Patent Application
20230414776
The disclosure belongs to the technical field of cell regulation, and in particular relates to small molecule-nanobody conjugate inducers of proximity (SNACIP) and preparation methods and use thereof.
Proximity-inducing mechanisms control many cellular processes, including protein-protein interactions, signaling cascades, enzymatic catalytic reactions, post-translational modifications, regulated protein degradation, etc. Chemical inducers of proximity (CIPs) or chemical inducers of dimerization (CIDs) use bifunctional small molecules to induce dimerization between two proteins, and further realizes regulation of cellular processes, including cell signal transduction, selective autophagy, localization control of proteins and organelles, axonal transport and cell-cell adhesion, as well as use in cell therapy, etc. However, CIPs generally require an additional binding tag to be fused to a protein to be regulated by exogenous gene expression. Therefore, the CIP technology has the disadvantages that endogenous proteins, in particular those proteins without ligand binding sites are difficult to directly regulate, background activity of endogenous proteins to be regulated has interference, and CIP inducers are difficult to be converted into drug molecules, because genetic augmentation and modification of individuals are generally not allowed during therapeutic intervention due to ethical and risk issues.
It is difficult for a small molecule-nanobody conjugate to penetrate a cell, so it cannot be directly used for regulating intracellular processes. The small molecule-nanobody conjugate needs to be chemically functionalized to penetrate a cell. Conventional intracellular delivery vectors, such as linear cell-penetrating peptides (CPP), and other relatively novel intracellular delivery vectors, such as engineered C3 protein toxins, mostly achieve intracellular delivery by endocytosis. In addition to being relatively slow, endocytosis is inevitably accompanied by processes such as endosome entrapment and lysosomal degradation. Recently, cyclic cell-penetrating peptides have been found to deliver cargos into cells more rapidly in a non-endocytic form. Microtubule nucleation in spindle assembly is important for sustaining life, and dysregulation of this nucleation process is implicated in a variety of diseases. Although microtubule targeting agents (MTAs) that directly bind to microtubules have been successfully used in cancer treatment in chemotherapy, the development of agents that regulate the microtubule nucleation process remains challenging. The microtubule nucleation process involves concerted actions of multiple protein complexes and several intrinsically disordered protein factors, which make it difficult to develop corresponding small-molecule regulatory agents via, e.g. structure-guided drug design (SGDD).
The objective of the disclosure is to develop a new type of intracellular inducers of proximity with core advantages for regulating intracellular processes, which have the value of drug development.
The disclosure provides small molecule-nanobody conjugate inducers of proximity, i.e., SNACIP inducers, including a small molecule binding motif, a nanobody targeting moiety, an intracellular delivery moiety and a linker, the general formula of the inducers being as follows: small molecule binding motif-nanobody targeting moiety-linker-intracellular delivery moiety.
More specifically, the small molecule binding motif is directly introduced by chemical ligation, or is indirectly introduced based on a post-translational modification mechanism after entering a cell; the nanobody is a mono-valent or bivalent nanobody; and the intracellular delivery moiety is a cyclic cell-penetrating peptide (CPP) or a linear CPP.
More specifically, the intracellular delivery moiety is cyclic decaarginine or a Tat polypeptide sequence.
More specifically, the cyclic cell-penetrating peptide has a structure containing a cyclic (KrRrRrRrRrRE) moiety or cR10* for short, wherein the K and E residues are preferably cyclic with an amide bond, and the C-terminal end is preferably a —CONH2 group.
More specifically, Cys-(Gly)n-cyclic(KrRrRrRrRrRE)-NH2, n being zero or a natural number, r: L-Arg, R: L-Arg, has a structural formula as follows:
More specifically, in Cys-(Gly)n-cyclic(KrRrRrRrRrRE)-NH2, n=5.
More specifically, the nanobody is a fluorescent protein nanobody or a nanobody for an intracellular target that mediates cellular processes.
More specifically, the fluorescent protein nanobody is a green fluorescent protein nanobody (GBP) or a red fluorescent protein nanobody (RBP); and the nanobody for an intracellular target that mediates cellular processes is a nanobody for a relevant target of a cell division pathway, a nanobody for a relevant target of a tumor cell invasion pathway, a nanobody for relevant targets of various pathways of ferroptosis, or a nanobody for relevant targets related to cytoskeleton functions.
More specifically, the small molecule binding motif is a protein tag binding ligand or an intracellular binding moiety capable of being introduced through post-translational modification of protein.
More specifically, the protein tag binding ligand is trimethoprim (TMP) or chlorohexyl; and the intracellular binding moiety capable of being introduced through post-translational modification of protein is prenyl or myristoyl.
More specifically, the linker is a disulfide bond, a thioether bond, or a peptide bond.
More specifically, the small molecule binding motif is trimethoprim (TMP), the intracellular delivery moiety is cyclic decaarginine cR10*, and the linker is a reducible broken disulfide bond, that is, the inducer is cR10*-GBP-TMP.
More specifically, the inducer is a latent SNACIP inducer, and is converted into a functional farnesyl-cRTC inducer after entering cells, the nanobody is a TPX2 binding protein (TBP), the small molecule binding motif is a CAAX-box polypeptide sequence capable of being prenylated, the intracellular delivery moietymodule is cyclic decaarginine cR10*, and the linker is a thioether bond generated via the reaction between maleimide and sulfhydryl, that is, the inducer is cR10*-TBP-CAAX.
More specifically, the inducer is a latent SNACIP inducer, and is converted into a functional farnesyl-CTTC inducer after entering cells, the nanobody is a bivalent TBP nanobody, the small molecule binding motif is a CAAX-box polypeptide sequence capable of being prenylated, the intracellular delivery moiety is cyclic decaarginine cR10*, and the linker is a peptide bond —NHCO—, that is, the inducer is mCherry-CPP-2×TBP-CAAX.
The disclosure provides a method for inducing proximity inside a cell, including the following steps:
The disclosure provides use of the SNACIP inducers in regulating cellular processes.
More specifically, the use is for preparation of antitumor drugs.
More specifically, the use is for activation and deactivation of intracellular proteins.
The disclosure provides a kit for regulating cellular processes, including any of the aforementioned SNACIP inducers, for regulating cellular processes.
The disclosure provides a nanobody drug for treating tumors, including any of the aforementioned SNACIP inducers, and blocking cell division by targeting and deactivating TPX2, thereby inhibiting tumor proliferation.
The disclosure provides a nanobody drug for treating tumors, including any of the aforementioned SNACIP inducers, and blocking cell division by targeting and deactivating TPX2, thereby inhibiting tumor proliferation.
The disclosure provides a method for inhibiting cell division by targeting a microtubule nucleator TPX2 protein to deactivate the TPX2, and a means derived therefrom for developing drugs for treating tumor.
The disclosure provides a method for activating and deactivating intracellular proteins using a nanobody conjugate by using any of the aforementioned SNACIP inducers, which achieves activation by localizing a protein to be regulated to a functional location of a plasma membrane, or achieves deactivation by localizing a protein to be regulated in a non-functional location of a plasma membrane.
The disclosure provides a method for regulating ferroptosis by using any of the aforementioned SNACIP inducers, which localizes GPX4 to a peroxisome, such as a PEX3 sequence, to induce ferroptosis, as a new strategy for the treatment of tumors.
In the disclosure, three different SNACIP inducers are specifically demonstrated, which represent different application types respectively and have their own characteristics.
The first is an inducer cR10*-GBP-TMP, cRGT for short, which can quickly penetrate a cell (t1/2=7.3 min), and induce dimerization between an intracellular green fluorescent protein (GFP) mutant and E. coli dihydrofolate reductase (eDHFR), thereby realizing regulation of intracellular cellular processes. The cRGT features as a general SNACIP for regulating cellular processes, and has the advantages of being fast, reversible, no-wash, dose-dependent, and complete in regulation, which will be described in the Examples. cRGT can control cell localization, regulate the cell signal transduction process, regulate the transport of intracellular cargo, and regulate one of the current research fronts and hotspots—ferroptosis.
The second is a latent SNACIP inducer, cR10*-TBP-CAAX, cRTC for short, which is developed for the important microtubule nucleation process. With the help of the post-translational modification mechanism of cells, the latent cRTC can be linked with farnesyl after entering the cell, thereby being converted into a functional farnesyl-cRTC inducer of proximity. cRTC deactivates TPX2 by localizing an intrinsically disordered protein TPX2, which is also a key microtubule nucleator, to a non-functional location of the plasma membrane, thereby inhibiting microtubule nucleation, blocking cell division, and inhibiting cancer cell proliferation. The cRTC is valued as the first regulator of microtubule nucleation and for its capability of inhibiting cancer cell proliferation, and is also an important example of direct regulation of endogenous targets without ligand binding.
