NLRP3 variants and library screening of NLRP3 inhibitors using said NLRP3 variants

Abstract

The present invention relates to an NLRP3 variant comprising an at least partial deletion of PYD. The invention also relates to a method for screening the compound for inhibiting the NLRP3 inflammasome. The invention further provides a method comprising the step of administering to a subject suffering from an NLRP3-related inflammatory condition a compound identified by such a screening method.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 587,779, filed October 4, 2023. The entire contents of the above-mentioned patent application are incorporated herein by reference in their entirety. Background Technology

[0003] The NLRP3 inflammasome, containing the NOD-like receptor (NLR) family and a heat protein domain, plays a central role in innate immune responses and inflammation. Many diseases are associated with aberrant NLRP3 activity. Modulating the NLRP3 pathway by inhibiting NLRP3 may potentially provide an effective way to alter adverse clinical pathways in certain diseases. Numerous small-molecule NLRP3 inhibitors have been identified, and these compounds exhibit a range of NLRP3 binding sites and reversible or irreversible mechanisms of action. Despite these advances, new NLRP3 inhibitors and methods for identifying such inhibitors remain needed. Summary of the Invention

[0004] In one aspect, this disclosure provides a variant containing a nucleotide-binding oligomerization domain, a leucine-rich repeat sequence, and a thermoprotein domain 3 (NLRP3) variant comprising at least a partial deletion of the amino-terminal thermoprotein domain (PYD).

[0005] In some embodiments, the NLRP3 variant contains the deletion of amino acids 1-142 of the amino acid sequence of SEQ ID NO: 1.

[0006] In some embodiments, the NLRP3 variant contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 2.

[0007] In some implementations, the NLRP3 variant is linked to one or more polypeptide domains.

[0008] In some implementations, one or more polypeptide domains include a solubility-enhancing domain.

[0009] In some embodiments, one or more polypeptide domains are selected from the group consisting of maltose-binding protein (MBP), glutathione S-transferase (GST), N-utilization substance A (NusA), and small ubiquitin-associated modifier (SUMO).

[0010] In some implementations, one or more polypeptide domains include an affinity purification domain.

[0011] In some embodiments, the affinity purification domains include a His domain, a 6xHis domain, biotin, streptavidin, glutathione S-transferase (GST), FLAG (DYKDDDDK; SEQ ID NO: 4), and an antibody Fc domain.

[0012] In some embodiments, the NLRP3 variant contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

[0013] In some implementations, the NLRP3 variant retains ATPase activity relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0014] In some embodiments, the NLRP3 variant retains at least about 25% of the ATPase activity relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0015] In some implementations, the NLRP3 variant retains the ability to form NLRP3 inflammasomes, relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0016] In some embodiments, the NLRP3 variant retains the ability to induce the release of IL-1β in cells, relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0017] In one aspect, this disclosure provides a nucleic acid encoding the NLRP3 variant described herein.

[0018] In one aspect, this disclosure provides a vector comprising the nucleic acid described herein.

[0019] In one aspect, this disclosure provides a host cell comprising the nucleic acids described herein.

[0020] In some implementations, the host cell is an E. coli cell, a yeast cell, an insect cell, or a mammalian cell.

[0021] In some implementations, the insect cells are Sf21 cells.

[0022] In one aspect, this disclosure provides a method for purifying the NLRP3 variant described herein, the method comprising: 1) introducing a vector encoding the NLRP3 variant into a host cell; 2) culturing the host cell under conditions that allow the NLRP3 variant to be expressed in the host cell; and 3) isolating the NLRP3 variant from the host cell.

[0023] In some embodiments, separation step 3) includes contacting the lysate of the host cells with one or more affinity purification resins.

[0024] In some embodiments, one or more affinity purification resins are selected from the group consisting of: immobilized metal affinity chromatography (IMAC) resins, maltose or amylose affinity chromatography resins, glutathione affinity chromatography resins, protein A affinity chromatography resins, and anti-FLAG affinity chromatography resins.

[0025] In one aspect, this disclosure provides a method for identifying compounds capable of binding to polypeptides containing nucleotide-binding oligomerization domains, leucine-rich repeat sequences, and thermoprotein domain 3 (NLRP3), the method comprising: 1) contacting a compound suspected of being capable of binding the NLRP3 polypeptide with an NLRP3 variant described herein, wherein the NLRP3 variant is immobilized on a surface; 2) washing the compound and the immobilized NLRP3 variant with a buffer solution; and 3) detecting the compound, wherein if the compound is detected, the compound is capable of binding the NLRP3 polypeptide.

[0026] In some implementations, the compound is linked to a polynucleotide sequence.

[0027] In some implementations, the detection of the compound includes the detection of a polynucleotide sequence.

[0028] In some implementations, detecting polynucleotide sequences involves sequencing the polynucleotide sequence.

[0029] In some implementations, the polynucleotide sequence contains at least 5 nucleotides.

[0030] In some implementations, the length of the polynucleotide sequence is between 5 and 50 nucleotides.

[0031] In some implementations, the polynucleotide sequence is single-stranded DNA (ssDNA).

[0032] In some implementations, the concentration of the NLRP3 variant is between 1 nM and 300 nM.

[0033] In some implementations, the concentration of the NLRP3 variant is between 25 nM and 250 nM.

[0034] In some embodiments, the NLRP3 variant is contained in a buffer containing: 1) 5 mM to 500 mM Tris-HCl; 2) 15 to 1500 mM NaCl; 3) 1 to 100 mM MgCl2; 4) 1% to 20% glycerol; and 5) 0.0005% to 0.05% Tween-20.

[0035] In some embodiments, the buffer also contains: 1) 1 to 500 mM imidazole; and 2) 0.03 to 3 mg / mL single-stranded DNA (ssDNA).

[0036] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 50 mM Tris-HCl; 2) 150 mM NaCl; 3) 10 mM MgCl2; 4) 10% glycerol; and 5) 0.005% Tween-20.

[0037] In some implementations, the buffer also contains: 1) 10 mM imidazole; and 2) 0.3 mg / mL ssDNA.

[0038] In some embodiments, contact step 1) includes incubating the compound with the NLRP3 variant and 0.01 mM to 50 mM ATP.

[0039] In some embodiments, contact step 1) includes incubating the compound with the NLRP3 variant and 0.1 mM to 1 mM ATP.

[0040] In some implementations, the NLRP3 variant is immobilized on a solid carrier.

[0041] In some embodiments, the solid carrier comprises nickel-immobilized resin.

[0042] In some embodiments, the method further includes: 4) incubating a compound capable of binding the NLRP3 peptide with cells; and 5) measuring at least one pro-inflammatory cytokine secreted from the cells, wherein a decrease in the secretion of at least one pro-inflammatory cytokine from the cells indicates that the compound is an inhibitor of NLRP3.

[0043] In some implementations, at least one pro-inflammatory cytokine is IL-1β.

[0044] In some implementations, the cells are human mononuclear cells (THP-1 cells).

[0045] In one aspect, this disclosure provides a method for identifying an inhibitor containing a nucleotide-binding oligomerization domain, a leucine-rich repeat sequence, and a heat protein domain 3 (NLRP3), the method comprising: 1) contacting a compound suspected of being capable of binding an NLRP3 peptide with an NLRP3 variant described herein, wherein the NLRP3 variant is immobilized on a surface; 2) washing the compound and the immobilized NLRP3 variant with a buffer solution; 3) detecting the compound, wherein if the compound is detected, the compound is capable of binding an NLRP3 peptide; 4) incubating the NLRP3-binding compound from step 3) with cells; and 5) measuring at least one pro-inflammatory cytokine secreted from the cells, wherein a decrease in the secretion of at least one pro-inflammatory cytokine from the cells indicates that the compound is an inhibitor of NLRP3.

[0046] In some implementations, at least one pro-inflammatory cytokine is IL-1β.

[0047] In some implementations, the cells are human mononuclear cells (THP-1 cells).

[0048] In one aspect, this disclosure provides a method for treating an inflammatory condition, the method comprising administering to a subject an NLRP3 inhibitor identified herein.

