143D, a novel selective KRASG12C inhibitor exhibits potent antitumor activity in preclinical models.

The KRASG12C mutant has emerged as an important therapeutic target in recent years. Covalent inhibitors have shown promising antitumor activity against KRASG12C-mutant cancers in the clinic. In this study, a structure-based and focused chemical library analysis was performed, which led to the identification of 143D as a novel, highly potent and selective KRASG12C inhibitor. The antitumor efficacy of 143D in vitro and in vivo was comparable with that of AMG510 and of MRTX849, two well-characterized KRASG12C inhibitors. At low nanomolar concentrations, 143D showed biochemical and cellular potency for inhibiting the effects of the KRASG12C mutation. 143D selectively inhibited cell proliferation and induced G1-phase cell cycle arrest and apoptosis by downregulating KRASG12C-dependent signal transduction. Compared with MRTX849, 143D exhibited a longer half-life and higher maximum concentration (Cmax) and area under the curve (AUC) values in mouse models, as determined by tissue distribution assays. Additionally, 143D crossed the blood‒brain barrier. Treatment with 143D led to the sustained inhibition of KRAS signaling and tumor regression in KRASG12C-mutant tumors. Moreover, 143D combined with EGFR/MEK/ERK signaling inhibitors showed enhanced antitumor activity both in vitro and in vivo. Taken together, our findings indicate that 143D may be a promising drug candidate with favorable pharmaceutical properties for the treatment of cancers harboring the KRASG12C mutation.

Discovery and characterization of a novel cGAS covalent inhibitor for the treatment of inflammatory bowel disease.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, acts as a nucleotidyl transferase that catalyzes ATP and GTP to form cyclic GMP-AMP (cGAMP) and plays a critical role in innate immunity. Hyperactivation of cGAS-STING signaling contributes to hyperinflammatory responses. Therefore, cGAS is considered a promising target for the treatment of inflammatory diseases. Herein, we report the discovery and identification of several novel types of cGAS inhibitors by pyrophosphatase (PPiase)-coupled activity assays. Among these inhibitors, 1-(1-phenyl-3,4-dihydro-1H-pyrrolo[1,2-a]pyrazin-2-yl)prop-2-yn-1-one (compound 3) displayed the highest potency and selectivity at the cellular level. Compound 3 exhibited better inhibitory activity and pathway selectivity than RU.521, which is a selective cGAS inhibitor with anti-inflammatory effects in vitro and in vivo. Thermostability analysis, nuclear magnetic resonance and isothermal titration calorimetry assays confirmed that compound 3 directly binds to the cGAS protein. Mass spectrometry and mutation analysis revealed that compound 3 covalently binds to Cys419 of cGAS. Notably, compound 3 demonstrated promising therapeutic efficacy in a dextran sulfate sodium (DSS)-induced mouse colitis model. These results collectively suggest that compound 3 will be useful for understanding the biological function of cGAS and has the potential to be further developed for inflammatory disease therapies.

AlphaFold2 versus experimental structures: evaluation on G protein-coupled receptors.

As important drug targets, G protein-coupled receptors (GPCRs) play pivotal roles in a wide range of physiological processes. Extensive efforts of structural biology have been made on the study of GPCRs. However, a large portion of GPCR structures remain unsolved due to structural instability. Recently, AlphaFold2 has been developed to predict structure models of many functionally important proteins including all members of the GPCR family. Herein we evaluated the accuracy of GPCR structure models predicted by AlphaFold2. We revealed that AlphaFold2 could capture the overall backbone features of the receptors. However, the predicted models and experimental structures were different in many aspects including the assembly of the extracellular and transmembrane domains, the shape of the ligand-binding pockets, and the conformation of the transducer-binding interfaces. These differences impeded the use of predicted structure models in the functional study and structure-based drug design of GPCRs, which required reliable high-resolution structural information.

Discovery and identification of a novel small molecule BCL-2 inhibitor that binds to the BH4 domain.

