Structural and signaling mechanisms of TAAR1 enabled preferential agonist design.

Trace amine-associated receptor 1 (TAAR1) senses a spectrum of endogenous amine-containing metabolites (EAMs) to mediate diverse psychological functions and is useful for schizophrenia treatment without the side effects of catalepsy. Here, we systematically profiled the signaling properties of TAAR1 activation and present nine structures of TAAR1-Gs/Gq in complex with EAMs, clinical drugs, and synthetic compounds. These structures not only revealed the primary amine recognition pocket (PARP) harboring the conserved acidic D3.32 for conserved amine recognition and "twin" toggle switch for receptor activation but also elucidated that targeting specific residues in the second binding pocket (SBP) allowed modulation of signaling preference. In addition to traditional drug-induced Gs signaling, Gq activation by EAM or synthetic compounds is beneficial to schizophrenia treatment. Our results provided a structural and signaling framework for molecular recognition by TAAR1, which afforded structural templates and signal clues for TAAR1-targeted candidate compounds design.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

Structure of GPR101-Gs enables identification of ligands with rejuvenating potential.

GPR101 is an orphan G protein-coupled receptor actively participating in energy homeostasis. Here we report the cryo-electron microscopy structure of GPR101 constitutively coupled to Gs heterotrimer, which reveals unique features of GPR101, including the interaction of extracellular loop 2 within the 7TM bundle, a hydrophobic chain packing-mediated activation mechanism and the structural basis of disease-related mutants. Importantly, a side pocket is identified in GPR101 that facilitates in silico screening to identify four small-molecule agonists, including AA-14. The structure of AA-14-GPR101-Gs provides direct evidence of the AA-14 binding at the side pocket. Functionally, AA-14 partially restores the functions of GH/IGF-1 axis and exhibits several rejuvenating effects in wild-type mice, which are abrogated in Gpr101-deficient mice. In summary, we provide a structural basis for the constitutive activity of GPR101. The structure-facilitated identification of GPR101 agonists and functional analysis suggest that targeting this orphan receptor has rejuvenating potential.

Azaphilone pigments from the marine-derived Penicillium sclerotium UJNMF 0503 and their neuroprotective potential against H2O2-induced cell apoptosis through modulating PI3K/Akt pathway.

Azaphilones represent a particular group of fascinating pigments from fungal source, with easier industrialization and lower cost than the traditional plant-derived pigments, and they also display a wide range of pharmacological activities. Herein, 28 azaphilone analogs, including 12 new ones, were obtained from the fermentation culture of a marine fungus Penicillium sclerotium UJNMF 0503. Their structures were elucidated by MS, NMR and ECD analyses, together with NMR and ECD calculations and biogenetic considerations. Among them, compounds 1 and 2 feature an unusual natural benzo[d][1,3]dioxepine ring embedded with an orthoformate unit, while 3 and 4 represent the first azaphilone examples incorporating a novel rearranged 5/6 bicyclic core and a tetrahydropyran ring on the side chain, respectively. Our bioassays revealed that half of the isolates exhibited neuroprotective potential against H2O2-induced injury on RSC96 cells, while compound 13 displayed the best rescuing capacity toward the cell viability by blocking cellular apoptosis, which was likely achieved by upregulating the PI3K/Akt signaling pathway.

Design, synthesis and biological evaluation of novel (E)-2-benzylidene-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)hydrazine-1-carboxamide derivatives as α-glucosidase inhibitors.

In this present study, a series of novel (E)-2-benzylidene-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)hydrazine-1-carboxamide derivatives against α-glucosidase were designed and synthesized, and their biological activities were evaluated in vitro and in vivo. Most of the designed analogues exhibited better inhibitory activity than the marketed acarbose, especially the most potent compound 7 with an IC50 value of 9.26 ± 1.84 µM. The direct binding of 7 and 8 with α-glucosidase was confirmed by fluorescence quenching experiments, and the kinetic and molecular docking studies revealed that 7 and 8 inhibited α-glucosidase in a non-competitive manner. Cytotoxicity bioassay indicated compounds 7 and 8 were non-toxic towards LO2 and HepG2 at 100 µM. Furthermore, both compounds were demonstrated to have in vivo hypoglycemic activity by reducing the blood glucose levels in sucrose-treated rats.

Nosiheptide analogues as potential antibacterial agents via dehydroalanine region modifications: Semi-synthesis, antimicrobial activity and molecular docking study.