[0049] In one aspect, this disclosure provides a composition comprising: 1) the NLRP3 variant described herein; 2) 5 mM to 500 mM Tris-HCl; 3) 15 to 1500 mM NaCl; 4) 1 to 100 mM MgCl2; 5) 1% to 20% glycerol; and 6) 0.0005% to 0.05% Tween-20.

[0050] In some embodiments, the composition further comprises: 1) 1 to 500 mM imidazole; and 2) 0.03 to 3 mg / mL single-stranded DNA (ssDNA).

[0051] In some embodiments, the composition comprises: 1) the NLRP3 variant described herein; 2) 50 mM Tris-HCl; 3) 150 mM NaCl; 4) 10 mM MgCl2; 5) 10% glycerol; and 6) 0.005% Tween-20.

[0052] In some implementations, the buffer also contains: 1) 10 mM imidazole; and 2) 0.3 mg / mL ssDNA.

[0053] In some embodiments, the composition further comprises 0.01 mM to 50 mM ATP. Attached Figure Description

[0054] Figure 1 Domain architecture of the MBP-ΔNLRP3-HIS protein used in DEL screening.

[0055] Figure 2 An overview of the construction of the key screening library.

[0056] Figure 3 The general structure of the best ligands found in the DEL screening.

[0057] Figure 4 Synthesis of an analogue of compound 1 for evaluating NLRP3 inhibition in THP-1. NLRP3 inflammasome activation assay.

[0058] Figure 5 The luminescence of the NLRP3 variant protein in this application's buffer was compared to that in the literature buffer. This application's buffer: 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 10% glycerol, 0.005% Tween-20, pH=7.5. The literature buffer: 20 mM Tris-HCl, 133 mM NaCl, 20 mM MgCl2, 0.56 mM EDTA, 3 mM KCl, pH=7.8. Each buffer also contains 10 µM ATP and NLRP3 ranging from 0 nM to 250 nM.

[0059] Figure 6 The luminescence of NLRP3 variant protein in the buffer of this application containing 0.3 mg / mL ssDNA, 10 mM imidazole or both (selection buffer). Detailed Implementation

[0060] This document provides a method for identifying compounds (such as peptides, inhibitors, and small molecules) capable of binding to a nucleotide-binding oligomerization domain, a leucine-rich repeating sequence, and a thermoprotein domain 3 (NLRP3). As used herein, the terms "nucleotide-binding oligomerization domain, leucine-rich repeating sequence, and thermoprotein domain 3," "protein 3 containing NOD, LRR, and thermoprotein domains," or "NLRP3" refer to the amino acid sequence of UNIPROT reference number Q96P20 and SEQ ID NO: 1.

[0061] NLRP3

[0062] NLRP3 is a member of the Nod-like receptor (NLR) protein family. NLRP3 is an intracellular sensor that detects a wide range of danger signals and environmental damage, thereby generating a protective pro-inflammatory response aimed at damaging pathogens and repairing tissue damage through the formation and activation of NLRP3 inflammasomes (Coll, RC et al.). Trends Pharmacol. Sci. 2022 43(8) NLRP3 is highly expressed in subsets of peripheral leukocytes and microglia in the central nervous system.

[0063] NLRP3 exhibits a triplet structure consisting of a thermoprotein domain (PYD), a central nucleotide-binding and oligomerizing domain of the NACHT subfamily of NTP enzymes, and a leucine-rich repeating sequence domain (LRR). The NACHT domain possesses ATPase activity, which is essential for inducing the assembly of inactive ADP-binding decameric molecules in cells (Hochheiser, I. et al.). Nature , 2022, 604 , 184-189), (Brinkschulte, R. et al., Commun. Biol. , 2022, 5 , 1176).

[0064] Assembly of the NLRP3 inflammasome leads to caspase-1-dependent secretion and release of pro-inflammatory cytokines IL-1β and IL-18, as well as gasdermin D-mediated pyroptosis.

[0065] NLRP3 variant

[0066] This disclosure relates to NLRP3 variants that can be used to screen NLRP3 inhibitors, such as those described herein.

[0067] In one aspect, this disclosure provides an NLRP3 variant comprising at least a partial deletion of the amino-terminal thermoprotein domain (PYD).

[0068] As used herein, “partial deletion” includes the deletion of at least one amino acid that reduces or eliminates the activity of PYD or maintains or increases the stability of the protein compared to the wild-type protein of SEQ ID NO: 1.

[0069] As used herein, “wild-type protein” includes the amino acid sequence of SEQ ID NO: 1 and naturally occurring variants or isotypes of NLRP3.

[0070] In some implementations, partial deletions include the deletion of at least 1, 5, 10, 25, 50, 75, 100, 125, 142, 150, 175, 200, or 216 amino acids of the PYD of NLRP3.

[0071] In some embodiments, the NLRP3 variant comprises amino acids 1-50, 1-90, 1-100, 1-142, 1-216, 3-110, 6-90, 7-12, and 8-108 deleted from the N-terminus of the amino acid sequence of SEQ ID NO: 1.

[0072] In some embodiments, the NLRP3 variant comprises amino acids 6-16, 17-20, 21-31, 32-42, 43-48, 49-50, 51-62, 62-63, 64-78, 79-80, and 81-90 deleted from the N-terminus of the amino acid sequence of SEQ ID NO: 1.

[0073] In some embodiments, the NLRP3 variant contains an amino acid sequence that has at least 80% identity with SEQ ID NO: 2 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0074] In some embodiments, the NLRP3 variant contains an amino acid sequence that has at least 80% identity with SEQ ID NO: 3 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0075] In some embodiments, the NLRP3 variant retains ATPase activity relative to the wild-type NLRP3 of SEQ ID NO: 1. Those skilled in the art can readily identify methods for measuring ATPase activity. For example, but not limited to, methods include measuring the release of inorganic phosphates produced by the hydrolysis of ATP using a colorimetric method. For example, but not limited to, ATPase activity can be measured by ADP formed by a kinase reaction that converts ADP to ATP, which is used to generate light in a luciferase reaction. The resulting luminescence is correlated with kinase activity.

[0076] In some embodiments, the NLRP3 variant retains at least about 25% of the ATPase activity relative to the wild-type NLRP3 of SEQ ID NO: 1. In some embodiments, the NLRP3 variant has more than 100% of the ATPase activity relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0077] In some embodiments, the NLRP3 variant retains at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ATPase activity relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0078] In some implementations, the NLRP3 variant retains the ability to form NLRP3 inflammasomes, relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0079] In some embodiments, the NLRP3 variant retains the ability to induce the release of IL-1β in cells, relative to the wild-type NLRP3 of SEQ ID NO: 1.

[0080] This disclosure also provides nucleic acids encoding the NLRP3 variants disclosed herein. In some embodiments, this disclosure provides a vector comprising a nucleic acid sequence encoding an NLRP3 variant. In some embodiments, this disclosure provides a host cell comprising a vector encoding an NLRP3 variant. In some embodiments, the host cell is an E. coli cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is an insect Sf21 cell.

[0081] Purification of NLRP3 variants

[0082] The present invention is characterized by a method for purifying the NLRP3 variant disclosed herein.

[0083] In one aspect, this disclosure provides a method for purifying the NLRP3 variant described herein, the method comprising: 1) introducing a vector encoding the NLRP3 variant into a host cell; 2) culturing the host cell under conditions that allow the NLRP3 variant to be expressed in the host cell; and 3) isolating the NLRP3 variant from the host cell.

[0084] In some embodiments, separation step 3) includes contacting the host cell lysate with one or more affinity purification resins. In other embodiments, the NLPR3 variant is secreted into the host cell culture medium. In such embodiments, the host cell culture medium containing the NLPR3 variant is contacted with one or more affinity purification resins.

[0085] In some embodiments, one or more affinity purification resins are selected from the group consisting of: immobilized metal affinity chromatography (IMAC) resins, maltose or amylose affinity chromatography resins, glutathione affinity chromatography resins, protein A affinity chromatography resins, and anti-FLAG affinity chromatography resins.

