Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Zika Virus NS3 Protease Pharmacophore Anchor Model and Drug Discovery.

Zika virus (ZIKV) of the flaviviridae family, is the cause of emerging infections characterized by fever, Guillain-Barré syndrome (GBS) in adults and microcephaly in newborns. There exists an urgent unmet clinical need for anti-ZIKV drugs for the treatment of infected individuals. In the current work, we aimed at the promising virus drug target, ZIKV NS3 protease and constructed a Pharmacophore Anchor (PA) model for the active site. The PA model reveals a total of 12 anchors (E, H, V) mapped across the active site subpockets. We further identified five of these anchors to be critical core anchors (CEH1, CH3, CH7, CV1, CV3) conserved across flaviviral proteases. The ZIKV protease PA model was then applied in anchor-enhanced virtual screening yielding 14 potential antiviral candidates, which were tested by in vitro assays. We discovered FDA drugs Asunaprevir and Simeprevir to have potent anti-ZIKV activities with EC50 values 4.7 µM and 0.4 µM, inhibiting the viral protease with IC50 values 6.0 µM and 2.6 µM respectively. Additionally, the PA model anchors aided in the exploration of inhibitor binding mechanisms. In conclusion, our PA model serves as a promising guide map for ZIKV protease targeted drug discovery and the identified 'previr' FDA drugs are promising for anti-ZIKV treatments.

Anchor-based classification and type-C inhibitors for tyrosine kinases.

Tyrosine kinases regulate various biological processes and are drug targets for cancers. At present, the design of selective and anti-resistant inhibitors of kinases is an emergent task. Here, we inferred specific site-moiety maps containing two specific anchors to uncover a new binding pocket in the C-terminal hinge region by docking 4,680 kinase inhibitors into 51 protein kinases, and this finding provides an opportunity for the development of kinase inhibitors with high selectivity and anti-drug resistance. We present an anchor-based classification for tyrosine kinases and discover two type-C inhibitors, namely rosmarinic acid (RA) and EGCG, which occupy two and one specific anchors, respectively, by screening 118,759 natural compounds. Our profiling reveals that RA and EGCG selectively inhibit 3% (EGFR and SYK) and 14% of 64 kinases, respectively. According to the guide of our anchor model, we synthesized three RA derivatives with better potency. These type-C inhibitors are able to maintain activities for drug-resistant EGFR and decrease the invasion ability of breast cancer cells. Our results show that the type-C inhibitors occupying a new pocket are promising for cancer treatments due to their kinase selectivity and anti-drug resistance.

Evaluation of the antitumor effects of BPR1J-340, a potent and selective FLT3 inhibitor, alone or in combination with an HDAC inhibitor, vorinostat, in AML cancer.

Overexpression or/and activating mutation of FLT3 kinase play a major driving role in the pathogenesis of acute myeloid leukemia (AML). Hence, pharmacologic inhibitors of FLT3 are of therapeutic potential for AML treatment. In this study, BPR1J-340 was identified as a novel potent FLT3 inhibitor by biochemical kinase activity (IC50 approximately 25 nM) and cellular proliferation (GC50 approximately 5 nM) assays. BPR1J-340 inhibited the phosphorylation of FLT3 and STAT5 and triggered apoptosis in FLT3-ITD(+) AML cells. The pharmacokinetic parameters of BPR1J-340 in rats were determined. BPR1J-340 also demonstrated pronounced tumor growth inhibition and regression in FLT3-ITD(+) AML murine xenograft models. The combination treatment of the HDAC inhibitor vorinostat (SAHA) with BPR1J-340 synergistically induced apoptosis via Mcl-1 down-regulation in MOLM-13 AML cells, indicating that the combination of selective FLT3 kinase inhibitors and HDAC inhibitors could exhibit clinical benefit in AML therapy. Our results suggest that BPR1J-340 may be further developed in the preclinical and clinical studies as therapeutics in AML treatments.

Optimization of ligand and lipophilic efficiency to identify an in vivo active furano-pyrimidine Aurora kinase inhibitor.

Ligand efficiency (LE) and lipophilic efficiency (LipE) are two important indicators of "drug-likeness", which are dependent on the molecule's activity and physicochemical properties. We recently reported a furano-pyrimidine Aurora kinase inhibitor 4 (LE = 0.25; LipE = 1.75), with potent activity in vitro; however, 4 was inactive in vivo. On the basis of insights obtained from the X-ray co-crystal structure of the lead 4, various solubilizing functional groups were introduced to optimize both the activity and physicochemical properties. Emphasis was placed on identifying potential leads with improved activity as well as better LE and LipE by exercising tight control over the molecular weight and lipophilicity of the molecules. Rational optimization has led to the identification of Aurora kinase inhibitor 27 (IBPR001; LE = 0.26; LipE = 4.78), with improved in vitro potency and physicochemical properties, resulting in an in vivo active (HCT-116 colon cancer xenograft mouse model) anticancer agent.

EGFR and myosin II inhibitors cooperate to suppress EGFR-T790M-mutant NSCLC cells.

