Discovery of a novel pyroptosis inhibitor acting though modulating glutathionylation to suppress NLRP3-related signal pathway.

Pyroptosis is a proinflammatory type of regulated cell death and has been involved in many pathological processes. Inhibition of pyroptosis is thought to be a promising strategy for the treatment of related diseases. Here, we performed a phenotypic screening against NLRP3-dependent pyroptosis and obtained the novel compound N77 after structure optimization. N77 showed a half-maximal effective concentration (EC50) of 0.070 ± 0.008 µM against cell pyroptosis induced by nigericin, and exhibited a remarkable ability to prevent NLRP3-dependent inflammasome activation and the release of IL-1ß. Chemical proteomics revealed the biological target of N77 to be glutathione-S-transferase Mu 1 (GSTM1); our mechanism of action studies indicated that GSTM1 might act as a negative regulator of NLRP3 inflammasome activation by modulating the glutathionylation of caspase-1. In vivo, N77 substantially alleviated the inflammatory reaction in a pyroptosis-related acute keratitis model. Overall, we identified a novel pyroptosis inhibitor and revealed a new regulatory mechanism of pyroptosis. Our findings suggest an alternative potential therapeutic strategy for pyroptosis-related diseases.

Discovery of (S)-3-(4-(benzyloxy)phenyl)-2-(2-phenoxyacetamido)propanoic acid derivatives as a new class of GPR34 antagonists.

GPR34 is a rhodopsin-like class G protein-coupled receptor (GPCR) that is involved in the development and progression of several diseases. Despite its importance, effective targeting strategies are lacking. We herein report a series of (S)-3-(4-(benzyloxy)phenyl)-2-(2-phenoxyacetamido)propanoic acid derivatives as a new class of GPR34 antagonists. Structure-activity relationship (SAR) studies led to the identification of the most potent compound, 5e, which displayed an IC50 value of 0.680 µM in the GloSensor cAMP assay and 0.059 µM in the Tango assay. 5e demonstrated low cytotoxicity and high selectivity in vitro, and it was able to dose-dependently inhibit Lysophosphatidylserine-induced ERK1/2 phosphorylation in CHO cells expressing GPR34. Furthermore, 5e displayed excellent efficacy in a mouse model of neuropathic pain without any apparent signs of toxicity. Collectively, this study has identified a promising compound, which shows great potential in the development of potent antagonists with a new chemical scaffold targeting GPR34.

A new generation Mpro inhibitor with potent activity against SARS-CoV-2 Omicron variants.

Emerging SARS-CoV-2 variants, particularly the Omicron variant and its sublineages, continually threaten the global public health. Small molecule antivirals are an effective treatment strategy to fight against the virus. However, the first-generation antivirals either show limited clinical efficacy and/or have some defects in pharmacokinetic (PK) properties. Moreover, with increased use of these drugs across the globe, they face great pressure of drug resistance. We herein present the discovery and characterization of a new generation antiviral drug candidate (SY110), which is a potent and selective inhibitor of SARS-CoV-2 main protease (Mpro). This compound displayed potent in vitro antiviral activity against not only the predominant SARS-CoV-2 Omicron sublineage BA.5, but also other highly pathogenic human coronaviruses including SARS-CoV-1 and MERS-CoV. In the Omicron-infected K18-hACE2 mouse model, oral treatment with SY110 significantly lowered the viral burdens in lung and alleviated the virus-induced pathology. Importantly, SY110 possesses favorable PK properties with high oral drug exposure and oral bioavailability, and also an outstanding safety profile. Furthermore, SY110 exhibited sensitivity to several drug-resistance Mpro mutations. Collectively, this investigation provides a promising new drug candidate against Omicron and other variants of SARS-CoV-2.

Discovery of 4-(1,2,4-Oxadiazol-5-yl)azepan-2-one Derivatives as a New Class of Cannabinoid Type 2 Receptor Agonists for the Treatment of Inflammatory Pain.

Selectively targeting the cannabinoid receptor CB2 is an attractive therapeutic strategy for the treatment of inflammatory pain without psychiatric side effects mediated by the cannabinoid receptor CB1. Herein, we report the discovery of 4-(1,2,4-oxadiazol-5-yl)azepan-2-one derivatives as a new class of CB2 agonists. Systematic structure-activity relationship investigations resulted in the identification of the most potent compound 25r. This compound displayed high selectivity for CB2 against CB1 (CB2 EC50 = 21.0 nM, Emax = 87%, CB1 EC50 > 30 µM, ratio CB1/CB2 > 1428) with favorable pharmacokinetic properties. Especially, 25r demonstrated significant efficacy in the analgesic model of rodent inflammatory pain. All the results suggest that compound 25r could serve as a lead compound for treating inflammatory pain and deserves further in-depth studies.

