Identification of a new class of activators of the Hippo pathway with antitumor activity in vitro and in vivo.

The Hippo pathway is a key regulator of tissue growth, organ size, and tumorigenesis. Activating the Hippo pathway by gene editing or pharmaceutical intervention has been proven to be a new therapeutic strategy for treatment of the Hippo pathway-dependent cancers. To now, a number of compounds that directly target the downstream effector proteins of Hippo pathway, including YAP and TEADs, have been disclosed, but very few Hippo pathway activators are reported. Here, we discovered a new class of Hippo pathway activator, YL-602, which inhibited CTGF expression in cells irrespective of cell density and the presence of serum. Mechanistically, YL-602 activates the Hippo pathway via MST1/2, which is different from known activators of Hippo pathway. In vitro, YL-602 significantly induced tumor cell apoptosis and inhibited colony formation of tumor cells. In vivo, oral administration of YL-602 substantially suppressed the growth of cancer cells by activation of Hippo pathway. Overall, YL-602 could be a promising lead compound, and deserves further investigation for its mechanism of action and therapeutic applications.

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.

Cryo-EM structures of human GPR34 enable the identification of selective antagonists.

GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.

Discovery of [1,2,3]Triazolo[4,5-c]quinoline Derivatives as a New Class of Ataxia-Telangiectasia Mutated Kinase Inhibitors.

Ataxia-telangiectasia mutated (ATM) is an atypical serine/threonine protein kinase which is implicated in the repair of DNA double-strand breaks. Numerous reports have shown that ATM inhibition is an attractive target for radiotherapy and chemotherapy sensitization. Herein we report a new series of ATM kinase inhibitors containing the 1H-[1,2,3]triazolo[4,5-c]quinoline scaffold, which was obtained by virtual screening, structural optimization, and structure-activity relationship studies. Among the inhibitors, A011 was one of the most potent, with an IC50 value of 1.0 nM against ATM. In colorectal cancer cells (SW620 and HCT116), A011 was able to inhibit activation of ATM signaling induced by irinotecan (CPT-11) and ionizing radiation and then increased the sensitivity of colorectal cancer cells to irinotecan and ionizing radiation through increasing G2/M arrest and inducing apoptosis. In the SW620 human colorectal adenocarcinoma tumor xenograft model, A011 sensitized SW620 to CPT-11 by inhibiting ATM activity. Collectively, this work has identified a promising lead in the discovery of potent inhibitors against ATM.

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.

Erratum: HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing.

[This corrects the article DOI: 10.1016/j.omtn.2022.05.036.].

Discovery of Small Molecule Agonist of Gonadotropin-Releasing Hormone Receptor (GnRH1R).

The gonadotrophin-releasing hormone (GnRH) is a central regulator of the human reproductive system and exerts physiological effects by binding to GnRH1R. The GnRH-GnRH1R system is a promising therapeutic target for the maintenance of reproductive function. There are several GnRH1R agonists on the market, but like GnRH, they are all peptide compounds and are limited by their way of administration (subcutaneous or intramuscular injection). To date, no published GnRH1R small molecule agonists have been reported. In this paper, the HTRF-based screening method has been used to screen our in-house chemical library, and we found and confirmed CD304 as a hit compound. Subsequently, structure optimization led to the discovery of compound 6d, exhibited with a certain GnRH1R activation activity (EC50: 1.59 ± 0.38 µM). Further molecular dynamics simulation experiments showed that 6d can well bind to the orthosteric site of GnRH1R through forming a hydrogen-bonding interaction with Y2836.51. Binding of 6d further induces conformational changes in TM6 and TM7, promoting the formation of a continuous water channel in GnRH1R, thereby promoting GnRH1R activation. This well-characterized hit compound will facilitate the further development of novel small molecule agonists of GnRH1R.

HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing.

Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE). With this system, we screened an in-house small-molecule library and identified a group of histone deacetylase inhibitors (HDACi) increasing prime editing. We also found that HDACi increased the efficiency of both cytosine base editing (CBE) and adenine base editing (ABE). Moreover, HDACi increased the purity of cytosine base editor products, which was accompanied by an upregulation of the acetylation of uracil DNA glycosylase (UNG) and UNG inhibitor (UGI) and an enhancement of their interaction. In summary, our work demonstrated that HDACi improves Cas9-mediated prime editing and base editing.

Molecular mechanism of allosteric modulation for the cannabinoid receptor CB1.

Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.

An orally available Mpro inhibitor is effective against wild-type SARS-CoV-2 and variants including Omicron.

Emerging SARS-CoV-2 variants continue to cause waves of new infections globally. Developing effective antivirals against SARS-CoV-2 and its variants is an urgent task. The main protease (Mpro) of SARS-CoV-2 is an attractive drug target because of its central role in viral replication and its conservation among variants. We herein report a series of potent α-ketoamide-containing Mpro inhibitors obtained using the Ugi four-component reaction. The prioritized compound, Y180, showed an IC50 of 8.1 nM against SARS-CoV-2 Mpro and had oral bioavailability of 92.9%, 31.9% and 85.7% in mice, rats and dogs, respectively. Y180 protected against wild-type SARS-CoV-2, B.1.1.7 (Alpha), B.1.617.1 (Kappa) and P.3 (Theta), with EC50 of 11.4, 20.3, 34.4 and 23.7 nM, respectively. Oral treatment with Y180 displayed a remarkable antiviral potency and substantially ameliorated the virus-induced tissue damage in both nasal turbinate and lung of B.1.1.7-infected K18-human ACE2 (K18-hACE2) transgenic mice. Therapeutic treatment with Y180 improved the survival of mice from 0 to 44.4% (P = 0.0086) upon B.1.617.1 infection in the lethal infection model. Importantly, Y180 was also highly effective against the B.1.1.529 (Omicron) variant both in vitro and in vivo. Overall, our study provides a promising lead compound for oral drug development against SARS-CoV-2.

