dMXP: A De Novo Small-Molecule 3D Structure Predictor with Graph Attention Networks.

Generating the three-dimensional (3D) structure of small molecules is crucial in both structure- and ligand-based drug design. Structure-based drug design needs bioactive conformations of compounds for lead identification and optimization. Ligand-based drug design techniques, such as 3D shape similarity search, 3D pharmacophore model, 3D-QSAR, etc., all require high-quality small-molecule ligand conformations to obtain reliable results. Although predicting a small molecular bioactive conformer requires information from the receptor, a crystal structure of the molecule is a proper approximation to its bioactive conformer in a specific receptor because the binding pose of a small molecule in its receptor's binding pockets should be energetically close to the crystal structures. This study presents a de novo small molecular structure predictor (dMXP) with graph attention networks based on crystal data derived from the Cambridge Structural Database (CSD) combined with molecular electrostatic information calculated by density-functional theory (DFT). Two featuring strategies (topological and atomic partial change features) were employed to explore the relation between these features and the 3D crystal structure of a small molecule. These features were then assembled to construct the holistic 3D crystal structure of a molecule. Molecular graphs were encoded using a graph attention mechanism to deal with the issues of the inconsistencies of local substructures contributing to the entire molecular structure. The root-mean-square deviation (RMSDs) of approximately 80% dMXP predicted structures and the native binding poses within receptors are less than 2.0 Å.

Identifying eleven new ferroptosis inhibitors as neuroprotective agents from FDA-approved drugs.

With increasing evidence that ferroptosis is associated with diverse neurological disorders, targeting ferroptosis offers a promising avenue for developing effective pharmaceutical agents for neuroprotection. In this study, we identified ferroptosis inhibitors as neuroprotective agents from US Food and Drug Administration (FDA)-approved drugs. 1176 drugs have been screened against erastin-induced ferroptosis in HT22 cells, resulting in 89 ferroptosis inhibitors. Among them, 26 drugs showed significant activity with EC50 below10 µM. The most active ferroptosis inhibitor is lumateperone tosylate at nanomolar level. 11 drugs as ferroptosis inhibitors were not reported previously. Further mechanistic studies revealed that their mechanisms of actions involve free radical scavenging, Fe2+ chelation, and 15-lipoxygenase inhibition. Notably, the active properties of some drugs were firstly revealed here. These ferroptosis inhibitors increase the chemical diversity of ferroptosis inhibitors, and offer new therapeutic possibilities for the treatments of related neurological diseases.

Biochemical and structural characterization of chlorhexidine as an ATP-assisted inhibitor against type 1 methionyl-tRNA synthetase from Gram-positive bacteria.

Methionyl-tRNA synthetase (MetRS) catalyzes the attachment of l-methionine (l-Met) to tRNAMet to generate methionyl-tRNAMet, an essential substrate for protein translation within ribosome. Owing to its indispensable biological function and the structural discrepancies with human counterpart, bacterial MetRS is considered an ideal target for developing antibacterials. Herein, chlorhexidine (CHX) was identified as a potent binder of Staphylococcus aureus MetRS (SaMetRS) through an ATP-aided affinity screening. The co-crystal structure showed that CHX simultaneously occupies the enlarged l-Met pocket (EMP) and the auxiliary pocket (AP) of SaMetRS with its two chlorophenyl groups, while its central hexyl linker swings upwards to interact with some conserved hydrophobic residues. ATP adopts alternative conformations in the active site cavity, and forms ionic bonds and water-mediated hydrogen bonds with CHX. Consistent with this synergistic binding mode, ATP concentration-dependently enhanced the binding affinity of CHX to SaMetRS from 10.2 µM (no ATP) to 0.45 µM (1 mM ATP). While it selectively inhibited two representative type 1 MetRSs from S. aureus and Enterococcus faecalis, CHX did not show significant interactions with three tested type 2 MetRSs, including human cytoplasmic MetRS, in the enzyme inhibition and biophysical binding assays, probably due to the conformational differences between two types of MetRSs at their EMP and AP. Our findings on CHX may inspire the design of MetRS-directed antimicrobials in future.

