Application of reagent for blocking and / or negatively regulating SARM1 in preparation of medicine for treating chemotherapy-induced neuropathy

Abstract

The invention belongs to the technical field of anti-neuropathy drugs, and particularly relates to application of a reagent for blocking and / or negatively regulating SARM1 in preparation of drugs for treating chemotherapy-induced neuropathy. The invention provides application of a reagent for blocking and / or negatively regulating and controlling SARM1 in preparation of a medicine for treating chemotherapy-induced neuropathy. The amino acid sequence of the SARM1 is as shown in SEQ ID NO. 1. The construction of SARM1 gene knockout and mutant mice shows that the nerve cell death can be inhibited by blocking the combination of the SARM1 and DNA (Deoxyribose Nucleic Acid). The invention provides a new application of an activator for blocking SARM1, and the reagent for blocking combination of SARM1 and DNA can be used for preparing potential drugs for treating chemotherapy-induced neuropathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-neuropathy drugs, and particularly relates to the use of an agent that blocks and / or negatively regulates SARM1 in the preparation of a drug for treating chemotherapy-induced neuropathy. Background Art

[0002] With the increasing incidence of malignant tumors, chemotherapy drugs play an important role in their treatment. Chemotherapy has been well-documented to alter DNA to kill cancer cells. Unfortunately, side effects are almost unavoidable. 30% to 68% of cancer survivors experience chemotherapy-induced neuropathy, and up to 90% of patients struggle with neuropathy with certain chemotherapy regimens, such as oxaliplatin.

[0003] Sterile α and Toll / interleukin-1 receptor domain-containing protein 1 (SARM1) is a key neural actuator belonging to the Toll / interleukin-1 receptor (TIR) ​​domain-containing protein family. TIR domain-containing proteins are conserved throughout life and can degrade nicotinamide adenine dinucleotide (NAD) into nicotinamide (NAM) and cyclic adenosine diphosphate ribose (cADPR), triggering infected cell death to limit infection. However, little is known about the substances that activate SARM1, and there are no reports of agents that block SARM1 activation for chemotherapy-induced neuropathy. Summary of the Invention

[0004] The present invention aims to provide a method for treating chemotherapy-induced neuropathy by using an agent that blocks and / or negatively regulates SARM1. Blocking SARM1 activation can inhibit cell death and thus play a role in treating neuropathy.

[0005] The present invention provides use of an agent for blocking and / or negatively regulating SARM1 in the preparation of a drug for treating chemotherapy-induced neuropathy; the amino acid sequence of the SARM1 is shown in SEQ ID NO.1.

[0006] As a preferred embodiment, the reagents for blocking SARM1 include reagents for blocking the binding of SARM1 to DNA; the reagents for negatively regulating SARM1 include reagents for knocking out and / or knocking down SARM1.

[0007] As a preferred embodiment, the DNA comprises double-stranded DNA with a size of ≥40 bp.

[0008] As a preferred embodiment, the agent that blocks the binding of SARM1 to DNA includes an agent that blocks the binding of the TIR region of SARM1 to DNA.

[0009] As a preferred embodiment, the agent for blocking the binding of the TIR region of SARM1 to DNA includes an agent that induces multiple point mutations at positions 602, 628 and 636 of the amino acid sequence shown in SEQ ID NO.1.

[0010] As a preferred embodiment, the multiple point mutations include: the 602nd lysine mutation to glutamic acid, the 628th lysine mutation to glutamic acid and the 636th lysine mutation to glutamic acid in the amino acid sequence shown in SEQ ID NO.1.

[0011] As a preferred embodiment, the chemotherapy drug includes oxaliplatin.

[0012] As a preferred embodiment, the neuropathy includes neuropathy associated with axonal mutation.

[0013] As a preferred embodiment, the axonal mutation-related neuropathy includes sensory neuropathy and / or motor neuropathy.

[0014] The present invention also provides a drug for treating chemotherapy-induced neuropathy, comprising an active component and a pharmaceutically acceptable carrier; the active component comprises an agent that blocks and / or negatively regulates SARM1, and the amino acid sequence of the SARM1 is shown in SEQ ID NO.1.

[0015] Beneficial effects:

[0016] The present invention provides the use of agents that block and / or negatively regulate SARM1 in the preparation of drugs for treating chemotherapy-induced neuropathy. SARM1 can sense and bind to DNA in the body, leading to SARM1 activation and cell death. The present invention, through the construction of SARM1 knockout and mutant mice, demonstrates that blocking SARM1 binding to DNA can inhibit neuronal cell death. The present invention provides a new application for agents that block SARM1 activators. Agents that block SARM1 binding to DNA can be used as potential drugs for treating chemotherapy-induced neuropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1Figure 1 shows the results of DNA activation of SARM1 in Example 1; A shows the cADPR level generated by mixing hSARM1 with different substances; B and C show the NAD, NAM, and cADPR levels in the reaction mixture of 0.1 μM hSARM1 and 0.1 μM 90 bp DNA analyzed by MS; D shows the cADPR, NAM, and NAD levels in the mixture of 0.1 μM hSARM1 protein and 0.1 μM DNA; *** in the figure indicates significant differences in the data, p < 0.001;

[0019] Figure 2 Figures 2 and 3 are the domain results of SARM1 binding to DNA in Example 2; wherein, A is a representative gel electrophoresis image and binding fraction of EMSA determination of DNA binding to hSARM1 protein; B is a fluorescence quenching analysis of hSARM1 and Ctrl (GFP) proteins at different concentrations; C is a modified Stern-Volmer curve used for DNA binding constant in (B); D is MST determination of hSARM1 binding to DNA; E is Coomassie staining and circular dichroism analysis of in vitro purified hSARM1 full-length (FL), ARM-, SAM-, and TIR domain-truncated hSARM1 proteins; F is a representative gel electrophoresis image and binding fraction of 0.05 μM Cy3-labeled DNA binding to a series of dilutions of different proteins; G is MST determination of hSARM1 truncated proteins binding to DNA; H is a fluorescence quenching analysis of different concentrations of DNA on 0.1 μM of the protein; I is a modified Stern-Volmer curve used to estimate the DNA binding constant in (H);

