Patent Application
20190391160
The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 31, 2019, is named B12425_SL.txt and is 3,672 bytes in size.
Cystic fibrosis (CF) is caused by mutations in the CFTR gene, which encodes a 1480-amino acid transmembrane protein with a symmetrical structure composed of two membrane-spanning domains (MSD1 and MSD2), each with six transmembrane helices, and two nucleotide binding domains (NBD1 and NBD2) separated by a hydrophilic regulatory domain (R)1. The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a unique chloride (Cl—) channel that links ATP hydrolysis to channel gating and regulates transepithelial fluid transport2,3. A deletion of phenylalanine at position 508 (DF508) in the NBD1 domain is present in at least one allele in 90% of patients suffering from cystic fibrosis and gives rise to an incorrectly folded protein which is rapidly degraded and cannot reach the plasma membrane4. This defect leads to reduced intrinsic Cl— membrane channel conductance in CF cells, compared with wild type CFTR5.
A number of studies have been conducted to search for a pharmacological approach to correct the dysfunction of the mutated proteins6. For the DF508 mutation, small molecule compounds have been developed to facilitate trafficking and delivery of the abnormal protein to the plasma membrane (correctors) and to improve channel gating (potentiators). To this end, two strategies have been employed. The first is to stabilize ΔF508CFTR using a high-throughput screening approach to identify compounds that are able to correct ΔF508CFTR dysfunction. The best examples are the corrector VX-809 and the potentiator VX-770 (ivacaftor), the latter being the most successful example of this approach. Today Ivacaftor is used to treat G551D CF patients, but neither VX-809 nor ivacaftor are sufficiently active in ΔF508 CF patients. The second strategy is a hypothesis-driven approach, which has led to the identification of correctors such as curcumin and resveratrol derived from plants Z. This strategy also suggested that the site of interaction between cytokeratin-8 (K8) and DF508CFTR should constitute a therapeutic target 2. However, there is still a major need for the development of new selective and high affinity compounds acting as dual modulators. The inventions disclosed herein meet these and other needs.
In an attempt to identify a new class of correctors/modulators of ΔF508 CFTR natural, multifunctional proteins present in snake venom were studied. In particular, the phospholipase A2 (PLA2) CB subunit of crotoxin from the South American rattlesnake Crotalus durissus terrificus was investigated. During investigation of the cytokeratin 8-NBD1 interaction10 and using CB as a negative control, a surprisingly high binding affinity of CB for NBD1 was discovered. Viperidae snake venom PLA2 (structurally homologous to inflammatory, non-pancreatic human sPLA2-IIA) are known to possess a large spectrum of pharmacological functions. However, no effect on CFTR was taught or suggested in the art. Numerous studies have shown that neurotoxic phospholipases A2 can enter into cells and interact with various protein targets, exhibiting different pharmacological effects, sometimes independently of their enzymatic activity11 12 13 14 15. In particular, crotoxin from the South American rattlesnake Crotalus durissus terrificus, a heterodimeric CA-CB presynaptic toxin with PLA2 activity16 exhibits bactericidal, antiviral, anti-cytotoxic properties against various tumor cells and can also regulate Ca2+ channel currents17; 18; 19; 20. Ollivier-Bousquet et al21 showed that the CB subunit of crotoxin, alone or in combination with CA, was able to adsorb onto the membrane of epithelial cells and to be internalized to induce lectin secretion. More recently Lomeo et al20 reported that the CB subunit of crotoxin is internalized within less than 5 min in cerebellar granule cells and that CB internalization does not depend on the presence of CA and does not depend on PLA2 activity. Both subunits of crotoxin exist in four major natural isoforms (acidic CA1-4 and basic CBa2/b/c/d) and represent interesting models to identify new PLA2-binding targets14; 16. None of these studies pointed to a role for crotoxin in modulating the molecular mechanisms that underly cystic fibrosis.
The interaction of the CB subunit of crotoxin with CFTR and the potential effect of CB on Cl− channel activity was investigated. Experimental evidence is provided that CB directly interacts with the wild-type (WT) and mutated NBD1 domain of human CFTR and corrects the functional defect of ΔF508CFTR. It is shown that CB behaves as a dual modulator of CFTR activity as a potentiator, increasing the Cl− channel current, and as a corrector, facilitating transport and insertion of DF508CFTR into the plasma membrane.
Accordingly, in a first aspect this disclosure provides methods of increasing CFTR activity in a cell, comprising contacting the cell with a peptide modulator of CFTR to thereby increase CFTR activity in the cell; wherein the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, and a polypeptide comprising a functional variant of SEQ ID NO: 2; a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, and a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
In another aspect this disclosure provides methods of treating cystic fibrosis in a subject in need thereof, comprising administering an effective amount of a peptide modulator of CFTR to the subject to thereby increase CFTR activity in the subject; wherein the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, and a polypeptide comprising a functional variant of SEQ ID NO: 2; and a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
In another aspect this disclosure provides pharmaceutical compositions comprising a peptide modulator of CFTR; wherein the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, a polypeptide comprising a functional variant of SEQ ID NO: 2; and a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, and a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
In another aspect this disclosure provides uses of a peptide modulator of CFTR for the manufacture of a medicament for use in treating cystic fibrosis; wherein the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom.
In another aspect this disclosure provides peptide modulators of CFTR for use in treating cystic fibrosis; wherein the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom.
In another aspect this disclosure provides methods of characterizing a CFTR modulator. In some embodiments the methods comprise contacting a cell that expresses CFTR with a peptide modulator of CFTR that increases CFTR activity in the cell; contacting the cell with a candidate agent; and determining whether the candidate agent modulates the effect of the peptide modulator of CFTR on CFTR activity; wherein the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, and a polypeptide comprising a functional variant of SEQ ID NO: 2; and a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the candidate agent modulates the effect of the peptide modulator of CFTR on CFTR activity and the candidate agent is identified as a CFTR modulator.
(B) Effect of CA-CB on CFTR-Cl− current in X. laevis oocytes. Current-voltage (I/V) relationships were determined in CFTR-expressing oocytes injected with 1 ng of CFTR cRNA and in CFTR-expressing oocytes injected with 2.5 ng CA-CB, before and after superfusion with PKA-activating cocktail (1 μM forskolin plus 100 μM IBMX). Error bars represent the standard error of the mean for each data point (n=8). Black circles correspond to I/V curves obtained for oocytes expressing CFTR alone; white circles for oocytes expressing CFTR and injected with CACB; green lines correspond to I/V curves obtained in the presence of PKA-activating cocktail; red lines to I/V curves obtained in the presence of 10 μM of Inh-172; black lines to the I/V curves obtained in control conditions (without PKA-activating cocktail). Results are shown as means±SEM, with n as the number of oocytes from different donors (n=8), N=3.