CDK1 promotes the phosphorylation of KIFC1 to regulate the tumorgenicity of endometrial carcinoma.

OBJECTIVE: This study aims to clarify the mechanical action of cyclin-dependent protein kinase 1 (CDK1) in the development of endometrial carcinoma (EMCA), which may be associated with the phosphorylation of kinesin family member C1 (KIFC1) and further activate the PI3K/AKT pathway. METHODS: The protein and gene expression of CDK1 in EMCA tissues and tumor cell lines were evaluated by western blot, quantitative polymerase chain reaction, and immunohistochemistry staining. Next, Cell Counting Kit-8 and colony formation assay detected cell survival and proliferation. Cell migration and invasion were measured by Transwell assay. Cell apoptosis and cell cycle were tested by flow cytometry. Immunofluorescence staining of γH2AX was used to evaluate DNA damage, respectively. Subsequently, a co-immunoprecipitation assay was used to detect the interaction between CDK1 and KIFC1. The phosphorylated protein of KIFC1 and PI3K/AKT was detected by western blot. Finally, the effect of CDK1 on the tumor formation of EMCA was evaluated in a nude mouse xenograft model. RESULTS: CDK1 was highly expressed in EMCA tumor cell lines and tissues, which contributed to cell survival, proliferation, invasion, and migration, inhibited cell apoptosis, and induced DNA damage of EMCA cells dependent on the phosphorylation of KIFC1. Moreover, the CDK1-KIFC1 axis further activated PI3K/AKT pathway. Finally, CDK1 knockdown repressed tumor formation of EMCA in vivo. CONCLUSION: We report that increased CDK1 promotes tumor progression and identified it as a potential prognostic marker and therapeutic target of EMCA.

Discovery of neo-Clerodane Diterpenoids from Ajuga campylantha as Neuroprotective Agents against Ferroptosis and Neuroinflammation.

Twelve new neo-clerodane diterpenoids, eight undescribed methoxy/ethoxy acetal analogues, and one new nor-iridane monoterpenoid were isolated from Ajuga campylantha. Their structures were elucidated using a combination of spectroscopic data, quantum chemical calculations, and X-ray crystallography. This research reveals the distinctive structural features of A. campylantha diterpenes, including distinct C rings and 4,18-double bonds, distinguishing them from diterpenes of other plants in the Ajuga genus. Compound 2 represents the first example of a 19(5→6)-abeo-clerodane formed through a Wagner-Meerwein rearrangement. The isolated compounds were assessed for their neuroprotective effects against RSL3-induced ferroptosis in HT22 cells and LPS-induced neuroinflammation in BV-2 cells. Notably, compound 7 inhibits ferroptosis (EC50 = 10 µM) with a potentially new mechanism of action. The preliminary structure-activity relationship studies revealed that the furan-clerodane diterpenoids possess potential ferroptosis inhibitory activity, while the lactone-clerodanes do not. This study represents the first report of furan-containing clerodanes within the Ajuga genus, providing fresh insights into the phytochemistry and pharmacological potential of A. campylantha.

Atractylodinol prevents pulmonary fibrosis through inhibiting TGF-ß receptor 1 recycling by stabilizing vimentin.

Pirfenidone and nintedanib are only anti-pulmonary fibrosis (PF) drugs approved by the FDA. However, they are not target specific, and unable to modify the disease status. Therefore, it is still desirable to discover more effective agents against PF. Vimentin (VIM) plays key roles in tissue regeneration and wound healing, but its molecular mechanism remains unknown. In this work, we demonstrated that atractylodinol (ATD) significantly inhibits TGF-ß1-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transition in vitro. ATD also reduces bleomycin-induced lung injury and fibrosis in mice models. Mechanistically, ATD inhibited TGF-ß receptor I recycling by binding to VIM (KD = 454 nM) and inducing the formation of filamentous aggregates. In conclusion, we proved that ATD (derived from Atractylodes lancea) modified PF by targeting VIM and inhibiting the TGF-ß/Smad signaling pathway. Therefore, VIM is a druggable target and ATD is a proper drug candidate against PF. We prove a novel VIM function that TGF-ß receptor I recycling. These findings paved the way to develop new targeted therapeutics against PF.