[0020] Figure 3 Figures 1 and 2 show the results of DNA length control for DNA binding and SARM1 activation in Example 3; wherein, A is a representative gel electrophoresis image and binding fraction of EMSA assay for the binding of DNA of different lengths to hSARM1 protein; B is MST analysis of the binding of 0.05 μM Cy5-labeled DNA to hSARM1 protein; C is the levels of cADPR and NAD in a mixture of hSARM1 protein and the DNA; D is the molecular weight distribution of the hSARM1, 45 bp DNA-, and 90 bp DNA-hSARM1 complex peaks;

[0021] Figure 4 Representative gel electrophoresis images and binding fraction graphs of DNA of different lengths binding to hSARM1 protein in Example 3;

[0022] Figure 5 Schematic diagram of the DNA and SARM1 complex in Example 3;

[0023] Figure 6Figure 4 shows the results of SARM1 sensing DNA to promote cell death; A is a representative IF image of mouse neurons transfected with DNA for 6 hours, green: DNA stained with anti-dsDNA antibody; red: mSARM1 stained with anti-SARM1 antibody; blue: DAPI; scale bar = 10 μm; B (left) is a quantitative graph of the colocalization of hSARM1 and DNA in (A), and the right is a MS analysis to assess the level of cADPR in the cells described in (A); C is a representative IF image showing axonal degeneration in neurons transfected with DNA for 36 hours, green: Tubb3; red: mSARM1; BF: bright field photograph; Morge: composite image; scale bar = 10 μm; D is the cell ratio (left) and percentage of live cells (right) in neurons transfected with 2 μg / mL DNA every 16 hours for 60 hours;

[0024] Figure 7 Figure 5 shows the detection of SARM1 sensing chemotherapy-induced cytoplasmic DNA to promote cell death in Example 5; wherein, A is a representative IF image of mouse neurons treated with oxaliplatin for 36 h, green: DNA stained with anti-dsDNA antibody; red: mSARM1 stained with anti-SARM1 antibody; blue: DAPI; scale bar = 10 μm; B is mSARM1 DNA colocalization (left) and cADPR level (right) in the cells described in (A), n = 6; C is axon degeneration in neurons treated with 20 μM oxaliplatin for 48 h, green: Tubb3; red: mSARM1; BF: bright field photograph; Morge: composite image; scale bar = 10 μm; D is quantitative data of cell number (left) and axon degeneration (right) in cells treated with 20 μM oxaliplatin (Oxa) for 60 h;

[0025] Figure 8 Figures 1 and 2 show that the SARM1 mutation attenuates chemotherapy-induced neuropathy in mice in Example 5, wherein A is a representative image of hindlimb clenching in mice injected intraperitoneally with 6 mg / kg Oxa once daily for three consecutive days, n=10; B is the limb clenching score of mice; C is mechanical allodynia in mice, with the left image showing the mechanical force response curve generated by the two hind paws and fitted by nonlinear regression, and the right image showing the half-maximal effective force (EF50) value determined by nonlinear regression, n=10; D is thermal allodynia in mice injected with oxaliplatin. DETAILED DESCRIPTION

[0026] The present invention provides use of an agent for blocking and / or negatively regulating SARM1 in the preparation of a medicament for treating chemotherapy-induced neuropathy; the amino acid sequence of the SARM1 is shown in SEQ ID NO. 1: MVLTLLLSAYKLCRFFAMSGPRPGAERL.

[0027] In one embodiment, the agent that blocks SARM1 includes an agent that blocks SARM1 binding to DNA. In one embodiment, the DNA of the present invention includes double-stranded DNA of 40 bp or greater. In one embodiment, the agent that blocks SARM1 binding to DNA includes an agent that blocks the binding of the TIR region of SARM1 to DNA. SARM1 is a nucleic acid sensor with three domains: an N-terminal allosteric regulatory repeat (ARM) domain, a sterile alpha motif (SAM) domain, and a C-terminal catalytic TIR domain. The catalytic site of SARM1 spans both TIR regions and can recognize double-stranded DNA of 40 bp or greater, regardless of DNA sequence. DNA binds to SARM1 and activates it, releasing NADase activity, degrading nicotinamide adenine dinucleotide (NAD), and promoting neuronal cell death. Therefore, blocking the binding of the TIR region of SARM1 to DNA can block SARM1 activation and thereby reduce neuronal cell death.

[0028] In one embodiment, the present invention provides an agent for blocking DNA binding to the TIR region of SARM1, including an agent that induces multiple point mutations at positions 602, 628, and 636 of the amino acid sequence set forth in SEQ ID NO. 1. In one embodiment, the multiple point mutations may include: lysine 602 to glutamic acid, lysine 628 to glutamic acid, and lysine 636 to glutamic acid in the amino acid sequence set forth in SEQ ID NO. 1. The present invention constructs SARM1 mutant mice and finds that DNA binding by SARM1 is primarily dependent on three lysine residues in the amino acid sequence set forth in SEQ ID NO. 1. Multiple point mutations in these three lysine residues render SARM1 unable to recognize DNA, reduce neuronal cell death, and attenuate chemotherapy-induced neuropathy. As an embodiment, the present invention uses CRISPR / Cas9 to achieve multiple point mutations at positions 602, 628 and 636 of the amino acid sequence shown in SEQ ID NO.1; in a specific embodiment of the present invention, the sgRNAs for achieving multiple point mutations at positions 602, 628 and 636 of the amino acid sequence shown in SEQ ID NO.1 by CRISPR / Cas9 are as shown in SEQ ID NO.24 to SEQ ID NO.27: sgRNA2-A1 (SEQ ID NO.24): 5′-TGAGGAGTACCGGCATTTAC-CGG-3′; sgRNA2-A2 (SEQ ID NO.25): 5′-AGCCAGGGCTGAGATAATCA-GGG-3′; sgRNA2-B1 (SEQ ID NO.26): 5′-TGGTAAATGCCGGTAAATGC-CGG-3′; sgRNA2-B2 (SEQ ID NO.27): 5′-AGGGCTGAGATAA TCAGGGT-AGG-3′, the present invention uses homologous recombination to simultaneously transfer donor DNA containing the expected mutation sequence into the target cells together with Cas9mRNA and sgRNA. When double-strand breaks occur in the cells, homologous recombination is performed using the donor DNA containing the expected mutation sequence as a template to integrate the specific mutation into the genome.