(C) Summary of the results obtained in Figure S1B. at −100 mV and +40 mV in the CFTR—expressing oocytes. ND96, Ringer's solution. The experimental conditions are indicated above or below the bars. 10 μM Inh-172 was used.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Certain references and other documents cited herein are expressly incorporated herein by reference. Additionally, all Genbank or other sequence database records cited herein are hereby incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).
Before the present compositions, methods, and other embodiments are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
The term “comprising” as used herein is synonymous with “including” or “containing”, and is inclusive or open-ended and does not exclude additional, unrecited members, elements or method steps.
As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe.
As used herein, “subject” means any mammal including mice or primates. In a preferred embodiment the subject is a human.
As used herein, the terms “treat,” “treatment,” “treating,” and “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down and/or stop the progression or severity of a condition associated with a disease or disorder. The terms include reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The terms “treat,” “treatment,” “treating,” and “amelioration” in reference to a disease also include providing relief from the symptoms or side-effects of the disease (including palliative treatment).
As used herein, an “effective amount” is an amount of a chemical entity that is effective when administered following a dosing schedule over a therapeutic dosing period.
As used herein, a “therapeutic dosing period” is a period of time during which a chemical entity is administered to a subject following a defined dosing schedule.
The human CFTR gene and protein are known in the art. The human CFTR sequence is available at www.uniprot.org/uniprot/P13569.
Peptide modulators of CFTR are provided. The peptide modulators bind to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR.
A template-based modelling approach was used to select peptides to effectively disrupt interactions between delF508-NBD1 and CB subunit of Crotoxin. As a starting point, a structural 3D model of the delF508NBD1/CBb complex were created. A molecular docking protocol consisting of the following steps was used: (a) an initial, rigid body 3D search based on fast a Fourier transform algorithm; (b) primary rescoring with a linear weighted scoring function implemented in ZRANK; (c) structural refinement by Monte Carlo methods; (d) secondary rescoring with ZRANK function optimized for refined complexes. The highest scored model of the delF508-NBD1/CB complex structure, has been used for identification of protein-protein interaction interface and then to proposed final peptide sequences on the basis of the native sequence of CB. This approach was used to identify the following peptides: HLLQFNK (SEQ ID NO: 1), NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), NGYMFYPDS (SEQ ID NO: 3).
In some embodiments the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator comprises the amino acid sequence HLLQFNK (SEQ ID NO: 1) or a functional variant of SEQ ID NO: 1. In some embodiments the peptide modulator consists of the amino acid sequence SEQ ID NO: 1 or a functional variant of SEQ ID NO: 1.
In some embodiments the peptide modulator comprises the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2) or a functional variant of SEQ ID NO: 2. In some embodiments the peptide modulator consists of the amino acid sequence SEQ ID NO: 2 or a functional variant of SEQ ID NO: 2.
In some embodiments the peptide modulator comprises the amino acid sequence NGYMFYPDS (SEQ ID NO: 3) or a functional variant of SEQ ID NO: 3. In some embodiments the peptide modulator consists of the amino acid sequence SEQ ID NO: 3 or a functional variant of SEQ ID NO: 3.
In some embodiments the peptide modulator comprises the amino acid sequence NGYMFYPDSR CRG (SEQ ID NO: 4) or a functional variant of SEQ ID NO: 4. In some embodiments the peptide modulator consists of the amino acid sequence SEQ ID NO: 4 or a functional variant of SEQ ID NO: 4.
In some embodiments the peptide modulator comprises the amino acid sequence NAVPFYAFYG CYSGWGGQGR (SEQ ID NO: 5) or a functional variant of SEQ ID NO: 5. In some embodiments the peptide modulator consists of the amino acid sequence SEQ ID NO: 5 or a functional variant of SEQ ID NO: 5.
In some embodiments the peptide modulator comprises the amino acid sequence HLLQFNKMIK FET (SEQ ID NO: 6) or a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator consists of the amino acid sequence SEQ ID NO: 6 or a functional variant of SEQ ID NO: 6.
In some embodiments the peptide modulator is recombinant. In some embodiments the peptide modulator is synthetic. In some embodiments the peptide modulator is isolated. In some embodiments the peptide modulator is purified.
The properties of the peptide modulator can be readily verified by techniques known to those skilled in the art, such as those described in the examples of the present application.
“Functional” with respect to a peptide modulator refers to a peptide which is able to bind to CFTR protein and increase CFTR activity in a cell. A “functional variant” of an amino acid sequence is an amino acid sequence that has at least one sequence modification in comparison to a reference sequence; and that is able to bind to CFTR protein and increase CFTR activity in a cell. In some embodiments the functional variant increases CFTR activity in a cell by at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the increase in CFTR activity in a cell that is achieved by a peptide modulator comprising the reference sequence. In some embodiments the functional variant increases CFTR activity in a cell by at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the increase in CFTR activity in a cell that is achieved by the CB subunit of crotoxin from Crotalus durrissus terrificus venom. Suitable assays for making this comparison are provided, for example, in the examples of this application.
Functional variants may be derived from wild-type amino acid sequences by the introduction of one or more mutations (deletion, insertion, and/or substitution) at specific amino acid positions. In some embodiments the functional variant differs from the wild-type amino acid sequence by the deletion, insertion, and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. In some embodiments the functional variant comprises one or more deletion, and/or one or more insertion, and/or one or more substitution relative to the wild-type amino acid sequence. In a particular embodiment functional variants are obtained by insertion of amino acid residues at the N- or C-terminal end of the peptide. These variants may in particular result from addition of amino acid residues which are adjacent to those of the peptide in the CB unit in particular in the CBb sequence of SEQ ID NO: 7. Examples of such constructs are disclosed herein, in particular for peptides of less than 25 amino acid residus.
In some embodiments a functional variant comprises an amino acid sequence which is “substantially homologous” or “substantially similar” to the sequence of the reference peptide from which it is derived. Two amino acid sequences are “substantially homologous” or “substantially similar” when one or more amino acid residues are replaced by a biologically similar residue and/or when the sequences are at least 80% identical and/or at least 90% similar.
The percent amino acid sequence identity/similarity is defined as the percent of amino acid residues in a Compared Sequence that are identical/similar to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity. The Percent identity is then determined according to the following formula: Percent identity=100×[1−(C/R)], wherein C is the number of differences between the Reference Sequence and the Compared sequence over the entire length of the Reference Sequence, wherein (i) each amino acid in the Reference Sequence that does not have a corresponding aligned amino acid in the Compared Sequence, (ii) each gap in the Reference Sequence, and (iii) each aligned amino acid in the Reference Sequence that is not identical/similar to an amino acid in the Compared Sequence constitutes a difference; and R is the number amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as an amino acid.
Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-), FASTA, the GCG (Genetics computer Group, Program Manual for the GCG Package, version 7, Madison, Wis.) pileup program, or any of the programs known in the art. When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10.
Conservative substitution refers to the substitution of one amino acid with another, without altering the overall conformation and function of the peptide, including but not limited to the replacement of an amino acid with one which has similar chemical or physical properties (size, charge or polarity), which generally does not modify the functional properties of the protein. Amino acids with similar properties are well known in the art. A non-limitative example of conservative substitution(s) comprises the five following groups: Group 1-small aliphatic, non-polar or slightly polar residues (A, S, T, P, G); Group 2-polar, negatively charged residues and their amides (D, N, E, Q); Group 3-polar, positively charged residues (H, R, K); Group 4-large aliphatic, nonpolar residues (M, L, I, V, C); and Group 5-large, aromatic residues (F, Y, W). Alternative, examples of conservative substitutions are known in the art.
In some embodiments the functional variant comprises or consists of an amino acid sequence which is at least 70%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 1, 2 3, 4, 5 or 6. In some embodiments the functional variant differs from SEQ ID NO: 1, 2 or 3 by one or more conservative substitutions.
In some embodiments the peptide modulator comprises no more than 100, 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, or 7 amino acids. In a particular embodiment, the peptide modulator that comprises the amino acid sequence of SEQ ID NO: 1 is the peptide of amino acid sequence SEQ ID NO: 6 or a peptide which comprises the amino acid sequence SEQ ID NO: 6, especially having at most a number of amino acid residues as disclosed herein. In a particular embodiment, the peptide modulator that comprises the amino acid sequence of SEQ ID NO: 2 is the peptide of amino acid sequence SEQ ID NO: 5 or a peptide which comprises the amino acid sequence SEQ ID NO: 5, especially having at most a number of amino acid residues as disclosed herein. In a particular embodiment, the peptide modulator that comprises the amino acid sequence of SEQ ID NO: 3 is the peptide of amino acid sequence SEQ ID NO: 4 or a peptide which comprises the amino acid sequence SEQ ID NO: 4, especially having at most a number of amino acid residues as disclosed herein. In a particular embodiment the peptide modulator consists in a sequence of 7 to 30 or 7 to 25 amino acid residues.
In some embodiments the peptide modulator comprises a first amino acid sequence selected from SEQ ID NOS: 1-6 or in particular SEQ ID NOS: 1-3 or 4-6, or a functional variant thereof, fused to a second amino acid sequence. The second amino acid sequence may be fused to the N-terminal and/or C-terminal end(s) of the amino acid sequence selected from SEQ ID NOS: 1-6 or in particular SEQ ID NOS: 1-3 or 4-6. The second amino acid sequence may be selected to facilitate the purification, detection, immobilization, and/or cellular targeting of the peptide modulator, and/or to increase the affinity of the peptide modulator for CFTR, the bioavailability of the peptide modulator, the production in expression systems of the peptide modulator and/or the stability of the peptide modulator. The second amino acid sequence may be selected from: (i) a cell-penetrating moiety, (ii) a labeling moiety such as a fluorescent protein (GFP and its derivatives, BFP and YFP), (iii) a reporter moiety such as an enzyme tag (luciferase, alkaline phosphatase, glutathione-S-transferase (GST), β-galactosidase), (iv) a binding moiety such as an epitope tag (polyHis6 (SEQ ID NO: 8), FLAG, HA, myc.), a DNA-binding domain, a hormone-binding domain, a poly-lysine tag for immobilization onto a support, (v) a stabilization moiety, and (vi) a targeting moiety for addressing the peptide modulator to a specific cell type or subcellular compartment. In addition, the amino acid sequence selected from SEQ ID NOS: 1-6, or a functional derivative thereof, may be separated from the second amino acid sequence by a linker which is long enough to avoid inhibiting interactions between the amino acid sequence selected from SEQ ID NOS: 1-6, or a functional derivative thereof, and the second amino acid sequence. The linker may comprise a recognition site for a protease, for example, for removing affinity tags and/or stabilization moieties.
In some embodiments the second amino acid sequence is a cell-penetrating peptide (CPP), also known as protein transduction domains (PTDs), membrane translocation sequences (MTSs), transport peptides, carrier peptides or Trojan peptides are well-known in the art. In some embodiments, the CPP aids translocation of the peptide modulator into cells at significantly higher levels than passive diffusion, without causing substantial membrane damage, and can be used as vectors of other molecules when linked to them.
In some embodiments the peptide modulator comprises a chemical modification. In some embodiments all or substantially all of the amino acids of a peptide modulator comprise a similar or identical chemical modification. In some embodiments a subset of at least one of the amino acids of a peptide modulator comprise a similar or identical chemical modification. In some embodiments the chemical modification comprises at lease one of: the substitution of a natural amino acid with a non-proteinogenic amino acid (D amino acid or amino acid analog); the modification of the peptide bond, in particular with a bond of the retro or retro-inverso type or a bond different from the peptide bond; the cyclization, and the addition of a chemical group to the side chain or the end(s) of the peptide, in particular for coupling an agent of interest. These modifications may be used to label the peptide, and/or to increase its stability and/or its resistance to proteolysis.
In some embodiments the at least one chemical modification protects the peptide modulator against proteolysis.
In some embodiments the N- and/or C-terminus of the peptide modulator is protected against proteolysis.
In some embodiments the N-terminus is in the form of an acetyl group and/or the C-terminus in the form of an amide group.
In some embodiments the peptide modulator is protected against proteolysis by internal modifications such as the replacement of at least one —CONH— peptide bond by a (CH2NH) reduced bond, a (NHCO) retro-inverso bond, a (CH2-O) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO—CH) cetomethylene bond, a (CHOH—CH2) hydroxyethylene bond, a (N—N) bond, a E-alcene bond, or a —CH═CH— bond.
In some embodiments the peptide modulator is modified by at least one of acetylation, acylation, amidation, cross-linking, cyclization, disulfide bond formation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, and the like.
In some embodiments the peptide modulator comprises at least one amino acid in D configuration.
In some embodiments the peptide modulator is stabilised by intramolecular crosslinking, by modifying at least two amino acid residues with olefinic side chains, preferably C3-C8 alkenyl chains, more preferably penten-2-yl chains, followed by crossliking of the chains according to the so-called ‘“stapled-peptide technology” described in Walensky et al., Science, 2004, 305, 1466-1470.