An asymmetric structure of bacterial TrpRS supports the half-of-the-sites catalytic mechanism and facilitates antimicrobial screening.

Tryptophanyl-tRNA synthetase (TrpRS) links tryptophan to tRNATrp, thereby playing an indispensable role in protein translation. Unlike most class I aminoacyl-tRNA synthetases (AARSs), TrpRS functions as a homodimer. Herein, we captured an 'open-closed' asymmetric structure of Escherichia coli TrpRS (EcTrpRS) with one active site occupied by a copurified intermediate product and the other remaining empty, providing structural evidence for the long-discussed half-of-the-sites reactivity of bacterial TrpRS. In contrast to its human counterpart, bacterial TrpRS may rely on this asymmetric conformation to functionally bind with substrate tRNA. As this asymmetric conformation is probably a dominant form of TrpRS purified from bacterial cells, we performed fragment screening against asymmetric EcTrpRS to support antibacterial discovery. Nineteen fragment hits were identified, and 8 of them were successfully cocrystallized with EcTrpRS. While a fragment named niraparib bound to the L-Trp binding site of the 'open' subunit, the other 7 fragments all bound to an unprecedented pocket at the interface between two TrpRS subunits. Binding of these fragments relies on residues specific to bacterial TrpRS, avoiding undesired interactions with human TrpRS. These findings improve our understanding of the catalytic mechanism of this important enzyme and will also facilitate the discovery of bacterial TrpRS inhibitors with therapeutic potential.

The binding mode of orphan glycyl-tRNA synthetase with tRNA supports the synthetase classification and reveals large domain movements.

As a class of essential enzymes in protein translation, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are organized into two classes of 10 enzymes each, based on two conserved active site architectures. The (αß)2 glycyl-tRNA synthetase (GlyRS) in many bacteria is an orphan aaRS whose sequence and unprecedented X-shaped structure are distinct from those of all other aaRSs, including many other bacterial and all eukaryotic GlyRSs. Here, we report a cocrystal structure to elucidate how the orphan GlyRS kingdom specifically recognizes its substrate tRNA. This structure is sharply different from those of other aaRS-tRNA complexes but conforms to the clash-free, cross-class aaRS-tRNA docking found with conventional structures and reinforces the class-reconstruction paradigm. In addition, noteworthy, the X shape of orphan GlyRS is condensed with the largest known spatial rearrangement needed by aaRSs to capture tRNAs, which suggests potential nonactive site targets for aaRS-directed antibiotics, instead of less differentiated hard-to-drug active site locations.

Chemical constituents of industrial hemp roots and their anti-inflammatory activities.

OBJECTIVE: Although the chemical constituents of the aerial parts of Cannabis have been extensively studied, phytochemicals of Cannabis roots are not well characterized. Herein, we investigated the chemical constituents of industrial hemp (Cannabis sativa L.) roots and evaluated the anti-inflammatory activities of phytochemicals isolated from the hemp roots extract. METHODS: An ethyl acetate extract of hemp roots was subjected to a combination of chromatographic columns to isolate phytochemicals. The chemical structures of the isolates were elucidated based on spectroscopic analyses (by nuclear magnetic resonance and mass spectrometry). The anti-inflammatory effects of phytochemicals from hemp roots were evaluated in an anti-inflammasome assay using human monocyte THP-1 cells. RESULTS: Phytochemical investigation of hemp roots extract led to the identification of 32 structurally diverse compounds including six cannabinoids (1-6), three phytosterols (26-28), four triterpenoids (22-25), five lignans (17-21), and 10 hydroxyl contained compounds (7-16), three fatty acids (29-31), and an unsaturated chain hydrocarbon (32). Compounds 14-21, 23, 27, and 32 were identified from the Cannabis species for the first time. Cannabinoids (1-5) reduced the level of cytokine tumor necrosis-alpha (by 38.2, 58.4, 47.7, 52.2, and 56.1%, respectively) and 2 and 5 also decreased the interleukin-1ß production (by 42.2 and 92.4%, respectively) in a cell-based inflammasome model. In addition, non-cannabinoids including 11, 13, 20, 25, 29, and 32 also showed selective inhibition of interleukin-1ß production (by 23.7, 22.5, 25.6, 78.0, 24.1, 46.6, and 25.4%, respectively) in THP-1 cells. CONCLUSION: The phytochemical constituent of a hemp roots extract was characterized and compounds from hemp roots exerted promising anti-inflammatory effects.