The third is a bivalent SNACIP for in vivo use, mCherry-CPP-2×TBP-CAAX, CTTC for short. The inducer includes a bivalent TBP nanobody, so it is more suitable for use in vivo. CTTC can also be post-modified and converted into farnesyl-CTTC after entering cells, deactivate the microtubule nucleator TPX2, and inhibit cancer cell proliferation, showing an effect of inhibiting tumor proliferation in vivo. This result confirms that SNACIP inducers may not only directly regulate endogenous proteins, but also may be developed into nanobody drugs for treatment of diseases.
Compared to the relevant chemical inducers of proximity (CIPs), SNACIP have several advantages and are summarized in the following table.
As can be seen from the above table, SNACIPs could be advantageous over traditional CID/CIP molecules in several aspects including: i) the ability to directly regulate endogenous proteins, ii) translational potential, iii) high binding affinity, iv) tag-size, v) versatility and others. Also, SNACIP shares some appreciable features of CIP, e.g., reversibility, ease to use, dose-dependent response, and others.
The SNACIP examples disclosed herein have the corresponding beneficial effects as follows:
Mammalian cell culture: HeLa (Cat #CL-0101) and HepG2 (Cat #CL0103) cells are purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells are identified by short tandem repeats (STR) and proved free of HIV-1, HBV, HCV, mycoplasma and other microorganisms prior to culture. Other required reagents, e.g., DMEM and PBS for cell culture, also need to be confirmed free of mycoplasma infection before use. Cells are cultured in 5% carbon dioxide with high glucose (4.5g·L−1) Dulbecco's modified Eagle's complete medium (DMEM, Cat #SH30243.01 purchased from HyClone), containing 4 mM L-glutamine and 1× sodium pyruvate, supplemented with fetal bovine serum (Cat #5V30087.03 purchased from HyClone), 1% non-essential amino acids (NEAA 100×) and 1% penicillin-streptomycin (100×) premix. During cell passaging, cells are digested using EDTA-trypsin (purchased from HyClone, Cat #5H30042.01) and phosphate buffered saline (PBS) (Cat #5H30256.01, purchased from HyClone). HeLa cells are subcultured at a ratio of 1:(5-10), while HepG2 cells are subcultured at a ratio of 1:(4-6).
Animal Welfare: Mice are kept under specific pathogen-free (SPF) grade clean conditions, and are handled with the approval of the Institutional Animal Care and Use Committee of Harbin Institute of Technology (IACUC/HIT), with the license number IACUC-2021052. Mice are reared under controlled conditions of light (12 h light/12 h dark cycle), temperature (24±2° C.) and humidity (50±10%), and are fed normal chow and water ad libitum. The rearing, maintenance and oocyte collection of Xenopus are carried out with the approval of the IACUC/HIT, with the license number IACUC-2020020. Briefly, the feeding equipment for female (2-3 years old) and male Xenopus is purchased from Lingyun Boji (Beijing, China), and parameters such as water quality (deionized water, ID-H2O), pH (7.2), temperature (18° C.), and conductivity (1600 μS/cm) are set as recommended by the manufacturer's manual. Xenopus is fed qualified Xenopus chow twice a week. Sperm nuclei from male Xenopus are prepared for spindle assays, while female Xenopus is induced spawning. According to the method described in CSH protocols (Shaidani et al., 2021), female Xenopus is injected with an appropriate amount of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in sequence, wherein PMSG promotes oocyte maturation, and hCG promotes ovulation.
Establishment and drug treatment of xenograft tumor mice model: Immunodeficient BALB/c nude mice are purchased from Liaoning Changsheng Biotechnology Co., Ltd., and female mice are injected with HepG2 cells after 4-δ weeks of age for tumorigenesis. Before injection, HepG2 cells are cultured in a standard Φ˜85 mm culture dish, and collected after entering exponential growth. The cells are first rinsed with 10 ml of PBS, then 1 ml of trypsin is added for digestion for 5-10 min to detach the cells, and then 3 ml of PBS is added for suspending the separated cells. The cell suspension is centrifuged at 1000 rpm for 8 min at 4° C., the supernatant is removed, and the cells are resuspended in a freshly prepared mixture (v/v=1:1) of PBS/Matrigel (Solarbio, Cat #M8370). The final cell concentration is approximately 50 million cells per milliliter. For establishing the HepG2 xenograft mice model, ˜5 million HepG2 cells in 0.1 ml PBS/Matrigel solution are injected subcutaneously into the axillary region of BALB/c nude mice. A stable tumor would appear within 1-2 weeks. To evaluate the effectiveness of a TPX2 nanobody conjugate, PBS (pH 7.2, containing additional 1 mM TCEP, 0.5 M NaCl and 3% glycerol) is used as blank control, and the CTTC nanobody conjugated drug is dissolved in the PBS for administration by injecting 100 μl into the mice through the tail vein. The mean tumor size [Φ=(ΦL+ΦS)/2] is monitored daily using vernier calipers. The calculation formula of tumor volume is as follows: V=1/6 (ϕΦ3).
Plasmid construction: Plasmid vectors pTXB1, pET28a(+), EGFP-C1, EGFP-N1, etc., are purchased from commercial suppliers. These vectors may be further designed and modified by, e.g., introducing His6 or His8 affinity tags, adding TEV or TEV″ protease cleavage sites, changing restriction enzyme cleavage sites, or replacing EGFP with mTagBFP2, mTurquoise 2, mEYFP, DsRed, mScarlet or mCherry to obtain vectors expressing other fluorescent proteins for performing subsequent cloning. Regarding cloning methods, subcloning, Gibson assembly or modified Gibson assembly is employed to construct the desired plasmids. For subcloning, appropriate restriction enzymes are used for cleaving the relevant fragment directly from a vector plasmid, or perfusion high-fidelity polymerase (APExBIO, CAT #1032) is used for amplifying the corresponding gene fragment from the plasmid containing the desired gene by performing PCR, and then gel purification and restriction enzyme cleavage are performed. The obtained gene fragment is ligated into an appropriate vector with T4 DNA ligase. Cloning methods involving insertion of multiple fragments may be accomplished by stepwise subcloning or multi-fragment Gibson one-step assembly. Most genes are obtained by means of gene synthesis, and the gene exchange service is provided by Comate Bioscience Co., Ltd. (Changchun, China).
These genes include E. coli codon-optimized human TPX2 (i.e., codon-optimized hTPX2), E. coli codon-optimized GFP nanobody (GBP), mScarlet, etc. The non-codon-optimized human TPX2 gene is amplified from the plasmid pLenti-EF1a-EGFP-P2A-Puro-CMV-TPX2-3Flag, which is purchased from Obio Technology (Shanghai) Co., Ltd., Cat #H10559. Plasmids encoding human KIF5B, Rac1, Rab1b, Rab5a and other genes are purchased from the MiaoLing Plasmid Sharing Platform.
Transfection: Cells are typically seeded and transiently transfected in Thermo Scientific 8-well dishes (Cat #155409) or 4-well dishes (Cat #155382) Lab-Tek®II. DNA (0.25 μg) is dissolved in 12.5 μl of gibco opti-MEM (Cat #31985-062), and then 0.5 μl of ExFect®2000 transfection reagent (Cat #T202, Vazyme Biotech Co., Ltd., Nanjing, China) is dissolved in 12.5 μl of gibco opti-MEM. The two solutions are first incubated at room temperature for 5 min. Then, the DNA-containing opti-MEM solution is added to the ExFect®2000-containing opti-MEM solution and mixed gently. The opti-MEM containing the DNA/ExFect®2000 mixture is incubated at room temperature for 5-10 min (usually 7.5 min), and gently dropped into a 8-well dish containing 250 μl of complete DMEM, wherein the dish is seeded with 15000-20000 cells. The cells are incubated at 37° C. under 5% carbon dioxide for about 2 h to allow the cells to adhere. Then, the previous medium is replaced with fresh warm complete DMEM, and the cells are incubated at 37° C. under 5% carbon dioxide for 20 h or more. For co-transfecting multiple plasmids, the number of DNAs used in this solution refers to the total mass of plasmids.