In some embodiments the peptide modulator is stabilised by covalent binding to a polyethylene glycol (PEG) molecule, preferably a PEG of 1500 Da or 4000 Da, advantageously bound to their C-terminus or a lysine residue. Such coupling has the advantage to decrease urinary clearance and therapeutic doses and increase half-life in blood plasma.
In some embodiments the peptide modulator is stabilised and its half-life increased by incorporation into a biodegradable and biocompatible polymer material for drug delivery system forming microspheres, such as for instance poly(D, L-lactide-co-glycolide (PLGA) and nanoparticules.
Also provided are pharmaceutical compositions comprising a peptide modulator of this disclosure. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, and a polypeptide comprising a functional variant of SEQ ID NO: 2; a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, and a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSR CRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYG CYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; a polypeptide comprising the amino acid sequence HLLQFNKMIK FET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
Typically a pharmaceutical composition comprises a peptide modulator as described above, and a pharmaceutically acceptable carrier. In some embodiments the pharmaceutical composition is formulated for administration by a route selected from oral, parenteral and local.
The pharmaceutical composition comprises a therapeutically effective amount of the peptide modulator, e.g., sufficient to show benefit to the subject to whom it is administered. The pharmaceutically effective dose depends upon the composition used, the route of administration, the type of subject being treated, the physical characteristics of the subject, concurrent medication, cystic fibrosis disease state and other factors, that those skilled in the art will recognize.
Methods of increasing CFTR activity in a cell are also provided. The methods comprise contacting the cell with a peptide modulator of CFTR to thereby increase CFTR activity in the cell. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, and a polypeptide comprising a functional variant of SEQ ID NO: 2; and a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
The cell may be contacted with the peptide modulator using any technique known in the art. In general the peptide modulator will be provided to the cell in a form and/or using a method such that at least some of the peptide modulator enters the cell and becomes intracellular. In some embodiments this is achieved by incorporating a polypeptide sequence that ius a cell penetrating peptide into the peptide modulator. In some embodiments this is achieved by introducing a nucleic acid sequence into the cell that encodes the peptide modulator under conditions such that the peptide modulator is synthesized intracellularly to thereby provide the peptide modulator to the cell.
Also provided are methods of treating cystic fibrosis in a subject in need thereof. In some embodiments the methods comprise administering an effective amount of a peptide modulator of CFTR to the subject to thereby increase CFTR activity in the subject. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, a polypeptide comprising a functional variant of SEQ ID NO: 2; and a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, and a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
Typically an effective amount of the peptide modulator is administered to the subject for a therapeutic dosing period. The therapeutic dosing period is chosen to allow improvement in at least one symptom or feature of cystic fibrosis in a subject. In some embodiments the at least one feature is use of a concurrent medication and improvement is a reduction in the amount and/or frequency of administration of a second cystic fibrosis therapy. In some embodiments the second cystic fibrosis therapy is an antibiotic. In some embodiments the second cystic fibrosis therapy is a mechanical lung treatment or therapy.
In some embodiments the subject is a human.
In some embodiments the subject is heterozygous for the ΔF508CFTR allele. In some embodiments the subject is homozygous for the ΔF508CFTR allele. In some embodiments the subject does not comprise a ΔF508CFTR allele.
Also provided are uses of a peptide modulator of CFTR for the manufacture of a medicament for use in treating cystic fibrosis. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, and a polypeptide comprising a functional variant of SEQ ID NO: 2; a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
Also provided are peptide modulators of CFTR for use in treating cystic fibrosis in a subject. In some embodiments the peptide modulator binds to the nucleotide binding domain 1 (NBD1) of CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments binding of the peptide modulator to CFTR increases CFTR activity by increasing Cl− channel current in a cell comprising the CFTR and increasing the plasma membrane fraction of CFTR in a cell comprising the CFTR. In some embodiments the CFTR is ΔF508CFTR. In some embodiments the peptide modulator comprises or consists of an amino acid fragment of the CB subunit of crotoxin from Crotalus durrissus terrificus venom. In some embodiments the peptide modulator is selected from: a polypeptide comprising the amino acid sequence HLLQFNK (SEQ ID NO: 1), a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, and a polypeptide comprising a functional variant of SEQ ID NO: 1; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYCGWGGQ (SEQ ID NO: 2), a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, and a polypeptide comprising a functional variant of SEQ ID NO: 2; a polypeptide comprising the amino acid sequence NGYMFYPDS (SEQ ID NO: 3), a polypeptide consisting of the amino acid sequence SEQ ID NO: 3, a polypeptide comprising a functional variant of SEQ ID NO: 3; a polypeptide comprising the amino acid sequence NGYMFYPDSRCRG (SEQ ID NO: 4); a polypeptide consisting of the amino acid sequence SEQ ID NO: 4, and a polypeptide comprising a functional variant of SEQ ID NO: 4; a polypeptide comprising the amino acid sequence NAVPFYAFYGCYSGWGGQGR (SEQ ID NO: 5), a polypeptide consisting of the amino acid sequence SEQ ID NO: 5; and a polypeptide comprising a functional variant of SEQ ID NO: 5; and a polypeptide comprising the amino acid sequence HLLQFNKMIKFET (SEQ ID NO: 6), a polypeptide consisting of the amino acid sequence SEQ ID NO: 6, and a polypeptide comprising a functional variant of SEQ ID NO: 6. In some embodiments the peptide modulator comprises a chemical modification.
Biological Material:
Snake venom PLA2s (heterodimeric crotoxin [CACB complex], its nonenzymatic CA subunit and three isoforms of the CB subunit [CBa2, CBb and CBc] were purified as previously described16; recombinant human PLA2 was produced as previously described28 and stored at −20° C. All chemicals were purchased from Sigma. Anti-CFTR antibodies MM13-R were from EMD Millipore (Ma, USA). CFTR correctors, Corr4a and VX-809 were kindly provided by Cystic Fibrosis Foundation Therapeutics. CFTR inhibtor glyH101 was from EMD Millipore (Ma, USA) and Inh172 was from Calbiochem. Glibenclamide, amiloride, bumetanide were from Sigma-Aldrich.
Preparation of Oocytes:
After the partial ovariectomy of an anesthetized animal, oocytes were defolliculated by gentle shaking in calcium-free ND96 solution containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES pH 7.5 supplemented with 0.4 U/ml collagenase (Type lA, Sigma). Healthy stage V-VI oocytes were selected for experiments. All animal protocols were approved by the Necker Faculty of Medicine Animal Care and Use Committee (University of Paris Descartes); authorization no. 7514, Ministry of Agriculture and Fishery and conformed to European Community regulations for the use of animals in research (authorization no. P2.AE.092.09). Animals (male and females) used in this study were from FVB/N strain. Mice were obtained from CDTA (Orleans, France, provided by Erasmus University, Rotterdam, Netherland) and housed at the SPF Animal Care Facility of Necker Faculty of Medicine.