[0086] In some embodiments, the host cell used to express the NLRP3 variant for purification is an E. coli cell, yeast cell, insect cell, or mammalian cell. In some embodiments, the insect cell is an Sf21 cell.

[0087] NLRP3 variant fusion

[0088] NLRP3 variants can be linked to one or more non-NLRP3 peptide domains. These additional peptide domains confer non-native activities to the NLRP3 variants (i.e., activities not found in WT NLRP3).

[0089] In some embodiments, one or more polypeptide domains include a solubility-enhancing domain. As used herein, the term "solubility-enhancing domain" refers to a polypeptide that enhances the solubility of the protein to which it is attached.

[0090] In some embodiments, one or more polypeptide domains (e.g., solubility-enhancing domains) are selected from the group consisting of maltose-binding protein (MBP), glutathione S-transferase (GST), N-utilization substance A (NusA), and small ubiquitin-associated modifier (SUMO). Solubility-enhancing domains are further described in detail in Berneir et al., Protein Expr Purif. 2018. 152:92-106.

[0091] In some implementations, one or more polypeptide domains include an affinity purification domain.

[0092] In some embodiments, the affinity purification domains include a His domain, a 6xHis domain, biotin, streptavidin, glutathione S-transferase (GST), FLAG (DYKDDDDK; SEQ ID NO: 4), and an antibody Fc domain.

[0093] Identification of compounds that can bind to NLRP3

[0094] The NLRP3 variants disclosed herein can be used to identify compounds capable of binding to NLRP3. These binding compounds can be further tested to determine whether they are capable of inhibiting NLRP3. For example, but not limited to, inhibition of NLRP3 can be reflected in the ability to form NLRP3 inflammasomes or to trigger the release of IL-1β. Inhibition of NLRP3 inflammasome activity can be accomplished using any method known to those skilled in the art.

[0095] As used herein, the term "DNA-encoded chemical library" or "DEL" refers to a mixture of small molecules conjugated to unique polynucleotide sequence tags, where the structural information of each small molecule is encoded into its corresponding sequence. See (Brenner, S., Lerner, RA, Proc. Natl. Acad. Sci. , 1992, 89 (5381-5383), (Gartner, ZJ) Science , 2004, 305 (1601-1605), (Buller F. et al., Bioconjug. Chem. , 2010, 21 References 1571-1580, which are incorporated herein by reference. When DEL is applied to proteins of interest under selected conditions, the library compounds with the highest affinity are enriched after multiple washes, and DNA sequencing is used to elucidate their structural identity (Clark MA et al., Nat. Chem. Biol., 2009). 5 , 647-654).

[0096] In some embodiments, the NLRP3 variant described herein is contacted with a DNA-encoded chemical library (DEL) in a buffer. In some embodiments, the NLRP3 variant DEL library composition is applied to a solid support, such as Ni-banded magnetic beads. In some embodiments, the Ni-banded beads containing the bound NLRP3 variant and DEL library are washed once or multiple times, and subsequently the NLRP3 variant and the DNA-encoded library member (DEL) are released into the supernatant (Chen, Q. et al., SLASDiscov., 2020). 25(5) , 523-529).

[0097] In some embodiments, the resulting DEL library composition is identified. In some embodiments, identification is determined after PCR amplification and DNA sequencing.

[0098] Therefore, in one aspect, this disclosure provides a method for identifying compounds capable of binding to polypeptides containing nucleotide-binding oligomerization domains, leucine-rich repeat sequences, and thermoprotein domain 3 (NLRP3), the method comprising: 1) contacting a compound suspected of being capable of binding the NLRP3 polypeptide with an NLRP3 variant described herein, wherein the NLRP3 variant is immobilized on a surface; 2) washing the compound and the immobilized NLRP3 variant with a buffer solution; and 3) detecting the compound, wherein if the compound is detected, the compound is capable of binding the NLRP3 polypeptide.

[0099] In some implementations, the compound is linked to a polynucleotide sequence.

[0100] In some implementations, the detection of the compound includes the detection of a polynucleotide sequence.

[0101] In some implementations, detecting polynucleotide sequences involves sequencing the polynucleotide sequence.

[0102] In some implementations, the polynucleotide sequence contains at least 5 nucleotides.

[0103] In some embodiments, the length of the polynucleotide sequence is between 5 and 100 nucleotides (e.g., lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 4...). 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides). In some embodiments, the length of the polynucleotide sequence is between 5 and 50 nucleotides.

[0104] In some implementations, the polynucleotide sequence is single-stranded DNA (ssDNA).

[0105] In some embodiments, multiple compounds suspected of binding to the NLRP3 peptide are contacted with the NLRP3 variant described herein, each of the multiple compounds being linked to a unique polynucleotide sequence. Therefore, each of the multiple compounds can be identified by sequencing the unique polynucleotide sequence.

[0106] In some embodiments, the concentration of the NLRP3 variant is between 1 nM and 300 nM (e.g., 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, or 300 nM). In some embodiments, the concentration of the NLRP3 variant is between 25 nM and 250 nM.

[0107] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 5 mM to 500 mM Tris-HCl (e.g., 5 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM); 2) 15 to 1500 mM NaCl (e.g., 15 mM, 50 mM, 100 mM, 125 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 165 mM, 150 mM, 155 mM, 160 mM, 165 mM, 150 mM, 150 mM, 155 mM, 160 mM, 15 ... 3) 1 to 100 mM MgCl2 (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 4) 1% to 20% glycerol (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%); and 0.0005% to 0.05% Tween-20 (e.g., 0.0005%, 0.001%, 0.005%, 0.01% or 0.05%).

[0108] In some embodiments, the buffer also contains 1 to 500 mM imidazole (e.g., 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM or 500 mM).

[0109] In some embodiments, the buffer also contains 0.03 to 3 mg / mL of single-stranded DNA (ssDNA) (e.g., 0.03, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg / mL).

[0110] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 25-75 mM Tris-HCl; 2) 140-160 mM NaCl; 3) 5-15 mM MgCl2; 4) 5%-10% glycerol; and 5) 0.001%-0.01% Tween-20.

[0111] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 50 mM Tris-HCl; 2) 150 mM NaCl; 3) 10 mM MgCl2; 4) 10% glycerol; and 5) 0.005% Tween-20.

[0112] In some implementations, the buffer also contains: 1) 10 mM imidazole; and 2) 0.3 mg / mL ssDNA.

[0113] In some embodiments, contact step 1) includes incubating the compound with the NLRP3 variant and 0.01 mM to 50 mM ATP.

[0114] In some embodiments, contact step 1) includes incubating the compound with the NLRP3 variant and 0.1 mM to 1 mM ATP.

[0115] In some implementations, the NLRP3 variant is immobilized on a solid carrier.

[0116] In some embodiments, the solid support is conjugated with a ligand that binds to an NLRP3 variant. In some embodiments, the solid support comprises a nickel-immobilized resin. In some embodiments, the solid support comprises an immobilized metal affinity (IMAC) resin, a maltose or amylose affinity resin, a glutathione affinity resin, a protein A affinity resin, or an anti-FLAG affinity resin.

[0117] In some embodiments, the solid carrier comprises nickel-immobilized resin.

[0118] In some embodiments, the method further includes: 4) incubating a compound capable of binding the NLRP3 peptide with cells; and 5) measuring at least one pro-inflammatory cytokine secreted from the cells, wherein a decrease in the secretion of at least one pro-inflammatory cytokine from the cells indicates that the compound is an inhibitor of NLRP3.

[0119] In some implementations, at least one pro-inflammatory cytokine is IL-1β.

[0120] In some implementations, the cells are human mononuclear cells (THP-1 cells).

[0121] In some embodiments, the compound identified as an NLRP3 inhibitor is able to reduce IL-1β secretion by cells that come into contact with the compound identified as an NLRP3 inhibitor. In some embodiments, the cells are human monocytes THP-1 cells.