Confocal microscopy and super-resolution imaging: 24 h after transfection, cells are imaged by confocal microscopy. With an 8-well or 4-well dish as described above, and phenol red-free DMEM medium (REF: 21063-29) containing additional 10% fetal bovine serum, 1% sodium pyruvate, 1% NEAA, 1% penicillin-streptomycin and 15 mM HEPES-Na (final pH 7.0), cells are cultured at 37° C. under 5% carbon dioxide and observed using a ZeissLSM880 inverted scanning confocal microscope. In most cases, a Zeiss Plan-APOHROMAT 100×/1.4 DIC oil immersion lens is used for microscopic imaging, and a Zeiss Plan-APOCHROMAT 60×/1.4 DIC oil immersion lens may also be used. For a larger field of view, a Zeiss PlanAPOCHROMAT 40×/0.95 DICIII objective lens (as in EdU detection) is used. The obtained image is generally 12-bit in depth and 512×512 in resolution, and scanning is performed 8 times on average. 405 nm laser is used for exciting mTagBFP2, DAPI or Hoechest; 458 nm laser is used for exciting mTurquoise2; 488 nm argon laser is used for exciting EGFP or fluorescein; 514 nm argon laser is used for exciting mEYFP; HeNe 543 nm laser is used for exciting an Apollo 567 dye in EdU assays; HeNe laser 543 nm or HeNe laser 594 nm is used for exciting mScarlet-I or mCherry; and HeNe laser 647 nm is used for exciting far-infrared HiLyte647. In most cases, basic imaging setup parameters are set with the aid of the “smart setup” function. To obtain super-resolution images, an Airyscan module may be used for imaging with the ChA channel typically at 1024×1024 resolution.
Treatment of living cells with an SNACIP inducer of dimerization for microscopic imaging: Unless otherwise specified, first a DMEM complete medium of the cells is replaced with a phenol red-free imaging medium of the SNACIP inducer of dimerization of the corresponding concentration, and then imaging is performed after incubation for a given period of time. For cRGT, the concentration represents an effective ratio of cRGT; and near-complete dimerization regulation may be achieved without washing off excess cRGT before imaging. For reversible regulation with TMP, a freshly prepared phenol red-free imaging medium with a final concentration of 10 μM TMP is replaced for the previous imaging medium containing the SNACIP inducer of dimerization, and hence resulted in rapid near-complete reversible regulation, with microscopic imaging starting after 10 min.
EdU cell proliferation assay: EdU cell proliferation assay is performed using an EdU cell proliferation assay kit from RiboBio (Cat #R11053.9). Briefly, a 8-well imaging dish is seeded with 50×103 HepG2 or 20×103 HeLa cells in the exponential growth phase and the cells are allowed to grow overnight. On the next morning, a PBS solution containing a drug (e.g., cRTC) is added to each well at a final concentration of 10 μM, while the same volume of PBS solution is added to the control cell wells. On the third morning (usually 24 h later), EdU is added at a final concentration of 50 μM to all imaging wells for incubation for 2 h at 37° C. under 5% carbon dioxide. This method is suitable for general cancer cell lines. Then each well is rinsed with PBS (2×5 min) to remove excess EdU, and 100 μl of cell fixative (4% PMA in PBS) is added for incubation at room temperature (RT) for 30 min. Then 100 μl of 2 mg·ml−1 glycine solution is added to each well and shaken for 5 min at room temperature to neutralize the fixative. The glycine solution in each well is pipetted, and each well is shaken and rinsed with 200 μl of PBS at room temperature for 5 min. The PBS is pipetted, and 200 μl of plasma membrane penetrating solution (0.5% TritonX-100 in PBS) is added to each well and shaken at room temperature for 10 min. The fixed cells are washed again with PBS (1×5 min) before the assay. Before fluorescent labeling is performed by a click reaction, a freshly prepared 1×Apollo labeling solution containing a red Apollo567 dye (Cat #C10310-1), a catalyst and other necessary reagents need to be prepared according to reagent instructions. For example, 1 ml of 1×Apollo labeling solution may be prepared by sequentially mixing and adding 938 μl of DI-H2O, 50 μl of Apollo reaction buffer (reagent B), 10 μl of Apollo catalyst solution (containing Cu2+, buffer C), 3 μl of Apollo567 dye (reagent D) and ˜9 mg of Apollo supplement (ascorbate sodium salt, reagent E). 200 μl of freshly prepared 1×Apollo labeling solution is added to each well, and shaken for 30 min at room temperature in the dark to complete labeling. The labeling solution is removed, and the cells in each well are washed again with the plasma membrane penetrating solution (0.5% TritonX-100PBS) (3×10 min). The osmotic solution is pipetted and the cells are washed with PBS (1×5 min). Finally, fresh PBS is added, and the labeled cells can be imaged by fluorescence confocal microscopy.
Isothermal titration calorimetry (ITC): ITC measurements are performed using a MicroCal ITC200 device from GE Malvern. TPX2 nanobody and hTPX2 protein are dissolved in freshly prepared PBS (pH 7.2, containing additionally added 1 mM TCEP, 0.5 M NaCl and 3% glycerol). 21 μl of hTPX2 solution is added to a sample pool, and 51 μM of TPX2C nanobody is pipetted by a syringe and injected 2.0 μl×18 times into the sample pool at an interval of 3 min (except for the first injection of 0.8 μl of nanobody at an interval of 2.5 min) . The titration data is processed using Origin software, and parameters such as Kd and binding stoichiometric values are calculated.
Fröster resonance energy transfer (FRET) measurement (measurement method in
Immunodepletion (ID) (research mechanism,
Spindle assembly assay and microtubule nucleation assay performed in Xenopus oocyte extract (research mechanism, assays in
Design and preparation of cyclic cell-penetrating peptide Cys-cR10*: The cyclic peptide Cys-cR10* is characterized by a cyclic rR ring (r=D-Arg, R=L-Arg), a (Gly)5 linker, a free N-terminal cysteine and a C-terminal containing a —CONH2 group. Solid phase peptide synthesis (SPPS) is performed with Rink amide resin. After an R10 fragment is synthesized, intramolecular cyclization is performed, and a ring is formed by condensing a lysine side chain (—NH2 group) and glutamic acid (—COON group) to form an intramolecular amide bond. Cys-(Gly)5 moieties are then sequentially added to the cyclic r10 moiety, and Cys-cR10* is finally deprotected and purified. The Cys-cR10* cyclic peptide is 98.8% pure and its structure is identified by mass spectrometry. C84H160N50O19S, exact molecular weight: 2205.28; molar mass M.W.: 2206.56; measured mass-to-charge ratio m/z: 736.4[M+3H]3+, 552.6[M+4H]4+, 442.3[M+5H]5+.
Universal expressed protein ligation (EPL) method: A protein for EPL is expressed as a fusion chimera with a pTXB1 plasmid, the C-terminal end of the chimera will contain MxeGyrA intein, and the gene for expressing the protein will be cloned into the pTXB1 vector. Then, the fusion-expressed chimera is purified and exchanged into buffer A (PBS (pH 8.0), 0.5 M sodium chloride, 3% of glycerol), and the concentration is adjusted to 8.5 mg·L−1. At the start of the ligation reaction, 1/4 volume of sodium 2-mercaptoethanesulfonate (MENSNa, 2M) stock (pH 8.0) is added as a proteolytic cleavage reagent, and 1/4 volume of 4-mercaptoacetic acid (MPAA, 1.1 M) stock (pH 8.0) is added as a catalyst to speed up the ligation. Finally, a small molecule reagent containing N-terminal cysteine is added to the reaction solution at a concentration of 0.5-1 mM. The reaction mixture is incubated on ice for several days, and the ligation process is monitored by SDS-PAGE. Generally, most proteins are converted to ligation products after 2-4 days of incubation. The ligation products may be further purified by a step gradient (0-500 mM imidazole) gravity column using high affinity nickel resin FF (GenScript, Cat #). If necessary, non-ligated products containing CBD-fusions may be further removed using chitin resin (NEB, Cat #S6651L).