Cloning:
Human CFTR cDNA was subcloned into pT7TS plasmid flanked by the 3′ and 5′ untranslated regions of the Xenopus β-globin gene. The cDNA coding for ΔF508CFTR was obtained from the wild-type construct using the Quick Change Site-Directed Mutagenesis Kit (Stratagene). Capped cRNAs were synthesized in vitro from the constructs, linearized by Sma I, using Riprobe in Vitro Transcription System Kit (mMESSAGE mMACHINE, Ambion). Stage V-VI oocytes were injected (Inject+Matic microinjector, Geneva, Switzerland) with 1 ng of CFTR cRNA or ΔF508-CFTR cRNA solubilised in 50 nl water, or with water alone (control oocytes)29; 30 and incubated at 18° C. in ND96 supplemented with penicillin-streptomycin for 3-4 days before further experiments.
Two-electrode voltage-clamp experiments (TEVC): Oocytes were placed in a microchamber and punctured with two low-resistance (0.5-3 Mohm), 3M KCl-filled microelectrodes. To reduce series resistance-induced errors during voltage clamp measurements, a virtual ground amplifier (VG2-A 100, Axon Inst, Union City, Calif., USA) was connected to the current-voltage amplifier (Axoclamp 2B, Union City, Calif., USA) and to bath electrodes (an agar-3M KCl bridge electrode and an Ag—AgCl pellet)31. Current voltage (I/V) relationships were obtained by applying from the resting membrane potential+20 mV voltage steps using Clampex9-generated protocol (Axon Instruments). Results were analysed using the program P-Clamp 9 (Axon Instruments). During the experiements, oocytes were superfused with ND96 Ringer's solution or with solutions differing from each other by a single parameter. Solution changes were commanded electronically using a laboratory-made device. Protein Kinase A (PKA) stimulation was achieved using stimulating cocktail consisting of ND96 supplemented with forskolin (Fsk, 1 μM) and IsoButylMethylXanthine (IBMX, 100 μM). CFTR-induced currents was taken as the difference in whole cell currents measured before and after exposure to the PKA-stimulating cocktail solution and the selective CFTR blocker (Inh172)32 obtained from Calbiochem. Other chemicals were purchased from Sigma. On the day of the electrophysiological experiment, the various PLA2 were injected into oocytes (CFTR- or DF508-CFTR-expressing oocytes, or control oocytes). Crotoxin (CACB complex, CA, and CB subunits) and human sPLA2 were solubilized in buffer mimicking an intracellular solution: potassium Glutamate 128 mM, NaCl 5 mM, MgSO4 7 mM, Hepes/KOH 20 mM, pH 7.0)30 at protein concentrations of 50 μg/ml, 10 μg/ml and 12.5 μg/ml, respectively. After injecting 50 nl of the PLA2-containing buffer, TEVC experiments were performed as described above. CFTR-mediated currents were measured before and after injection of PLA2 using the same oocyte.
Short-Circuit Current Measurements:
Mice were 32 to 38 weeks old and weighed 23 to 36 g. Pups were weaned at 21 days and fed solid mouse chow (Teklad 2018S, Harland). All mice were allowed food and water ad libitum until the time of sacrifice by cervical dislocation. Colons were dissected, cut in 4 pieces from rectum to lower intestine. The fragments from colon were introduced immediately into Ussing chambers for subsequent short-circuit experiments.
Ussing Chamber:
Transepithelial transport measurements were performed as described previously33 in the short-circuit mode (6 mice and 17 tissus). Mice had a cervical dislocation before excising intestine for isolated tissue preparations. The excised colon were stripped of external muscle layers and mounted in Ussing chambers (0.018 cm2 aperture). Hemichambers were connected to a DVC-1000 voltage clamp (World Precision Instruments, Inc., Sarasota, Fla.) via Ag/AgCl electrodes and 3 M KCl agar bridges for recording of short-circuit current. Currents were stored on a computer using analog-to-digital converter (PowerLab) and LabChart software 5.0. Prior to the experiment, prostaglandin generation was blocked with 10 μM indomethacin on the apical and basolateral sides (incubation of tissue for 30 min before the beginning of the experiment). Transepithelial Isc was calculated as μEq cm−2 tissue surface area. The apical and basolateral solutions contained (in mM): 116 NaCl, 25 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, 2,4 H2HPO4, 0,4 KH2PO4, 10 glucose and was gassed with 95% O2-5% CO2 (pH 7.4).
Patch-Clamp Experiments on HeLa Cells:
HeLa cells stably transfected with WT-CFTR were kindly provided by Pascale Fanen (INSERM U955, Créteil, France). The intracellular side of the membrane patch was exposed to a solution containing (in mM): 150 N-methyl-D-gluconate chloride (NMDG-Cl), 0.5 MgCl2, 2 EGTA, 10 HEPES, 10 glucose, pH 7.4, adjusted to pH 7.4 with NMDG. The patch pipettes were filled with (in mM): 150 NMDG-Cl, 0.5 MgCl2, 2 EGTA, 10 HEPES, 10 glucose, 1 ATP-Mg adjusted to pH 7.4 with NMDG. CFTR channels were activated by addition of 40 nM PKA+1 mM ATP-Mg at the intracellular side of the patches. The presence of CFTR channels was verified by addition of 100 μM glibenclamide and 10 μM inh172. The effect of CB was tested by perfusion of 1 nM of CB in the intracellular solution in the presence of 40 nM PKA+0.05, 0.5, or 1 mM ATP-Mg. The currents were recorded as previously described34. Briefly, patch-clamp pipettes were pulled in two stages with a Kopf puller (Tujunga, Calif., USA) using borosilicate glass (GC150T, Harvard Apparatus, Edenbridge, Kent, UK). They were coated with Sylgard and polished just before use. Currents were recorded with Bio-logic RK 400 patch-clamp amplifiers (Bio-Logic, Claix, France), monitored using PClamp9 software (Axon Instruments, Foster City, Calif., USA) and stored on a computer. The experiments were carried out at room temperature (22-27° C.). For analysis of channel activity, current recordings were filtered at 300 Hz low-pass using an 8-pole Bessel filter (LPBF-48DG, NPI Electronic, Tamm, Germany) and digitized at a sampling rate of 1-2 kHz using a Digidata 1200 analog-to-digital converter and PClamp9 software (Axon Instruments, Foster City, Calif., USA). We calculated channel activity (NPo) as <I>/i, where <I> is the time-averaged current passing through the channels on the patch with the closed current level as reference and i is the unit current amplitude.