Discovery of Spiro[pyrrolidine-3,3'-oxindole] LXRß Agonists for the Treatment of Osteoporosis.

Osteoclasts have an additional demand for cholesterol compared to normal cells. Liver X receptors (LXRs) are famous for regulation of lipid and cholesterol metabolism. Therefore, we propose that the LXR ß agonist can regulate the cholesterol balance in osteoclasts to inhibit osteoclast differentiation. Here, we designed and synthesized a novel LXRß agonist by introduction of the privileged fragments from anti-osteoporosis agents to the spiro[pyrrolidine-3,3'-oxindole] scaffold which is a novel scaffold of LXR agonists in our previous research. As a result, seven LXRß agonists inhibited osteoclastogenesis with IC50 values ranging from 0.078 to 0.36 µM. Especially, the most potent LXRß agonist B9 significantly inhibited RANKL-induced osteoclast differentiation and bone resorption in vitro and in vivo. Furthermore, B9 selectively activated LXRß to promote intracellular cholesterol exclusion in osteoclasts and reduce extracellular cholesterol uptake and thereby inhibited osteoclast production. This study provides a new strategy to develop LXRß agonists for osteoporosis.

Discovery and optimization of 2-(trifluoromethyl)benzimidazole derivatives as novel ferroptosis inducers in vitro and in vivo.

Ferroptosis is implicated in diverse human diseases. Ferroptosis inducers hold great potential for cancer therapy. The existing ferroptosis inducers, however, lack structural diversity, and only a few of them are suitable for in vivo applications. Herein, by phenotypic screenings, we discovered a new ferroptosis inducer FA-S, a 2-(trifluoromethyl)benzimidazole derivative, from which a series of its analogs were designed and synthesized to improve the activity. This produced the most potent compound FA16 with single-digit micromolar activity of ferroptosis induction and satisfactory metabolic stability. Further studies demonstrated that FA16 induced ferroptosis by inhibiting cystine/glutamate antiporter (system Xc-). It is noteworthy that analogue FA16 has more favorable metabolic stability than the classic system Xc- inhibitor erastin, which is not suitable for in vivo studies. FA16 significantly inhibited tumor growth in the HepG2 xenograft model by inducing ferroptosis. This work provides new ferroptosis inducers with a novel scaffold, but also a promising lead for hepatocellular carcinoma treatment. Our work reveals a suitable in vivo ferroptosis-inducing tool to explore the mechanisms underlying ferroptosis and the relevance of ferroptosis to pathogenesis of human diseases.

Release of hepatitis B virions is positively regulated by glucose-regulated protein 78 through direct interaction with preS1.

In this study, we investigated the mechanism of hepatitis B virus (HBV)-enveloped particle release. Specifically, we used preS1 as a bait protein to screen host proteins using mass spectroscopy, with the results of immunofluorescence, western blot, co-immunoprecipitation, isothermal titration calorimetry, and pull-down assays identifying glucose-regulated protein (GRP)78 as a specific target for preS1 binding. We employed transcriptome sequencing, enzyme-linked immunosorbent assays, and particle gel assays to investigate the mechanism of GRP78-mediated positive regulation of HBV-enveloped particle release. Additionally, we performed phage-display, surface plasmon resonance, and molecular-docking assays to assess peptides inhibiting enveloped-particle release. We found that HBV upregulated GRP78 expression in liver cell lines and the serum of patients with chronic hepatitis B. Furthermore, GRP78 promoted the release of HBV-enveloped particles in vitro and in vivo within an HBV transgenic mouse model. Moreover, we identified interactions of preS1 peptides with GRP78 via hydrogen bonding and hydrophobic interactions, which effectively inhibited its interaction with HBV-enveloped particles and their subsequent release. These findings provide novel insights regarding HBV virion release, and demonstrated that GRP78 interacted with preS1 to positively regulate the release of HBV-enveloped particles, suggesting GRP78 as a potential therapeutic target for inhibiting HBV infection.