General steps for protein expression and purification: pET28a(+) or genetically engineered pET28a (+) vectors, e.g., pET28b (TEV), may be used as vectors for expression of most proteins, and pTXB1 vectors are used for expressing GFP nanobody fused to an intein-chitin binding domain (intein-CBD) tag for a subsequent expressed protein ligation (EPL) reaction. The protein-expressing plasmids are first transformed with E. coli Rosetta2a competent cells, and then screened on an ampicillin (100 mg·L−1) or kanamycin (50 mg·L−1) agarose plate according to the resistance of the plasmids. 50-100 mL of LB medium containing the corresponding 100 mg·L−1 ampicillin or 50 mg·L−1 kanamycin is inoculated with a single colony. First the cells are pre-cultured at 37° C. with shaking at 240 rpm for 8-12 h or overnight. Then ˜1.8 L of fresh LB medium containing 100 mg·L−1 ampicillin or 50 mg·L−1 kanamycin is inoculated with 30-50 mL of the pre-cultured bacterial solution, and additional chloramphenicol (33 mg·L−1) is added. The competent cells in the LB medium are shaken at 180 rpm at 37° C. for several hours (usually 2-3 h) until the OD600 (absorbance at 600 nm) is 0.05-0.1. Then 0. 5 ml of 1 M IPTG solution (final 0.27 mM) is added to induce protein expression at 37° C. for 3-5 h, or at 16° C. overnight. Sometimes, the expression time and temperature of the protein need to be optimized through assays to achieve a more ideal protein expression level.
Subsequently, cells are collected by centrifugation (8000 rpm, 4° C., 15 min) and washed once with PBS (4700 rpm, 10 min). Bacterial pellets are resuspended in a lysis buffer (PBS (pH 8.0), containing additional 0.5 M sodium chloride, 3% glycerol, 3 mM BEM added as appropriate, and 1 mM PMSF). A small volume of bacterial cell suspensions (<40 ml) is usually lysed on ice with 80 W sonication for 30 min or 60 W sonication for 45 min (1 s sonication followed by a 3 s interval). For batch treatment or a large volume of cell suspension, cells are usually lysed using an ultra-high pressure homogenizer at 4° C. at 800-900 bar for 2-3 cycles, wherein the ultra-high pressure homogenizer needs to be equipped with a desktop circulating condensate machine for providing condensate water. The obtained lysate is centrifuged at high speed (25000 rpm, 45 min, 4° C.), and the obtained supernatant is purified by a gravity Ni-NTA column (2-5 ml resin filler). The labeled protein is washed and then eluted with a gradient of imidazole (50, 100, . . . , up to 500 mM). In addition, gradient elution (0→500 mM imidazole) may also be performed using GE's AKTAPure equipped with a HisTrapFF column, e.g., gradient elution is achieved using a buffer A (PBS (pH 8.0), containing additional 0.5 M NaCl, 3% glycerol, and 3 mM BEM added as appropriate) combined with a buffer B (solution A with the same pH, containing additional 0.5 M imidazole dissolved). If the protein purified according to this procedure requires further purification, ion exchange or size exclusion chromatography may be applied. The obtained protein typically needs to be concentrated, exchanged with buffer A, aliquoted, quickly frozen in liquid nitrogen, and stored at 80° C.
Synthesis and characterization of key small molecule compounds: A 1H-NMR or 13 C-NMR nuclear magnetic spectrum is obtained by a 400 MHz or 600 MHz Bruker BioSpin GmbH magnetic resonance spectrometer. The relevant parameters of the 1H-NMR spectrum are as follows: chemical shift δ is expressed in parts per million (ppm); multiplicities are expressed as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants J are expressed in Hertz (Hertz or Hz); the integral (n) of the hydrogen spectrum is expressed in nH. High resolution mass spectra (HR-MS) are obtained using an Agilent 6540 Q-TOF mass spectrometer by electrospray ionization (ESI).
5-(2-(2-(2-(2-(tert-butoxycarbonypethoxy)ethoxy)ethoxy)ethylamino)-5-oxopentanoic acid (BocNH-PEG4-Glu-COON, 3). BocNH-PEG4-NH2 (1.2 g, 4.11 mmol), and glutaric anhydride (468 mg, 4.11 mmol) are dissolved in anhydrous tetrahydrofuran (THF) (21 ml), and then DIEA (636 mg, 4.93 mmol) is added. The reaction is performed under stirring at room temperature overnight. The reaction solution is aliquoted into EtOAc/aq. NaH2PO4 (2 M), then the organic layers are separated and the aqueous layer is extracted twice with ethyl acetate (EtOAc). All organic layers are mixed, washed once with saturated brine, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and subjected to silica gel column chromatography (EtOAc/MeOH 8/1, Rf 0.4-0.6 (tailing), followed by EtOAc/MeOH 5/1) to obtain 1.5 g of product with a yield of 90%. 1 H-NMR (CDCl3, 400 MHz): δ 6.51 (s, 1H), 5.16 (s, 1H), 3.63 (br, 8H), 3.53 (m, 4H), 3.44 (m, 2H), 3.29 (br, 2H), 2.37 (t, J=6.8Hz, 2H), 2.28 (t, J=7.4Hz, 2H), 1.94 (m, 2H), 1.42 (s, 9H); 13C-NMR (CDCl3, 101 MHz): δ 176.00, 172.91, 156.30, 79.53, 77.36, 70.61, 70.50, 70.29, 69.95, 40.41, 39.33, 35.32, 32.92, 28.51; HRMS: C18H35N2O8+ [M+H]+ calc. 407.2393, found 407.2393.
5-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethylamino)-5-oxopentanoicacid (H2 N-PEG4-Glu-COOH, 4). BocNH-PEG4-Glu-COOH (406 mg, 1 mmol) is dissolved in anhydrous dichloromethane (DCM) (2 ml), then trifluoroacetic acid (1 ml) is added, and the reaction is performed under stirring at room temperature for 30 min for deprotection. DCM, TFA and other volatile components are removed under high vacuum to obtain ˜423 mg of the deprotected product nTFA·H2N-PEG4-Glu-COON (˜1 mmol) with an almost quantitative yield. 1 H-NMR (CDCl3, 400 MHz): δ 7.94 (br, 1H), 7.90 (br, 1H), 4.66 (br, 3H), 3.79 (m, 2H), 3.70 (m, 2H), 3.62 (m, 6H), 3.56 (m, 2H), 3.43 (br, 2H), 3.21 (br, 2H), 2.37 (m, 2H), 2.29 (m, 2H), 1.93 (m, 2H); 13C-NMR (CDCl3, 101 MHz): δ 176.88, 174.36, 77.36, 70.33, 70.22, 69.96, 69.89, 69.75, 39.94, 39.48, 35.12, 33.09, 20.98; HRMS: C13H27N2O6+ [M+H]+ calc. 307.1869, found 307.1868.
(R)-2-(tert-butoxycarbonyl)-3-(tritylthio)propanoic acid N-hydroxysuccinimidyl ester (BocCys(Trt)-OSu, 6). BocCys(Trt)-OH (464 mg, 1 mmol) and HBTU (417 mg, 1.1 mmol) are dissolved in anhydrous dichloromethane (DCM) (10 ml) and stirred at RT for 10 min. Then weak base DIEA (206 mg, 1.6 mmol) is added and the reaction is continued under stirring for 10 min. Finally, N-hydroxysuccinimide (NHS) (127 mg, 1.1 mmol) is added, and the reaction is continued for about 2-3 h. Thin layer chromatography (TLC) (cyclohexane/EtOAc 2/1, Rf 0.4) shows that the reaction is complete. The reaction solution is aliquoted in EtOAc/aq. NaH2PO4 (2M), then the organic layers are separated and the aqueous layer is extracted once with ethyl acetate EtOAc. All organic layers are mixed, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried in vacuum to obtain an NHS ester product. Gradient silica gel column chromatography (cyclohexane-cyclohexane/EtOAc 4/1, 3/1, up to 2/1) is performed to obtain 459 mg of a white foamy solid as the final product with a yield of 82%. 1 H-NMR (DMSO-d6, 400 MHz): δ 7.67 (d, J=8.32Hz, 1H), 7.33 (m, 12H), 7.26 (m, 3H), 3.91 (m, 1H), 3.32 (s, 2H), 2.75 (s, 4H), 1.38 (s, 9H); 13C-NMR (DMSO-d6, 101 MHz): δ 169.60, 167.00, 154.83, 143.94, 129.00, 128.15, 126.89, 78.93, 66.71, 51.67, 32.28, 28.03, 25.37; HRMS: C31H33N2O6S+ [M+H]+ calc. 561.2059, found 561.2057.