Whole-Cell Patch-Clamp Recordings:
Nystatin-perforated whole-cell patch-clamp experiments were performed as previously described9. Detailed information is provided in the legend of
Analysis of protein expression by Western blot: After being washed three times in cold PBS buffer, oocytes were lysed in an ice cold solution containing 250 mM sucrose, 0.5 mM EDTA, 5 mM Tris-HCl, pH 7.4. supplemented with a protease inhibitor cocktail solution (Complete Mini, Roche, Indianapolis). For microsmal preparations enriched in plasma membranes, cell lysates were centrifuged at 200, 400 and 800 g (10 min each, 4° C.). The final supernatant was centrifuged at 100 000 g (1 hour, 4° C.). The resulting pellet, containing total membrane proteins, was resuspended in the lysis buffer. Equal protein samples were separated by SDS-8% PAGE and subjected to Western blotting. The blots were incubated with anti-CFTR MM13.4 monoclonal antibody (Millipore, Ma, USA) 1000-fold diluted. This antibody recognizes fully glycosylated CFTR (band C, 170-190 kDa), and immature CFTR (band B, 150-155 kDa). Anti mouse IgG, 5000-fold diluted (GE Healthcare) coupled to horseradish peroxidase was used as secondary antibody. Stained proteins were detected using an enhanced chemoluminescence ECL system (GE Healthcare).
Surface Plasmon Resonance (SPR) Analysis:
Protein-protein interactions were studied in real time using a SPR Biacore 2000 system and CM5 sensor chips (Biacore AB, GE Healthcare). NBD1 (WT or mutant) was purified as previously described by35. It was covalently immobilized at 15° C. via primary amino groups on the sensor chip surface as follows: the carboxymethylated dextran matrix was activated with 35 μl of EDC/NHS (1/1) mixture, 10 μl of NBD1 at a concentration of 50 μg/ml in 10 mM sodium acetate, pH 5.0, was injected and unreacted groups were blocked with 35 μl of ethanolamine (pH 8.5). A separate flow channel on the same sensor chip, reserved for control runs, was subjected to a blank immobilization run by preparing it in the same way but without NBD1. The running and dilution buffer had the following composition: 50 mM Tris, 150 mM NaCl, 5 mM MgCl2 (pH 7.6), 0.005% P20, 1 mM DTT. The interaction between different sPLA2 (crotoxin, CA, CB, hGIIA, GIB PLA2s) and the immobilized NBD1 was monitored at 20 or 25° C. by injecting different concentrations (20, 10, 5, 2.5, 1.25 μg/ml) of the purified proteins, with a flow rate of 30 μl/min, and recording the refractive index changes at the sensor surface. The subsequent dissociation phase was followed after each association run by injecting the running buffer alone. Surfaces were regenerated by three washes with 20 μl of 5 mM NaOH followed by two washes with 20 μl of 1M NaCl. All association and dissociation curves were corrected for non-specific binding by subtraction of control curves obtained from injection of the analyte concentrations through the blank flow channel. The kinetic constants, kon and koff, were calculated using Biacore BIAEVALUATION 4.1 software (Biacore AB, GE Healthcare), assuming a simple two-component model of interaction. Each run consisted of three independent measurements (three different immobilization flowpaths and one control flowpath).
Effect of Inhibitor PMS 1062 on the Interaction of CBa2 with hNBD1:
PLA2 activity was measured by fluorometric assay36 using 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol ammonium salt (β-py-C10-PG) (Molecular probes USA) as substrate. The effect of PMS 106222 on inhibition of PLA2 activity of CBd and the CBd/NBD1 complex was determined in reaction mixtures containing 50 mM TrisHCl pH 7.5, 0.05M NaCl, 1 mM EGTA, 0.1% BSA and 10 mM CaCl2, with β-py-C10-PG concentrations of 5.0 and 10.0 μM in the presence of 0-8.0 μM concentrations of inhibitor. The inhibition type of the enzymatic reaction was determined by graphical analysis using the Dixon method37 and the inhibition constant (Ki) values were calculated by Dixon plot.
Molecular Modeling of the ΔF508-NBD1/CBb Complex:
For molecular docking studies, crystal structures of ΔF508-NBD1 (1XMJ)38 and crotoxin (3ROL)24 were obtained from PDB39. To study the interactions between F508-NBD1 and CBb, we applied a previously described procedure25 slightly modified. Briefly, the initial stage docking algorithm of ZDOCK 3.0.240 was used with rotational sampling density set to 60. Fifty four thousand complexes generated by ZDOCK were rescored and reranked according to optimized energy-based function for initial-stage docking implemented in ZRANK41. Prior to rescoring, polar hydrogen atoms were added to the initial structures of both binding partners using SYBYLX2.0 (Tripos International). For each of the top 20 models proposed by ZDOCK/ZRANK combination, 300 refined structures were generated by the Monte Carlo (MC) refinement method of RosettaDock implemented in Rosetta v. 3.4 package42. This step was performed with extra chi1 and chi2-aromatic rotamers and with MC rigid-body perturbation parameters set to 4A and 0.20. Each of the 20 sets of 300 refined models was further treated separately. All complexes within the groups were rescored with the ZRANK scoring function optimized for refinement complexes. For each set, only the top scored model was selected, resulting in twenty new refined docking models, which were ordered according to the ZRANK score. Subsequently, all complexes were superimposed onto the structural model proposed by Serohijos43. Structures in which CBb occupies the ΔF508-NBD1 inter-domain interfaces of CFTR were discarded. The final top structure among the remaining complexes was analyzed by Protein Interaction Calculator44. All figures of the complex model were generated with the PyMOL Molecular Graphics System Version 1.5.0.445.
