Discovery of a Potent and Cell-Active Inhibitor of DNA 6mA Demethylase ALKBH1.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 µM and 1.39 ± 0.13 µM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 µM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

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.

Discovery of α-Ketoamide inhibitors of SARS-CoV-2 main protease derived from quaternized P1 groups.

Although the SARS-CoV-2 pandemic has ended, multiple sporadic cases still exist, posing a request for more antivirals. The main protease (Mpro) of SARS-CoV-2, a key enzyme for viral replication, is an attractive target for drug development. Here, we report the discovery of a new potent α-ketoamide-containing Mpro inhibitor, N-((R)-1-cyclohexyl-2-(((R)-3-methoxy-1-oxo-1-((1-(2-oxo-2-((thiazol-2-ylmethyl)amino)acetyl)cyclobutyl)amino)propan-2-yl)amino)-2-oxoethyl)-4,4-difluorocyclohexane-1-carboxamide (20j). This compound presented promising enzymatic inhibitory activity against SARS-CoV-2 Mpro with an IC50 value of 19.0 nM, and an excellent antiviral activity in cell-based assay with an EC50 value of 138.1 nM. This novel covalent inhibitor may be used as a lead compound for subsequent drug discovery against SARS-CoV-2.

Discovery of a potent inhibitor targeting the cap-binding domain of the PB2 subunit of influenza RNA-dependent RNA polymerase.

Influenza pandemics have emerged as a significant global public health and security concern. PB2, a crucial subunit of the influenza RNA-dependent RNA polymerase (RdRP), has been identified as a promising target for influenza treatment. We herein report the discovery of a potent novel PB2 inhibitor, 7-51A, with a KD value of 1.64 nM as determined by ITC. The high activity of 7-51A was elucidated by the co-crystal structure of the PB2-7-51A complex, and comparative analysis revealed unique interactions that had never been observed before. The preliminary pharmacological evaluation indicated that 7-51A exhibited commendable cellular safety, hepatic microsomal metabolic safety and stability. Collectively, 7-51A was found to be an effective PB2 inhibitor and could be used as a lead compound for further studies.

Discovery of 5-((1H-indazol-3-yl) methylene)-2-thioxoimidazolidin-4-one derivatives as a new class of AHR agonists with anti-psoriasis activity in a mouse model.

Aryl hydrocarbon receptor (AHR) is a ligand dependent transcription factor and participates in the regulation of the immune balance of Th17/22 and Treg cells. It has been found to be widely expressed in the skin, and involved in the pathology of psoriasis. Therefore, AHR is thought as a potential intervention target for psoriasis. Here, we report the discovery of 5-((1H-indazol-3-yl) methylene)-2-thioxoimidazolidin-4-one derivatives as a new class of AHR agonists. Structure-activity relationship analyses led to the identification of the most active compound, 5- ((1H-indazol-3-yl)methylene) -3- (prop-2-yn-1-yl) -2-thiooimidazolidin-4-one (24e), which exhibited an EC50 value of 0.015 µM against AHR. Mechanism of action studies showed that 24e regulated the expression of CYP1A1 by activating the AHR pathway. Topical administration of 24e substantially alleviated imiquimod (IMQ)-induced psoriasis-like skin lesion. Overall, compound 24e could be a good lead compound for drug discovery against psoriasis, and hence deserving further in-depth studies.

Discovery of 3-phenyl-1,2,4-oxadiazole derivatives as a new class of SARS-CoV-2 main protease inhibitors.

The ongoing COVID-19 pandemic has led to massive infections and deaths and caused tremendous grief among the people. Although vaccines have played an important role in fighting COVID-19, the situation that the protective effect of current vaccines significantly decreases against mutated strains reminds us of the pressing need for developing effective antiviral therapeutics. The main protease (Mpro) is a key enzyme for SARS-CoV-2 viral replication and transcription and an attractive target for drug development. In this research, we report a new series of Mpro inhibitors containing 3-phenyl-1,2,4-oxadiazole. Structure-activity relationship (SAR) studies led to the discovery of the most active compound, 16d, which showed an IC50 value of 5.27 ± 0.26 µM. Collectively, we obtained a new small molecular inhibitor targeting SARS-CoV-2 Mpro, which contains a new scaffold. This compound could be taken as a lead compound for subsequent drug discovery against SARS-CoV-2.

Discovery of benzo[d]oxazol-2(3H)-one derivatives as a new class of TNIK inhibitors for the treatment of colorectal cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancer types and Traf2- and Nck-interacting kinase (TNIK) has been thought as a potential target for CRC treatment. Herein we report the discovery and structure-activity relationship (SAR) of benzo[d]oxazol-2(3H)-one derivatives as a new class of TNIK inhibitors. The most potent compound 8g showed an IC50 value of 0.050 µM against TNIK. It effectively suppressed proliferation and migration of colorectal cancer cells. Western blot analysis indicated that 8g could inhibit aberrant transcription activation of Wnt signaling. Collectively, this study provides a potential lead compound for subsequent drug discovery targeting TNIK.