The frequent and inappropriate use of antibiotics aggravate the variation and evolution of multidrug-resistant bacteria, posing a serious threat to public health. Nosiheptide (NOS) has excellent lethality against a variety of Gram-positive bacteria, however the physical and chemical drawbacks hamper its routine application in clinical practice. In this study, by using NOS as the starting material, a total of 15 NOS analogues (2a-4e) were semi-synthesized via its dehydroalanine residue reacting with monosubstituted anilines. In vitro antimicrobial susceptibilities of NOS and its analogues against two methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) clinical isolates were determined by broth microdilution assay to determine the minimum inhibitory concentration (MIC). Antimicrobial susceptibility testing data shown that most of the NOS analogues had a better antibacterial effect than the parent compound, with compound 3c exhibiting the highest antibacterial activity against VRE (MIC = 0.0078 mg/L) and MRSA (MIC < 0.0039 mg/L). Molecular docking of synthetic compounds was also performed to verify the binding interactions of NOS analogues with the target. Our data indicated that compound 3c possesses stronger and more complex intermolecular force than other analogues, which is consistent with the results of the biological activity evaluation. Overall, this study identified a number of potential antibacterial NOS analogues that could act as potent therapeutic agents for multidrug-resistant bacterial infections.

Rational design and synthesis of 6-aryl-6H-benzo[c]chromenes as non-steroidal progesterone receptor antagonists for use against cancers.

Progesterone receptor (PR) antagonists have been found to be effective for treating certain human cancers. However, the steroidal structure of PR antagonists could bind to other hormone receptors, thus leading to serious side effects. On the other hand, non-steroidal PR antagonists have rarely been evaluated for their anti-cancer efficacy. Therefore, identifying novel non-steroidal PR antagonists possessing potent anti-cancer efficacy would be an attractive project to pursue. In this study, we presented a new metal-free oxidative CH arylation method to rapidly synthesize a series of 6-aryl-6H-benzo[c]chromene derivatives. Multiple cancer cell lines were used for their anti-cancer activity screening. An extensive analysis of structure-activity relationships (SAR) of the derivatives revealed that compounds 32 and 34 markedly inhibited the proliferation of MCF-7 cells with IC50 values of 6.32 ± 0.52 µM and 5.71 ± 0.49 µM, respectively. Further investigation indicated that derivatives 32 and 34 could elevate the expression of p21 and decrease the expressions of CDK4 and cyclin D1, leading to cell cycle arrest at G0/G1 phase. In addition, derivatives 32 and 34 could induce apoptosis of MCF-7 cells in both dose- and time-dependent manners by activation of p53 pathway, i.e., activation of Cleaved Caspase-3, p53 and P-p53 as well as elevation of the Bax/Bcl-2 ratio. Docking of derivatives 32 and 34 into a PR homology model exhibited potent PR antagonistic activity indicating the 6-aryl-6H-benzo[c]chromene derivatives are promising PR antagonists. We envisioned that derivatives 32 and 34 might be potential anti-cancer drug candidates as novel therapeutic treatment for breast cancer.

Diterpenoid glucosides with cystathionine γ-lyase inhibitory activity from Tinospora sinensis.

Fifteen previously undescribed nor-clerodane diterpenoid glucosides tinosinesides C-Q (1-15), along with four known analogues (16-19), were isolated from the stems of Tinospora sinensis. The structures of the new compounds were elucidated by spectroscopic means, and their absolute configurations were established on the basis of time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculation and chemical methods. All the isolates were evaluated for their inhibitory effects on cystathionine γ-lyase (CSE), a natural enzyme responsible for the synthesis of H2S. Compounds 4 and 5 represent rare examples of natural CSE inhibitors and the possible binding mode to CSE was further probed by molecular docking experiment.

Discovery of new 2-phenyl-1H-benzo[d]imidazole core-based potent α-glucosidase inhibitors: Synthesis, kinetic study, molecular docking, and in vivo anti-hyperglycemic evaluation.

