Identification of novel GSPT1 degraders by virtual screening and bioassay.

GSPT1 plays crucial physiological functions, such as terminating protein translation, overexpressed in various tumors. It is a promising anti-tumor target, but is also considered as an "undruggable" protein. Recent studies have found that a class of small molecules can degrade GSPT1 through the "molecular glue" mechanism with strong antitumor activity, which is expected to become a new therapy for hematological malignancies. Currently available GSPT1 degraders are mostly derived from the scaffold of immunomodulatory imide drug (IMiD), thus more active compounds with novel structure remain to be found. In this work, using computer-assisted multi-round virtual screening and bioassay, we identified a non-IMiD acylhydrazone compound, AN5782, which can reduce the protein level of GPST1 and obviously inhibit the proliferation of tumor cells. Some analogs were obtained by a substructure search of AN5782. The structure-activity relationship analysis revealed possible interactions between these compounds and CRBN-GSPT1. Further biological mechanistic studies showed that AN5777 decreased GSPT1 remarkably through the ubiquitin-proteasome system, and its effective cytotoxicity was CRBN- and GSPT1-dependent. Furthermore, AN5777 displayed good antiproliferative activities against U937 and OCI-AML-2 cells, and dose-dependently induced G1 phase arrest and apoptosis. The structure found in this work could be good start for antitumor drug development.

Discovery of potential novel TRPC5 inhibitors by virtual screening and bioassay.

The transient receptor potential canonical channel 5 (TRPC5), a member of the TRPC family, plays a crucial role in the regulation of various physiological activities and diseases, including those related to the central nervous system, cardiovascular system, kidney, and cancer. As a nonselective cation channel, TRPC5 mainly controls the influx of extracellular Ca2+ into cells, thereby modulating cellular depolarization and intracellular ion concentration. Inhibition of TRPC5 by small molecules presents a promising approach for the treatment of TRPC5-associated diseases. In this study, we conducted a comprehensive virtual screening of more than 1.5 million molecules from the Chemdiv database (https://www.chemdiv.com) to identify potential inhibitors of hTRPC5, utilizing the published structures and binding sites of hTRPC5 as a basis. Lipinski's rule, Veber's rule, PAINS filters, pharmacophore analysis, molecular docking, ADMET evaluation and cluster analysis methods were applied for the screening. From this rigorous screening process, 18 candidates exhibiting higher affinities to hTRPC5 were subsequently evaluated for their inhibitory effects on Ca2+ influx using a fluorescence-based assay. Notably, two molecules, namely SML-1 and SML-13, demonstrated significant inhibition of intracellular Ca2+ levels in hTRPC5-overexpressing HEK 293T cells, with IC50 values of 10.2 µM and 10.3 µM, respectively. These findings highlight SML-1 and SML-13 as potential lead molecules for the development of therapeutics targeting hTRPC5 and its associated physiological activities and diseases.

Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition.

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2-7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

Small-Molecule Drugs in Immunotherapy.

Immunotherapy has been increasingly used in the treatment of cancer. Compared with chemotherapy, immunotherapy relies on the autoimmune system with fewer side effects. Small molecule immune-oncological medicines usually have good bioavailability, higher tissue and tumor permeability, and a reasonable half-life. In this work, we summarize the current advances in the field of small molecule approaches in tumor immunology, including small molecules in clinical trials and preclinical studies, containing PD1/PD-L1 small molecule inhibitors, IDO inhibitor, STING activators, RORγt agonists, TGF-ß inhibitors, etc. PD-1/DP-L1 is the most attractive target at present. Some small molecule drugs are being in clinical trial studies. Among them, CA-170 has attracted much attention as an oral small molecule drug. IDO is another popular target after PD-1/PDL1. The dual IDO and PD-1 inhibitor can improve the low response of PD-1 and has a good synergistic effect. STING is a protein that occurs naturally in the human body and can enhance the body's immunity. RORγt is mainly expressed in cells of the immune system. It promotes the differentiation of Th17 cells and produces the key factor IL-17, which plays a key role in the development of autoimmune diseases. TGFß signaling exhibits potent immunosuppressive activity on the coordinate innate and adaptive immunity, impairing the antitumor potential of innate immune cells in the tumor microenvironment. It is worth mentioning that immunotherapy drugs can often achieve better effects when used in combination, which will help defeat cancer.