(R)-5-(2-(2-(2-(2-(2-(tert-butoxycarbonyl)-3-(tritylthio)propanamido)ethoxy)ethoxy)ethoxy) ethylamino)-5-oxopentanoic acid (BocCys(Trt)-PEG4-Glu-COOH, 7). BocCys(Trt)-OSu (440 mg, 0.78 mmol) is dissolved in THF (4.5 ml), which is then added dropwise to a stirred solution of nTFA·H2N-PEG4-Glu-COON (423 mg, ˜1 mmol) and DIEA (387 mg, 3 mmol) in basic THF (3.5 ml). The reaction is performed under stirring at room temperature for 8 h. Then the reaction solution is aliquoted into EtOAc/aq. NaH2PO4 (2 M). The organic layers are separated and the aqueous layer is extracted twice with ethyl acetate (EtOAc). All organic layers are mixed and washed four times with 2 M aq. NaH2PO4 to substantially remove NHS by-products and excess H2N-PEG4-Glu-COOH starting material. The organic layers are washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to gradient silica gel column chromatography (EtOAc→EtOAc/MeOH 20/1, finally EtOAc/MeOH 15/1) to obtain 235 mg of a white foamy solid as the product with the yield of 40%. 1H-NMR (DMSO-d6, 400 MHz): δ 12.00 (s, br, 1H), 7.84 (t, J=5.76Hz, 1H), 7.74 (t, J=5.40Hz, 1H), 7.36-7.20 (m, 15H), 6.87 (d, J=8.36Hz, 1H); 3.91 (m, 1H), 3.48 (m, 10H), 3.38 (t, J=5.92Hz, 2H), 3.17 (m, 4H), 2.32 (d, J=7.08Hz, 2H), 2.19 (t, J=7.44Hz, 2H), 2.09 (t, J=7.44Hz, 2H), 1.69 (m, 2H), 1.37 (s, 9H); 13C-NMR (DMSO-d6, 101MHz): δ 174.16, 171.70, 170.08, 144.32, 129.08, 128.02, 126.75, 78.37, 69.71, 69.60, 69.53, 69.10, 68.86, 65.86, 53.39, 38.44, 34.37, 32.99, 28.11, 20.67; HRMS: C40H54N3O9S+ [M+H]+ calc. 752.3581, found 752.3582.
tert-butyl4-(44(2,4-diaminopyrimidin-5-yOmethyl)-2,6-dimethoxyphenoxy)butylcarbamate (TMP-Bu-NHBoc, 9). Dimethoprim, i.e. TMP-OH (8), may be obtained by demethylating trimethoprim (TMP), ref. (Chen et al., Chem. Commun. 2015, 51, 16537). Then TMP-OH (1.1 g, 4 mmol), 4-Boc-1-bromobutylamine (1.06 g, 4.2 mmol), Cs2CO3 (2.74 g, 8.4 mmol) and NaI·2H2O (0.6 g, 4 mmol) are suspended/dissolved in anhydrous DMF (20 ml). The reaction solution is stirred at room temperature for 20 h in the presence of argon. The reaction solution is aliquoted into EtOAc/H2O. After the organic layers are separated, the aqueous layer is extracted three times with ethyl acetate. All organic layers are mixed, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to gradient silica gel column chromatography (EtOAc→CHCl3→CHCl3/MeOH 10/1)) to obtain a crude product. Secondary silica gel column chromatography (CHCl3/MeOH 10/1, Rf 0.4) is performed to obtain 570 mg of pale yellow high-purity solid product with a yield of 32%. 1H-NMR (DMSO-d6, 400 MHz): δ 7.51 (s, 1H), 6.77 (t, J=5.84, 1H), 6.54 (s, 2H), 6.08 (s, 2H), 5.69 (s, 2H), 3.77 (t, J=7.25Hz, 2H), 3.71 (s, 6H), 3.52 (s, 2H), 2.95 (m, 2H), 1.53 (m, 3H), 1.37 (s, 9H); 13C-NMR (DMSO-d6, 101 MHz): δ 162.22, 162.19, 155.69, 155.58, 152.85, 135.86, 134.64, 105.86, 105.78, 77.28, 72.02, 55.82, 32.97, 28.25, 27.01, 26.09; HRMS: C22H34N5O5+ [M+H]+ calc. 448.2560, found 448.2560.
5-(4-(4-aminobutoxy)-3,5-dimethoxybenzyl)pyrimidine-2,4-diamine (TMP-Bu-NH2, 10). TMP-Bu-NHBoc (186 mg, 0.416 mmol) is dissolved in 1 ml of anhydrous dichloromethane (DCM), then 0.5 ml of trifluoroacetic acid (TFA) is added, and the mixture is stirred at room temperature for 1 h for deprotection. Volatile components such as DCM and TFA are removed in vacuum to obtain 144 mg of a trifluoroacetate salt product. The product is aliquoted into EtOAc/aq. Na2CO3, the organic layers are separated, and the aqueous layer is extracted several times with ethyl acetate. All organic layers are mixed, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and dried in vacuum to obtain 122 g of a white solid product with an almost quantitative yield. 1H-NMR (DMSO-d6, 400 MHz): δ 7.51 (s, 1H), 6.53 (s, 1H), 6.06 (s, 1H), 5.67 (s, 1H), 3.77 (t, J=6.36Hz, 2H), 3.71 (s, 6H), 2.55 (t, J=6.72Hz, 2H), 1.61 (m, 2H), 1.45 (m, 2H); 13C-NMR (DMSO-d6, 101 MHz): δ 162.24, 162.17, 155.72, 152.86, 135.60, 134.96, 105.90, 105.77, 72.36, 55.85, 41.41, 32.95, 29.72, 27.15; HRMS: C17H26N5O3+ [M+H]30 calc. 348.2036, found 348.2036.
(R)-tert-butyl 1-(2-(2-(2-(2-(5-(4-(44(2,4-diaminopyrimidin-5-yl) methyl)-2,6-dimethoxyphenoxy)butylamino)-5-oxopentanamido)ethoxy)ethoxy)ethoxy)ethylamino)-1-oxo-3-(tritylthio)propan-2-ylcarbamate (BocCys(Trt)-TMP, 11). BocCys(Trt)-PEG4-Glu-COOH (75.2 mg, 0.1 mmol) and DIEA (36 mg, 0.28 mmol) are added, the reaction solution is stirred for 5-10 min, and then TMP-Bu-NH2 (35 mg, 0.1 mmol) is added. The reaction mixture is stirred at room temperature overnight to obtain a settled solution. The reaction solution is aliquoted into EtOAc/aq. Na2CO3, and then extracted twice with ethyl acetate. All organic layers are combined and washed with aq. NaH2PO4 (2 M), aq. Na2CO3, and saturated brine successively, then dried with anhydrous sodium sulfate, filtered, concentrated, and subjected to gradient silica gel column chromatography (CHCl3/MeOH 10/1, 8/1, finally 5/1, Rf (CHCl3/MeOH 5/1)=0.5) to obtain 61.2 mg of a pale yellow product with a yield of 57%. 1H-NMR (DMSO-d6, 600 MHz): δ 7.84 (t, J=5.88Hz, 1H), 7.77 (m, 2H), 7.49 (s, 1H), 7.32 (t, J=7.5Hz, 6H), 7.28 (d, J=7.92Hz, 6H), 7.24 (t, J=7.08Hz, 3H), 6.92 (d, J=8.52Hz, 1H), 6.54 (s, 1H), 6.38 (br, 2H), 5.97 (s, 2H), 4.12 (s, 2H), 3.92 (m, 1H), 3.78 (t, J=6.0Hz, 2H), 3.70 (s, 6H), 3.52 (s, 2H), 3.47 (br, 4H), 3.45 (br, 4H), 3.19 (m, 1H), 3.12 (m, 1H), 3.05 (m, 2H), 2.04 (m, 2H), 1.55 (m, 2H), 1.53 (m, 2H), 1.37 (s, 9H); 13C-NMR (DMSO-d6, 151 MHz): δ 171.88, 171.58, 170.15, 162.51, 161.00, 154.92, 152.91, 144.35, 135.28, 134.88, 129.12, 128.08, 128.79, 106.31, 105.88, 78.39, 72.00, 69.74, 69.63, 69.56, 69.16, 68.89, 55.85, 53.41, 40.06, 38.68, 38.45, 38.17, 34.84, 34.77, 34.06, 32.88, 28.15, 27.16, 25.76, 21.63; HRMS: C57H77N8O11S+ [M+H]+ calc. 1081.5427, found 1081.5471.