[0029] As an embodiment, the negative regulation agent of SARM1 includes an agent for knocking out and / or knocking down SARM1. In a specific embodiment of the present invention, the negative regulation agent of SARM1 may include an agent for knocking out or knocking down SARM1. In a specific embodiment of the present invention, CRISPR / Cas9 is used to knock out SARM1; the sgRNA sequence information in the CRISPR / Cas9 is shown in SEQ ID NO.16 to SEQ ID NO.19: sgRNA1-A1 (SEQ ID NO.16): 5′-CTTAGGCTTCACGCGTCTGT-AGG-3′; sgRNA1-A2 (SEQ ID NO.17): 5′-CTAATTATTCCACGTTCACC-TGG-3′; sgRNA1-B1 (SEQ ID NO.18): 5′-TCCATTTCCTACCGCTCGCT-CGG-3′; sgRNA1-B2 (SEQID NO.19): 5′-AGCCACCATCATGACGCCCC-TGG-3′. The present invention utilizes multiple sgRNAs to guide the Cas9 protein to cut at different sites. Different sgRNAs act simultaneously to create double-strand breaks at different key positions of the SARM1 gene, making the gene fragments more susceptible to degradation by nucleases in the cell, thereby effectively achieving gene knockout.

[0030] In one embodiment, the chemotherapy drug includes oxaliplatin. In one embodiment, the neuropathy includes axonal mutation-related neuropathy. In a specific embodiment of the present invention, the axonal mutation-related neuropathy includes sensory neuropathy and / or motor neuropathy; the sensory neuropathy is primarily manifested by numbness, tingling, discomfort, and other cold-related sensory abnormalities in the extremities; and the motor neuropathy is primarily manifested by muscle weakness, muscle atrophy, and quadriplegia and spasticity.

[0031] The present invention also provides a drug for treating chemotherapy-induced neuropathy, comprising an active ingredient and a pharmaceutically acceptable carrier; the active ingredient includes an agent that blocks and / or negatively regulates SARM1; the amino acid sequence of SARM1 is shown in SEQ ID NO.1. In a specific embodiment of the present invention, the active ingredient may include an agent that induces multiple point mutations at positions 602, 628, and 636 of the amino acid sequence shown in SEQ ID NO.1, and may also include an agent that knocks out or reduces SARM1. In one embodiment, the active ingredient may include an sgRNA that induces multiple point mutations at positions 602, 628, and 636 of the amino acid sequence shown in SEQ ID NO.1; the sgRNA sequence information is shown in SEQ ID NO.24 to SEQ ID NO.27; and may also include an sgRNA that knocks out or reduces SARM1, the sgRNA sequence information being shown in SEQ ID NO.16 to SEQ ID NO.19.

[0032] To further illustrate the present invention, the use of the agent for blocking and / or negatively regulating SARM1 provided by the present invention in the preparation of a medicament for treating chemotherapy-induced neuropathy is described in detail below with reference to the accompanying drawings and examples. However, these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] SARM1 senses DNA and activates, releasing its NADase activity

[0035] 1) Visualization of the SARM1 octamer model

[0036] The inactive hSARM1 octamer model (Protein Data Bank (PDB): 7ANW) was obtained from the Protein DataBank database (https: / / www.rcsb.org). The cryo-electron microscopy (Cryo-EM) map of the active SARM1 octamer model (accession number: EMD-24274) was obtained from the Electron Microscopy Data Bank (https: / / www.emdataresource.org / ). The active TIR domain octamer model (PDB: 7NAK) was then fitted to the Cryo-EM map of the active SARM1 octamer model (EMD-24274) using the ChimeraX "Fit Plot" tool. The flexible peptide chain between the SAM and TIR domains (residues 546–560) was modeled using the SWISS-MODEL server. The SARM1 model was visualized using PyMOL (version 2.5.2) using default parameters.

[0037] 2) Expression and purification of SARM1 protein

[0038] The plasmid encoding the SARM1 protein (NP_055892.2) was used to transform Escherichia coli strain BL21 (DE3). The resulting protein was concentrated and buffer exchanged using an Amicon Ultrafree centrifugal filter with a 10 kD cutoff (Millipore) to produce the SARM1 protein (hSARM1). The amino acid sequence of the SARM1 protein is shown in SEQ ID NO. 1.

[0039] 3) Mass spectrometry (MS) analysis of cADPR levels in the reaction mixture

[0040] ① Prepare different samples: A, 90 bp DNA, the nucleotide sequence of one strand of the 90 bp DNA is shown in SEQ ID NO. 2: 5′-AGTACATGTCTAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTATCTAGTGATTATCTAGACATGGACTC-3′. B. Metal ions: CaCl2, CoCl2, CrCl3, CuCl2, FeCl2, MgCl2, NiCl2, ZnCl2; C. Amino acids: Alanine, Arginine, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Serine; D. Glycometabolism: Aconitate, Citrate, Fumarate, Glucose, Isocitrate, Lactate, Malate, Pyruvate, Succinate; E. Lipid metabolites: Glycerin, Lauric acid, Linoleic acid, Nervonic acid, Oleic acid acid, palmitic acid; F, nucleoside: adenine, cytidine, guanosine, uridine, inosine.