Targeting Noncanonical Pyroptosis With a Small Molecular Inhibitor Alleviates Inflammation in the LPS-Induced Keratitis Mouse Model.

Purpose: Pyroptosis, a novel proinflammatory programmed cell death, has been implicated in some ocular diseases. Of special note is the noncanonical pyroptosis that has recently been recognized to play a critical role in microbial keratitis. We previously discovered a new potent small molecular pyroptosis inhibitor, J114. In this investigation, we will explore whether J114 is able to inhibit the noncanonical pyroptosis and the underlying mechanism. Then a lipopolysaccharide (LPS)-induced keratitis mouse model will be used to evaluate the therapeutic effect of J114 in vivo. Methods: In vitro, macrophages originating from humans or mice were stimulated with intracellular LPS to induce noncanonical pyroptosis activation. in vivo, acute keratitis in mouse was induced by LPS intrastromal injection. We verified the protective effect of J114 on noncanonical pyroptosis. Clinical scoring, histological observation, macrophage localization, and quantification of pyroptotic markers in the cornea were used to characterize the therapeutic effects. Results: J114 substantially inhibited the noncanonical pyroptosis and the release of inflammatory cytokines by suppressing the activation of caspase-4/5/11 and the noncanonical NLRP3 inflammasome through blocking the NLRP3-ASC interaction. in vivo, J114 protected against LPS-induced noncanonical pyroptosis of acute keratitis, as manifested by alleviated clinical manifestations and histological disorders, and relieved inflammatory reactions. Conclusions: In this study, we found that J114 could efficiently inhibit LPS-induced noncanonical pyroptosis and revealed the underlying mechanism. This compound displayed significant anti-inflammatory activity in the LPS-induced keratitis mouse model. All the findings indicated that J114 could be a potential lead compound for drug development against inflammatory ocular surface diseases.

Discovery of a potent and highly selective inhibitor of ataxia telangiectasia mutated and Rad3-Related (ATR) kinase: Structural activity relationship and antitumor activity both in vitro and in vivo.

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is an important regulator of the DNA damage response (DDR), especially in response to replication stress (RS). Tumor cells with ataxia-telangiectasia mutated (ATM) kinase loss of function or DDR defects that promote replicative stress are often more reliant on ATR for survival, highlighting ATR as a good antitumor target under the principle of synthetic lethality. Herein we report the discovery of a potent and highly selective ATR inhibitor, SKLB-197, which was obtained through structural optimization and structure-activity relationship (SAR) studies towards a hit compound (Cpd-1). SKLB-197 showed an IC50 value of 0.013 µM against ATR but very weak or no activity against other 402 protein kinases. It displayed potent antitumor activity against ATM-deficent tumors both in vitro and in vivo. In addition, this compound exhibited good pharmacokinetic properties. Overall, SKLB-197 could be a promising lead compound for drug discovery targeting ATR and deserves further in-depth studies.

Discovery of a novel and potent inhibitor with differential species-specific effects against NLRP3 and AIM2 inflammasome-dependent pyroptosis.

The NLRP3 inflammasome, which regulated a proinflammatory programmed cell death form termed pyroptosis, is involved in the pathological process of various human diseases, such as multiple sclerosis, type 2 diabetes, and gout. Thus, compounds inhibiting activation of the NLRP3 inflammasome can be promising treatments for these diseases. In this study, we conducted a phenotypic screening against NLRP3-dependent pyroptosis and discovered the hit compound 1, which showed moderate antipyroptotic activity. Chemistry efforts to improve potency of 1 resulted in a novel compound 59 (J114), which exhibited a half-maximal inhibitory concentration (IC50) of 0.077 ± 0.008 µM against cell pyroptosis. Interestingly, unlike all pyroptosis inhibitors currently reported, the activity of J114 showed significant differences in human- and mouse-derived cells. The IC50 of J114-mediated inhibition of IL-1ß secretion by human THP-1 macrophages was 0.098 µM, which was nearly 150-fold and 500-fold more potent than that of J774A.1 (14.62 µM) and bone marrow-derived macrophages (BMDMs) (48.98 µM), respectively. Further studies showed that J114 displayed remarkable inhibitory activity against NLRP3- and AIM2-but not NLRC4-dependent activation of caspase-1 and the release of IL-1ß in human THP-1 macrophages. Mechanistically, J114 disturbed the interaction of NLRP3 or AIM2 with the adaptor protein ASC and inhibited ASC oligomerization. Overall, our study identified a unique molecule that inhibits NLRP3 and AIM2 inflammasome activation and has species differences, which is worthy of further research to understand the differential regulation of the NLRP3 and AIM2 inflammasomes in humans and mice.