Hydrogen Deuterium Exchange (HDX):
The HDX-MS experiments were performed essentially as described in (doi: 10.1016/j.jsb.2015.10.001). In the first step of the analysis, the list of peptic peptides for both NBDs were established using a non-deuterated sample. An aliquot (5 μL) of the protein stock solution was diluted 10 times by adding to 45 μL of H2O Reaction Buffers at room temperature (50 mM Tris-HCl, 150 mM NaCl, pH 7.6, 25° C.). The sample was then acidified by mixing with 10 μL of H2O Stop Buffer (2 M Glycine buffer, pH 2.5). In the case of the NBD-CB complex, the samples were first mixed in 1:1 ratio before the HDX experiments were carried out. The sample was digested online using a 2.1 mm×30 mm immobilized pepsin resin column (Porozyme, ABI, Foster City, Calif.) with 0.07% formic acid in water as the mobile phase (200 μL/min flow rate). Peptic peptides were passed directly to the 2.1 mm×5 mm C18 trapping column (ACQUITY BEH C18 VanGuard precolumn, 1.7 μm resin, Waters, Milford, Mass.). Trapped peptides were eluted onto a reversed phase column (Acquity UPLC BEH C18 column, 1.0×100 mm, 1.7 m resin, Waters, Milford, Mass.) using a 6-40% gradient of acetonitrile in 0.1% formic acid at 40 μL/min, controlled by the nanoACQUITY Binary Solvent Manager. Total time of a single run was 13.5 minutes. All fluidics, valves, and columns were maintained at 0.5° C. using the HDX Manager (Waters, Milford, Mass.), with the exception of the pepsin digestion column which was kept at 20° C. inside the temperature controlled digestion column compartment of the HDX manager. The C18 column outlet was coupled directly to the ion source of SYNAPT G2 HDMS mass spectrometer (Waters, Milford, Mass.) working in Ion Mobility mode. Lock mass was activated and carried out using Leucine-enkephalin (Sigma). For protein identification, mass spectra were acquired in MSE mode over the m/z range of 50-2000. The spectrometer parameters were as follows: ESI positive mode, capillary voltage 3 kV, sampling cone voltage 35 V, extraction cone voltage 3 V, the source temperature 80° C., desolvation temperature 175° C. and desolvation gas flow 800 L/h. The spectrometer was calibrated using standard calibrating solutions. Peptides were identified using ProteinLynx Global Server software (Waters, Milford, Mass.). The list of identified peptides containing peptide m/z, charge, retention time and ion mobility drift time was passed to the DynamX 2.0 hydrogen-deuterium data analysis program (Waters, Milford, Mass.). HDX experiments were carried out as described for the non-deuterated samples, with the Reaction Buffer prepared using D2O (99.8% Cambridge Isotope Laboratories, Inc.) and pH (uncorrected meter reading) adjusted using DCl (Sigma). After mixing 5 μL protein stock with 45 μL D2O Reaction buffer, the exchange reactions were carried out for varied time periods as specified in the text, at room temperature. The exchange was quenched by reducing the pH to 2.5 by adding the reaction mixture to Stop Buffer (2 M Glycine buffer, pH 2.5) and cooling on ice. Immediately after quenching in the Stop Buffer, the sample was manually injected into the nanoACQUITY (Waters, Milford, Mass.) UPLC system. Subsequently, pepsin digestion and LC and MS analyses were carried out exactly as described above for non-deuterated samples. Two control experiments were carried out to account for in- and out-exchange artifacts, as described previously (doi: 10.1016/j.jsb.2015.10.001). In brief, to assess minimum exchange (in-exchange control), D2O Reaction Buffer was added to Stop Buffer that had been cooled on ice prior to addition of protein stock, and this mixture was immediately subjected to pepsin digestion and LC-MS analysis as described above. The deuteration level in an in-exchange experiment was calculated as described below and denoted as 0% exchange (Mex0). For out-exchange analysis, 5 μL of protein stock was mixed with 45 μL of D2O Reaction Buffer, incubated for 24 h, mixed with Stop Buffer, and analyzed as described above. The deuteration level in an out-exchange experiment was calculated and denoted as 100% exchange (Mex100).
The above experimental scheme enabled us to obtain the same set of fragments from the control and HD exchange experiments. Each experiment was repeated twice and the results represent the mean of these replicates. The Y-error bars represent the range between the douplicate data points (doi:10.1021/bi3008998).
HDX data analysis: The deuteration level for each peptide resulting from exchange was calculated in an automated way using DynamX 2.0 software, based on the peptic peptide list obtained from the PLGS program, and further filtered by the DynamX 2.0 program with the following acceptance criteria: minimum intensity threshold −3000, minimum products per amino acids −0.3. The analysis of the isotopic envelopes after exchange was carried out by the DynamX 2.0 program with the following parameters: RT deviation+15 s, m/z deviation+12.5 ppm, drift time deviation+2 time bins. The average masses of peptides in the exchange experiment (Mex) and the two control experiments (Mex0 and Mex100) obtained from the automated analysis were then verified by visual inspection. Ambiguous or overlapping isotopic envelopes were discarded from further analysis. Whenever a split isotopic envelope was observed, the separate Mex values corresponding to each envelope were calculated using the MassLynx program. Final data were exported to Excel (Microsoft) spreadsheets for calculation of HD exchange mass shifts and fraction of exchange calculations. The percentage of relative deuterium uptake (% Deuteration) of a given peptide was calculated by taking into account both control values, following the formula: % Deuteration=((Mex−Mex0)/(Mex100−Mex0))*100. Error bars for fraction of exchange were calculated for two independent experiments. The differences in exchange (ΔHDX−% Difference in deuteration) were obtained by subtracting the fraction of exchange under different conditions.
Statistics:
Except when stated, results were expressed as means±SEM, with n as the number of oocytes. Significance of the results was assessed by paired Student t-test using SigmaPlot (Systat software Inc., San Jose, Calif.). The difference was considered significant for P values <0.05.
Using surface plasmon resonance (SPR), we investigated the possible interaction between the recombinant, purified hNBD1 of CFTR, covalently attached to a sensor chip, and heterodimeric crotoxin, its nonenzymatic CA subunit and two isoforms (CBa2 and CBc) of the PLA2 CB subunit. We demonstrated for the first time that CA-CB complex (
Based on SPR experiments (
According to our hypothesis, CA, which does not interact with NBD1 (
To provide additional evidence that CB, which binds to hNBD1, (
To investigate if CB increases the expression of CFTR, we performed immunoblotting on microsomal proteins from CFTR-expressing oocytes in the presence or absence of CB (
To test for acute potentiator activity of CB, we performed a series of experiments on HeLa cells expressing WTCFTR. First, we performed current recordings using the inside-out configuration of the patch-clamp technique. A representative experiment is shown in
To verify if CB could be active on living epithelium, we performed short circuit current (Isc) measurements in mouse colon tissue. Addition of 1 μM CB to the basal and apical side of the epithelium after the exposure of tissues increased cAMP-dependent Isc previously activated by Fsk plus IBMx cocktail by 4.9±2.8 (N=6 mice (n=17 colon fragments)). This increase was inhibited by the addition of 100 μM bumetanide, Na—K—Cl inhibitor, to the basolateral side (
Since the deletion of Phe508 in NBD1 is the most frequent mutation leading to CF, we studied binding of the CB subunit of crotoxin to the ΔF508-NBD1 mutant.