[0041] ② The 0.1 μM SARM1 protein (hSARM1) prepared in step 2) was directly mixed with buffer as a control group (ctrl); the 0.1 μM SARM1 protein (hSARM1) prepared in step 2) was mixed with different samples (100 nM) from step 1) in a buffer containing 2 mM NAD (Sigma-Aldrich, Cat. number: N8285), 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM MgCl2, and incubated at 37°C for 1 hour.

[0042] ③ The sample mixture was separated using an ACQUITY UPLC system (Waters) in multiple reaction monitoring (MRM) mode, monitoring the precursor ion to the product ion. Standards of NAD, NAM, and cADPR were used to capture the precursor and product ions, determine the collision energy (CE) and declustering potential (DP), and collect the corresponding ions. Mass spectrometry (MS) detection was then performed using a Qtrap5500 triple quadrupole mass spectrometer (ABSciex) in positive electrospray ionization mode. Multiple reaction monitoring (MRM) mode was applied to monitor the precursor ions to products: NAD (precursor ion to product ion: nucleocytoplasmic ratio (M / z) 664 to 428; collision energy (CE): 90.4 V; declustering potential (DP): 34.1 V), NAM (M / z 123.0 to 80.1; CE 24.3 V; DP 93.9 V) and cADPR (M / z 542 to 136; CE 26.8 V; DP 90.3 V). The flow rate was set to 0.6 mL / min. Solvent A consisted of H2O and 5 mM ammonium formate; solvent B consisted of 100% methanol. Analytical software for Windows (version, 1.6.1) was used for peak area quantification and data processing, and the results are shown in Figure 2. Figure 1 As shown in A, the levels of NAD, cA DPR and NAM after 90bp DNA binds to hSARM1 are as follows Figure 1 As shown in B and C.

[0043] The cADPR level in the reaction mixture of hSARM1 protein, NAD, and DNA samples was analyzed by mass spectrometry (MS), and it was found that double-stranded DNA significantly released the NADase activity of hSARM1 ( Figure 1 Middle A), hSARM1 protein mixed with DNA increased the levels of cADPR and NAM in the mixture, while decreasing the level of NAD ( Figure 1 B and C).

[0044] 4) Different sequences activate SARM1

[0045] ① Follow the procedure in step ① of step 3), except that: in the experimental group, 0.1 μM SARM1 protein (hSARM1) was mixed with 0.1 μM 90bp DNA, 0.1 μM poly(dC:dG), and 0.1 μM poly(dA:dT) in buffer. The nucleotide sequence of one strand of the 90bp DNA is shown in SEQ ID NO. 2; the poly(dC:dG) is a DNA sequence (~1000 bp) consisting of repeating guanine (G) and cytosine (C); and the poly(dA:dT) is a DNA sequence (~4000 bp) consisting of repeating adenine (A) and thymine (T).

[0046] ②Follow the steps ② in step 3) and the test results are as follows Figure 1 As shown in D. The results showed that 90bp random sequence, poly(dC:dG) and poly(dA:dT) DNA all effectively released hSARM1 activity, indicating that DNA activation of hSARM1 is sequence-independent.

[0047] Example 2

[0048] SARM1 binds DNA through its TIR domain.

[0049] 1) Electrophoretic mobility shift assay (EMSA) of SARM1 binding to DNA

[0050] Based on the principle that the electrophoretic mobility of protein-DNA complexes is lower than that of free DNA, an electrophoretic mobility shift assay (EMSA) was used to assess hSARM1-DNA binding. Lower DNA mobility during electrophoresis indicates stronger protein binding. Experimental group (FL): 0.05 μM Cy3-labeled DNA (90 bp DNA from Example 1) was mixed with a series of hSARM1 protein dilutions (0 μM, 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM) in a buffer containing 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Control group (Ctrl): The procedure was identical to the experimental group, except that the DNA was mixed with a series of GFP protein dilutions. After incubation at 37°C for 30 minutes, the mixtures were resolved on 1%-1.5% agarose gels using 40 mM Tris-HCl (pH 9.2) running buffer at a constant voltage of 110 V. Images were then acquired using ChemiDoc XRS+ (Bio-Rad) and analyzed using ImageJ. The results are shown in Figure 2. Figure 2 As shown in A.

[0051] 2) Metabolite Affinity Response Target Fluorescence Quenching (MARTFQ)

[0052] Experimental group (FL): 0.05 μM SARM1 protein was mixed with a series of diluted DNA (90 bp DNA in Example 1, 0 nM, 20 nM, 50 nM, 100 nM); the control group (Ctrl) was operated in the same way as the experimental group, except that the DNA was mixed with a series of dilutions of GFP protein. After the experimental and control groups were incubated for 5 minutes, the fluorescence intensity was analyzed under 280 nm excitation light. The binding constant was estimated according to the modified Stern-Volmer equation as follows: RF0 / ΔRF=(1 / fK)×(1 / [Q])+(1 / f), where ΔRF is equal to RF0 (protein fluorescence intensity in the absence of metabolites)-RF (intensity in the presence of metabolites); f is the fractional maximum fluorescence intensity of the protein; K is the quenching constant, which is used as the binding constant, and [Q] is the concentration of DNA. The results are shown in Figure 2. Figure 2 As shown in B and C.

[0053] 3) Microscale thermophoresis (MST)

[0054] At room temperature, 0.05 μM Cy5-labeled DNA (90 bp DNA in Example 1) was diluted and mixed with a series of SARM1 protein concentrations (0 nM, 20 nM, 50 nM, and 100 nM), and then subjected to an MST experiment in a buffer containing 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The control group (Ctrl) was operated in the same manner as the experimental group, except that the DNA was mixed with a series of GFP protein dilutions. The thermophoretic movement of DNA and protein was analyzed using a Monollith NT.115 instrument (Nanotetemper Technologies). The results are shown in Figure 2. Figure 2 As shown in D.