[0122] Identification of NLRP3 inhibitors

[0123] In one aspect, this disclosure provides a method for identifying an inhibitor containing a nucleotide-binding oligomerization domain, a leucine-rich repeat sequence, and a heat protein domain 3 (NLRP3), the method comprising: 1) contacting a compound suspected of being capable of binding an NLRP3 peptide with an NLRP3 variant described herein, wherein the NLRP3 variant is immobilized on a surface; 2) washing the compound and the immobilized NLRP3 variant with a buffer solution; 3) detecting the compound, wherein if the compound is detected, the compound is capable of binding an NLRP3 peptide; 4) incubating the NLRP3-binding compound from step 3) with cells; and 5) measuring at least one pro-inflammatory cytokine secreted from the cells, wherein a decrease in the secretion of at least one pro-inflammatory cytokine from the cells indicates that the compound is an inhibitor of NLRP3.

[0124] In some implementations, the compound is linked to a polynucleotide sequence.

[0125] In some implementations, the detection of the compound includes the detection of a polynucleotide sequence.

[0126] In some implementations, detecting polynucleotide sequences involves sequencing the polynucleotide sequence.

[0127] In some implementations, the polynucleotide sequence contains at least 5 nucleotides.

[0128] In some embodiments, the length of the polynucleotide sequence is between 5 and 100 nucleotides (e.g., lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 4...). 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides). In some embodiments, the length of the polynucleotide sequence is between 5 and 50 nucleotides.

[0129] In some implementations, the polynucleotide sequence is single-stranded DNA (ssDNA).

[0130] In some embodiments, multiple compounds suspected of binding to the NLRP3 peptide are contacted with the NLRP3 variant described herein, each of the multiple compounds being linked to a unique polynucleotide sequence. Therefore, each of the multiple compounds can be identified by sequencing the unique polynucleotide sequence.

[0131] In some embodiments, the concentration of the NLRP3 variant is between 1 nM and 300 nM (e.g., 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, or 300 nM). In some embodiments, the concentration of the NLRP3 variant is between 25 nM and 250 nM.

[0132] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 5 mM to 500 mM Tris-HCl (e.g., 5 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM); 2) 15 to 1500 mM NaCl (e.g., 15 mM, 50 mM, 100 mM, 125 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 165 mM, 150 mM, 155 mM, 160 mM, 165 mM, 150 mM, 150 mM, 155 mM, 160 mM, 15 ... 3) 1 to 100 mM MgCl2 (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 4) 1% to 20% glycerol (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%); and 0.0005% to 0.05% Tween-20 (e.g., 0.0005%, 0.001%, 0.005%, 0.01% or 0.05%).

[0133] In some embodiments, the buffer also contains 1 to 500 mM imidazole (e.g., 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM or 500 mM).

[0134] In some embodiments, the buffer also contains 0.03 to 3 mg / mL of single-stranded DNA (ssDNA) (e.g., 0.03, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg / mL).

[0135] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 25-75 mM Tris-HCl; 2) 140-160 mM NaCl; 3) 5-15 mM MgCl2; 4) 5%-10% glycerol; and 5) 0.001%-0.01% Tween-20.

[0136] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 50 mM Tris-HCl; 2) 150 mM NaCl; 3) 10 mM MgCl2; 4) 10% glycerol; and 5) 0.005% Tween-20.

[0137] In some implementations, the buffer also contains: 1) 10 mM imidazole; and 2) 0.3 mg / mL ssDNA.

[0138] In some embodiments, contact step 1) includes incubating the compound with the NLRP3 variant and 0.01 mM to 50 mM ATP.

[0139] In some embodiments, contact step 1) includes incubating the compound with the NLRP3 variant and 0.1 mM to 1 mM ATP.

[0140] In some implementations, the NLRP3 variant is immobilized on a solid carrier.

[0141] In some embodiments, the solid support is conjugated with a ligand that binds to an NLRP3 variant. In some embodiments, the solid support comprises a nickel-immobilized resin. In some embodiments, the solid support comprises an immobilized metal affinity (IMAC) resin, a maltose or amylose affinity resin, a glutathione affinity resin, a protein A affinity resin, or an anti-FLAG affinity resin.

[0142] In some embodiments, the solid carrier comprises nickel-immobilized resin.

[0143] In some implementations, at least one pro-inflammatory cytokine is IL-1β.

[0144] In some implementations, the cells are human mononuclear cells (THP-1 cells).

[0145] NLRP3 variant buffer

[0146] This application provides NLRP3 variant buffer compositions that enhance the stability of NLRP3 variants.

[0147] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 5 mM to 500 mM Tris-HCl (e.g., 5 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM); 2) 15 to 1500 mM NaCl (e.g., 15 mM, 50 mM, 100 mM, 125 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 165 mM, 150 mM, 155 mM, 160 mM, 165 mM, 150 mM, 150 mM, 155 mM, 160 mM, 15 ... 3) 1 to 100 mM MgCl2 (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 4) 1% to 20% glycerol (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%); and 0.0005% to 0.05% Tween-20 (e.g., 0.0005%, 0.001%, 0.005%, 0.01% or 0.05%).

[0148] In some embodiments, the buffer also contains 1 to 500 mM imidazole (e.g., 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM or 500 mM).

[0149] In some embodiments, the buffer also contains 0.03 to 3 mg / mL of single-stranded DNA (ssDNA) (e.g., 0.03, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg / mL).

[0150] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 25-75 mM Tris-HCl; 2) 140-160 mM NaCl; 3) 5-15 mM MgCl2; 4) 5%-10% glycerol; and 5) 0.001%-0.01% Tween-20.

[0151] In some embodiments, the NLRP3 variant is contained in a buffer solution comprising: 1) 50 mM Tris-HCl; 2) 150 mM NaCl; 3) 10 mM MgCl2; 4) 10% glycerol; and 5) 0.005% Tween-20.

[0152] In some implementations, the buffer also contains: 1) 10 mM imidazole; and 2) 0.3 mg / mL ssDNA.

[0153] Treatment

[0154] In one aspect, this document provides a method for inhibiting the NLRP3 inflammasome in a subject of need, the method comprising administering to the individual a therapeutically effective amount of a compound disclosed herein (i.e., an NLRP3 inhibitor identified in the screening method disclosed herein).

[0155] In another aspect, this document provides a method for treating inflammation in a subject in need, the method comprising administering to the individual a therapeutically effective amount of the compound disclosed herein.

[0156] In another aspect, this document provides a method for treating inflammation in a subject in need, the method comprising administering to the individual a therapeutically effective amount of the compound disclosed herein.

[0157] In another aspect, this article provides a method for treating cryptothermal protein-associated periodic syndrome (CAPS) in subjects of need, the method comprising administering to an individual a therapeutically effective amount of the compound disclosed herein.

[0158] In one implementation, CAPS is selected from the group consisting of: familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal episodic multisystem inflammatory disease.

[0159] In another aspect, this article provides a method for treating a skin condition in a subject in need, the method comprising administering to the individual a therapeutically effective amount of the compound disclosed herein.

[0160] In one implementation, the skin disease is selected from the group consisting of: psoriasis, urticaria, photoaging of the skin, and eczema.

[0161] In another aspect, this article provides a method for treating sensory nerve disorders in subjects in need, the method comprising administering to an individual a therapeutically effective amount of the compound disclosed herein.

[0162] In one implementation, sensory neurological diseases are selected from the group consisting of: amyotrophic lateral sclerosis (ALS), traumatic brain injury, Parkinson's disease, and Alzheimer's disease.