Structural Optimization and Structure-Activity Relationship Studies of 6,6-Dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one Derivatives as A New Class of Potent Inhibitors of Pan-Trk and Their Drug-Resistant Mutants.

Tropomyosin receptor kinases (TrkA, TrkB, and TrkC) are attractive therapeutic targets for multiple cancers. Two first-generation small-molecule Trks inhibitors, larotrectinib and entrectinib, have just been approved to use clinically. However, the drug-resistance mutations of Trks have already emerged, which calls for new-generation Trks inhibitors. Herein, we report the structural optimization and structure-activity relationship studies of 6,6-dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one derivatives as a new class of pan-Trk inhibitors. The prioritized compound 11g exhibited low nanomolar IC50 values against TrkA, TrkB, and TrkC and various drug-resistant mutants. It also showed good kinase selectivity. 11g displayed excellent in vitro antitumor activity and strongly suppressed Trk-mediated signaling pathways in intact cells. In in vivo studies, compound 11g exhibited good antitumor activity in BaF3-TEL-TrkA and BaF3-TEL-TrkCG623R allograft mouse models without exhibiting apparent toxicity. Collectively, 11g could be a promising lead compound for drug discovery targeting Trks and deserves further investigation.

Discovery and structure-activity relationship studies of 1-aryl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione derivatives as potent dual inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and trytophan 2,3-dioxygenase (TDO).

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO), which mediate kynurenine pathway of tryptophan degradation, have emerged as potential new targets in immunotherapy for treatment of cancer because of their critical role in immunosuppression in the tumor microenvironment. In this investigation, we report the structural optimization and structure-activity relationship studies of 1-phenyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione derivatives as a new class of IDO1/TDO dual inhibitors. Among all the obtained dual inhibitors, 1-(3-chloro-4-fluorophenyl)-6-fluoro-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (38) displayed the most potent IDO1 and TDO inhibitory activities with IC50 (half-maximal inhibitory concentration) values of 5 nM for IDO1 and 4 nM for TDO. It turned out that compound 38 was not a PAINS compound. Compound 38 could efficiently inhibit the biofunction of IDO1 and TDO in intact cells. In LL2 (Lewis lung cancer) and Hepa1-6 (hepatic carcinoma) allograft mouse models, this compound also showed considerable in vivo anti-tumor activity and no obvious toxicity was observed. Therefore, 38 could be a good lead compound for cancer immunotherapy and deserving further investigation.

Discovery of small molecule FLT3 inhibitors that are able to overcome drug-resistant mutations.

Herein we report the discovery of 1-(5-(tert-butyl)isoxazol-3-yl)-3- (3-fluorophenyl)urea derivatives as new FLT3 inhibitors that are able to overcome the drug resistance mutations: the secondary D835Y and F691L mutations on the basis of the internal tandem duplications (ITD) mutation of FLT3 (FLT3-ITD/D835Y and FLT3-ITD/F691L, respectively). The most potent compound corresponds to 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3- fluorophenyl)urea (4d), which showed IC50s (half maximal inhibitory concentrations) of 0.072 nM, 5.86 nM and 3.48 nM against FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y, respectively. Compound 4d also showed good selectivity for FLT3 in a kinase profiling assay. Collectively, 4d could be a good lead compound and deserves further in-depth studies.

Discovery of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors.

Herein, we report the discovery of a series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies of these compounds led to the identification of the most potent compound, 3-(3-methoxybenzyl)-6-(1H-pyrrolo[2,3-b]pyridin-4-yl)thieno[2,3-d]pyrimidin-4(3H)-one (8k), which showed IC50 values of 0.004 µM and 0.001 µM against ROCK Ⅰ and ROCK Ⅱ, respectively. In vitro, 8k significantly reduced the phosphorylation level of ROCK downstream signaling protein and induce changes in cell morphology and migration. Overall, this study provides a promising lead compound for drug discovery targeting ROCKs.