To explore selective FXIa inhibitors, a series of compounds was rationally designed using computer-aided drug design (CADD) and synthesized by introducing hydrophobic P1 fragments, which could strengthen the interaction with the S1 pocket of FXIa and weaken the interaction with plasma kallikrein. All the compounds were tested for the inhibition of FXIa, and some of them were further evaluated via plasma kallikrein selectivity and clotting assays. Compound 4c exhibited excellent in vitro potency, good membrane permeability and higher selectivity than asundexian. Furthermore, 4c showed an excellent pharmacokinetic property in rats after intravenous or oral administration. These results indicate that 4c can serve as a novel FXIa inhibitor that has potential clinical applications in patients.

Nitric oxide (NO) modifies protein structure and plays a critical role in promoting tumor growth, metastasis, and resistance to therapy. In inflammatory tumors, multiple key proteins, including p53, are susceptible to S-nitrosylation (SNO) by NO. In this study, we investigated the effects of different NO donors on melanoma cell growth and p53 modification. Biotin switch assays demonstrated that treatment with S-nitrosoglutathione (GSNO) or diethylenetriamine (DETA) NONOate significantly increased total S-nitrosylated proteins in A375 and SB2 melanoma cells. p53 was confirmed to undergo SNO under nitrosative stress, as evidenced by biotin switch assays and mass spectrometry. Proteomic analysis identified Cys242, Cys275, and Cys277 as specific SNO sites on p53. Structural modelling revealed that SNO of Cys242 and Cys277 disrupts p53 conformation and impairs its DNA-binding ability. These findings uncover a critical mechanism by which nitrosative stress alters the function of p53 through site-specific SNO, laying a foundation for further exploration of how SNO affects p53-mediated transcriptional regulation in melanoma cells.

Atropisomerism and restricted amide CO–N bond rotation are commonly encountered structural characteristics in drug discovery and development. Inspired by the unusual NMR spectra of soticlestat (S-1), a CYP46A1 inhibitor currently under development in phase 3 clinical trials, soticlestat and its 14 structurally close analogs were designed, synthesized and studied by variable-temperature 13C NMR, molecular mechanics, quantum mechanics and HPLC to explore the structural characteristics that affect the restricted bond rotations and the number of stable conformations. It is concluded that there are four stable conformations at room temperature which exist as two diastereomers each as a pair of enantiomers, and the interconversion rates of these conformations are intermediate on the NMR time scale but are inseparable by HPLC, justifying that soticlestat can be developed as a mixture of four rotational isomers.

The 1R-alkoxygenipins exhibit significantly enhanced bioactivity in comparison to their S-isomers; however, the challenge lies in the selective synthesis of these compounds. A streamlined and highly effective approach to achieve stereoselective 1R-alkyloxygenipins involved the use of a carefully balanced mixture of Lewis acid catalytic reagents, specifically BF3·Et2O and H2O. This method emerged as the optimal choice after evaluating various synthetic routes, encompassing 1-OH and 10-OH substitutions, different Lewis acids, and their respective ratios as catalysts. And study the possible mechanism of the catalyst for genipin. particularly, the method with non-strict conditions for the synthesis of 1R-alkoxygenipins was underscored by its operational simplicity, stability, and low energy consumption. This approach not only streamlined the synthetic process but also established a robust foundation for future bioactivity studies on 1R-alkyloxygenipins.

DNA repair is strongly associated with tumor resistance to radiotherapy and chemotherapy. WD repeat and HMG-box DNA binding protein 1 (WDHD1) is a key adaptor for homologous recombination repair of DNA, and its overexpression is relevant to the poor prognosis of many tumor patients. We previously have identified and validated bazedoxifene (BZA), which had 60% inhibitory rate on WDHD1 in MCF7 cells at 10 μM, from the Food and Drug Administration-approved compound library. Here, we initially established the binding model of BZA, synthesized and evaluated eight BZA analogs. Further, we detailed the use of molecular dynamics simulations to provide insights into the basis for activity against WDHD1. This binding mode will be instructive for the development of new WDHD1 degraders.