[0163] This article also provides a method for treating or improving aging-related disorders that negatively impact lifespan or quality of life using the compounds provided herein, wherein the aging-related disorders negatively impacting lifespan or quality of life are selected from the group consisting of: inflammation, anemia, hyperglycemia, dyslipidemia, hyperinsulinemia, insulin resistance, immunosuppression, liver disease, iron overload, hypertriglyceridemia, impaired skin integrity, wound healing, scarring, pain, allergies, sleep disorders and problems, gastrointestinal disorders and problems, Th1 inflammation, Th2 inflammation, inflammatory diseases involving T-cell-dependent B-cell proliferation, T-cell-dependent B-cell proliferation, allergies, asthma, atherosclerosis, autoimmune diseases, and high blood pressure. Hypercholesterolemia, chronic inflammation, chronic obstructive pulmonary disease (COPD), Crohn's disease, skin response to tissue damage, fibrosis, hematologic oncology, metabolic diseases, cardiovascular diseases, organ transplantation, psoriasis, liver fibrosis, dermatitis, pulmonary fibrosis, lung response to respiratory infections, restenosis, rheumatoid arthritis, sarcoidosis, tumor matrix biology, systemic lupus erythematosus (SLE), ulcerative colitis, vasculitis, and diseases driven or exacerbated by one or more factors selected from the group consisting of: α-smooth muscle actin (αSMA), CD40, CD69, collagen I, collagen III, core proteoglycan, e-selectin, eosinophil activating chemokine 3. (CCL26), fibroblast proliferation, human leukocyte antigen-DR isotype (HLA-DR), immunoglobulin G, interferon-γ inducible protein 10 (IP-10 / CXCL10), interferon-induced T cell α chemokine (I-TAC / CXCL11), interleukin (IL)-1, IL-1α, IL-2, IL-6, IL-8 (CXCL8), IL-10, IL-17A, IL-17F, keratin 8 / 81, macrophage colony-stimulating factor (M-CSF), matrix metalloproteinase (MMP)-1, MMP-9, monocyte chemoattractant protein 1 (MCP-1), interferon-γ induced mononuclear factor (MIG / CXCL9), plasminogen activator inhibitor 1 The method comprises administering the compounds provided herein to a subject in need of treatment with (PAI-1), prostaglandin E2 (PGE2), serum amyloid A, T or B cell proliferation, tissue plasminogen activator (tPA), tumor necrosis factor α (TNF.α.), vascular cell adhesion molecule (VCAM-1), and vascular endothelial growth factor 2 (VEGFR2).

[0164] In one aspect, this article provides a method for reversing the natural aging process of a subject, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

[0165] In another aspect, this article provides a method for reversing the natural aging process of a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

[0166] In another aspect, this article provides a method for prolonging the life of a subject, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

[0167] In another aspect, this article provides a method for prolonging the life of a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

[0168] In another aspect, this article provides a method for slowing down and mitigating the aging process in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

[0169] In another aspect, this document provides a method for inhibiting or modulating pro-inflammatory pathways in cells, the method comprising contacting the cells with a compound provided herein or a pharmaceutically acceptable salt thereof. In yet another aspect, this document provides a method for inhibiting or modulating NLRP3 in cells, the method comprising contacting the cells with a compound provided herein or a pharmaceutically acceptable salt thereof.

[0170] Treatment of cells expressing the NLRP3 inflammasome (in vitro or in vivo) with the compounds provided in this article can inhibit pro-inflammatory pathways and suppress downstream events related to signaling pathways, such as inflammation or inflammatory senescence.

[0171] In another aspect, this article provides a method for treating sensory nerve disorders in subjects in need, the method comprising administering to an individual a therapeutically effective amount of the compound disclosed herein.

[0172] In one implementation, sensory nerve diseases are selected from the group consisting of: hearing loss, hearing impairment, and eye diseases. In one implementation, eye diseases are retinal and optic nerve damage.

[0173] In another aspect, this article provides a method for treating inflammatory conditions in a subject in need, the method comprising administering to the individual a therapeutically effective amount of the compound disclosed herein.

[0174] In one implementation scheme, inflammatory conditions are selected from the group consisting of: allergies, asthma, atopic dermatitis, atherosclerosis, autoimmune diseases, celiac disease, chronic inflammation, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, SARS-CoV-2 infection, transplant rejection, heart disease, diabetes, arthritis, Crohn's disease, ulcerative colitis, non-alcoholic steatohepatitis (NASH), gout, coronary artery disease, rheumatoid arthritis, intestinal diseases, and acute respiratory distress syndrome (ARDS).

[0175] In another implementation, the inflammatory condition is a neuroinflammatory disease. In yet another implementation, the inflammatory condition is inner ear inflammation.

[0176] In one implementation, chronic inflammation includes tissue inflammation. Tissue inflammation is chronic inflammation limited to a specific tissue or organ. In one implementation, tissue inflammation includes, for example, inflammation of the skin, eyes, muscles, tendons, ligaments, bones, cartilage, lungs, heart, liver, pancreas, kidneys, bladder, stomach, intestines, neurons, and brain.

[0177] In another implementation, chronic inflammation includes systemic inflammation. While the processes involved are the same as in tissue inflammation, systemic inflammation is not limited to a specific tissue but rather damages the entire body, involving the endothelium and other organ systems. The term sepsis is appropriate when it is attributed to infection; bacteremia is appropriate for bacterial sepsis, and viremia is appropriate for viral sepsis. Vasodilatory dysfunction and organ dysfunction are serious problems associated with widespread infection and can lead to septic shock and death.

[0178] In yet another implementation, chronic inflammation includes arthritis. Arthritis encompasses a group of conditions involving damage to joints in the body due to inflammation of the synovium, including but not limited to osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis-like ankylosing spondylitis, reactive arthritis (Reiter's syndrome), psoriatic arthritis, enteropathic arthritis associated with inflammatory bowel disease, Whipple's disease, and Behcet's disease, septic arthritis, gout (also known as gouty arthritis, crystalline synovitis, or metabolic arthritis), pseudogout (calcium pyrophosphate deposition disease), and Still's disease. Arthritis can affect a single joint (monoarthritis), two to four joints (oligoarthritis), or five or more joints (polyarthritis) and can be an autoimmune or non-autoimmune disease.

[0179] In another aspect, this article provides a method for treating age-related conditions in subjects in need, the method comprising administering to an individual a therapeutically effective amount of the compound disclosed herein.

[0180] In one implementation plan, age-related conditions are selected from the group consisting of: neurodegeneration, cardiovascular disease, insulin resistance, diabetes, osteoporosis, osteoarthritis, cognitive decline, dementia, fragility, cataracts, arthritis, obesity, hypertension, angina pectoris, congestive heart failure, dyslipidemia, myocardial infarction, vascular disease, respiratory disease, kidney disease, cerebrovascular disease, peripheral vascular disease, Alzheimer's disease, diastolic dysfunction, benign prostatic hyperplasia, aortic aneurysm, and emphysema.

[0181] In another aspect, this document provides a method for treating metabolic disorders in subjects of need, the method comprising administering to an individual a therapeutically effective amount of the compound disclosed herein.

[0182] In one implementation scheme, metabolic disorders are selected from the group consisting of: diabetes, obesity, cystic fibrosis, and hyperthyroidism.

[0183] In another aspect, this document provides a method for treating neurodegenerative diseases in a subject of need, the method comprising administering to the individual a therapeutically effective amount of the compound disclosed herein.

[0184] In one implementation scheme, neurodegenerative diseases are selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Batten disease.

[0185] In one aspect, this article provides a method for treating an inner ear disease or condition in a subject of need, the method comprising administering to the individual a therapeutically effective amount of the compound disclosed herein.

[0186] In one implementation, the inner ear disease or condition is selected from the group consisting of: hearing loss, hearing impairment, vertigo, Meniere's disease, and tinnitus. In another implementation, the inner ear disease is hearing loss. In yet another implementation, the inner ear disease is hearing impairment.

[0187] In another implementation, the hearing loss is age-related, noise-induced, or a result of a viral infection. In yet another implementation, the viral infection is Zika virus or coronavirus.

[0188] It can also be done through IC 50The value determines the potency of the inhibitor. This is relative to an inhibitor with a higher IC50 value. 50 Compounds with lower IC50 values, as determined under substantially similar conditions, are considered to have lower IC50 values. 50 The compounds with the highest value are more effective inhibitors.

[0189] In one embodiment of the method, the subject is a human being.

[0190] In another aspect, this disclosure provides a compound disclosed herein or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for treating or preventing diseases in which the NLRP3 inflammasome plays a role.