Discovery of 6,7-dihydro-5H-pyrrolo[3,4-d] pyrimidine derivatives as a new class of ATR inhibitors.

Ataxia telangiectasia and Rad3-related (ATR) kinase is a key regulating protein within the DNA damage response (DDR), responsible for sensing replication stress (RS), and has been considered as a potential target for cancer therapy. Herein, we report the discovery of a series of 6,7-dihydro-5H-pyrrolo[3,4-d]-pyrimidine derivatives as a new class of ATR inhibitors. Among them, compound 5g exhibits an IC50 value of 0.007 µM against ATR kinase. In vitro, 5g displays good anti-tumor activity and could significantly reduce the phosphorylation level of ATR and its downstream signaling protein. Overall, this study provides a promising lead compound for subsequent drug discovery targeting ATR kinase.

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.

Discovery of 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one derivatives as a new class of ROCK inhibitors for the treatment of glaucoma.

The Rho-associated protein kinases (ROCKs) are associated with the pathology of glaucoma and discovery of ROCK inhibitors has attracted much attention in recent years. Herein, we report a series of 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies led to the discovery of compound 12b, which showed potent activities against ROCK I and ROCK Ⅱ with IC50 values of 93 nM and 3 nM, respectively. 12b also displayed considerable selectivity for ROCKs. The mean IOP-lowering effect of 12b in an ocular normotensive model was 34.3%, and no obvious hyperemia was observed. Overall, this study provides a good starting point for ROCK-targeting drug discovery against glaucoma.

Discovery of 3,6-disubstutited-imidazo[1,2-a]pyridine derivatives as a new class of CLK1 inhibitors.

Inhibition of cdc2-like kinase1 (CLK1) could efficiently induce autophagy and it has been thought as a potential target for treatment of autophagy-related diseases. Herein we report the discovery of a series of 3,6-disubstutited-imidazo[1,2-a]pyridine derivatives as a new class of CLK1 inhibitors. Among them, compound 9e is the most potent one, which exhibits an IC50 value of 4 nM against CLK1 kinase. In vitro, this compound reduces the phosphorylation level of the typical downstream substrates of CLK1 and affects their subcellular redistribution. Further study indicates that 9e is efficient to induce autophagy. Overall, this study provides a promising lead compound for drug discovery targeting CLK1 kinase.

SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

Structure-Guided Discovery of a Potent and Selective Cell-Active Inhibitor of SETDB1 Tudor Domain.

SET domain bifurcated protein 1 (SETDB1) is a histone lysine methyltransferase that promotes the silencing of some tumour suppressor genes and is overexpressed in many cancers. SETDB1 contains a unique tandem tudor domain (TTD) that recognizes histone H3 sequences containing both methylated and acetylated lysines. Beginning with the identification of a hit compound (Cpd1), we discovered the first potent and selective small molecule SETDB1-TTD inhibitor (R,R)-59 through stepwise structure-guided optimization. (R,R)-59 showed a KD value of 0.088±0.045 µM in the ITC assay. The high potency of (R,R)-59 was well explained by the cocrystal structure of the (R,R)-59-TTD complex. (R,R)-59 is an endogenous binder competitive inhibitor. Evidence has also demonstrated its cellular target engagement. Interestingly, the enantiomer (S,S)-59 did not show activity in all the assays, highlighting the potential of (R,R)-59 as a tool compound in exploring the biological functions of SETDB1-TTD.

Discovery of Potent Small-Molecule SIRT6 Activators: Structure-Activity Relationship and Anti-Pancreatic Ductal Adenocarcinoma Activity.

SIRT6 activation is thought to be a promising target for the treatment of many diseases, particularly cancer. Herein, we report the discovery of a series of new small-molecule SIRT6 activators. Structure-activity relationship analyses led to the identification of the most potent compound, 2-(1-benzofuran-2-yl)-N-(diphenylmethyl) quinoline-4-carboxamide (12q), which showed an EC1.5 value of 0.58 ± 0.12 µM and an EC50 value of 5.35 ± 0.69 µM against SIRT6-dependent peptide deacetylation in FLUOR DE LYS assay. It exhibited weak or no activity against other HDAC family members as well as 415 kinases, indicating good selectivity for SIRT6. 12q significantly inhibited the proliferation and migration of pancreatic ductal adenocarcinoma (PDAC) cells in vitro. It also markedly suppressed the tumor growth in a PDAC tumor xenograft model. This compound showed attractive pharmacokinetic properties. Overall, 12q could be a good lead compound for the treatment of PDAC, and it is worthy of further study.