Overcoming the acquired C797S mutation in nonsmall cell lung cancer (NSCLC) represents a great challenge for EGFR tyrosine kinase inhibitors (TKIs). BLU-945 is a promising fourth-generation EGFR TKI undergoing clinical trials, with different binding conformation from other known EGFR TKIs. In this study, we explored the binding mode of a BLU-945 analog (BLU) with wild-type and mutant EGFRs using [Formula: see text]s-scale molecular dynamics. The results show that Met793 at the hinge region is the critical binding site for BLU as observed in other known EGFR inhibitors. The occupancy of hydrogen bonds with Met793 is lower in wild-type EGFRs than in mutant EGFRs. Meanwhile, Thr854 contributes largely to the hydrogen bond formation for triple mutant EGFR compared to wild-type EGFR. Energy decomposition reveals that Leu718 and Leu844 dominate the energy contribution. T790M mutation plays an important role in BLU binding with wild-type and mutant EGFRs. Dynamic network analysis indicates that Arg841 behaves differently in triple mutant EGFR compared to wild-type, single and double mutant EGFRs, confirmed by further analysis of salt-bridge formation. Our study may provide beneficial information for developing fourth-generation EGFR TKIs.

A general approach for regioselective deacetylation at sugar 3-OH of peracetylated 6-deoxy-C-glucopyranosides mediated by BCl3 was developed. The approach could be extended to other sugar-derived 6-deoxy-C-glycopyranosides, such as those derived from mannose, galactose, and rhamnose, with deacetylation occurring at varied sugar hydroxyl groups, and further extended to 4-deoxy-C-glucopyranosides with deacetylation occurring at sugar 3-OH. The approach would enable access to synthetically challenging carbohydrate derivatives. A possible mechanism of the regioselectivity was proposed.

A series of 6-chloro-quinolin-2-one derivatives were designed and synthesized as FXIa inhibitors by exploration of P1, P1 prime and P2 prime groups. Each compound was accessed for inhibitory effect on FXIa and some of them were evaluated in the clotting assay. 14c demonstrated excellent in-vitro potency (FXIa IC50: 15 nM, 2 x aPTT: 6.8 μM) and good in-vivo efficacy (prolonged in-vivo aPTT by more than 1-fold but not PT). Moreover, the pharmacokinetics property of 14c were evaluated following intravenous administration in rats, which indicated that 14c probably will be a clinical candidate for intravenous administration.

Background: SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Hence the inhibition of TMPRSS2 may block or decrease the severity of SARSCoV- 2, making TMPRSS2 an attractive target for COVID-19. fXIa has a similar binding pocket as TMPRSS2, implying the possibility of fXIa inhibitors being TMPRSS2 inhibitors. Methods: In order to find potential TMPRSS2 inhibitors, molecular docking of known fXIa inhibitors was performed. Molecular dynamics simulations and MM/GBSA were conducted on representative compounds with characteristic binding modes. R-group enumeration was used to generate compounds with better binding interactions. Results: Three scaffolds can make hydrogen bonds with Gly439 and Ser441, and form the chloride– Tyr474 interactions at S1 pocket as well. Further structure optimization of one scaffold found that two compounds have better docking scores and lower binding free energies. Conclusion: Compounds R1a and R1b can be taken as potentially reversible inhibitors of TMPRSS2. Our results could provide insight into both the discovery and lead optimization of TMPRSS2 inhibitors.