(R)-N1-(2-(2-(2-(2-(2-amino-3-mercaptopropanamido)ethoxy)ethoxy)ethoxy)ethyl)-N5-(4-(44(2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)butyl)glutaramide (CysTMP, 1). BocCys(Trt)-TMP (25 mg, 0.023 mmol) is dissolved in 1 ml of TFA, and then 25 μl of TIS is added. The reaction is performed in the presence of argon for 1 h at room temperature. Volatile components such as TFA and TIS are removed in vacuum to obtain a product aliquoted in EtOAc/H2O. The organic layers are washed twice with ethyl acetate, concentrated, and dried in vacuum to obtain a white foam product with a yield of 86%. 1H-NMR (DMSO-d6, 400 MHz): δ 8.54 (t, J=5.7Hz, 1H, 8.29 (br, 3H), 7.84 (t, J=5.6Hz, 1H), 7.77 (t, J=5.6Hz, 1H), 7.74 (s, 1H), 7.64 (s, br, 2H), 7.44 (s, 1H), 6.60 (s, 2H), 3.95 (t, J=5.6Hz, 1H), 3.79 (t, J=6.28Hz, 2H), 3.73 (s, 6H), 3.59 (s, 2H), 3.51 (br, 4H), 3.50 (br, 4H), 3.39 (t, J=6.1Hz, 2H), 3.34 (m, 1H), 3.26 (m, 1H), 3.18 (m, 2H), 3.06 (m, 2H), 2.90 (br, 2H), 2.05 (m, 4H), 1.68 (m, 2H), 1.57 (m, 4H); 13C-NMR (DMSO-d6, 101 MHz): δ 171.88, 171.57, 166.72, 164.06, 154.28, 153.06, 139.77, 135.33, 132.79, 108.92, 106.30, 71.99, 69.71, 69.58, 69.53, 69.09, 68.71, 55.92, 53.90, 38.40, 38.13, 34.83, 34.77, 32.08, 30.75, 27.12, 25.72, 21.60; HRMS: C33H55N8O9S+ [M+H]+ calc. 739.3807, found 739.3820.
5-(4-((21-chloro-3,6,9,12,15-pentaoxahenicosypoxy)-3,5-dimethoxybenzyl) pyrimidine-2,4-diamine (TMP-Cl, 14). Dimethoprim (TMP-OH, 12) and TsO-PEG5-Cl (13) are synthesized according to a previously reported scheme (Chen, et al. Angew. Chem. Int. Ed. 2017, 56, 5916). Then, TMP-OH (12, 50 mg, 0.18 mmol), TsO-PEG5-Cl (13, 97.2 mg, 0.19 mmol) and Cs2CO3 (76.3 mg, 0.234 mmol) are added into a two-neck round bottom flask, anhydrous DMF (1.8 ml) is added, and the reaction suspension is rapidly stirred at room temperature overnight in the presence of argon to complete the coupling reaction. DMF is removed under vacuum. A small amount of methanol is added and dissolved, and then vacuum is applied, which process is repeated about three times to remove DMF more thoroughly. The dried residue is aliquoted into EtOAc/Na2CO3 (aq.), then the organic layers are separated, and the aqueous layer is extracted twice with ethyl acetate. All organic layers are combined, washed twice with saturated brine, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and subjected to gradient silica gel column chromatography (EtOAc:MeOH 10:1→8:1→DCM:MeOH 10:1)) to obtain 60.4 mg of a white solid product with a yield of 54%. The NMR and MS characterization data are consistent with the above-mentioned literature. 1H-NMR (DMSO-d6, 600 MHz): δ 7.50 (s, 1H), 6.54 (s, 2H), 6.11 (s, 2H), 5.72 (s, 2H), 3.90 (t, 2H, J=5.1Hz), 3.71 (s, 6H), 3.63-3.69 (m, 4H), 3.56 (m, 2H), 3.48-3.53 (m, 14H), 3.45 (m, 2H), 3.35 (t, 2H, t, J=6.48Hz), 1.70 (m, 2H), 1.47 (m, 2H), 1.37 (m, 2H), 1.29 (m, 2H). MS(ESI): C29H48O8N4Cl+ [M+H]+ , calcd. 615.32, found 615.42.
tert-Butyl (18-chloro-3,6,9,12-tetraoxaoctadecyl)carbamate (BocNH-PEG4-Cl, 17). alcohol (15) (293 mg, 1.0 mmol) and Iodide (16) (278 mg, 1.05 mmol) starting materials were dissolved in THF (4 ml), and then KOH (72.4 mg, 85%) was also added. The reaction suspension was stirred at RT overnight. The next day, aq. NaH2PO4 solution was added to quench the reaction and the reaction mixture was extracted three times by EtOAc. EtOAc was removed under reduced pressure and the crude product was purified via silica gel chromatography (cyclohexane→cyclohexane/EtOAc 3/2→1/1→2/3) to give 193.5 mg oil product in a yield of 47%. 1H-NMR (CDCl3, 600 MHz): δ 5.07 (s, 1H), 3.67-3.62 (m, 8H), 3.62-3.59 (m, 2H), 3.59-3.56 (m, 2H), 3.54-3.50 (m, 4H), 3.44 (t, 2H, J=6.67Hz, 2H), 3.30 (m, 2H), 1.76 (m, 2H), 1.59 (m, 2H), 1.45 (m, 2H), 1.43 (s, 9H), 1.36 (m, 2H). 13C-NMR (CDCl3, 600 MHz): δ 156.15, 79.26, 71.36, 70.74, 70.72, 70.65, 70.36, 70.22, 45.19, 40.48, 32.67, 29.57, 28.55, 26.82, 25.55; HRMS(ESI): C19H38ClNO6Na+, calcd. 434.2285, found 434.2286 [M+Na]+.
18-Chloro-3,6,9,12-tetraoxaoctadecan-1-amine (H2N-PEG4-Cl, 18). BocNH-PEG4-Cl (17) (182.5 mg, 0.443 mmol) was dissolved in DCM (1 ml) and TFA (0.5 ml) was added. The reaction solution was stirred at RT for 20 min. DCM and TFA were removed under high vacuum to give 209 mg (0.44 mmol) deprotected H2N-PEG4-Cl (18) in a quantitative yield. 1H-NMR (CDCl3, 600 MHz): δ 8.05 (s, 3H), 5.70 (s, 2H), 3.85 (m, 2H), 3.76 (m, 2H), 3.67 (m, 2H), 3.65-3.60 (m, 8H), 3.53 (t, 2H, J=6.67H), 3.50 (t, J=7.08Hz, 2H), 3.14 (m, 2H), 1.76 (m, 2H), 1.58 (m, 2H), 1.44 (m, 2H), 1.33 (m, 2H); 13C-NMR (CDCl3, 600 MHz): δ 71.62, 70.78, 70.44, 70.11, 70.08, 69.97, 67.56, 45.10, 40.24, 32.54, 29.27, 26.66, 25.21; HRMS(ESI): C14H31ClNo4+, calcd. 312.1936, found 312.1940 [M+H]+.
tert-Butyl (R)-(24-chloro-5-oxo-1,1,1-triphenyl-9,12,15,18-tetraoxa-2-thia-6-azatetracosan-4-yl)carbamate (BocCys(Trt)-Cl, 19). BocCys-OH (93 mg, 0.2 mmol), HBTU (83 mg, 0.22 mmol), HOBt (13.5 mg, 0.1 mmol), and DIEA (171 μl, 1 mmol) were dissolved in DMF and stirred at RT for 10 min. Then H2N-PEG4-Cl (100 mg, 0.21 mmol) was added. The reaction solution was stirred at RT overnight. Aq. Na2CO3 solution was added to quench the reaction and the reaction mixture was extracted three additional times by EtOAc. Organic layers were combined, washed with brine for two times, dried over anhydrous Na2SO4, and the organic solution was directed subjected to silica gel chromatography (cyclohexane→cyclohexane/EtOAc 2/1″1/1→1/2→EtOAc) to give 143 mg white foamy solid as the product in a yield of 94%. 1H-NMR (CDCl3, 600 MHz): δ 7.40 (s, 3H), 7.38 (s, 3H), 7.29 (m, 6H), 7.23 (m, 3H), 6.46 (s, 1H), 4.88 (m, 1H), 3.88 (m, 1H), 3.67-3.62 (m, 4H), 3.61-3.58 (m, 2H), 3.58-3.52 (m, 6H), 3.52-3.45 (m, 6H), 3.45-3.40 (m, 1H), 3.38-3.30 (m, 1H), 2.71 (m, 1H), 2.51 (dd, J1=13.1 Hz, J2=5.34 Hz, 1H), 1.73 (m, 2H), 1.58 (m, 2H), 1.42 (s, 9H), 1.39 (m, 2H), 1.31 (m, 2H); 13C-NMR (CDCl3, 600 MHz): δ 171.77, 155.54, 144.48, 129.67, 128.21, 127.10, 80.53, 71.55, 70.42, 70.10, 69.88, 69.63, 69.37, 67.28, 53.73, 45.30, 39.39, 38.74, 33.97, 32.60, 29.01, 28.40, 26.78, 25.28; HRMS(ESI): C41H57ClN2O7SNa+, calcd. 779.3473, found 779.3468 [M+Na]+.