In the present study, a series of 2-phenyl-1H-benzo[d]imidazole-based α-glucosidase inhibitors were synthesized and evaluated for their in vitro and in vivo anti-diabetic potential. Screening of an in-house library revealed a moderated α-glucosidase inhibitor, 6a with 3-(1H-benzo[d]imidazol-2-yl)aniline core, and then the structural optimization was performed to obtain more efficient derivatives. Most of these derivatives showed increased activity than 6a, and the most promising inhibitors were found to be compounds 15o and 22d with IC50 values of 2.09 ± 0.04 and 0.71 ± 0.02 µM, respectively. Fluorescence quenching experiment confirmed the direct binding of compounds 15o and 22d with α-glucosidase. Kinetic study revealed that both compounds were non-competitive inhibitors, that was consistent with the result of molecular docking studies where they located at the allosteric site of the enzyme. Cell viability evaluation demonstrated the non-cytotoxicity of 15o and 22d against LO2 cells. Furthermore, the in vivo pharmacodynamic study revealed that compound 15o showed significant hypoglycemic activity and improved oral sucrose tolerance, comparable to the positive control acarbose.

Novel tetrahydrobenzo[b]thiophen-2-yl)urea derivatives as novel α-glucosidase inhibitors: Synthesis, kinetics study, molecular docking, and in vivo anti-hyperglycemic evaluation.

α-Glucosidase inhibitors, which can inhibit the digestion of carbohydrates into glucose, are one of important groups of anti-type 2 diabetic drugs. In the present study, we report our effort on the discovery and optimization of α-glucosidase inhibitors with tetrahydrobenzo[b]thiophen-2-yl)urea core. Screening of an in-house library revealed a moderated α-glucosidase inhibitors, 5a, and then the following structural optimization was performed to obtain more efficient derivatives. Most of these derivatives showed increased inhibitory activity against α-glucosidase than the parental compound 5a (IC50 of 26.71 ± 1.80 µM) and the positive control acarbose (IC50 of 258.53 ± 1.27 µM). Among them, compounds 8r (IC50 = 0.59 ± 0.02 µM) and 8s (IC50 = 0.65 ± 0.03 µM) were the most potent inhibitors, and showed selectivity over α-amylase. The direct binding of both compounds with α-glucosidase was confirmed by fluorescence quenching experiments. Kinetics study revealed that these compounds were non-competitive inhibitors, which was consistent with the molecular docking results that compounds 8r and 8s showed high preference to bind to the allosteric site instead of the active site of α-glucosidase. In addition, compounds 8r and 8s were not toxic (IC50 > 100 µM) towards LO2 and HepG2 cells. Finally, the in vivo anti-hyperglycaemic activity assay results indicated that compounds 8r could significantly decrease the level of plasma glucose and improve glucose tolerance in SD rats treated with sucrose. The present study provided the tetrahydrobenzo[b]thiophen-2-yl)urea chemotype for developing novel α-glucosidase inhibitors against type 2 diabetes.

A New Fusicoccane-Type Norditerpene and a New Indone from the Marine-Derived Fungus Aspergillus aculeatinus WHUF0198.

A new norditerpene named aculeaterpene A (1) and a new indone named aculeaindone A (2), along with eight known compounds 3-10 were isolated from the culture extract of Aspergillus aculeatinus WHUF0198. The structural characterization of compounds 1 and 2 were performed by spectroscopic analysis, including 1D and 2D NMR and HR-ESI-MS experiments, whereas the absolute configurations were determined by comparing their experimental or calculated ECD spectra. Compound 1 was the first report of fusicoccane-based norditerpene, in which the C-20 was degraded and tured into a hydroxy group.

Identification and Assessments of Novel and Potent Small-Molecule Inhibitors of EED-EZH2 Interaction of Polycomb Repressive Complex 2 by Computational Methods and Biological Evaluations.

Polycomb repressive complex 2 (PRC2) is an attractive drug target for anti-cancer treatment. Among the three core subunits (EZH2, EED and SUZ12) of PRC2, EZH2 is the catalytic subunit that methylates histone H3 lysine 27 (H3K27), while EED is the regulatory subunit. Besides the small-molecule inhibitors of EZH2, those targeting the protein-protein interaction (PPI) between EZH2 and EED have also been reported. Here, for the first time, we have identified the key residues that contributed most to the EED-EZH2 binding affinity by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations based on the 200 ns molecular dynamics simulation. Moreover, we report the identification of two novel and potent small-molecule inhibitors (35 and 49) of EZH2-EED interaction (bottom interaction surface) by virtual screening and biological evaluations. Binding modes of the two identified molecules with EED were probed by molecular docking. Additionally, 35 and 49 displayed cellular antiproliferative activity against diffuse large B-cell lymphoma (DLBCL) cancer cell line Toledo whose cell growth was driven by aberrant PRC2 activity. Our findings have provided structural insights for the design of novel EZH2-EED interaction inhibitors to regulate the activity of PRC2 complex.