Structure-Guided Design of Halofuginone Derivatives as ATP-Aided Inhibitors Against Bacterial Prolyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are promising antimicrobial targets due to their essential roles in protein translation, and expanding their inhibitory mechanisms will provide new opportunities for drug discovery. We report here that halofuginone (HF), an herb-derived medicine, moderately inhibits prolyl-tRNA synthetases (ProRSs) from various pathogenic bacteria. A cocrystal structure of Staphylococcus aureus ProRS (SaProRS) with HF and an ATP analog was determined, which guided the design of new HF analogs. Compound 3 potently inhibited SaProRS at IC50 = 0.18 µM and Kd = 30.3 nM and showed antibacterial activities with an MIC of 1-4 µg/mL in vitro. The bacterial drug resistance to 3 only developed at a rate similar to or slower than those of clinically used antibiotics in vitro. Our study indicates that the scaffold and ATP-aided inhibitory mechanism of HF could apply to bacterial ProRS and also provides a chemical validation for using bacterial ProRS as an antibacterial target.

Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis.

One of the major challenges for discovering protein-protein interaction inhibitors is identifying selective and druggable binding sites at the protein surface. Here, we report an approach to identify a small molecular binding site to selectively inhibit the interaction of soluble RANKL and RANK for designing anti-osteoporosis drugs without undesirable immunosuppressive effects. Through molecular dynamic simulations, we discovered a binding site that allows a small molecule to selectively interrupt soluble RANKL-RANK interaction and without interfering with the membrane RANKL-RANK interaction. We describe a highly potent inhibitor, S3-15, and demonstrate its specificity to inhibit the soluble RANKL-RANK interaction with in vitro and in vivo studies. S3-15 exhibits anti-osteoporotic effects without causing immunosuppression. Through in silico and in vitro experiments we further confirm the binding model of S3-15 and soluble RANKL. This work might inspire structure-based drug discovery for targeting protein-protein interactions.

Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease.

Oridonin Inhibits SARS-CoV-2 Oridonin, a natural product extracted from Rabdosia rubescens, possesses a wide range of pharmacological properties, including anti-inflammatory, anti-cancer, anti-microbial, neuroprotection, immunoregulation, etc. In article number 2100124, Baisen Zhong, Litao Sun, and co-workers demonstrate that Oridonin targets the SARS-CoV-2 3CL protease by covalently binding to cysteine145 in its active pocket to exert an anti-SARS-CoV-2 effect, which provides a novel candidate for the treatment of COVID-19.

Ferroptosis Inhibitory Aromatic Abietane Diterpenoids from Ajuga decumbens and Structural Revision of Two 3,4-Epoxy Group-Containing Abietanes.

Two new 3,4-epoxy group-containing abietane diterpenoids (1 and 2), together with five known diterpenoids (3-7), were isolated from Ajuga decumbens. Their structures were elucidated by spectroscopic data analysis, NMR calculations, and X-ray diffraction experiments. The structures of two known abietane diterpenoids were revised based on NMR calculations and X-ray diffraction data. Notably, compound 4 specifically inhibited RSL3-induced ferroptosis with an EC50 of 56 nM by antioxidation. Moreover, 4 significantly decreased RSL3-induced lipid and cytosolic ROS accumulation and ferroptosis marker gene PTGS2 mRNA expression. This work reports the most potent natural inhibitor against ferroptosis found so far.