[0055] Further testing of whether SARM1 directly binds to DNA revealed that hSARM1 reduced the electrophoretic mobility of DNA, indicating that hSARM1 binds to DNA ( Figure 2 Based on the metabolite-induced intrinsic fluorescence quenching of target proteins, metabolite affinity-responsive target fluorescence quenching (MARTFQ) analysis showed that the intrinsic fluorescence of hSARM1 was quenched by DNA ( Figure 2 Consistently, hSARM1-DNA binding was also observed in microscale thermophoresis analysis (MST) ( Figure 2 The above results all indicate that hSARM1 binds to DNA.

[0056] 5) In order to determine which domain of hSARM1 is responsible for DNA binding, the present invention further expressed and purified truncated proteins of the ARM-, SAM- and TIR-domains, respectively. The amino acid sequences are shown in SEQ ID NOs. 3 to 5.

[0057] ARM-domain truncated protein (SEQ ID NO.3):MVLTLLLSAYKLCRFFAMSGPRPGAERLAVPGPDGGGGTGPWWAAGGRGPREVSPGAGTEVQDALERALPELQQALSALKQAGGARAVGAGLAEVFQLVEEAWLLPAVGREVAQGLCDAIRLDGGLDLLLRLLQAPELETRVQAARLLEQILVAENRDRVARIGLGVILNLAKEREPVELARSVAGILEHMFKHSEETCQRL VAAGGLDAVLYWCRRTDPALLRHCALALGNCALHGGQAVQRRMVEKRAAEWLFPLAFSKEDELLRLHACLAVAVLATNKEVEREVERSGTLALVEPLVASLDPGRFARCLVDASDTSQGRGPDDLQRLVPLLDSNRLEAQCIGAFYLCAEAAIKSLQGKTKVFSDIGAIQSLKRLVSYSTNGTKSALAKRALRLLGEEVPRPILPS*;

[0058] SAM-domain truncated protein (SEQ ID NO. 4): MVPSWKEAEVQTWLQQIGFSKYCESFREQQVDG DLLLRLTEEELQTDLGMKSGITRKRFFRELTELKTFANYSTCDRSNLADWLGSLDPRFRQYTYGL VSCGLDRSLLHRVSEQQLLEDCGIHLGVHRARILTAAREMLHSPLPCTGGKPSGD*;

[0059] TIR-domain truncated protein (SEQ ID NO. 5): MTPDVFISYRRNSGSQLASLLKVHLQLHGFSVFIDVEKLEAGKFEDKLIQSVMGARNFVLVLSPGALDKCMQDHDCKDWVHKEIVTALSCGKNIVPIIDG FEWPEPQVLPEDMQAVLTFNGIKWSHEYQEATIEKIIRFLQGRSSRDSSAGSDTSLEGAAPMGPT*.

[0060] The hSARM1 full-length sequence (FL), ARM-, SAM- and TIR domain-truncated hSARM1 proteins prepared in Example 1 were subjected to Coomassie staining and circular dichroism analysis. The results are as follows: Figure 2 As shown in E.

[0061] 6) FL, ARM-, SAM- and TIR domain proteins were mixed with 0.05 μM Cy3-labeled DNA (90 bp DNA in Example 1) and subjected to EMSA analysis. The operation method was the same as that in step 1). The results were as follows: Figure 2 As shown in F. EMSA analysis showed that TIR domain proteins effectively reduced the electrophoretic mobility of DNA, while ARM and SAM domain proteins had little effect on the mobility, indicating that the TIR domain is responsible for binding to DNA.

[0062] 7) The mixture in step 6) was subjected to MST and MARTFQ analysis, and the operation method was the same as that in step 2) and step 3). The results were as follows: Figure 2 As shown in G to I. MST and MARTFQ analyses also showed that hSARM1 binds to DNA through its TIR domain.

[0063] Example 3 DNA length controls DNA binding and activation of SARM1

[0064] 1) Size Exclusion Chromatography (SEC)

[0065] ①SEC separates molecules or complexes based on molecular weight. hSARM1 was mixed with 20bp, 30bp, 45bp, and 90bp DNA, respectively, in a buffer containing 20mM Tris (pH 7.4) and 200mM NaCl. The molar ratio of hSARM1:20bp DNA was 9:1; the molar ratio of hSARM1:30bp DNA was 9:1; the molar ratio of hSARM1:45bp DNA was 9:1; and the molar ratio of hSARM1:90bp DNA was 17:1. The nucleotide sequences of one strand of the 20bp, 30bp, and 45bp DNA are shown below, and the nucleotide sequence of one strand of the 90bp DNA is shown in SEQ ID NO. 2.

[0066] 20bp-F (SEQ ID NO.6): 5′-ATTAGTACATGTCTAGTCAG-3′;

[0067] 30bp-F (SEQ ID NO.7): 5′-TACAGATCTACTAGTGATCTATGACTGATC-3′;

[0068] 45bp-F (SEQ ID NO. 8): 5′-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3′.

[0069] ② Take 100 μL of the mixture from step ① and load it onto a Superose 6 incremental 10 / 300GL column equilibrated with 20 mM Tris (pH 7.4) and 100 mM NaCl buffer. The flow rate was set to 1 mL / min, and the separated peaks were analyzed using Wyatt-MiniDawn multi-angle light scattering (MALS). The molecular mass of the complex was determined using Astra software (Wyatt Technology); the DNA level in the separated peak was assessed by agarose gel electrophoresis; and the protein expression level in the separated peak was analyzed using the Micro-BCA protein assay kit from ThermoFisher Scientific (Waltham, MA, USA). The results are shown in Figure 2. Figure 3 As shown in A.