The B-cell lymphoma 2 (BCL-2) protein family plays a pivotal role in regulating the apoptosis process. BCL-2, as an antiapoptotic protein in this family, mediates apoptosis resistance and is an ideal target for cell death strategies in cancer therapy. Traditional treatment modalities target BCL-2 by occupying the hydrophobic pocket formed by BCL-2 homology (BH) domains 1-3, while in recent years, the BH4 domain of BCL-2 has also been considered an attractive novel target. Herein, we describe the discovery and identification of DC-B01, a novel BCL-2 inhibitor targeting the BH4 domain, through virtual screening combined with biophysical and biochemical methods. Our results from surface plasmon resonance and cellular thermal shift assay confirmed that the BH4 domain is responsible for the interaction between BCL-2 and DC-B01. As evidenced by further cell-based experiments, DC-B01 induced cell killing in a BCL-2-dependent manner and triggered apoptosis via the mitochondria-mediated pathway. DC-B01 disrupted the BCL-2/c-Myc interaction and consequently suppressed the transcriptional activity of c-Myc. Moreover, DC-B01 inhibited tumor growth in vivo in a BCL­2­dependent manner. Collectively, these results indicate that DC-B01 is a promising BCL-2 BH4 domain inhibitor with the potential for further development.

BCL-2 inhibitor synergizes with PI3Kδ inhibitor and overcomes FLT3 inhibitor resistance in acute myeloid leukaemia.

Inhibitors targeting the antiapoptotic molecule BCL-2 have therapeutic potential for the treatment of acute myeloid leukaemia (AML); however, BCL-2 inhibitors such as venetoclax exhibit limited monotherapy efficacy in relapsed or refractory human AML. PI3Kδ/AKT signalling has been shown to be constitutively active in AML patients. Here, we demonstrate that the combination of BCL-2 and PI3Kδ inhibitors exerts synergistic antitumour effects both in vitro and in vivo in AML. Cotreatment with venetoclax and the specific PI3Kδ inhibitor idelalisib significantly enhanced antiproliferative effects and induced caspase-dependent apoptosis in a panel of AML cell lines. The synergistic effects were mechanistically based on the inactivation of AKT/4E-BP-1 signalling and the reduction of MCL-1 expression, which diminished the binding of Bim to MCL-1. Notably, compared with the parental FLT3-ITD-positive MV-4-11, the acquired FLT3 inhibitor quizartinib-resistant xenograft model carrying the F691L mutation, exhibited a markedly higher sensitivity to venetoclax. Furthermore, venetoclax combined with idelalisib led to tumour regression in all animals in this quizartinib-resistant AML model. Thus, these data indicate that combined inhibition of BCL-2 and PI3Kδ may be a promising strategy in AML, especially for patients with FLT3-ITD and/or FLT3-TKD mutations.

Safety, tolerability, and pharmacokinetics of VV116, an oral nucleoside analog against SARS-CoV-2, in Chinese healthy subjects.

VV116 (JT001) is an oral drug candidate of nucleoside analog against SARS-CoV-2. The purpose of the three phase I studies was to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116. Three studies were launched sequentially: Study 1 (single ascending-dose study, SAD), Study 2 (multiple ascending-dose study, MAD), and Study 3 (food-effect study, FE). A total of 86 healthy subjects were enrolled in the studies. VV116 tablets or placebo were administered per protocol requirements. Blood samples were collected at the scheduled time points for pharmacokinetic analysis. 116-N1, the metabolite of VV116, was detected in plasma and calculated for the PK parameters. In SAD, AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25-800 mg. T1/2 was within 4.80-6.95 h. In MAD, the accumulation ratio for Cmax and AUC indicated a slight accumulation upon repeated dosing of VV116. In FE, the standard meal had no effect on Cmax and AUC of VV116. No serious adverse event occurred in the studies, and no subject withdrew from the studies due to adverse events. Thus, VV116 exhibited satisfactory safety and tolerability in healthy subjects, which supports the continued investigation of VV116 in patients with COVID-19.

Discovery of potential small molecular SARS-CoV-2 entry blockers targeting the spike protein.

An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant KD) of 0.0947 µM and anti-virus IC50 of 85.75 µM.

A multi-targeting drug design strategy for identifying potent anti-SARS-CoV-2 inhibitors.

The COVID-19, caused by SARS-CoV-2, is threatening public health, and there is no effective treatment. In this study, we have implemented a multi-targeted anti-viral drug design strategy to discover highly potent SARS-CoV-2 inhibitors, which simultaneously act on the host ribosome, viral RNA as well as RNA-dependent RNA polymerases, and nucleocapsid protein of the virus, to impair viral translation, frameshifting, replication, and assembly. Driven by this strategy, three alkaloids, including lycorine, emetine, and cephaeline, were discovered to inhibit SARS-CoV-2 with EC50 values of low nanomolar levels potently. The findings in this work demonstrate the feasibility of this multi-targeting drug design strategy and provide a rationale for designing more potent anti-virus drugs.