The Flexible Loop is a New Sweetness Determinant Site of the Sweet-Tasting Protein: Characterization of Novel Sweeter Mutants of the Single-Chain Monellin (MNEI).

The single-chain monellin (MNEI) displays same sweet potency as the natural monellin protein. To identify critical residues determining its sweetness, residues located at the loops region were selected for mutagenesis analysis. Mutations of positive-charge residues R31, R53, and R82 consistently led to obvious decrease of sweetness, whereas mutations of negative-charge residues resulted in variable sweet potency. Of note, the E50N mutant in the loop region linking the 2 natural chains showed significantly increased sweetness. Mutations of this residue to M or K led to similar effects, in accordance with the so-called wedge model for explanation of the sweet protein-receptor interaction. Homology modeling was carried out with the firstly reported crystal structure of sweet taste receptor (from medaka fish) as the template, and molecular docking and dynamics simulations suggested that flexible conformations of specific residues located in the loops region play essential roles for the interaction with the receptor and the sweetness of the protein. Moreover, obvious additive effects were found for the sweetness as 2 double-site mutants (E50N/Y65R and E2N/E50N) displayed increased sweetness than their single-site mutants. Our results revealed the flexible loop L23 linking the 2 natural chains as a novel sweetness determinant site of the sweet protein monellin and raised a series of new sweeter mutants, which could provide helpful guidance for molecular designing the sweet-tasting proteins.

Development of hydroxamate-based histone deacetylase inhibitors containing 1,2,4-oxadiazole moiety core with antitumor activities.

Histone deacetylases (HDACs) has proved to be promising target for the development of antitumor drugs. In this study, we reported the design and synthesis of a class of novel hydroxamate-based bis-substituted aromatic amide HDAC inhibitors with 1,2,4-oxadiazole core. Most newly synthesized compounds displayed excellent HDAC1 inhibitory effects and significant anti-proliferative activities. Among them, compounds 11a and 11c increased acetylation of histone H3 and H4 in dose-dependent manner. Furthermore, 11a and 11c remarkably induced apoptosis in HepG2 cancer cells. Finally, the high potency of compound 11a was rationalized by molecular docking studies.

Rational Design, synthesis and biological evaluation of novel triazole derivatives as potent and selective PRMT5 inhibitors with antitumor activity.

Protein arginine methyltransferase 5 (PRMT5) is responsible for the mono-methylation and symmetric dimethylation of arginine, and its expression level and methyl transferring activity have been demonstrated to have a close relationship with tumorigenesis, development and poor clinical outcomes of human cancers. Two PRMT5 small molecule inhibitors (GSK3326595 and JNJ-64619178) have been put forward into clinical trials. Here, we describe the design, synthesis and biological evaluation of a series of novel, potent and selective PRMT5 inhibitors with antiproliferative activity against Z-138 mantle cell lymphoma cell line. Among them, compound C_4 exhibited the highest potency with enzymatic and cellular level IC50 values of 0.72 and 2.6 µM, respectively, and displayed more than 270-fold selectivity toward PRMT5 over several other isoenzymes (PRMT1, PRMT4 and PRMT6). Besides, C_4 demonstrated obvious cell apoptotic effect while reduced the cellular symmetric arginine dimethylation levels of SmD3 protein. The potency, small size, and synthetic accessibility of this compound class provide promising hit scaffold for medicinal chemists to further explore this series of PRMT5 inhibitors.

Discovery of new multifunctional selective acetylcholinesterase inhibitors: structure-based virtual screening and biological evaluation.

Although the mechanism of Alzheimer's disease (AD) is still not fully understood, the development of multifunctional AChE inhibitors remains a research focus for AD treatment. In this study, 48 AChE candidate inhibitors were picked out from SPECS database through a pharmacophore- and molecular docking-based virtual screening. The biological evaluation results indicated that four compounds 7, 29, 41 and 48 with different scaffolds exhibited potent and selective AChE inhibitory activity, with the best IC50 value of 1.62 ± 0.11 µM obtained for 48. Then their mechanism of action, the inhibition on Aß aggregation, neurotoxicity, and neuroprotective activity against Aß-induced nerve cell injury were well studied. The binding mode of 48 with AChE was also proposed. The present bioassay results indicated that these multifunctional AChE inhibitors were worth for further structural derivatization to make them the anti-AD lead compounds.