Discovery of 2-vinyl-10H-phenothiazine derivatives as a class of ferroptosis inhibitors with minimal human Ether-a-go-go related gene (hERG) activity for the treatment of DOX-induced cardiomyopathy.

Ferroptosis was an iron-dependent, nonapoptotic form of regulated cell death. In our previous study, we discovered a potent ferroptosis inhibitor with phenothiazine scaffold (1), but subsequent investigation showed that this compound had potent hERG binding affinity. Herein, we report the discovery of a series of 2-vinyl-10H-phenothiazine derivatives as new class of ferroptosis inhibitors. Structure-activity relationship (SAR) analyses led to the identification of compound 7j, which exhibited significantly reduced hERG inhibition (IC50 > 30 µM) while maintaining high ferroptosis inhibitory activity (EC50 = 0.001 µM on the erastin-induced HT1080 cell ferroptosis model). Further studies confirmed 7j acted as a ROS scavenger and could relieve DOX-induced cardiomyopathy. 7j also displayed favorable pharmacokinetic properties and exhibited no obvious toxicity in vivo and vitro. Overall, this study provides a promising lead compound for drug discovery targeting ferroptosis.

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 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.

Isarubrolone C Promotes Autophagic Degradation of Virus Proteins via Activating ATG10S in HepG2 Cells.

Isarubrolone C is a bioactive polycyclic tropoloalkaloid from Streptomyces. Our previous study showed that isarubrolone C could trigger autophagy. Here, we report isarubrolone C potential in broad-spectrum antiviral effect and its antiviral mechanism in vitro. Our results show that isarubrolone C activated autophagy and reduced levels of viral proteins in the cells harboring HCV-CORE/NS5B, HBx, ZIKV-NS5, and HIV-RT, respectively. The role of isarubrolone C in suppression of the viral proteins was via an autophagic degradation pathway rather than a proteasome pathway. Co-immunoprecipitation assays revealed that isarubrolone C promoted both autophagy flux opening and the viral proteins being enwrapped in autolysosomes. PCR assays showed that isarubrolone C elevated the transcription levels of ATG10/ATG10S and IL28A. Further, ATG10S high expression could efficiently enhance IL28A expression and the ability of isarubrolone C to degrade the viral proteins by promoting the colocalization of viral proteins with autolysosomes. Additionally, knockdown of endogenous IL28A caused both losses of the isarubrolone C antiviral effect and autolysosome formation. These results indicate that the role of isarubrolone C antiviruses is achieved by triggering the autophagic mechanism, which is mediated by endogenous ATG10S and IL28A activation. This is the first report about isarubrolone C potential of in vitro broad-spectrum antiviruses.

Neutrophil membrane engineered HucMSC sEVs alleviate cisplatin-induced AKI by enhancing cellular uptake and targeting.

Human umbilical cord mesenchymal stem cells-derived small extracellular vesicles (hucMSC-sEVs) have been demonstrated as a therapeutic agent to prevent and treat cisplatin-induced acute kidney injury (AKI). However, hucMSC-sEVs still face many problems and challenges in the repair and treatment of tissue injury, including short circulation time, insufficient targeting, and low therapeutic efficacy. Therefore, we constructed engineered hybrid vesicles fused with nanovesicles derived from human neutrophil membranes and hucMSC-sEVs, named neutrophil membrane engineered hucMSC-sEVs (NEX). NEX significantly enhanced the targeting of hucMSC-sEVs to injured kidney tissues, improved the impaired renal function via reducing pro-inflammatory cytokines expression, promoted the proliferation of renal tissue cells, and inhibited renal cell apoptosis in vivo. In addition, NEX enhanced hucMSC-sEVs uptake by NRK52E cells, but inhibited its uptake by RAW264.7 cells. Moreover, administration of NEX reduced cellular oxidative stress and promoted proliferation of NRK52E cells treated with cisplatin in vitro. In summary, our findings indicate that this design of a universal approach enhances the targeting and therapeutic efficacy of hucMSC-sEVs in kidney tissue regeneration, and provides new evidence promoting its clinical application.

Discovery of selective BPTF bromodomain inhibitors by screening and structure-based optimization.