[0070] 2) The mixture in step 1) was subjected to EMSA and MST analysis, and the operating steps were the same as those in step 1) and step 3) in Example 2. The results were as follows: Figure 3 The molecular weights determined by SEC were used to further analyze the complex characteristics of 45 bp DNA and 90 bp DNA with hSARM1 protein. Figure 3 As shown in D.

[0071] 3) In a buffer containing 20 mM Tris (pH 7.4) and 200 mM NaCl, hSARM1 was mixed with 45 bp-1, 45 bp-2, 90 bp-1, 90 bp-2, 500 bp-1, 500 bp-2, and 500 bp-3 DNA, wherein the sequence information of one strand of the DNA is shown in Table 1. The molar ratio of hSARM1:45 bp DNA was 9:1; the molar ratio of hSARM1:90 bp DNA was 17:1; and the molar ratio of hSARM1:500 bp DNA was 17:1. EMSA and MST analyses were performed, respectively, using the same procedures as steps 1) and 3) of Example 2. The results for 45 bp-1, 90 bp-1, and 500 bp-1 were as shown in Table 1. Figure 4 shown.

[0072] Table 1 DNA sequence information

[0073]

[0074]

[0075] Given that DNA activates some DNA sensors in a length-dependent manner, the present invention evaluated the effect of DNA length on DNA binding and activation of SARM1. EMSA analysis showed that hSARM1 significantly reduced the electrophoretic mobility of 45 bp and 90 bp DNA, but did not reduce the mobility of 20 bp or 30 bp DNA ( Figure 3 A), indicating that DNA length controls the potential of DNA binding to hSARM1, and SARM1 can bind to ≥40bp DNA. Consistently, MST experimental analysis also showed that DNA length controls the potential of DNA binding to hSARM1 ( Figure 3 Consistent with the binding results, 45bp and 90bp DNA can effectively activate hSARM1, while 20bp and 30bp DNA cannot activate hSARM1 ( Figure 3 We further designed different sequences of 45bp, 90bp and 500bp DNA sequences and found that different sequences of DNA ≥40bp could bind to SARM1 and significantly reduce the electrophoretic mobility of DNA ( Figure 4 ).

[0076] In addition, the molecular weight measured by SEC was used to further analyze the characteristics of the DNA-hSARM1 complex. The SARM1 protein structure is an octamer, and the isolated peak of hSARM1 is 621 kD ( Figure 3 D), the molecular weights of the 45 bp DNA-hSARM1 complex and the 90 bp DNA-hSARM1 complex were 1238 and 2356 kD, respectively ( Figure 3 These results indicate that the 45 bp DNA-hSARM1 complex contains two hSARM1 octamers (1238 ≈ 2 × 621), and the 90 bp DNA-hSARM1 complex contains four hSARM1 octamers (2356 ≈ 4 × 621).

[0077] From the above analysis, we can see that the 45bp DNA-hSARM1 complex contains two DNAs and two hSARM1 octamers, and the 90bp DNA-hSARM1 complex contains two DNAs and four hSARM1 octamers ( Figure 3 In general, DNA ≤ 30 bp cannot effectively bind and activate SARM1. DNA binds and activates SARM1 in a length-dependent manner. The DNA-hSARM1 complex is formed by two DNAs and 2n SARM1 octamers (n ≥ 1) (DNA2-SARM1 octamer 2n) ( Figure 5DNA and SARM1 form a DNA2-SARM1 octamer 2n (n ≥ 1) complex. The DNA length threshold for effective SARM1 binding is ~45 bp. As the length of the DNA double strand increases, 2n SARM1 octamers will bind to the DNA double strand (2n represents a double number).

[0078] Example 4 SARM1 senses DNA and promotes cell death.

[0079] 1) Mouse neuronal cell extraction

[0080] ① Using C57BL / 6 mice as the base, CRISPR / Cas9 was used to construct a SARM1 (NM_001168521) knockout mouse model (KO) and a SARM1 K602E / K628E / K636E mouse model (3KE).

[0081] KO model mice: Cas9 mRNA and sgRNA produced by in vitro transcription were injected into C57BL / 6 mouse fertilized eggs for the production of KO mice. After CRISPR / Cas9 editing, exons 2 to 7 of SARM1 were deleted, resulting in a frame shift of SARM1. Exon 2 starts at approximately 20.55% of the coding region, and exons 2 to 7 account for 68.63% of the coding region. The size of the effective KO region is approximately 15958 bp, and there are no other known functional genes in the KO region. DNA sequencing analysis is then performed on the edited individuals to screen positive individuals, and the positive individuals are bred to the next generation (F1), and then genotyped by PCR, DNA sequencing, and immunoblotting analysis to obtain KO model mice. The sgRNA sequence information of the KO model mouse is shown below, where the last 3 bases of the sgRNA are the PAM sequence.

[0082] SgRNA1-A1 (SEQ ID NO.16): 5′-CTTAGGCTTCACGCGTCTGT-AGG-3′;

[0083] SgRNA1-A2 (SEQ ID NO. 17): 5′-CTAATTATTCCACGTTCACC-TGG-3′;

[0084] SgRNA1-B1 (SEQ ID NO. 18): 5′-TCCATTTCCTACCGCTCGCT-CGG-3′;

[0085] SgRNA1-B2 (SEQ ID NO. 19): 5′-AGCCACCATCATGACGCCCC-TGG-3′.

[0086] The PCR primer sequence information is as follows:

[0087] WT-SARM1-F2 (SEQ ID NO. 20): 5′-GATAATCAGGGTAGGCTTCATGG-3′;

[0088] WT-SARM1-R2 (SEQ ID NO. 21): 5′-AGGTCTGAAGGCCAATGTAC-3′.

[0089] The F1 generation homozygote amplified a 759 bp band; the heterozygote amplified a 759 bp and a 858 bp band; the wild type amplified a 858 bp band.