Aberrant expression of inducible nitric oxide synthase (iNOS) and chronic nitrosative stress is correlated with growth, metastasis, resistance to therapy, and poor patient survival of metastatic melanoma. One physiological influence of nitric oxide (NO) is exerted directly through the post-translational modification of many proteins, including the unique S-nitrosylation (SNO). To date, the role of SNO in melanoma development has not been fully characterized. Herein, we focus on the mechanism of how nitrosative stress regulates the tumor suppressor p53 in melanoma cells. We first examined the effects of different levels of NO donors, S-nitrosoglutathione (GSNO) and diethylenetriamine (DETA) NONOate, on the growth of melanoma cells by the MTT assay. Low levels of nitrosative stress (NO donors ≤ 20 µM) did not suppress the growth of human melanoma A375 and SB2 cells, but high levels of nitrosative stress (NO donors ≥ 50 µM) inhibited melanoma cell growth. Our biotin switch assays (BSA) showed that the treatment of NO donors significantly increased total S-nitrosylated proteins in A375 and SB2 cells. Markedly, p53 is confirmed to be one of the S-nitrosylated proteins derived from BSA. Our mass spectrometry analysis of SNO proteins in melanoma cells confirms that p53 is S-nitrosylated in A375 cells under nitrosative stress. Further proteomic characterization identifies Cys242, Cys275, and Cys277 as the SNO sites of p53. We conducted a molecular dynamics (MD) simulation of p53-DNA binding under SNO modification. The structural analysis shows that the SNO of Cys277 reduces the hydrogen bonds between p53 and the targeted DNA, interfering with the recognition and binding of p53 to DNA. Moreover, the chelating ability of Cys242 for Zn2+ is weakened after SNO, indicating the SNO of p53 reduces p53 binding to Zn2+ and affects its active form. The electrophoretic mobility shift assay also confirmed that the treatment of 100 µM of DETA NONOate, significantly decreased the nuclear p53 of A375 and SB2 cells binding to the consensus p53-binding DNA. The induction of nitrosative stress by NO donors also significantly increased mouse double minute 2 homolog (MDM2), p21, and p53 expression but decreased p53 upregulated modulator of apoptosis (PUMA) expression.This study confirms that the crucial tumor suppressor, p53, is S-nitrosylated in melanoma cells under nitrosative stress. Notably, for the first time, the SNO sites of p53 and the effects of SNO on p53 functions were characterized. Our results describe an important mechanism of how nitrosative stress modifies crucial cysteines of p53, alters p53 DNA-binding activities, and regulates downstream gene expression through SNO. Our study provides much-needed insights into identifying novel NO-driven SNO proteins in melanoma as new biomarkers and targets for drug development. Citation Format: Jordan Winfield, Mariana Grigoruta, Yiliang Li, Fancui Meng, Yong Qin, Elizabeth Grimm, Leyuan Chen, Kevin Rosenblatt, Li Li. S-nitrosylation of p53 in melanoma alters p53-DNA binding and downstream gene expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4785.

Nitric oxide (NO) is known to modify protein structure and plays a critical role in supporting tumor growth, metastasis, and resistance to therapy. Multiple studies showed that various key proteins, including p53, are susceptible to S-nitrosylation (SNO) by NO in inflammatory tumors. Herein, we examined the effects of different levels of NO donors on the growth of melanoma cells. Our biotin switch assays showed that the treatment with S-nitrosoglutathione (GSNO) or diethylenetriamine (DETA) NONOate significantly increased total S-nitrosylated proteins in melanoma A375 and SB2 cells. p53 was confirmed to be S-nitrosylated in melanoma cells under nitrosative stress by the biotin switch assay and mass spectrometry. Proteomic characterization identified Cys242, Cys275, and Cys277 as the SNO sites of p53. The structural analysis demonstrated that the SNO of Cys277 and Cys242 could disrupt p53 conformation and its DNA-binding ability, further confirmed by the electrophoretic mobility shift assay. Our results describe an important mechanism of how nitrosative stress modifies crucial cysteines of p53 and alters p53 DNA-binding activities through SNO. Herein, we first-time present how the post-translational SNO of p53 alters its structure and DNA binding activities under nitrosative stress, which sheds light on a potential mechanism of nitrosative stress regulating p53 function.

目的:采用分子动力学模拟方法研究化合物AG-881(vorasidenib)抑制IDH1 R132 H突变蛋白的作用机制.方法:分别构建激活态的IDH1 R132 H突变蛋白结构和失活态的AG-881-IDH1 R132 H突变蛋白结构,置于立体水盒子内,进行100 ns的分子动力学模拟,比较不同体系的蛋白构象并分析氢键、能量和主成分等性质.结果:底物α-KG可使IDH1 R132 H突变蛋白稳定在活化状态的闭合构象.AG-881作用于蛋白二聚体界面的变构位点后,使IDH1 R132 H突变蛋白处于非活化状态的开放构象,发挥抑制作用.在作用过程中,氨基酸残基Gln277在AG-881的结合中起重要作用.同时,疏水作用和氢键在AG-881与IDH1 R132 H突变蛋白的结合中也做出重要贡献.此外,卤键也起到一定作用.结论:分子动力学模拟可以作为变构抑制剂设计的有效辅助手段,探索研究AG-881抑制IDH1 R132 H突变蛋白活性的潜在作用机制,进一步为以IDH1突变蛋白为靶点的新药研发提供一定的理论指导.