P300/CBP inhibition sensitizes mantle cell lymphoma to PI3Kδ inhibitor idelalisib.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder lacking reliable therapies. PI3K pathway contributes to the pathogenesis of MCL, serving as a potential target. However, idelalisib, an FDA-approved drug targeting PI3Kδ, has shown intrinsic resistance in MCL treatment. Here we report that a p300/CBP inhibitor, A-485, could overcome resistance to idelalisib in MCL cells in vitro and in vivo. A-485 was discovered in a combinational drug screening from an epigenetic compound library containing 45 small molecule modulators. We found that A-485, the highly selective catalytic inhibitor of p300 and CBP, was the most potent compound that enhanced the sensitivity of MCL cell line Z-138 to idelalisib. Combination of A-485 and idelalisib remarkably decreased the viability of three MCL cell lines tested. Co-treatment with A-485 and idelalisib in Maver-1 and Z-138 MCL cell xenograft mice for 3 weeks dramatically suppressed the tumor growth by reversing the unsustained inhibition in PI3K downstream signaling. We further demonstrated that p300/CBP inhibition decreased histone acetylation at RTKs gene promoters and reduced transcriptional upregulation of RTKs, thereby inhibiting the downstream persistent activation of MAPK/ERK signaling, which also contributed to the pathogenesis of MCL. Therefore, additional inhibition of p300/CBP blocked MAPK/ERK signaling, which rendered maintaining activation to PI3K-mTOR downstream signals p-S6 and p-4E-BP1, thus leading to suppression of cell growth and tumor progression and eliminating the intrinsic resistance to idelalisib ultimately. Our results provide a promising combination therapy for MCL and highlight the potential use of epigenetic inhibitors targeting p300/CBP to reverse drug resistance in tumor.

Natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 µM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.

Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with "short" and "long" inverse agonists.

A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the "short" inverse agonist 6. With the increase in the size of the 6-position substituents, the "short" inverse agonist 6 first reversed its function to agonists and then to "long" inverse agonists. The cocrystal structures of RORγt complexed with the representative "short" inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the "long" inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of "short" inverse agonist 6 and "long" inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that "short" or "long" inverse agonists led to different behaviors of helixes H11, H11', and H12 of RORγt. The "short" inverse agonist 6 destabilizes H11' and dislocates H12, while the "long" inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

The inhibitory mechanism of aurintricarboxylic acid targeting serine/threonine phosphatase Stp1 in Staphylococcus aureus: insights from molecular dynamics simulations.

Serine/threonine phosphatase (Stp1) is a member of the bacterial Mg2+- or Mn2+- dependent protein phosphatase/protein phosphatase 2C family, which is involved in the regulation of Staphylococcus aureus virulence. Aurintricarboxylic acid (ATA) is a known Stp1 inhibitor with an IC50 of 1.03 µM, but its inhibitory mechanism has not been elucidated in detail because the Stp1-ATA cocrystal structure has not been determined thus far. In this study, we performed 400 ns molecular dynamics (MD) simulations of the apo-Stp1 and Stp1-ATA complex models. During MD simulations, the flap subdomain of the Stp1-ATA complex experienced a clear conformational transition from an open state to a closed state, whereas the flap domain of apo-Stp1 changed from an open state to a semi-open state. In the Stp1-ATA complex model, the hydrogen bond (H-bond) between D137 and N142 disappeared, whereas critical H-bond interactions were formed between Q160 and H13, Q160/R161 and ATA, as well as N162 and D198. Finally, four residues (D137, N142, Q160, and R161) in Stp1 were mutated to alanine and the mutant enzymes were assessed using phosphate enzyme activity assays, which confirmed their important roles in maintaining Stp1 activity. This study indicated the inhibitory mechanism of ATA targeting Stp1 using MD simulations and sheds light on the future design of allosteric Stp1 inhibitors.

Identification of small molecule inhibitors targeting the SMARCA2 bromodomain from a high-throughput screening assay.