[0090] The PCR reaction system was as follows: 1.5 μL DNA template, 1.0 μL upstream primer, 1.0 μL downstream primer, 12.5 μL Premix Taq, and 9.0 μL ddH2O. The PCR reaction procedure was as follows: initial denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 60°C for 35 s, and extension at 72°C for 35 s, for 35 cycles; and final extension at 72°C for 5 min.

[0091] 3KE model mice: The bacterial artificial chromosome (BAC) clone RP23-356D12 was used to generate donors using PCR. Donor Cas9 mRNA, sgRNA, and a donor vector containing the "KI region-p.K602E (AAA to GAG)-p.K628E (AAG to GAG)-p.K636E (AAG to GAG)" cassette were injected into one-cell embryos (C57BL / 6) for the production of 3KE mice. Embryos were cultured in embryo-maximal KSOM medium (Sigma-Aldrich, MR-101, USA) to the two-cell stage and then transferred into the oviduct of the recipient 0.5 days after coitus to produce mice. The resulting mice were genotyped by PCR (3KE-F, SEQ ID NO. 22: 5'-TTATCAGTTACCGGAGGAACTCAG-3'; 3KE-R, SEQ ID NO. 23: 5'-CCCTGACATCACTGTACAACCTTAT-3') and DNA sequencing to obtain 3KE model mice. The PCR reaction system and procedure were the same as for the "KO model mice." The sgRNA sequence information for the 3KE model mice is shown below, where the last three bases of the sgRNA are the PAM sequence.

[0092] SgRNA2-A1 (SEQ ID NO.24): 5′-TGAGGAGTACCGGCATTTAC-CGG-3′;

[0093] <h2 style=";text-align:left;direction:ltr">SgRNA2-A2(SEQ ID NO.25):5′-AGCCAGGGCTGAGATAATCA-GGG-3′;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> SgRNA2-B1(SEQ ID NO.26):5′-TGGTAAATGCCGGTAAATGC-CGG-3′;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> SgRNA2-B2(SEQ ID NO.27):5′-AGGGCTGAGATAATCAGGGT-AGG-3′。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] The three lysine mutations K602E / K628E / K636E of SARM1 in the 3KE mouse model are shown in SEQ ID NO. 28: MVLTLLLSAYKLCRFFAMSGPRPGAERLAVPGPDG.

[0097] ② The brains of wild-type C57BL / 6 mice (WT), 3KE and KO embryonic mice were respectively digested in CMF-HBSS containing 0.125% trypsin, and then cut into pieces and filtered through a 75-μm filter to separate into single-cell suspensions.

[0098] 2) The neural cells extracted from step 1) were plated and maintained in Neurobasal medium containing 2% (w / v) B-27 and 0.5 mM glutamine at 37°C in a humidified atmosphere of 5% (v / v) CO. Neural cells extracted from mSARM1 WT, 3KE, and KO mice were transfected with 2 μg / mL FAM-labeled DNA (45 bp DNA from Example 3) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions for 6 h.

[0099] After the treatment, the cell slides were collected and washed with PBS 3 times / 1 min; 4% paraformaldehyde was used to cover the cell slides, and the slides were allowed to stand for 15 min, and then washed with PBS 3 times / 5 min; sufficient 0.2% TritonX-100 was added, and the slides were incubated on a shaker for 15 min, and then washed with PBS 3 times / 5 min; sufficient 2% BSA blocking solution was added, and the slides were incubated on a shaker for 30 min, and then washed with PBS 3 times / 5 min; according to the dilution ratio of the primary antibody (Abcam, ab309195) at 1:500, sufficient primary antibody dilution was added to the cell slides, and the slides were incubated at room temperature for 1 h; according to the dilution ratio of the fluorescent secondary antibody (Thermo, 2812001) at 1:500, sufficient secondary antibody dilution was added to the cell slides, and the slides were incubated at room temperature in the dark for 1 h; the slides were sealed with anti-fluorescence quenching solution containing DAPI, and after air-drying for 30 min in the dark, they were observed and photographed under a fluorescence microscope. The results are as follows Figure 6 As shown in A and B (left); Figure 6 Middle B (right) is MS analysis and evaluation Figure 6 cADPR levels in cells described in Figure 2A.

[0100] 36 h after the first transfection, axonal integrity was assessed by phase contrast microscopy and anti-tubulin β3 class III (Tubb3, a neural marker) immunostaining. Figure 6 As shown in C, the cell ratio and percentage of living cells in the neural cells are as follows Figure 6 As shown in D, the left figure shows the cell ratio in neural cells, and the right figure shows the percentage of living cells.

[0101] Experimental results: To determine whether DNA binds to and activates SARM1 in cells, we first evaluated the cellular colocalization between SARM1 and DNA. Immunofluorescence analysis showed that wild-type SARM1 colocalized with FAM-labeled DNA in neurons, indicating that DNA can effectively activate hSARM1 and that the 3KE mutation eliminates the ability of wild-type SARM1 to bind DNA. Figure 6 A and B), Figure 6Middle B shows the cells stained with anti-SARM1 antibody.

[0102] Based on the role of SARM1 in cell death, we then evaluated the effect of DNA on SARM1-induced cell death. The results showed that DNA effectively promoted the proliferation of neural cells ( Figure 6 A and B) and SARM13KE mutation reversed this phenomenon ( Figure 6 (C and D), indicating that DNA effectively activates endogenous SARM1, thereby promoting cell death.

[0103] Example 5 SARM1 senses chemotherapy-induced cytoplasmic DNA to promote cell death

[0104] 1) Oxaliplatin treatment of WT, 3KE and KO mouse neurons

[0105] The nerve cells of WT, 3KE and KO mice extracted in Example 4 were used for experiments. The nerve cells of WT, 3KE and KO mice were treated with 20 μM oxaliplatin (Oxa) for 36 h and 48 h, respectively. After 36 h of treatment, the cellular localization between SARM1 and cytoplasmic DNA was evaluated by immunofluorescence analysis. The operation steps were the same as those in step 1 of Example 4. After 48 h of treatment, the integrity of the axons was evaluated by phase contrast microscopy and anti-tubulin β3 class III (Tubb3) immunostaining. The results are as follows: Figure 7 shown.