Glycoprotein VI (GPVI) plays an important role in platelet aggregation, and inhibition of GPVI may block thrombosis with a low bleeding risk, thus making GPVI a promising antithrombotic target. In this paper, the binding mode of losartan, a known GPVI inhibitor, was investigated using molecular docking and molecular dynamics methods as well. Docking results can determine the location site of the phenyl tetrazole group of losartan, but the direction of the two substitute groups on imidazole ring is uncertain. Therefore, two sample conformations were selected, and long-time molecular dynamics simulations were carried out. The rotatable bond of C–N connecting the imidazole ring and the middle benzene ring was monitored during the simulation. The results showed that this dihedral angle is mostly distributed at about [Formula: see text] for both systems, implying that this conformation is the dominant conformation. The switchable conformation may be the reason for low activity of losartan to GPVI. Losartan1 system was well equilibrated and selected as the receptor to perform drug repurposing screening, and several drugs with a similar binding mode as losartan were identified. Our study may enhance the mechanism understanding of GPVI receptor, giving insights into the design and discovery of novel GPVI inhibitors.

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

BACKGROUND:Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Proteasome inhibitors (PIs) work as the backbone agent and have greatly improved the outcome in majority of newly diagnosed patients with myeloma. However, drug resistance remains the major obstacle causing treatment failure in clinical practice. Here, we investigated the effects of Indirubin-3'-monoxime (I3MO), one of the derivatives of Indirubin, in the treatment of MM. METHODS:MM patient primary samples and human cell lines were examined. I3MO effects on myeloma treatment and the underling molecular mechanisms were investigated via in vivo and in vitro study. FINDINGS:Our results demonstrated the anti-MM activity of I3MO in both drug- sensitive and -resistance MM cells. I3MO sensitizes MM cells to bortezomib-induced apoptosis. Mechanistically, I3MO acts as a multifaceted regulator of cell death, which induced DNA damage, cell cycle arrest, and abrogates NF-κB activation. I3MO efficiently down-regulated USP7 expression, promoted NEK2 degradation, and suppressed NF-κB signaling in MM. Our study reported that I3MO directly bound with and caused the down-regulation of PA28γ (PSME3), and PA200 (PSME4), the proteasome activators. Knockdown of PSME3 or PSME4 caused the inhibition of proteasome capacity and the overload of paraprotein, which sensitizes MM cells to bortezomib-mediated growth arrest. Clinical data demonstrated that PSME3 and PSME4 are over-expressed in relapsed/refractory MM (RRMM) and associated with inferior outcome. INTERPRETATION:Altogether, our study indicates that I3MO is agent triggering proteasome inhibition and represents a promising therapeutic strategy to improve patient outcome in MM. FUNDINGS:A full list of funding can be found in the acknowledgements.

Background: Neuropathic pain (NP) is a complex symptom related to nerve damage. The discovery of new drugs for treating chronic NP has been continuing for several decades, while more progress is still needed because of the unsatisfactory efficacy and the side effects of the currently available drugs. Among all the approved drugs for chronic NP, voltage- gated calcium channel (VGCC) α2δ subunit ligands, also known as gabapentinoids, are among the first-line treatment and represent a class of efficacious and relatively safe therapeutic agents. However, new strategies are still needed to be explored due to the unsatisfied response rate. Objectives: The aim of the study is to review the latest status of the discovery and development of gabapentinoids for the treatment of chronic NP by covering both the marketed and the preclinical/clinical ones. Moreover, it aims to analyze the structure-activity relationship (SAR) of gabapentinoids to facilitate the future design of structurally novel therapeutic agents targeting the VGCC α2δ subunit. Methods: We searched PubMed Central, Embase, Cochrane Library, Web of Science, Scopus, and Espacenet for the literature and patents on diabetic peripheral neuropathic pain, postherpetic neuralgia, fibromyalgia, voltage-gated calcium channel α2δ subunit and related therapeutic agents from incipient to June 10, 2021. The SAR of gabapentinoids was analyzed by pharmacophore modeling using the Phase module in the Schrödinger suite. Results: A variety of gabapentinoids were identified as VGCC α2δ ligands that have ever been under development to treat chronic NP. Among them, four gabapentinoids are marketed, one is in the active late clinical trials, and eight have been discontinued. Pharmacophore models were generated using the phase module in the Schrödinger suite, and common pharmacophores were predicted based on pharmacophoric features and analyzed. Conclusion: The latest progress in the discovery and development of gabapentinoids for the treatment of chronic NP was reviewed. Moreover, the structure-activity relationship (SAR) of gabapentinoids has been analyzed by pharmacophore modeling, which will be valuable for the future design of structurally novel therapeutic agents targeting the VGCC α2δ subunit.