SMARCA2 is a critical catalytic subunit of the switch/sucrose non-fermenting (SWI/SNF) chromatin remodeling complexes. Dysregulation of SMARCA2 is associated with several diseases, including some cancers. SMARCA2 is multi-domain protein containing a bromodomain (BRD) that specifically recognizes acetylated lysine residues in histone tails, thus playing an important role in chromatin remodeling. Many potent and specific inhibitors targeting other BRDs have recently been discovered and have been widely used for cancer treatments and biological research. However, hit discovery targeting SMARCA2-BRD is particularly lacking. To date, there is a paucity of reported high-throughput screening (HTS) assays targeting the SMARCA2-BRD interface. In this study, we developed an AlphaScreen HTS system for the discovery of SMARCA2-BRD inhibitors and optimized the physicochemical conditions including pH, salt concentrations and detergent levels. Through an established AlphaScreen-based high-throughput screening assay against an in-house compound library, DCSM06 was identified as a novel SMARCA2-BRD inhibitor with an IC50 value of 39.9±3.0 µmol/L. Surface plasmon resonance demonstrated the binding between SMARCA2-BRD and DCSM06 (Kd=38.6 µmol/L). A similarity-based analog search led to identification of DCSM06-05 with an IC50 value of 9.0±1.4 µmol/L. Molecular docking was performed to predict the binding mode of DCSM06-05 and to decipher the structural basis of the infiuence of chemical modifications on inhibitor potency. DCSM06-05 may be used as a starting point for further medicinal chemistry optimization and could function as a chemical tool for SMARCA2-related functional studies.

Benzbromarone aggravates hepatic steatosis in obese individuals.

As a widely used anti-gout drug, benzbromarone has been found to induce hepatic toxicity in patients during clinical treatment. Previous studies have reported that benzbromarone is metabolized via cytochrome P450, thus causing mitochondrial toxicity in hepatocytes. In this study, we found that benzbromarone significantly aggravated hepatic steatosis in both obese db/db mice and high fat diet (HFD)-induced obese (DIO) mouse models. However, benzbromarone had less effect on the liver of lean mice. It was found that the expression of mRNAs encoding lipid metabolism and some liver-specific genes were obviously disturbed in benzbromarone-treated DIO mice compared to the control group. The inflammatory and oxidative stress factors were also activated in the liver of benzbromarone-treated DIO mice. In accordance with the in vivo results, an in vitro experiment using human hepatoma HepG2 cells also confirmed that benzbromarone promoted intracellular lipid accumulation under high free fatty acids (FFAs) conditions by regulating the expression of lipid metabolism genes. Importantly, prolonged treatment of benzbromarone significantly increased cell apoptosis in HepG2 cells in the presence of high FFAs. In addition, in benzbromarone-treated hyperuricemic patients, serum transaminase levels were positively correlated with patients' obesity level. CONCLUSION: This study demonstrated that benzbromarone aggravated hepatic steatosis in obese individuals, which could subsequently contribute to hepatic cell injury, suggesting a novel toxicological mechanism in benzbromarone-induced hepatotoxicity.

The HNF1α-regulated lncRNA HNF1A-AS1 reverses the malignancy of hepatocellular carcinoma by enhancing the phosphatase activity of SHP-1.

BACKGROUND: Our previous study has demonstrated that hepatocyte nuclear factor 1α (HNF1α) exerts potent therapeutic effects on hepatocellular carcinoma (HCC). However, the molecular mechanisms by which HNF1α reverses HCC malignancy need to be further elucidated. METHODS: lncRNA microarray was performed to identify the long noncoding RNAs (lncRNAs) regulated by HNF1α. Chromatin immunoprecipitation and luciferase reporter assays were applied to clarify the mechanism of the transcriptional regulation of HNF1α to HNF1A antisense RNA 1 (HNF1A-AS1). The effect of HNF1A-AS1 on HCC malignancy was evaluated in vitro and in vivo. RNA pulldown, RNA-binding protein immunoprecipitation and the Bio-Layer Interferometry assay were used to validate the interaction of HNF1A-AS1 and Src homology region 2 domain-containing phosphatase 1 (SHP-1). RESULTS: HNF1α regulated the expression of a subset of lncRNAs in HCC cells. Among these lncRNAs, the expression levels of HNF1A-AS1 were notably correlated with HNF1α levels in HCC cells and human HCC tissues. HNF1α activated the transcription of HNF1A-AS1 by directly binding to its promoter region. HNF1A-AS1 inhibited the growth and the metastasis of HCC cells in vitro and in vivo. Moreover, knockdown of HNF1A-AS1 reversed the suppressive effects of HNF1α on the migration and invasion of HCC cells. Importantly, HNF1A-AS1 directly bound to the C-terminal of SHP-1 with a high binding affinity (KD = 59.57 ± 14.29 nM) and increased the phosphatase activity of SHP-1. Inhibition of SHP-1 enzymatic activity substantially reversed the HNF1α- or HNF1A-AS1-induced reduction on the metastatic property of HCC cells. CONCLUSIONS: Our data revealed that HNF1A-AS1 is a direct transactivation target of HNF1α in HCC cells and involved in the anti-HCC effect of HNF1α. HNF1A-AS1 functions as phosphatase activator through the direct interaction with SHP-1. These findings suggest that regulation of the HNF1α/HNF1A-AS1/SHP-1 axis may have beneficial effects in the treatment of HCC.