[0191] In one aspect, this article provides a method for treating diseases selected from the group consisting of: autoimmune diseases, inflammatory diseases, proliferative and hyperproliferative diseases, immune-mediated diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, hormone-related diseases, allergies, asthma, and Alzheimer's disease. In other embodiments, the diseases are selected from proliferative disorders and neurodegenerative disorders.

[0192] One aspect of this disclosure provides compounds that can be used to treat diseases, conditions, and disorders characterized by excessive or abnormal cell proliferation. Such diseases include, but are not limited to, proliferative or hyperproliferative disorders and neurodegenerative diseases. Examples of proliferative and hyperproliferative disorders include, but are not limited to, cancer.

[0193] Therefore, in one aspect, this document provides a method for treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the compound disclosed herein or a pharmaceutically acceptable salt thereof.

[0194] In one implementation scheme, cancers are selected from the group consisting of: breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, urogenital tract cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma, bone cancer, colon cancer, colorectal cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract cancer, kidney cancer, myeloid diseases, lymphoid diseases, Hodgkin's disease, pilomatric carcinoma, buccal and pharyngeal (oral) cancer, lip cancer, tongue cancer, oral cancer, pharyngeal cancer, small bowel cancer, colon cancer, rectal cancer, colorectal cancer, rectal cancer, brain cancer and central nervous system cancer, chronic myeloid leukemia (CML) and leukemia.

[0195] In another implementation, the cancer is selected from the group consisting of myeloma, lymphoma, or selected from the group consisting of: gastric cancer, kidney cancer, head and neck cancer, oropharyngeal cancer, non-small cell lung cancer (NSCLC), endometrial cancer, liver cancer, non-Hodgkin's lymphoma, and lung cancer.

[0196] In one implementation scheme, the cancer is selected from the group consisting of: prostate cancer, colon cancer, lung cancer, head and neck squamous cell carcinoma, esophageal cancer, hepatocellular carcinoma, melanoma, sarcoma, gastric cancer, pancreatic cancer, ovarian cancer, and breast cancer.

[0197] In one implementation, cancer is selected from the group consisting of: tumors, cysts, carcinomas, sarcomas, leukemias, lymphomas, etc. For example, cancer includes, but is not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, lymphomas associated with human T-cell lymphotropic virus (HTLV) such as adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, lymphoma and multiple myeloma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Other examples include myelodysplastic syndromes, childhood solid tumors such as brain tumors, neuroblastomas, retinoblastomas, Wilms' tumors, bone tumors and soft tissue sarcomas, common adult solid tumors such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal, and esophageal cancers), genitourinary cancers (e.g., prostate, bladder, kidney, uterine, ovarian, and testicular cancers), lung cancers (e.g., small cell and non-small cell carcinomas), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors associated with Gorlin syndrome (e.g., medulloblastomas, meningiomas, etc.), and liver cancer. Other exemplary forms of cancer that can be treated with the compounds of this invention include, but are not limited to, bone or smooth muscle cancers, stomach cancer, small bowel cancer, rectal cancer, salivary gland cancer, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

[0198] Other cancers for which the compounds described herein can be used to treat include, for example, colon cancer, familial adenomatous polyposis carcinoma, and hereditary nonpolyposis colorectal cancer or melanoma. Additionally, cancers include, but are not limited to, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), kidney cancer, renal parenchymal carcinoma, cervical cancer, uterine cancer, endometrial cancer, choriocarcinoma, testicular cancer, urethral cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, gallbladder cancer, bronchial cancer, multiple myeloma, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma.

[0199] In another aspect, this document provides for the use of one or more compounds of this disclosure in the manufacture of medicaments for treating cancer (including, but not limited to, the various types of cancer disclosed herein).

[0200] In some embodiments, the compounds of this disclosure can be used to treat cancers such as colorectal cancer, thyroid cancer, breast cancer, and lung cancer; and myeloproliferative disorders such as polycythemia vera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronic myeloid leukemia, chronic myelomonocytic leukemia, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mast cell disease. In some embodiments, the compounds of this disclosure can be used to treat hematopoietic disorders, particularly acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute promyelocytic leukemia, and acute lymphoblastic leukemia (ALL).

[0201] Serial ID number:

[0202] Example

[0203] The compounds and methods disclosed herein are further illustrated by the following examples, which should not be considered as further limitations. Unless otherwise indicated, the practice of this disclosure will utilize conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology, which are within the scope of the art.

[0204] To facilitate the discovery of DEL using NLRP3, a combinatorial screening library array containing >500 billion molecules was used. To further enhance this discovery, specific selections in the DEL library screening conditions enhanced the stability of NLRP3, preserving ATPase activity during DEL screening, and several NLRP3 site-blocking agents were employed to improve ligand analysis.

[0205] The following examples further illustrate various aspects of this disclosure. However, they in no way constitute a limitation on the teachings of this disclosure.

[0206] Example 1: Design and optimization of screening constructs

[0207] DEL screening used a protein construct called MBP-ΔNLRP3-HIS, which contains the human NLRP3 protein sequence lacking the N-terminal PYD domain. This protein also contains an N-terminal maltose-binding protein (MBP) domain and a C-terminal HIS tag consisting of a tandem repeat of six histidine residues to facilitate protein purification and binding to nickel-bearing magnetic beads, respectively. MBP-ΔNLRP3-HIS was purified from Sf21 (IPLB-Sf21-AE) insect cells and produced in a two-step purification process.

[0208] Peptide mass fingerprinting confirmed the protein identity, while integrity analysis confirmed the overall integrity of the protein and indicated the loss of the N-terminal methionine. The theoretical molecular weight and isoelectric point (pI) of MBP-NLRP3-HIS are 145.30 kDa and 6.2, respectively. After one freeze-thaw cycle, the protein showed a high-purity band eluted as a monodisperse sample in size exclusion chromatography (SEC). The functionality of the MBP-ΔNLRP3-HIS protein was evaluated based on ATPase activity using the ADP-GLO kinase assay (Promega). Based on these studies, the Km of ATP with respect to MBP-ΔNLRP3-HIS was determined to be 3.076 µM, and the MBP-ΔNLRP3-HIS protein showed sufficient activity in the ADP-GLO assay and DEL screening selection buffer. This protein construct demonstrated acceptable stability for up to 2 hours under simulated DEL screening conditions, as indicated by its ATPase activity. The intact structure of MBP-ΔNLRP3-HIS is shown in [the original text]. Figure 1 The information is provided in the text.

[0209] Example 2: DEL Filter Settings

[0210] DEL filter settings: DEL screening was organized into six separate screening tests identified in Table 1, with all DEL libraries screened in each test. The discovery that the presence of ATP led to the unusually high binding of NLRP3 to MCC950 (Coll, RC et al.) Nat.Chem Biol. , 2019, 15(6) , 556-559), ATP was included in experiments 2-5. In addition, MCC950, CY-09 and tranilast were used as blockers in separate screening assays to establish an unbiased screening method and to better understand the specific NLRP3 binding sites and mechanisms of action exhibited by various ligands (Jiang, H. et al., J.Exp.Med., 2017, 214(11), 3219-3238), (Huang, Y. et al., EMBO Mol. Med, 2018, 10(4), 8689). As standard screening practice, the MBP-ΔNLRP3-HIS protein construct was used in large molar overdoses exceeding the expected fM concentration of the individual screening compounds, and all three blockers were used at high uM levels to achieve maximum inhibition.

[0211] Table 1 - Examples of DEL screening for experiments using blocking ligands.

[0212]

[0213] Building a filter library: DEL screening employed a HitGen library containing over 500 billion compounds with diverse structures. The resulting screening data were analyzed to provide sequence counts as the number of specific DNA barcodes observed in sequencing results and characteristic strengths as calculated and normalized results of enrichment for any given compound series. In experiments 2–5, the arrays and distributions of bound sequence counts were very similar, indicating that none of the three blocking ligands significantly altered the binding of the novel ligand to NLRP3. Furthermore, the most favored screening library ligands were charge-neutral compared to the anionic NLRP3 inhibitors MCC950, CY-09, and tranilast used as ligand blockers. The most interesting and relevant background of the above bindings is demonstrated by compounds from a library of 98.8 million compounds, generated using a three-component synthetic scheme employing 521 aldehydes, 354 carboxylic acids, and 528 borates, as shown in the image. Figure 2 As shown.