(R)-2-Amino-N-(18-chloro-3,6,9,12-tetraoxaoctadecyl)-3-mercaptopropanamide (Cys-Cl, 20). Boc-Cys(Trt)-Cl (19) (141.2 mg, 0.187 mmol) was dissolved in TFA (2 ml) and TIPS (50 μl) was added. The reaction solution was stirred at RT for 4 hours to allow complete deprotection. TFA and TIPS were mostly removed under high vacuum and the residue was dissolved in DI-H2O, washed three times by EtOAc and the aqueous solution was concentrated under reduced pressure. The product was dried in vacuo to give 71.4 mg Cys-Cl (20) product in 72% yield. 1H-NMR (D2O, 600 MHz): 4.15 (t, J=6.2Hz, 1H), 3.69-3.64 (m, 15H), 3.60 (t, J=7.98 Hz, 2H), 3.53 (t, J=6.78 Hz, 2H), 3.51 (t, J=5.82 Hz, 1H), 3.43-3.38 (m, 1H), 3.05 (m, 2H), 1.76 (m, 2H), 1.58 (m, 2H), 1.43 (m, 2H), 1.35 (m, 2H); 13C-NMR (D2O, 600 MHz): 167.85, 70.93, 69.59, 69.56, 69.52, 69.47, 69.30, 69.03, 68.54, 54.42, 45.60, 39.08, 31.73, 28.30, 25.77, 24.81, 24.42; HRMS(ESI): C17H36ClN2O5S+ calcd. 415.2028, found 415.2036 [M+H]+.
3-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)-N-(2-cyanobenzo[d]thiazol-6-yl)propenamide (ACBT, 23). Azido-PEG 3 -acid (33.5 mg, 0.136 mmol, 21) is dissolved in 0.6 ml of anhydrous DMF in a thoroughly dried round bottom flask (RBF). HATU (57.5 mg, 0.136 mmol) and DIEA (38 mg, 0.29 mmol) are added and stirred in the presence of argon for several minutes, then amino-CBT (20 mg, 0.113 mmol, 22) is added, and the reaction is performed under stirring at room temperature for 1 day. The reaction solution is aliquoted into EtOAc/NaH2PO4 (2M), the organic layers are separated, and the aqueous layer is washed twice with ethyl acetate. All organic layers are combined, washed once with saturated sodium bicarbonate (sat. Na2CO3), filtered, concentrated, and subjected to silica gel column chromatography (EtOAc, Rf 0.25) to obtain 27.7 mg of a viscous yellowish oily product with a yield of 60%. 1H-NMR (DMSO-d6, 600 MHz): δ 10.47 (s, 1H), 8.76 (t, J=2.22Hz, 1H), 8.18 (dd, J1=8.94 Hz, J2=1.92 Hz, 1H), 7.73 (d, J=9 Hz, 1H), 3.73 (td, J1=6.24 Hz, J2=1.98 Hz, 2H), 3.48-3.55 (m, 11H), 3.33 (s, 2H), 2.63 (td, J1=6.02 Hz, J2=1.98 Hz, 2H); 13C-NMR (DMSO-d6, 151 MHz): 170.05, 147.55, 139.71, 136.79, 134.92, 124.83, 120.65, 113.66, 111.09, 69.81, 69.76, 69.74, 69.68, 69.24, 66.54, 49.97, 37.33; HRMS: C17H20N6O4SNa+ [M+Na]+ calcd. 427.1159, found 427.1151.
The general structural formula of the SNACIP inducers is as follows: small molecule binding motif-nanobody targeting moiety-linker-intracellular delivery moiety. The schematic diagram is shown in
The corresponding SNACIP inducers are prepared by a fusion expression method and a chemical coupling method according to the above general structural formula:
The intracellular delivery moiety can be one of the following: new cyclic cell-penetrating peptide—cR10*,
Since green fluorescent protein (GFP) is currently one of the most widely used fluorescent proteins (FPs), direct regulation of the function of GFP-fused proteins will be a general regulatory means. In addition, GFP is also a fluorescent molecule, which means that a protein of interest can be simultaneously regulated and imaged. So far, no small molecule ligands that directly bind GFP with high affinity have been reported, so GFP is also a target protein without small molecule ligands.
General SNACIP inducer of dimerization—cR10*-SS-GBP-TMP, or cRGT (
First, a CysTMP chemical small molecule was synthesized. The CysTMP was used for introducing a TMP ligand onto a GBP nanobody (a Cys-TMP synthesis method is shown in reaction scheme 1). CysTMP contained an N-terminal cysteine, a water-soluble PEG linker, and a TMP moiety for binding an eDHFR protein tag (the structure is shown in
The more detailed preparation steps are as follows:
It was confirmed by size exclusion chromatography (SEC) that a GBP-TMP nanobody conjugate can indeed induce dimerization between EGFP and eDHFR. It can be seen that in the presence of GBP-TMP, a stable EGFP/GBP-TMP/eDFHR ternary complex could be formed, while in the absence of GBP-TMP, eDHFR and EGFP could not form a protein complex (
A bicistronic vector was used for co-expressing EGFP-mito and mCherry-eDHFR in living HeLa cells and testing the regulatory effect of cRGT on intracellular dimerization (
The kinetics of cRGT penetrating into cells and inducing dimerization was subsequently investigated (
The localization of mCherry-eDHFR in the cytoplasm to the mitochondria was concentration-dependent, with 24 μM cRGT being an optimal concentration (
Many cellular processes are regulated by dynamic distribution of proteins in the cell. It was verified that cRGT could regulate the localization of EGFP to different subcellular structural regions, including mitochondria, Golgi apparatus and nuclear membrane subcellular regions. mScarlet-eDHFR-mito (mitochondria localized) and EGFP (distributed in cytoplasm) were co-expressed in HeLa cells. cRGT (24 μM, 1.5h) localized EGFP from the cytoplasm to the mitochondrial outer membrane where mScarlet-eDHFR-mito was, and the localization was very complete. The localization regulation process was rapidly reversible by adding TMP (10 μM, 10 min) (
Yellow fluorescent protein mEYFP and turquoise fluorescent protein mTurquoise2 are close mutants of the GFP. It was found that the localization of mEYFP could be efficiently regulated by cRGT, but the localization of mTurquoise2 was not (
Localization of proteins to the plasma membrane is a universal method for activating signaling cascades. To this end, we intended to use cRGT to regulate signal transduction. Rac1-mediated signal transduction plays a key role in the formation of lamellipodia, and also plays an important role in the metastasis and invasion of cancer cells. To this end, activation of the corresponding signaling transduction during lamellipodia formation by localizing Rac1 to the plasma membrane using cRGT was designed (
Next, SNACIP was used for studying biological issues. Kinesin-cargo specificity is an important issue during intracellular transport. However, many related issues remain unclear. To this end, it was first demonstrated that multiple reversible regulations of “off”-“on”-“off”-“on” of a kinesin-mediated cargo transport process can be achieved. This process could be achieved by washing out TMP small molecule inhibitors from the medium, further highlighting superior reversibility of SCNACID technology (
Ferroptosis is a recently discovered non-apoptotic form of programmed cell death with iron-dependent properties, which is also accompanied by morphological changes in mitochondria and an increase in lipid reactive oxygen species (ROS). Targeting ferroptosis is currently speculated to be a novel and promising approach to killing drug-resistant cancer cells, as cancer cells exhibit a higher ferroptosis dependence than normal cells. Inspired by this, we considered activation of ferroptosis with cRGT. Among many ferroptosis-related factors, glutathione peroxidase 4 (GPX4) is considered to be one of the most important factors, which plays a role in protecting plasma membranes from peroxidative damage (
The above-mentioned examples show that cRGT-based SNACIP represents a general tool for control of cellular processes. In fact, the SNACIP concept is not limited to regulate only EGFP variants or eDHFR fused proteins. For example, the GBP nanobody can be facilely replaced by other nanobodies to further extend the application potential. In order to demonstrate this possibility, we employed a mCherry red fluorescent protein binding protein (RBP) nanobody. Setup the ligation between RBP-Intein and Cys-TMP requires less than 10 min and coupling of Cys-cR10* requires a few hours of work (
A Cys-Cl ligand that features a cysteine moiety for EPL and a HaloTag ligand (chlorohexyl group) was prepared (Scheme 3). This was used to assemble a new SNACIP inducer called cR10*-SS-GBP-Cl, or (cRGC) (
Endogenous ligand-free binding proteins are target proteins that are difficult to regulate by conventional CID methods. Among these ligand-free target proteins, intrinsically disordered proteins (IDPs) are a major class, and are currently receiving increasing attention due to their important biological functions. Microtubule nucleation is an important issue in the field of cytoskeleton. The structure of the microtubule nucleator—γTuRC, i.e., a gamma-tubulin cyclic complex, has been resolved. However, the structures of many other key factors in microtubule nucleation, e.g., an augmin complex and several nucleation factors belonging to the IDP class, remain enigmatic. Further, key microtubule nucleators are essential for cell division. Strict gene regulation methods such as gene knockout will directly lead to division blocked and death of cells, cannot establish corresponding gene knockout cell lines, and hence are not suitable for studying the effect of nucleation factors on cellular functions.