Because (i) the regulation of CFTR current by CB in X. laevis oocytes was observed two hours after injection of CB, and (ii) CB binds to ΔF508-NBD1, we tested if CB could promote the functional rescue of ΔF508-CFTR in two experimental models (X. laevis oocytes and HeLa cells). In the first series of two electrode voltage clamp experiments, the effect of CBa2 injection on oocytes expressing ΔF508-CFTR was examined.
In another series of experiments, to test if CB potentiates CFTR currents in ΔF508CFTR, we used ΔF508CFTR expressing HeLa cells pre-treated with correctors and performed nystatin-perforated whole-cell patch-clamp recordings in which ΔF508CFTR was functionally rescued by treatment for 24 h with 1 μM of corr4a or 407 correctors. As shown in
Since CB is structurally homologous to human hsPLA2-IIA and our SPR experiments showed interaction of hsPLA2-IIA with hNBD1 (WT and mutated) (
Two-electrode voltage-clamp experiments in oocytes and inside-out patches in HeLa cells performed in the absence of calcium suggested that the potentiating effects of CB on CFTR-Cl− channel current were not calcium-dependent. To provide additional evidence that this potentiating effect is independent of the enzymatic activity of PLA2, we injected into CFTR-expressing oocytes the product of CB enzymatic activity, arachidonic acid. As shown in
To characterize the CB-NBD1 binding interface and to verify if access to the catalytic site of CB is masked by NBD1, we performed two series of experiments, SPR competition experiments and a spectrofluorimetric study using specific PLA2 inhibitor (PMS 1062)22 to access the inhibitory effect of PMS on PLA2-NBD1 interaction. As shown in
As we showed experimentally, the CB subunit of crotoxin can interact with WT and ΔF508NBD1, forming functional complexes resulting in increased CFTR Cl− channel current (
We have applied a molecular docking protocol consisting of the following steps: (a) an initial, rigid body 3D search based on fast a Fourier transform algorithm; (b) primary rescoring with a linear weighted scoring function implemented in ZRANK; (c) structural refinement by Monte Carlo methods; (d) secondary rescoring with ZRANK function optimized for refined complexes. The procedure used here25 has been shown to significantly improve structure prediction of protein-protein complexes.
The total solvent accessible surface buried at the ΔF508NBD1/CB interface for the predicted complex is ˜1600 Å2; contribution from the ΔF508NBD1 is ˜786.6 Å2 and that from the CB subunit is ˜813.7 Å2. The central part of the ΔF508NBD1 interface is characterized by a broad patch of hydrophobic residues including Y625, F626, Y627, L636, F669 and L671 (
To further characterize the CB-binding sites on WTNBD1/ΔF508NBD1, we performed HDX-MS experiments. The levels of hydrogen-deuterium exchange of amide protons in peptic peptides from WTNBD1 and ΔF508NBD1 were measured in the absence and presence of CB and CA crotoxin subunits. In
numerous peptides from the same region showing the same direction of change, weaker differences were also observed for WT. This is in agreement with the observations from SPR experiments that WTNBD1-CB complex dissociates much faster as compared to the ΔF508NBD1-CB complex. For WTNBD1, the stabilization is focused in two regions: 395-402, 434-447 (marked by red rectangles in
To gain further insight into the mechanisms involved in the interactions between CB and ΔF508CFTR, we performed SPR competition experiments, taking advantage of the known interaction between NBD1/ΔF508NBD1 and keratin 8 (K8)10. A typical experiment is shown in
This study shows for the first time the direct nanomolar binding of rattlesnake PLA2 CB to NBD1/ΔF508NBD1 of CFTR and that this interaction has functional consequences. CB behaves as a dual modulator of CFTR activity, it acts as corrector facilitating transport of mutated CFTR to the plasma membrane and as potentiator increasing the Cl− channel current of CFTR. It prompts the question of potential pharmacological applications.
The correcting effect of CB on ΔF508CFTR activity was revealed in two experimental models, X. leavis oocytes and HeLa cells (expressing ΔF508CFTR) using electrophysiological and biochemical assays (
Here, we present the first demonstration for a beneficial protein-protein interaction, CB-ΔF508CFTR, which leads to correction of the functional defect of ΔF508-CFTR (
CB is not only a corrector, but also acts as a potentiator of CFTR Cl− currents, fitting into a new class of modulators encompassing both correcting and potentiating activities26. The potentiating activity of CB was demonstrated in different cell models and in mouse colon tissue by electrophysiological assays (
Determination of the regions in CB responsible for functional effects may represent a fundamental step in the development of a new dual potentiator and corrector for DF508CFTR. To identify the binding interface between CB and NBD1, we studied the interaction of heterodimeric crotoxin complex with DF508NBD1 and observed that the affinity of the PLA2 subunit alone for hNBD1 is 30 times higher than that of the CA-CB complex. Even if the CA subunit does not directly interact with NBD1, it partially inhibits interaction between CB and NBD1 suggesting similar binding interfaces between CA-CB and CB/NBD1. Kinetic analyses revealed a critical mutation H1/S1CB24 located in the CB-NBD1 binding interface supporting our hypothesis. The fluorometric analysis showed that the access to the catalytic site of PLA2 is masked by NBD1.
Using molecular docking simulation and biophysical experimental data, we proposed a structural 3D model of the DF508NBD1/CBb complex (
The binding regions at ΔF508NBD1 proposed by molecular docking of ΔF508NBD1 and CB were confirmed in the HDX-MS experiments. For ΔF508NBD1, these experiments showed retarded exchange of amide protons in the peptides belonging to the F1-like ATP binding core sub-domain (boxed in yellow in
Our results open an exciting field of investigation as they evoke new and unexpected functions of secreted sPLA2 CB from rattlesnake venom. Since it has been proposed that effective pharmacotherapy could be based on a combination of two different types of drugs, correctors and potentiators in order to address trafficking and gating defects, respectively 27, the double effect of CB as corrector and potentiator described here provides an original perspective to develop new therapeutic molecules. The interface identified between rattlesnake sPLA2 and ΔF508NBD1 constitutes a promising target in the development of new anti-CF agents.
Peptides of SEQ ID NOS: 1-3 derived from CB and designed in accordance with the CB-DF508NBD1 model have been assessed for their interaction with the NBD1 domain of DF508CFTR. These peptides were shown to bind with mutated NBD1. The SPR profile has shown interaction of peptide having SEQ ID NO: 2 (NQ-18(16-33)) with DF508-NBD1 and has shown that CL-channel current is increased (
Three alternative peptides with slightly different sequences (having the sequences of SEQ ID NOS: 4, 5 and 6) have been designed and synthesized; they are shown on
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/000690 | 4/28/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62329875 | Apr 2016 | US |