Discovery of novel potential CRBN modulators through structure-based virtual screening and bioassay.

CRBN protein is an E3 ubiquitin ligase which plays an important role in the ubiquitin-proteasome system of eukaryotic cells. Small molecules can modulate CRBN and induce multiple target proteins to bind with CRBN, which contributes to ubiquitination and degradation of target proteins. Modulating the CRBN protein through small molecules provides a novel idea for treatment of tumors and immune system disease. Due to most of CRBN modulators containing glutarimide skeleton, we aimed to discover novel potent CRBN modulators. In this study, Lipinski's rule and Veber rule, pharmacophore based virtual screening, docking based virtual screening and ADMET screening methods were performed to discover potential CRBN modulators. The antitumor activity of 11 candidates were evaluated by MTS assay. AN7535 showed potent antitumor activity with IC50 = 0.72 µM against HL-60 and IC50 = 1.438 µM against SMMC-7721. AO6355 showed potent antitumor activity with IC50 = 7.469 µM against SMMC-7721. MD simulations and binding free energy calculations suggested that AN7535 and AO6355 could stabilize the CRBN protein and have favorable binding affinity with CRBN protein. Luciferase complementation assay suggested AN7535 could bind to CRBN with IC50 = 215.9 µM.

Discovery of potential novel CRBN modulators by virtual screening and bioassay.

The incidence of malignant tumor with high mortality is increasing yearly. CRBN E3 ubiquitin ligase was proved to be an antitumor target. It was found that thalidomide and its analogs could bind to CRBN E3 ubiquitin ligase and modulate CRBN. CRBN modulators could promote the binding of CRBN to specific target proteins or block the binding of CRBN to some endogenous proteins. In this way, CRBN modulators suppress various tumor cells by modulating the interactions between CRBN and various antitumor target proteins. However, almost all CRBN modulators reported include glutarimide scaffold. Therefore, the aim of this study is to developed novel CRBN modulators. Virtual screening methods and bioassay methods, including structural similarity search, molecular docking, substructure search, antitumor evaluation and apoptosis assay were used to search novel potential CRBN modulators in Specs database. Finally, 15 compounds exhibited strong inhibition activity against A549 cells. Among these active compounds, The IC50 value against A549 of AG6033 was 0.853 ± 0.030 µM. Apoptosis assay demonstrated that AG6033 could promote apoptosis of A549 cells. Further mechanism studies suggested that AG6033 caused remarkable decrease of GSPT1 and IKZF1, the substrates of CRBN, and AG6033 induced cytotoxic effects was CRBN-dependent.

Computational simulation studies on the binding selectivity of Wee1 and Checkpoint kinase 1 by molecular dynamics simulation combined with free energy calculations.

Wee1 kinase and Checkpoint kinase 1 (Chk1) kinase, which are well known to be involved in cancer, are promising targets for cancer therapy. Most of developed Wee1 inhibitors can inhibit activity of Chk1 kinase to different degrees as well. The poor selectivity brought side effects and selective inhibitor is needed. However, the selective mechanisms of Wee1 versus Chk1 are not clear. Therefore, the design of selective Wee1 and Chk1 inhibitors would provide a meaningful starting for the development of anticancer drugs with optimal efficacy. In this study, Wee1 inhibitors with different selectivity over Chk1 were chosen to analyze the selectivity mechanism by means of molecular docking, molecular dynamics simulations and binding free energy calculations. Two key residues of Wee1 kinase and two critical residues of Chk1 were mutated to detect their effect on ligand binding into protein. The results indicated that these residues play a pivotal role in the binding interactions of ligands to receptors through hydrogen bond and hydrophobic interaction with inhibitors. This may provide a better understanding of the selective mechanism of Wee1 and Chk1. It would be beneficial to the discovery and optimization of selective Wee1 and Chk1 inhibitors.Communicated by Ramaswamy H. Sarma.

A Cryptic Plant Terpene Cyclase Producing Unconventional 18- and 14-Membered Macrocyclic C25 and C20 Terpenoids with Immunosuppressive Activity.