[0106] 2) Mechanical hypersensitivity experiment

[0107] The WT, KO and 3KE mice of Example 4 were intraperitoneally injected with 6 mg / kg oxaliplatin (Oxa) once a day for three consecutive days to obtain Oxa-treated mice. The paw retraction threshold under mechanical stimulation was measured using a dynamic plantar tactile sensor (Touchtest sensory evaluator, manufacturer EXACTA) equipped with a series of von Frey fiber filaments. The Oxa-treated mice were acclimated on an acrylic grid platform in a plastic observation box for 60 minutes. The fiber contacted the mid-plantar surface of the hind paw until a slight bend of the fiber was observed, and then maintained for 2 seconds. Each fiber was tested 10 times for each mouse, with at least 30 seconds between the two tests. The percentage of paw retraction response was used for further analysis, and a force-response curve was established by plotting the relationship between force and response percentage. The half-maximal effective force (EF50) value was derived from the nonlinear regression of the force-response curve using Prism 8 (GraphPad software), and the results are shown in Figure 2. Figure 8 As shown in A and B.

[0108] 3) Thermal allodynia experiment

[0109] The WT, KO, and 3KE mice (n=10) of Example 4 were intraperitoneally injected with 6 mg / kg oxaliplatin once a day for four days to obtain Oxa-treated mice. The paw withdrawal latency to cold or radiant heat stimulation was assessed. The shorter the paw withdrawal latency, the more sensitive the mice were to thermal pain.

[0110] Before testing, mice treated with Oxa were acclimated for 60 min in a plastic observation box (plexiglass cylinder). For the heat stimulation test, the mid-plantar surface of the mouse hind paw was exposed to a radiant heat source through a glass floor until the paw was withdrawn. The heat source temperature was set to produce a standard baseline of about 8 s in WT mice, with a maximum cutoff time of 30 s. For the cold stimulation test, the cold source temperature was adjusted to produce a baseline of about 8 s in WT mice, with a maximum critical time of 30 s. Each mouse was tested three times, and the average value of the paw withdrawal latency was used for further statistical analysis. The results are shown in Figure 2. Figure 8 As shown in C and D.

[0111] Chemotherapy kills tumor cells by targeting DNA and induces cytoplasmic accumulation of DNA. Neuronal cells express high levels of SARM1, and 30-68% of cancer patients suffer from chemotherapy-induced neuropathy. First, it was determined whether endogenous SARM1 senses chemotherapy-induced cytoplasmic DNA to promote neuronal cell death. The results showed that SARM1 co-localized with chemotherapy-induced cytoplasmic DNA, and the 3KE mutation eliminated the ability of SARM1 to bind to cytoplasmic DNA. Chemotherapy-induced cytoplasmic DNA bound to and activated endogenous SARM1 in neuronal cells, which was reversed by SARM13KE and KO mutations ( Figure 7 Given the role of SARM1 in cell death, we then assessed the effect of chemotherapy-induced cytoplasmic DNA on SARM1's ability to promote cell death. Oxaliplatin treatment enhanced SARM1-induced neuronal cell death, suggesting that SARM1 senses chemotherapy-induced cytoplasmic DNA to promote cell death, and that the 3KE and KO mutations reduced neuronal cell death. Figure 7 Consistently, these SARM1 mutations attenuated chemotherapy-induced neuropathy in mice ( Figure 8 ), indicating a key role for SARM1 in chemotherapy-induced neuropathy.

[0112] In summary, SARM1 can sense double-stranded DNA (dsDNA) ≥40 bp and bind to its TIR domain, leading to SARM1 activation and cell death. The present invention, through the construction of SARM1 knockout and 3KE mutant mice, demonstrates that blocking SARM1 binding to DNA can inhibit neuronal cell death. These agents can be used to prepare drugs for treating chemotherapy-induced neuropathy.

[0113] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of an agent that blocks and / or negatively regulates SARM1 in the preparation of a medicament for treating chemotherapy-induced neuropathy; the amino acid sequence of the SARM1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The reagents that block SARM1 include reagents that block the binding of SARM1 to DNA; the reagents that negatively regulate SARM1 include reagents that knock out and / or knock down SARM1.

3. The use according to claim 2, characterized in that The DNA includes double-stranded DNA with a size of ≥40 bp.

4. The use according to claim 2, characterized in that The agent that blocks the binding of SARM1 to DNA includes an agent that blocks the binding of the TIR region of SARM1 to DNA.

5. The use according to claim 4, characterized in that The reagent for blocking the binding of the TIR region of SARM1 to DNA includes a reagent for inducing multiple point mutations at positions 602, 628 and 636 of the amino acid sequence shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that The multiple point mutations include: the 602nd lysine of the amino acid sequence shown in SEQ ID NO.1 is mutated into glutamic acid, the 628th lysine is mutated into glutamic acid, and the 636th lysine is mutated into glutamic acid.

7. The use according to claim 1, characterized in that The chemotherapy drugs include oxaliplatin.

8. The use according to claim 1, characterized in that The neuropathy includes axonal degeneration-related neuropathy.

9. The use according to claim 8, characterized in that The axonal degeneration-related neuropathy includes sensory neuropathy and / or motor neuropathy.

10. A drug for treating chemotherapy-induced neuropathy, characterized in that The invention comprises an active component and a pharmaceutically acceptable carrier; the active component comprises an agent for blocking and / or negatively regulating SARM1, and the amino acid sequence of the SARM1 is shown in SEQ ID NO.1.

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