Intrinsically disordered protein TPX2 is a key regulator of microtubule nucleation, which mediates the Ran signaling pathway during spindle assembly. As an oncoprotein, TPX2 is overexpressed in many cancer cells, including the most difficult-to-treat liver cancer (
Latent SNACIP inducer, cR10*-TBP-CAAX, or cRTC, was designed and constructed, which features by a nanobody containing a human TPX2 (hTPX2) binding protein (TBP), wherein the TBP nanobody has a cyclic cR10* cell-penetrating peptide at the N-terminal end and a CAAX box polypeptide sequence at the C-terminal end, and the CAAX box can be prenylated in a living cell (
TPX2 nanobodies were successfully screened by phage display technology. hTPX2 antigen for phage display were prepared by TEV protease cleavage. hTPX2 is difficult to express well in E. coli, so first a pET28b(TEV)_hTPX2-TEV-EGFP-His8 plasmid was constructed, which contained an EGFP tag, and can effectively promote expression of hTPX2. The plasmid has a His8 tag at the C-terminal end, and an EGFP tag and a TEV cleavage site at the C-terminal end of hTPX2. hTPX2-TEV-EGFP-His8 was expressed in E. coli following the general protein expression scheme described previously. More specific steps are as follows. After IPTG was added for induction, E. coli Rosetta 2a was cultured overnight at 30° C. After centrifugation, lysis, high-speed centrifugation and gradient Ni-IMAC purification are successively performed on the E. coli, hTPX2-TEV-EGFP-His8 dissolved in buffer A+ (pH 8.0, i.e., solution A with an additional 3 mM of BME) was obtained. Then, an appropriate amount of TEV protease was added, the protein solution was incubated at 2° C. overnight, and hTPX2-TEV-EGFP-His8 was cleaved by enzyme, so that hTPX2 was cleaved from EGFP-His8. The protein solution was subjected to Ni-IMAC purification again, and hTPX2 was eluted with an imidazole-free buffer A+. The cleaved His8-containing fragment and the His-tag-fused protease bind more tightly to the nickel column, and can only be eluted at a higher concentration of imidazole to separate hTPX2. hTPX2 protein fractions were mixed, concentrated by ultrafiltration, and subjected to size exclusion chromatography using PBS as the eluent by a Superdex200 10/300 increase GL column. The PBS solutions of hTPX2 were mixed and concentrated by ultrafiltration, aliquoted, quickly frozen in liquid nitrogen, stored at −80° C., and then used for nanobody screening in alpacas. To quantify protein concentration, typically 1 μl of protein sample is measured with a DS-11FX(+) DeNovix Spectrophotometer/Fluorometer. By measuring A280 and using M.W. and molar extinction coefficient ε, a relatively accurate protein concentration can be measured: c(mg·ml−1)=[A280×M.W.(g·moL−1)]/ε(L·moL−1cm−1).
Next, the nanobody was prepared by M13 phage display technology, and a nanobody TBP (TPX2_binding protein) with high binding ability was screened. After that, the prepared TBP nanobody was expressed, purified and used for ITC measurement. First, pET28b(TEV)_His8-mCherry-TEV-TBP was cloned and expressed according to the scheme described above, and cultured overnight at 30° C. with shaking after IPTG induction. The purified TBP nanobody was thoroughly cleaved with an appropriate amount of TEV protease overnight at 2° C. The protein solution was subjected to Ni-IMAC purification, and the TPX2 nanobody was eluted with an imidazole-free buffer A first and purified for subsequent analysis. The cleaved His8-mCherry fragment, His8-fused TEV protease and most impurities were removed due to high binding affinity to the nickel column, so a high-purity hTPX2 nanobody was prepared. Isothermal titration calorimetry (ITC) reveals that the binding Kd value between TBP and hTPX2 was 287 nM with an equivalence ratio of 1:5. A negative value of AS also implies that the binding process is accompanied by a significant conformational change (
Next, one-pot preparation of cRTC was achieved by a tandem bioorthogonal ligation reaction starting from azide-functionalized TBP-CAAX (
In a representative reaction, the previously prepared Cys-TBP-CAAX protein was first exchanged into a PBS solution (pH 7.2, 1.78 mg·ml−1), and then 4 μl of ACBT (˜10 mM, final concentration ˜0.5 mM) could be added. After incubation at 2° C. overnight, and being confirmed by SDS-PAGE to be completely labeled, Cys-TBP-CAAX was exchanged into buffer A+ to obtain an ACBT-TBP-CAAX intermediate (1.95 mg·ml−1, 71 μl, 97% yield). At the same time, 15 μl of Cys-cR10* (25 mM/DMSO, 0.375 μmol) and 10 μl of BCN-PEG2-maleimide (25 mM/DMSO, 0.25 μmol) were sequentially added to 80 μl of PBS solution, and incubated at room temperature for ˜1 h to complete a thiol-maleimide ligation reaction. 3.9 μl of the in situ ligation product cR10*-BCN (˜24 mM, ˜1.2 eq) was added to the ACBT-TBP-CAAX solution, and incubated for several hours to complete copper-free catalyzed click reaction labeling. After exchanging the solution into PBS, a cR10*-TBP-CAAX nanobody conjugate inducer of dimerization (1.52 mg·ml−1, 73 μl, 80% yield) was obtained, referred to as cRTC.
The cRTC inducer clearly localized hTPX2 protein to the plasma membrane in HepG2 cells (
Next, whether downregulation of TPX2 activity with cRTC could inhibit cell proliferation was investigated. The results of an EdU cell proliferation assay showed that the EdU positive ratio of cRTC-treated HepG2 cells decreased, and the nuclear fluorescence intensity also significantly decreased (
An example of linear cell penetrating peptide (CPP) is a Tat polypeptide sequence.
Based on the above results, we predicted that the SNACIP inducers that regulate hTPX2 could be developed as SNACIP inducer drugs of proximity for inhibiting tumor proliferation in vivo. To better adapt cRTC for in vivo assays, a bivalent nanobody latent SNACIP regulatory inducer, mCherry-CPP-2×TBP-CAAX (CTTC) was designed and prepared, which included a tandem bivalent TBP nanobody, 2×TBP (
CTTC, a SNACIP inducer, was prepared, whose structural elements included a bivalent TBP nanobody, a Tat linear cell penetrating peptide, and a CAAX-box polypeptide sequence. After entering a cell, CTTC could be modified by prenylation to introduce a farnesyl group, and then converted into a functional SNACIP (
Hepatocarcinoma xenograft mice model was obtained by injecting 5 million HepG2 hepatoma cells into the armpit of mice. It can be found that the tumor growth rate of the control group (PBS) was almost the same as that of the blank group. Only in the experimental group in which the mice were injected with CTTC, the tumor size began to decrease within 24 h after administration. At the same time, compared with the control and blank groups, tumor growth was also inhibited for a longer period of time (
M phase is considered to be the most critical period during cell separation, and correct assembly of the bipolar spindle determines whether M phase can proceed. It is now generally accepted that the spindle is assembled through three key pathways: 1) chromosome-based, 2) centrosome-based, and 3) microtubule-based three pathways (
As an efficient system for studying the mechanism of spindle assembly, a Xenopus cell-free system has many advantages, especially good biochemical accessibility, that is, without the barrier of plasma membrane, any regulatory reagents (e.g., antibodies) can be directly added to interfere with the relevant biochemical processes. A Xenopus oocyte extract completely depleted of TPX2 was obtained by immunodepletion (
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210495991X | May 2022 | CN | national |