A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18-/14-membered sesterterpenoids including five new ones and two 14-membered diterpenoids. These products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Two variants F516A and F516G producing approximately 3.3- and 2.5-fold, respectively, more sesterterpenoids than the wild-type enzyme were engineered. Both 18- and 14-membered ring sesterterpenoids displayed significant inhibitory activity on the IL-2 and IFN-γ production of T cells probably via inhibition of the MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well-trodden territory of plant terpenoid biosynthesis.

An efficient and concise synthesis of a selective small molecule non-peptide inhibitor of cathepsin L: KGP94.

KGP94 is a potent, selective, and competitive inhibitor of the lysosomal endopeptidase enzyme (Cathepsin L) currently in preclinical trials for the treatment of metastatic cancer, which is a leading cause of cancer-associated death. Herein, we report two new synthetic routes for synthesizing the target compound through four consecutive steps, using a Weinreb amide approach starting from a common 3-bromobenzoyl chloride. A key step in the approach is a coupling reaction of a readily available Grignard reagent with amide 4 to produce 6, a previously unreported coupling pattern. These new strategies offer an efficient and alternative approach to synthesis of target compound with an excellent overall yield.

Oligosaccharides from fucosylated glycosaminoglycan prevent breast cancer metastasis in mice by inhibiting heparanase activity and angiogenesis.

The invasion and metastasis of tumor cells are the hallmarks of malignant diseases and the greatest obstacle to overcome. Heparanase-mediated degradation of heparan sulfate (HS) is the critical process for tumor angiogenesis and metastasis, therefore, heparanase become an attractive target for cancer research. Herein, we reported a native fucosylated glycosaminoglycan (nHG) extracted from sea cucumber Holothuria fuscopunctata and a depolymerized nHG (dHG) and its contained oligosaccharides (hs17, hs14, hs11, hs8 and hs5), acting as heparanase inhibitors. nHG and its derivatives have the ability to bind with heparanase directly, leading to significant inhibition of heparanase activity. Moreover, their apparent binding affinity to heparanase was comparable to their inhibitory effect, which was elevated along with the increase of chain length, similar to the effect of heparins. In addition, oligosaccharides inhibited the migration and invasion of 4T1 mammary carcinoma cells and human umbilical vein endothelial cells (HUVECs) and also suppressed tube formation in Matrigel matrix and angiogenesis in the chick chorioallantoic membrane (CAM) assay. In the metastatic mouse model, oligosaccharides exhibited practical antimetastatic effects on 4T1 mammary carcinoma cells. According to the reported anticoagulant activity and the low bleeding tendency of dHG and its oligosaccharides, the use of the oligosaccharides may lead to better effects on tumor patients with thrombosis tendency.

Discovery of novel modulators targeting human TRPC5: Docking-based virtual screening, molecular dynamics simulation and binding affinity predication.

Canonical transient receptor potential channel 5 (TRPC5) plays a key role in the regulation of central nervous system, cardiovascular system, kidney disease, cancer, and could be also involved in liver function, arthritis, diabetes-associated complications and so on. However, evidence of TRPC5 function on cellular or organismic levels is sparse. There is still a need for identifying novel and efficient TRPC5 channel modulators to study TRPC5 function. In this study, based on the hTRPC5 structure obtained by homology modeling and the predicted binding site, we have performed virtual screening of 212,736 compounds from the specs database（http://www.specs.net) to find potential hTRPC5 modulators. Lipinski and Veber rules, ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) and PAINS (Pan Assay Interference structures) filters were used to screen the large database. Further, multi-software combination docking, cluster analysis and interaction analysis were used to select 20 potential active candidates with novel skeleton. 4 Hits, bearing appreciable binding affinity with hTRPC5 were selected for 40ns all-atom molecular dynamics (MD) simulations under explicit water conditions. The MD simulation results suggested that the 4 Hits binding induces a slight structural change and stabilizes the hTRPC5 structure. In addition, decomposition free energy demonstrated that residues TRP434, LEU437, MET438, ALA441, ILE484, ILE487, LEU488, LEU491, LEU515, ILE517, LEU518, LEU521, PHE531, THR607, VAL610, ILE611, VAL615 played the critical role on system stability. 4 Hits, as potential modulators of hTRPC5, may be potential leads to develop effective therapeutics hTRPC5-associated diseases.