An acquired mutation (T790M) in the epidermal growth factor receptor (EGFR) accounts for half of all relapses in non-small cell lung cancer (NSCLC) patients who initially respond to EGFR kinase inhibitors. In this study, we demonstrated for the first time that EGFR-T790M interacts with the cytoskeletal components, myosin heavy chain 9 (MYH9) and ß-actin, in the nucleus of H1975 cells carrying the T790M-mutant EGFR. The interactions of EGFR with MYH9 and ß-actin were reduced in the presence of blebbistatin, a specific inhibitor for the MYH9-ß-actin interaction, suggesting that the EGFR interaction with MYH9 and ß-actin is affected by the integrity of the cytoskeleton. These physical interactions among MYH9, ß-actin, and EGFR were also impaired by CL-387,785, a kinase inhibitor for EGFR-T790M. Furthermore, CL-387,785 and blebbistatin interacted in a synergistic fashion to suppress cell proliferation and induce apoptosis in H1975 cells. The combination of CL-387,785 and blebbistatin enhanced the down-regulation of cyclooxygenase-2 (COX-2), a transcriptional target of nuclear EGFR. Overall, our findings demonstrate that disrupting EGFR interactions with the cytoskeletal components enhanced the anti-cancer effects of CL-387,785 against H1975 cells, suggesting a novel therapeutic approach for NSCLC cells that express the drug-resistant EGFR-T790M.

Suppression of Stat3 activity sensitizes gefitinib-resistant non small cell lung cancer cells.

Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a small subset of non small cell lung cancer (NSCLC) harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR inhibitors, suggesting that other factors are implicated in survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) function as transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been discovered in a number of cancers. In this study, we found that a small molecule, reactivation of p53 and induction of tumor cell apoptosis (RITA), showed anti-cancer activity against gefitinib-resistant H1650 cells through a p53-independent pathway. Stat3 suppression by RITA attracted our attention to investigate the role of Stat3 in sustaining survival of H1650 cells. Pharmacological and genetic approaches were employed to down-regulate Stat3 in H1650 cells. WP1066, a known Stat3 inhibitor, was shown to exhibit inhibitory effect on the growth of H1650 cells. Meanwhile, apoptosis activation by siRNA-mediated down-regulation of Stat3 in H1650 cells provides more direct evidence for the involvement of Stat3 in viability maintenance of H1650 cells. Moreover, as a novel identified Stat3 inhibitor, RITA increased doxorubicin sensitivity of H1650 cells in vitro and in vivo, suggesting that doxorubicin accompanied with Stat3 inhibitors may be considered as an alternative strategy to treat NSCLC patients who have inherent resistance to doxorubicin. Overall, our observations reveal that targeting Stat3 may be an effective treatment for certain NSCLC cells with oncogenic addition to Stat3.

Fast-forwarding hit to lead: aurora and epidermal growth factor receptor kinase inhibitor lead identification.

A focused library of furanopyrimidine (350 compounds) was rapidly synthesized in parallel reactors and in situ screened for Aurora and epidermal growth factor receptor (EGFR) kinase activity, leading to the identification of some interesting hits. On the basis of structural biology observations, the hit 1a was modified to better fit the back pocket, producing the potent Aurora inhibitor 3 with submicromolar antiproliferative activity in HCT-116 colon cancer cell line. On the basis of docking studies with EGFR hit 1s, introduction of acrylamide Michael acceptor group led to 8, which inhibited both the wild and mutant EGFR kinase and also showed antiproliferative activity in HCC827 lung cancer cell line. Furthermore, the X-ray cocrystal study of 3 and 8 in complex with Aurora and EGFR, respectively, confirmed their hypothesized binding modes. Library construction, in situ screening, and structure-based drug design (SBDD) strategy described here could be applied for the lead identification of other kinases.

Isolation and characterization of neurogenic mesenchymal stem cells in human scalp tissue.

Recent studies have shown that adult tissues contain stem/ progenitor cells capable of not only generating mature cells of their tissue of origin but also transdifferentiating themselves into other tissue cells. Murine skin-derived precursor cells, for example, have been described as unique, nonmesenchymal-like stem cells capable of mesodermal and ectodermal neurogenic differentiation. Human-derived skin precursors are less well characterized. In this study, the isolation and characterization of adherent, mesenchymal stem cell-like cells from human scalp tissue (hSCPs) are described. hSCPs initially isolated by both medium-selection (ms-hSCPs) and single-cell (c-hSCPs) methods were cultured in medium containing epidermal growth factor and fibroblast growth factor-beta. Cultured ms-hSCPs and c-hSCPs demonstrated a consistent growth rate, continuously replicated in cell culture, and displayed a stable phenotype indistinguishable from each other. Both hSCPs expressed surface antigen profile (CDw90, SH2, SH4, CD105, CD166, CD44, CD49d-e, and HLA class I) similar to that of bone marrow mesenchymal stem cells (BM-MSCs). The growth kinetics, surface epitopes, and differentiation potential of c-hSCP cells were characterized and compared with BM-MSCs. In addition to differentiation along the osteogenic, chondrogenic, and adipogenic lineages, hSCPs can effectively differentiate into neuronal precursors evident by neurogenic gene expression of glial fibrillary acid protein, NCAM, neuron filament-M, and microtubule-associated protein 2 transcripts. Therefore, hSCPs may potentially be a better alternative of BM-MSCs for neural repairing, in addition to their other mesenchymal regenerative capacity. Our study suggests that hSCPs may provide an alternative adult stem cell resource that may be useful for regenerative tissue repair and autotransplantations.