Diverse polyacetylenes from Atractylodes chinensis and their anti-osteoclastogenesis activity.

The phytochemical investigation on Atractylodes chinensis afforded 15 polyacetylenes 1-15 and one meroterpenoid 16. Of the 16 isolates, compounds 4 and 9 are new ones, and compounds 8 and 16 are first reported from nature. In addition, the relative configuration of 1 and the available NMR data of compounds 1, 8, and 16 were first provided. Their structures were elucidated by extensive analysis of MS, UV, IR, and NMR spectroscopic data. Besides, all isolated compounds were evaluated for their effects on RANKL-induced osteoclastogenesis in BMMs. Among them, polyacetylenes 12-14 showed potent inhibitory activity with IC50 values of 0.67 ± 0.05 µM, 0.72 ± 0.31 µM, and 2.40 ± 0.41 µM, respectively. The current work demonstrates the polyacetylenes are the main active constituents of A. chinensis against osteoclastogenesis.

Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease.

The current COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an enormous threat to public health. The SARS-CoV-2 3C-like protease (3CLpro), which is critical for viral replication and transcription, has been recognized as an ideal drug target. Herein, it is identified that three herbal compounds, Salvianolic acid A (SAA), (-)-Epigallocatechin gallate (EGCG), and Oridonin, directly inhibit the activity of SARS-CoV-2 3CLpro. Further, blocking SARS-CoV-2 infectivity by Oridonin is confirmed in cell-based experiments. By solving the crystal structure of 3CLpro in complex with Oridonin and comparing it to that of other ligands with 3CLpro, it is identified that Oridonin binds at the 3CLpro catalytic site by forming a C-S covalent bond, which is confirmed by mass spectrometry and kinetic study, blocking substrate binding through a nonpeptidomimetic covalent binding mode. Thus, Oridonin is a novel candidate to develop a new antiviral treatment for COVID-19.

Saucerneol attenuates nasopharyngeal carcinoma cells proliferation and metastasis through selectively targeting Grp94.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is highly prevalent in southern China. The remote metastasis of advanced NPC requires chemotherapeutic treatments to reduce the mortality. Our previous work revealed that saucerneol (SN) showed cytotoxicity against several nasopharyngeal carcinoma (NPC) cells. This work aims to investigate the effect of SN in NPC growth and exploring the mechanism of action. STUDY DESIGN: Applying in vivo study, in vitro study and in silico study to indicate the mechanism of SN in inhibiting NPC growth. METHODS: Saucerneol (SN) toxicity was measured with MTT assay. NPC proliferation was measured with EdU and colony formation assays, cell cycle was detected with flow cytometry. NPC migration and invasion were measured with scratch assay and matrigel transwell method. Further, human NPC xenograft tumor models were established in nude mice to evaluate the therapeutic efficacy of SN in vivo. Toxicological analysis was performed on H&E staining and IHC. Quantitative real-time PCR and Western blot analyses were used to evaluate the expression levels of key molecules in PI3K/AKT/mTOR, MAPK, NF-κB, and HIF-1α signal pathways. Target predicting was conducted using computational method, and target identification was carried out by ATPase assay and TSA. RESULTS: SN, a potent NPC inhibitor that was previously isolated from Saururus chinensis in our lab, is proven to inhibit the proliferation and metastasis of HONE1 cell lines and inhibit the growth of human NPC xenografts in nude mice. Moreover, we further articulate the molecular mechanism of action for SN and, reveal that SN promotes the expression of cell cycle-dependent kinase inhibitory protein p21 Waf1/Cip1 through targeting Grp94 and then inhibiting PI3K/AKT signaling pathway as well as up-regulating p53 to disrupt the progression of HONE1 cells. CONCLUSION: SN significantly inhibits NPC cells proliferation and metastasis in vitro and in vivo via selectively inhibit Grp94 and then blocking PI3K/AKT/mTOR/HIF-1α signaling pathway. This study firstly provides a novel selective Grp94 inhibitor as a NPC candidate.