Ensemble-based virtual screening in discovering potent inhibitors targeting Von Hippel-Lindau (VHL) E3 ubiquitin ligase.

BACKGROUND: The Von Hippel-Lindau (VHL) E3 ubiquitin ligase, which mediates its substrate hypoxia-inducible factor 1α (HIF-1α) for ubiquitination and subsequent degradation, is an attractive drug target in various diseases, such as anemia, inflammation, neurodegeneration and cancer. Proteolysis targeting chimeras (PROTACs) containing a VHL ligand that can hijack the E3 ligase activity to degrade the target protein has also been studied in academic and in industry areas recently. METHODS: Herein, by developing and optimizing the Bayesian Model, we report ensemble-based virtual screening as an effective strategy to discover potential VHL inhibitors from Specs database. RESULTS: The virtual screening protocol was developed, ten representative molecules were obtained and five compounds were selected for subsequent binding mode analysis to be potent VHL inhibitors.

The discovery, design and synthesis of potent agonists of adenylyl cyclase type 2 by virtual screening combining biological evaluation.

Adenylate cyclases (ACs), play a critical role in the conversion of adenosine triphosphate (ATP) into the second messenger cyclic adenosine monophosphate (cAMP). Studies have indicated that adenylyl cyclase type 2 (AC2) is potential drug target for many diseases, however, up to now, there is no AC2-selective agonist reported. In this research, docking-based virtual screening with the combination of cell-based biological assays have been performed for discovering novel potent and selective AC2 agonists. Virtual screening disclosed a novel hit compound 8 as an AC2 agonist with EC50 value of 8.10 µM on recombinant human hAC2 + HEK293 cells. The SAR (structure activity relationship) based on the derivatives of compound 8 was further explored on recombinant AC2 cells and compound 73 was found to be the most active agonist with the EC50 of 90 nM, which is 160-fold more potent than the reported agonist Forskolin and could selectively activate AC2 to inhibit the expression of Interleukin-6. The discovery of a new class of AC2-selective agonists would provide a novel chemical probe to study the physiological function of AC2.

Discovery of a potent and selective adenylyl cyclase type 8 agonist by docking-based virtual screening.

Adenylyl cyclases (ACs), which are responsible for catalyzing the conversion of adenosine triphosphate (ATP) into the second messenger cyclic adenosine monophosphate (cAMP), play a critical role in cell signal transduction. In this study, a combined approach involving docking-based virtual screening, with the combination of homology modeling followed by an in-vitro, and cell-based biological assay have been performed for discovering a class of novel potent and selective isoform adenylyl cyclase type 8 (AC8) agonist. The computer-aided virtual screening was used to identify fourteen virtual cluster compounds as potential hits which were further subjected to rigorous bioassays. A novel hit compound VHC-7 (ethyl 3-(2,4-dichlorobenzyl)-2-oxoindoline-3-carboxylate) was identified as a highly potent selective AC8 agonist with EC50 value of 0.1052 ± 0.038 µM. Remarkably, the molecule herein reported can be explored further to discover greater number of hit compounds with better pharmacokinetic properties as well as to serve as a promising novel hit agonist of AC8 for the treatment of various central nervous system disorders and its associated diseases.

Ensemble docking-based virtual screening toward identifying inhibitors against Wee1 kinase.

Aim:Wee1 kinase plays a key role in the arrest of G2/M checkpoint that prevents mitotic entry in response to DNA damage. This work is to discover potent Wee1 inhibitors which can be considered valuable. Materials & Methods: Herein, Ensemble docking using multiple crystal structures was considered an effective strategy in the virtual screening. The performance of 17 scoring functions obtained from different docking software was evaluated for molecular docking. Results: Two novel compounds B1 and A2 were identified as Wee1 inhibitors with IC50 values of 10.23 ± 0.505 and 8.72 ± 0.323 µM, respectively. Further cell viability assay demonstrated that the two active compounds exhibited good anticancer activities. Conclusion: This provides a meaningful starting point for further structure optimization to discover more potent Wee1 inhibitors.

Design and synthesis of novel artemisinin derivatives with potent activities against colorectal cancer in vitro and in vivo.