[0214] Example 3: Ligands identified in DEL screening

[0215] This library provides a novel set of high-affinity neutral NLRP3 ligands that exhibit an indazole scaffold functionalized at C-7 via a 3-atom carbonylaminomethyl linker attached to the biphenyl group, as shown in General Structure 1. Figure 3 Typically, several substituents, namely R1, R2, R3, and R4, are present in favorable compounds that bind to NLRP3, and it is evident that the appropriate meta- or para-positioning of these substituents on the biphenyl unit is crucial for optimal binding. Specifically, DEL-derived SAR of the highest affinity compounds shows that R1 is an alkyl ether, and R2, R3, and R4 are typically substituents selected from amino, cyano, trifluoromethyl, methylaminocarboxyl, and fluorine. Notably, various shorter and longer 3-atom linker sequences are incorporated into the library; however, the resulting compounds consistently lack good binding potential.

[0216] Example 4. Novel Indazole Compounds

[0217] Synthesis of novel indazole compounds: To track the initial DEL screening, examples of these novel indazole compounds were directly prepared "de-DNA," and the functional activity of the resulting compounds was determined by measuring their ability to inhibit IL-1β secretion in THP-1 cells. The synthesis of these novel indazole analogs was similar to the DEL program and involved the reductive amination of 7-indazoleformaldehyde 1a to yield N-methyl derivative 1b. Figure 4 The secondary amine was acylated with a suitably derived acid 1c, and the resulting amide 1d was converted to biphenyl analog 1 by using a functionalized boric acid 1e in a Suzuki coupling.

[0218] Testing the potency of compounds in human cells: The efficacy of compounds in inhibiting NLRP3 activation was measured in the human monocyte THP-1 cell line, which expresses high levels of NLRP3 inflammasome components and readily forms fully active NLRP3 inflammasome complexes under standard cell culture conditions. Pretreatment of suspended THP-1 cells with phorbol 12-myristate 13-acetate (PMA) induced their adhesion and partial differentiation into macrophage-like cells. Subsequent lipopolysaccharide (LPS) treatment acted as “signal 1” to induce robust upregulation of pro-IL-1β, while further addition of nigericin (a bacterial ionocarrier) acted as “signal 2” to induce NLRP3 inflammasome formation and autolytic cleavage of caspase-1 to produce mature IL-1β. Under these conditions, the ability of compounds to inhibit NLRP3 activation was screened by measuring the level of mature IL-1β in the cell culture supernatant by ELISA (Zito, G. et al., Int. J. Mol. Sci., 2020, 21(12), 4294-4313).

[0219] Indazole compounds 2-8 (Table 2) were identified as high-affinity binders from the DEL screening and were the initial compounds prepared for evaluation in the THP-1 NLRP3 inflammasome activation assay. Advantageously, the phenyl analog (2), as well as the 3-fluoro-(3), 3-trifluoromethyl-(4), and 3-cyanophenyl-(5) analogs, all showed IC50 values ​​<100 nM for inhibiting IL-1β secretion. Despite their good binding properties in the DEL screening, analogs 6, 7, and 8 were inactive at 20 μM in the THP-1 assay. Furthermore, several alternatively functionalized terminal benzene ring analogs were prepared, and the lack of activity of those compounds in the THP-1 assay closely mirrored the lack of effective binding observed in the DEL screening.

[0220] Table 2 - SAR of indazole analogs at the R2 position. Compound potency (IC50) was determined by quantifying the decrease in IL-1β supernatant levels at various compound concentrations using a THP-1 NLRP3 inflammasome activation assay.

[0221]

[0222] The aforementioned cell-active indazole inhibitors are unprecedented in the NLRP3 literature, and their neutral charge characteristics suggest good potential for both oral bioavailability and CNS penetration.

[0223] Example 5. Buffer for improving the activity of NLRP3 variants

[0224] The above DEL screening was performed using a buffer optimized for the activity and stability of the NLRP3 variant. For example... Figure 5 As shown, the ATPase activity of the NLRP3 variant was measured in the ADP-Glo ​​activity assay using both the optimized buffer of this application and the literature buffer. The buffer used in this application consisted of 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 10% glycerol, and 0.005% Tween-20, pH 7.5. The literature buffer consisted of 20 mM Tris-HCl, 133 mM NaCl, 20 mM MgCl2, 0.56 mM EDTA, and 3 mM KCl, pH 7.8. Each buffer also contained 10 μM ATP and NLRP3 in the range of 0 nM to 250 nM.

[0225] Data showed that the optimized buffer produced an NLRP3 variant with increased ATPase activity.

[0226] Next, the inclusion of imidazole and ssDNA (which is part of the DEL screening) was tested. It was tested with 0.3 mg / mL ssDNA, 10 mM imidazole, or both. Figure 5 The assay buffer (the buffer of this application) was used. The test buffer also contained 3 µM ATP. Data showed that neither ssDNA nor imidazole had a negative impact on the activity of the NLRP3 variant. Figure 6 ).

Claims

1. A method for identifying compounds capable of binding to polypeptides containing nucleotide-binding oligomerization domains, leucine-rich repeat sequences, and thermoprotein domain 3 (NLRP3), the method comprising: 1) Contact a compound suspected of being able to bind an NLRP3 peptide with an NLRP3 variant, wherein the NLRP3 variant is immobilized on a surface; 2) Wash the compound and the immobilized NLRP3 variant with buffer solution; as well as 3) Detect the compound. If the compound is detected, it is capable of binding to the NLRP3 peptide. The NLRP3 variant contains at least a partial deletion of the amino-terminal thermoprotein domain (PYD).

2. The method of claim 1, wherein the compound is linked to a polynucleotide sequence.

3. The method of claim 2, wherein the detection of the compound comprises detecting the polynucleotide sequence.

4. The method of claim 3, wherein detecting the polynucleotide sequence comprises sequencing the polynucleotide sequence.

5. The method of any one of claims 2-4, wherein the polynucleotide sequence comprises at least 5 nucleotides.

6. The method of any one of claims 2-5, wherein the length of the polynucleotide sequence is between 5 and 50 nucleotides.

7. The method of any one of claims 2-6, wherein the polynucleotide sequence is single-stranded DNA (ssDNA).

8. The method of any one of claims 1-7, wherein the concentration of the NLRP3 variant is between 1 nM and 300 nM.

9. The method of any one of claims 1-8, wherein the concentration of the NLRP3 variant is between 25 nM and 250 nM.

10. The method of any one of claims 1-9, wherein the NLRP3 variant is contained in a buffer solution, the buffer solution comprising: 1) 5 mM to 500 mM Tris-HCl; 2) 15 to 1500 mM NaCl; 3) 1 to 100 mM MgCl2; 4) 1% to 20% glycerin; and 5) 0.0005% to 0.05% Tween-20.

11. The method of claim 10, wherein the buffer solution further comprises: 1) 1 to 500 mM imidazole; and 2) 0.03 to 3 mg / mL single-stranded DNA (ssDNA).

12. The method of any one of claims 1-11, wherein the NLRP3 variant is contained in a buffer solution, the buffer solution comprising: 1) 50 mM Tris-HCl; 2) 150 mM NaCl; 3) 10 mM MgCl2; 4) 10% glycerin; and 5) 0.005% Tween-20.

13. The method of claim 12, wherein the buffer solution further comprises: 1) 10 mM imidazole; and 2) 0.3 mg / mL ssDNA.

14. The method of any one of claims 1-13, wherein the contact step 1) comprises incubating the compound with the NLRP3 variant and 0.01 mM to 50 mM ATP.

15. The method of any one of claims 1-14, wherein the contact step 1) comprises incubating the compound with the NLRP3 variant and 0.1 mM to 1 mM ATP.