AZD3759 is an epidermal growth factor receptor inhibitor with good blood–brain barrier permeability, demonstrating encouraging activity against central nervous system metastases. However, the underlying mechanism was still unclear. In this study, the interaction between AZD3759 and membrane was studied with 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer as a model lipid. Both the cationic and neutral state of AZD3759 were considered in the simulations, and the results show that cationic AZD3759 forms more hydrogen bonds with bilayer than neutral AZD3759, and Coulombic interaction has great effects in the transmembrane process of cationic AZD3759. AZD3759 prefers to reside in the interface between the hydrophilic headgroup region and hydrophobic region of bilayer, and the chloroflurobenzene moiety plays a crucial role in the insertion of AZD3759. PMF results suggest that the hydrophobic region of DMPC bilayer is permeable by AZD3759. Understanding the transmembrane mechanism of AZD3759 at molecular level may provide useful information to the design and optimization of anti-tumor drugs with improved BBB penetration. • The penetration mechanism of AZD3759 with DMPC bilayer was studied by molecular dynamics simulations. • Neutral AZD3759 could penetrate deeper into DMPC bilayer than protonated AZD3759. • The chloroflurobenzene moiety plays a significant role in the insertion of AZD3759 into DMPC bilayer. • The electrostatic interaction is the driving force for the initial binding of AZD3759 to DMPC bilayer. • Our findings may enhance the mechanism understanding of drugs with good BBB permeability.

Glioblastoma (GBM) is a highly malignant primary brain tumor, and epidermal growth factor receptor (EGFR) is a well characterized biomaker on GBM. Treatment of GBM with EGFR inhibitors achieved limited efficacy due to low blood–brain barrier (BBB) permeability, and BBB-penetrant drugs are required. In this study, the BBB penetration of erlotinib and JN037 were studied using molecular dynamics method with explicit membrane model. The free energy profiles indicate that JCN037 has a lower central energy barrier than erlotinib, and it has a local minimum at lipid-water interface while erlotinib has not. Unconstrained MD simulations found that erlotinib prefers staying in water while JCN037 tends to interact with lipid molecules. Further analysis reveals that the Br atom of JCN037 plays an important role in its interaction with lipid molecules, and the adjacent F atom enhances the interaction of Br. The two flexible methoxyethoxy chains of erlotinib are responsible for its poor penetration. Our computational results agree well with the experimental results, providing useful information in the design and improvement of drugs with good BBB permeation.

Cholesterol plays a crucial role in modulating the physicochemical properties of membranes, thus influencing the membrane transport of drugs. In this paper, the effects caused by cholesterol on the membrane transport of chlorzoxazone (CZX), a centrally acting muscle relaxant drug, were probed through molecular dynamics simulations. POPC was selected as the model lipid, and three different cholesterol concentrations (0%, 20%, and 50% CHOL) were considered. The outcomes reveal that the area per lipid of POPC decreases and the order parameter increases with enhanced concentration of CHOL. CZX prefers to localize at the interface between the headgroup region and the hydrophobic tail region of POPC, and the main energy barrier occurs in the hydrophobic region. The impact of CHOL on the free energy profile is correlated with concentration: low concentration facilitates CZX permeation, while high concentration hinders CZX permeation. Our findings coincide with experimental results, enhancing the mechanism understanding of how drug molecules are transported through membranes in the presence of CHOL.