A series of novel derivatives of artemisinin-4-(arylamino)quinazoline have been designed and synthesized, and most of them showing potent in vitro cytotoxic activity against HCT116 and WM-266-4 cell lines. Compound 32 was the most active derivative against HCT116 cell line with an IC50 of 110 nM, and significantly improved the antitumor activity of the parent compounds dihydroartemisinin (DHA) (IC50 = 2.85 µM) and Gefitinib (IC50 = 19.82 µM). In vivo HCT116 xenografts assay showed that compound 32 exhibited potent antitumor activity with obvious tumor growth delay and tumor shrunken after 18 days treatment on xenografted mice, and especially without loss of body weight. Our results indicate that compounds 32 may represent a safe, novel structural lead for developing new chemotherapy of colorectal cancer.

Precise structures and anti-intrinsic tenase complex activity of three fucosylated glycosaminoglycans and their fragments.

Fucosylated glycosaminoglycan (FG), a glycosaminoglycan derivative containing distinct sulfated fucose (FucS) branches, displays potent anticoagulant activity by inhibiting the intrinsic tenase complex (iXase). Herein, AmFG, SvFG and HaFG from three species of sea cucumbers were isolated and depolymerized by ß-eliminative cleavage. Three series of fragments, A1-A4, S1-S4 and H1-H4, were purified from the depolymerized FGs. Based on structural analysis of these fragments, three FGs were deduced as -{→4)-[L-FucS-α(1→3)]-D-GlcA-ß(1→3)-D-GalNAc4S6S-ß(1}n-. The structures differed in sulfation types of FucS, namely, most of FucS in AmFG was Fuc3S4S, but the FucS in SvFG was Fuc2S4S, while the FucS in HaFG was Fuc3S4S, Fuc2S4S and Fuc4S. However, all FucS branches attached to C-3 of GlcA as monosaccharides. Anticoagulant and anti-iXase assays showed the octasaccharide is the minimum fragment for potent anticoagulant activity via anti-iXase irrespective of FucS types. Among FG fragments with same degree of polymerization, oligosaccharides containing Fuc2S4S had more potent anti-iXase activity.

Identification of potential AMPK activator by pharmacophore modeling, molecular docking and QSAR study.

AMP-activated protein kinase (AMPK) plays a major role in maintaining cellular energy homeostasis by sensing and responding to AMP/ADP concentrations relative to ATP. AMPK has attracted widespread attention as a potential therapeutic target for metabolic diseases such as cancer and cardiovascular diseases. The structure-based 3D pharmacophore model was developed based on the training set. The best pharmacophore model Hypo5 was proposed and validated using a decoy set, an external test set. Hypo5, with the correlation coefficient value of 0.936, cost difference value of 112.08 and low RMS value of 1.63, includes a ionizable positive, a hydrogen bond donor, a hydrogen bond acceptor and two hydrophobic features, which showed a high goodness of fit and enrichment factor. Thus it was used as a 3D query to find potential activator from the SPECS Database. Then the ADMET descriptors were used to filter all of 158 screening molecules. The 41 filtering compounds were subsequently subjected to molecular docking and Quantitative structure-activity relationship (QSAR) analysis. Finally, the compound H2 was picked out from those filtering compounds based on the receptor-ligand interaction analysis and the prediction of the QSAR models. And then it was submitted for molecular dynamics (MD) simulations to explore the stability of complex. The result indicates that the candidate could be considered a potential AMPK activator.

Discovery of novel wee1 inhibitors via structure-based virtual screening and biological evaluation.

Wee1 plays a critical role in the arrest of G2/M cell cycle for DNA repair before entering mitosis. Many cancer cells have been identified as overexpression of Wee1. In this research, pharmacophore modeling, molecular docking and molecular dynamics simulation approaches were constructed to identify novel potential Wee1 inhibitors. A compound 8 was found to have a novel skeleton against Wee1 with an IC50 value of 22.32 µM and a Ki value of 13.11 µM. Kinetic assays were employed to evaluate the compound 8 as a competitive inhibitor. Compound 8 was tested against A-549 tumor cell lines with IC50 value of 17.8 µM. To investigate the intermolecular interaction of Wee1 and compound 8, further molecular dynamics simulations were performed. It indicates that the binding mode of compound 8 and reference ligand is similar. The active core scaffold of compound 8 could represent a promising lead compound for studying Wee1 and be used for further structural optimization to design more potent Wee1 inhibitors.