16. The method of any one of claims 1-15, wherein the NLRP3 variant is fixed on a solid carrier.

17. The method of claim 16, wherein the solid carrier comprises a nickel-immobilized resin.

18. The method of any one of claims 1-17, further comprising: 4) Incubate the cells with the compound that can bind the NLRP3 peptide; as well as 5) Measure at least one pro-inflammatory cytokine secreted from said cells. The reduced secretion of at least one pro-inflammatory cytokine from the cells indicates that the compound is an inhibitor of NLRP3.

19. The method of claim 18, wherein the at least one pro-inflammatory cytokine is IL-1β.

20. The method of claim 18 or 19, wherein the cell is a human mononuclear cell THP-1 cell.

21. A method for identifying inhibitors containing a nucleotide-binding oligomerization domain, a leucine-rich repeat sequence, and a heat protein domain 3 (NLRP3), the method comprising: 1) Contact a compound suspected of being able to bind an NLRP3 peptide with an NLRP3 variant, wherein the NLRP3 variant is immobilized on a surface; 2) Wash the compound and the immobilized NLRP3 variant with buffer solution; 3) Detect the compound, wherein if the compound is detected, the compound is capable of binding the NLRP3 peptide; 4) Incubate the cells with the compound from step 3) that is capable of binding the NLRP3 peptide; as well as 5) Measure at least one pro-inflammatory cytokine secreted from said cells. The reduced secretion of at least one pro-inflammatory cytokine from the cells indicates that the compound is an inhibitor of NLRP3. The NLRP3 variant contains at least a partial deletion of the amino-terminal thermoprotein domain (PYD).

22. The method of claim 21, wherein the at least one pro-inflammatory cytokine is IL-1β.

23. The method of claim 21 or 22, wherein the cell is a human mononuclear cell THP-1 cell.

24. The method of any one of claims 1-23, wherein the NLRP3 variant comprises the deletion of amino acids 1-142 of the amino acid sequence of SEQ ID NO:

1.

25. The method of any one of claims 1-24, wherein the NLRP3 variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

2.

26. The method of any one of claims 1-25, wherein the NLRP3 variant is linked to one or more polypeptide domains.

27. The method of claim 26, wherein the one or more polypeptide domains comprise a solubility-enhancing domain.

28. The method of claim 26 or 27, wherein the one or more polypeptide domains are selected from the group consisting of maltose-binding protein (MBP), glutathione S-transferase (GST), N-utilization substance A (NusA), and small ubiquitin-associated modifier (SUMO).

29. The method of claim 26, wherein the one or more polypeptide domains comprise an affinity purification domain.

30. The method of claim 29, wherein the affinity purification domain comprises a His domain, a 6xHis domain, biotin, streptavidin, glutathione S-transferase (GST), FLAG (DYKDDDDK; SEQ ID NO: 4), and an antibody Fc domain.

31. The method according to any one of claims 1-30, wherein the method comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

3.

32. The method of any one of claims 1-31, wherein the NLRP3 variant retains ATPase activity relative to the wild-type NLRP3 of SEQ ID NO:

1.

33. The method of any one of claims 1-32, wherein the NLRP3 variant retains at least about 25% of the ATPase activity relative to the wild-type NLRP3 of SEQ ID NO:

1.

34. The method of any one of claims 1-33, wherein the NLRP3 variant retains the ability to form NLRP3 inflammasomes relative to the ability of wild-type NLRP3 in SEQ ID NO:

1.

35. The method of any one of claims 1-34, wherein the NLRP3 variant retains the ability to induce the release of IL-1β in cells relative to the ability of wild-type NLRP3 of SEQ ID NO:

1.

36. A method for treating an inflammatory condition, the method comprising administering to a subject an NLRP3 inhibitor identified in any one of claims 1-35.

37. A composition comprising: 1) NLRP3 variant; 2) 5 mM to 500 mM Tris-HCl; 3) 15 to 1500 mM NaCl; 4) 1 to 100 mM MgCl2; 5) 1% to 20% glycerin; and 6) 0.0005% to 0.05% Tween-20, The NLRP3 variant contains at least a partial deletion of the amino-terminal thermoprotein domain (PYD).

38. The composition of claim 37, wherein the composition further comprises: 1) 1 to 500 mM imidazole; and 2) 0.03 to 3 mg / mL single-stranded DNA (ssDNA).

39. The composition of claim 37 or 38, wherein the composition comprises: 1) The NLRP3 variant; 2) 50 mM Tris-HCl; 3) 150 mM NaCl; 4) 10 mM MgCl2; 5) 10% glycerin; and 6) 0.005% Tween-20.

40. The composition of claim 39, wherein the buffer solution further comprises: 1) 10 mM imidazole; and 2) 0.3 mg / mL ssDNA.

41. The composition of any one of claims 37-40, wherein the composition further comprises 0.01 mM to 50 mM of ATP.

42. A variant containing a nucleotide-binding oligomerization domain, a leucine-rich repeat sequence, and a thermoprotein domain 3 (NLRP3) variant, comprising at least a partial deletion of the amino-terminal thermoprotein domain (PYD).

43. The NLRP3 variant of claim 42, comprising the deletion of amino acids 1-142 of the amino acid sequence of SEQ ID NO:

1.

44. The NLRP3 variant of claim 42 or 43, comprising an amino acid sequence having at least 80% identity with SEQ ID NO:

2.

45. The NLRP3 variant of any one of claims 42-44, wherein the NLRP3 variant is linked to one or more polypeptide domains.

46. ​​The NLRP3 variant of claim 46, wherein one or more polypeptide domains comprise a solubility-enhancing domain.

47. The NLRP3 variant of claim 45 or 46, wherein the one or more polypeptide domains are selected from the group consisting of: maltose-binding protein (MBP), glutathione S-transferase (GST), N-utilization substance A (NusA), and small ubiquitin-associated modifier (SUMO).

48. The NLRP3 variant of claim 45, wherein one or more polypeptide domains comprise an affinity purification domain.

49. The NLRP3 variant of claim 48, wherein the affinity purification domain comprises a His domain, a 6xHis domain, biotin, streptavidin, glutathione S-transferase (GST), FLAG (DYKDDDDK; SEQ ID NO: 4), and an antibody Fc domain.

50. The NLRP3 variant of any one of claims 42-49, comprising an amino acid sequence having at least 80% identity with SEQ ID NO:

3.

51. The NLRP3 variant according to any one of claims 42-50, wherein the NLRP3 variant retains ATPase activity relative to the wild-type NLRP3 of SEQ ID NO:

1.

52. The NLRP3 variant according to any one of claims 42-51, wherein the NLRP3 variant retains at least about 25% of the ATPase activity relative to the wild-type NLRP3 of SEQ ID NO:

1.

53. The NLRP3 variant of any one of claims 42-52, wherein the NLRP3 variant retains the ability to form NLRP3 inflammasomes relative to the ability of wild-type NLRP3 of SEQ ID NO:

1.

54. The NLRP3 variant of any one of claims 42-53, wherein, relative to the ability of the wild-type NLRP3 of SEQ ID NO: 1, the NLRP3 variant retains the ability to induce the release of IL-1β in cells.

55. A nucleic acid encoding a variant of NLRP3 as described in any one of claims 42-54.

56. A vector comprising the nucleic acid of claim 55.

57. A host cell comprising the vector of claim 56.

58. The host cell of claim 57, wherein the host cell is an Escherichia coli cell, a yeast cell, an insect cell, or a mammalian cell.

59. The host cell of claim 58, wherein the insect cell is an Sf21 cell.

60. A method for purifying the NLRP3 variant according to any one of claims 42-54, the method comprising: 1) Introduce the vector encoding the NLRP3 variant into a host cell; 2) The host cells are cultured under conditions that allow the NLRP3 variant to be expressed in the host cells; as well as 3) Isolate the NLRP3 variant from the host cell.

61. The method of claim 60, wherein the separation step 3) comprises contacting the lysate of the host cell with one or more affinity purification resins.

62. The method of claim 61, wherein the one or more affinity purification resins are selected from the group consisting of: immobilized metal affinity chromatography (IMAC) resins, maltose or amylose affinity chromatography resins, glutathione affinity chromatography resins, protein A affinity chromatography resins, and anti-FLAG affinity chromatography resins.

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