. The government has certain rights in this invention.
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| WO2015/100277 | 7/2/2015 | WO | A |
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| Number | Date | Country | |
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