Development of a high-throughput fluorescence polarization assay for the discovery of EZH2-EED interaction inhibitors.

Aberrant activity of enhancer of zeste homolog 2 (EZH2) is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development (EED) is required for EZH2's catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening (HTS) assays for inhibitors acting at the EZH2-EED interface. In this study, we developed a fluorescence polarization (FP)-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z' factors (>0.9) at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds (apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate) as potential EZH2-EED interaction inhibitors.

Conformation and dynamics of the C-terminal region in human phosphoglycerate mutase 1.

Phosphoglycerate mutase 1 (PGAM1), an important enzyme in glycolysis, is overexpressed in a number of human cancers, thus has been proposed as a promising metabolic target for cancer treatments. The C-terminal portion of the available crystal structures of PGAM1 and its homologous proteins is partially disordered, as evidenced by weak electron density. In this study, we identified the conformational behavior of the C-terminal region of PGAM1 as well as its role during the catalytic cycle. Using the PONDR-FIT server, we demonstrated that the C-terminal region was intrinsically disordered. We applied the Monte Carlo (MC) method to explore the conformational space of the C-terminus and conducted a series of explicit-solvent molecular dynamics (MD) simulations, and revealed that the C-terminal region is inherently dynamic; large-scale conformational changes in the C-terminal segment led to the structural transition of PGAM1 from the closed state to the open state. Furthermore, the C-terminal segment influenced 2,3-bisphosphoglycerate (2,3-BPG) binding. The proposed swing model illustrated a critical role of the C-terminus in the catalytic cycle through the conformational changes. In conclusion, the C-terminal region induces large movements of PGAM1 from the closed state to the open state and influences cofactor binding during the catalytic cycle. This report describes the dynamic features of the C-terminal region in detail and should aid in design of novel and efficient inhibitors of PGAM1. A swing mechanism of the C-terminal region is proposed, to facilitate further studies of the catalytic mechanism and the physiological functions of its homologues.

High yield and efficient expression and purification of the human 5-HT3A receptor.

AIM: To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies. METHODS: Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM). RESULTS: The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure. CONCLUSION: With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor.

Identification of matrine as a promising novel drug for hepatic steatosis and glucose intolerance with HSP72 as an upstream target.

BACKGROUND AND PURPOSE: Matrine is a small molecule drug used in humans for the treatment of chronic viral infections and tumours in the liver with little adverse effects. The present study investigated its therapeutic efficacy for insulin resistance and hepatic steatosis in high-fat-fed mice. EXPERIMENTAL APPROACH: C57BL/J6 mice were fed a chow or high-fat diet for 10 weeks and then treated with matrine or metformin for 4 weeks. The effects on lipid metabolism and glucose tolerance were evaluated. KEY RESULTS: Our results first showed that matrine reduced glucose intolerance and plasma insulin level, hepatic triglyceride content and adiposity in high-fat-fed mice without affecting caloric intake. This reduction in hepatosteatosis was attributed to suppressed lipid synthesis and increased fatty acid oxidation. In contrast to metformin, matrine neither suppressed mitochondrial respiration nor activated AMPK in the liver. A computational docking simulation revealed HSP90, a negative regulator of HSP72, as a potential binding target of matrine. Consistent with the simulation results, matrine, but not metformin, increased the hepatic protein level of HSP72 and this effect was inversely correlated with both liver triglyceride level and glucose intolerance. CONCLUSIONS AND IMPLICATIONS: Taken together, these results indicate that matrine may be used for the treatment of type 2 diabetes and hepatic steatosis, and the molecular action of this hepatoprotective drug involves the activation of HSP72 in the liver.