Bromodomain and PHD finger containing transcription factor (BPTF) is a multidomain protein that regulates the transcription of chromatin and is related to many cancers. Herein, we report the screening-based discovery of Cpd1, a compound with micromolar affinity to the BPTF bromodomain. Through structure-guided optimization, we synthesized a variety of new inhibitors. Among these compounds, Cpd8 and Cpd10 were highly potent and selective inhibitors, with KD values of 428 nM and 655 nM in ITC assays, respectively. The high activity was explained by the cocrystal structure of Cpd8 in complex with the BPTF bromodomain protein. Cpd8 and Cpd10 were able to stabilize the BPTF bromodomain protein in cells in a cellular thermal shift assay (CETSA). Cpd8 downregulated c-MYC expression in A549 cells. All experiments prove that these two compounds are potential BPTF inhibitors.

Discovery of JMJD7 inhibitors with the aid of virtual screening and bioactivity evaluation.

Jumonji-C (JmjC) domain-containing 7 (JMJD7), which is a 2-oxoglutarate (2OG)-dependent oxygenase, has been demonstrated to play an important role in the occurrence and development of a number of diseases, particularly cancer. Discovery of JMJD7 inhibitors is thus of great importance. Herein consensus docking/scoring strategy and bioactivity evaluation were used to identify JMJD7 inhibitors from various chemical databases. Seven active compounds were retrieved. The most potent compound, Cpd-3, showed an IC50 value of 6.62 µM against JMJD7. Further biophysical assays confirmed that Cpd-3 could efficiently bind to JMJD7 in vitro. Flexible docking was used to predict the binding mode of Cpd-3 with JMJD7. In a cellular assay, Cpd-3 displayed good inhibitory activity against cancer cell lines expressing a high level of JMJD7. As far as we know, Cpd-3 is the first JMJD7 inhibitor reported so far. Overall, this study established a good starting point for drug discovery targeting JMJD7.

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.

Comparative analysis of mesenchymal stromal cells derived from rabbit bone marrow and Wharton's jelly for adipose tissue engineering.

Purpose: To investigate the proliferative, adipogenic, and immunological properties of rabbit Mesenchymal stromal cells (MSCs) derived from bone marrow and umbilical cord Wharton's jelly.Materials and Methods: We extracted rabbit MSCs from bone marrow (BMSCs) and umbilical cord Wharton's jelly (WJ-MSCs). Both BMSCs and WJ-MSCs underwent adipogenic differentiation for 2 weeks, and then were transferred to non-inductive complete medium. Their adipogenic capacities were examined by histomorphometry and quantitative RT-PCR (qRT-PCR). The immunological markers were determined by mRNA expression of MHC-Ia, MHC-II, and RLA-DRA by qRT-PCR and protein expression of MHC-II by immunofluorescent staining. The proliferative capacities of adipogenic MSCs were also examined by counting kit-8 experiment and cell population doubling time.Results: We found that adipogenic differentiation increased the mRNA expression levels of adipogenic and immunological markers. The protein expression levels of MHC-II also increased after adipogenic differentiation in both groups. The adipogenic BMSCs showed higher mRNA expression levels of adipogenic and immunological markers. Removal of adipogenic agents after 2 weeks of adipo-differentiation inversely decreased the expression of immunological and adipogenic markers. The adipo-differentiation could decreased the proliferative capacities of both MSCs, but the adipogenic WJ-MSCs showed significantly higher proliferative capacities than BMSCs.Conclusions: Adipogenic differentiation increased the immunogenicity of both BMSCs and WJ-MSCs, and dedifferentiation inversely decreased their immunogenicity. Adipogenic WJ-MSCs showed significantly higher proliferative and immunoprivileged capacities than BMSCs, and the dedifferentiated BMSCs showed almost the same adipogenic capacity as WJ-MSCs. WJ-MSCs were more suitable than BMSCs for adipose tissue engineering.

Discovery of 5-(4-methylpiperazin-1-yl)-2-nitroaniline derivatives as a new class of SIRT6 inhibitors.

SIRT6 is a deacetylase of histone H3 and inhibitors of SIRT6 have been thought as potential agents for treatment of diabetes. Herein we report the discovery of a series of new SIRT6 inhibitors containing the skeleton 1-phenylpiperazine. Among them, compound 5-(4-methylpiperazin-1-yl)-2-nitroaniline (6d) is the most potent one, which showed an IC50 value of 4.93 µM against SIRT6 in the Fluor de Lys (FDL) assay. It displayed KD values of 9.76 µM and 10 µM in surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays, respectively. In selectivity assay, 6d showed no activity against other members of the HDAC family (SIRT1-3 and HDAC1-11) at concentrations up to 200 µM. In a mouse model of type 2 diabetes, 6d could significantly increase the level of glucose transporter GLUT-1, thereby reducing blood glucose. Overall, this study provides a promising lead compound for subsequent drug discovery targeting SIRT6.

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.

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.