Octahydro-Protoberberine and Protoemetine-Type Alkaloids from the Stems of Alangium salviifolium and Their Cytotoxicity.

Two octahydro-protoberberine alkaloids, alangiifoliumines A (1) and B (2), and two new protoemetine derivatives, alangiifoliumines C (3) and D (4), together with 11 known compounds, have been isolated from the stems of Alangium salviifolium. While the structures of these compounds were elucidated by spectroscopic methods, the absolute configurations of the new alkaloids were determined by conformational analysis and time-dependent density functional theory-electronic circular dichroism spectra calculations on selected stereoisomers. Compounds 1 and 2 represent the first 5,8,8a,9,12,12a,13,13a-octahydro-protoberberine derivatives, in which the aromatic ring D was reduced to cyclohexene. All the compounds isolated were evaluated for their cytotoxic activity against three human cancer cell lines: A-549, HeLa, and SKOV-3. Alkaloids 1, 3, and 6-14 exhibited inhibitory effects against all three human cancer cell lines, with half-maximal inhibitory concentration (IC50) values in the range of 3 nM to 9.4 µM.

Germacrane-type sesquiterpenoids with cytotoxic activity from Sigesbeckia orientalis.

Eleven new highly oxygenated germacrane-type sesquiterpenoids (1-11) and 16 known analogues (12-27) were isolated from the aerial parts of Sigesbeckia orientalis. Their structures, including absolute configurations, were determined by comprehensive spectroscopic methods especially NMR and ECD analyses. Compounds 13, 21 and 23 possessing an 8-methacryloxy group showed stronger in vitro cytotoxicity against human A549 and MDA-MB-231 cancer cell lines than other co-metabolites, with IC50 values ranging from 6.02 to 10.77 µM comparable to the positive control adriamycin.

Molecular-docking-guided design and synthesis of new IAA-tacrine hybrids as multifunctional AChE/BChE inhibitors.

A series of new indole-3-acetic acid (IAA)-tacrine hybrids as dual acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) inhibitors were designed and prepared based on the molecular docking mode of AChE with an IAA derivative (1a), a moderate AChE inhibitor identified by screening our compound library for anti-Alzheimer's disease (AD) drug leads. The enzyme assay results revealed that some hybrids, e.g. 5d and 5e, displayed potent dual in vitro inhibitory activities against AChE/BChE with IC50 values in low nanomolar range. Molecular modeling studies in tandem with kinetic analysis suggest that these hybrids target both catalytic active site and peripheral anionic site of cholinesterase (ChE). Molecular dynamic simulations and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) calculations indicate that 5e has more potent binding affinity than hit 1a, which may explain the stronger inhibitory effect of 5e on AChE. Furthermore, their predicted pharmacokinetic properties and in vitro influences on mouse brain neural network electrical activity were discussed. Taken together, compound 5e can be highlighted as a lead compound worthy of further optimization for designing new anti-AD drugs.

Discovery of Novel PRMT5 Inhibitors by Virtual Screening and Biological Evaluations.

As an important epigenetics related enzyme, protein arginine methyltransferase 5 (PRMT5) has been confirmed as an anticancer therapeutic target in recent years. Among all the reported PRMT5 inhibitors, two small molecules (GSK-3326595 and JNJ-64619178) are currently being assessed in clinical trial. In this study, 40 PRMT5 inhibitor candidates were purchased from SPECS database supplier according to the pharmacophore and molecular docking based virtual screening results. Alpha linked immunosorbent assay (LISA) methylation assay was performed to test their inhibitory activity against PRMT5. The in vitro enzymatic assay results indicated that four compounds (2, 4, 10 and 37) showed PRMT5 inhibitory activity, while 4 and 10 displayed the most potent activity with IC50 values of 8.1 ± 1.1 and 6.5 ± 0.6 µM, respectively. The inhibitory activity results of 20 extra analogs of 4 further confirmed the potency of this scaffold. As expected, compounds 4 and 10 exhibited moderate anti-proliferative activity against mantle cell lymphoma Jeko-1 and leukemia cell MV4-11. Besides, Western blot assay results showed that 4 could reduce the H4R3me2s level in a dose-dependent manner, indicating that it could inhibit the activity of PRMT5 in cellular context. Detailed interactions between 4 and PRMT5 were characterized by binding mode analysis through molecular docking. The compounds discovered in this study will inspire medicinal chemists to further explore this series of PRMT5 inhibitors.