Acute Regulation of Cardiac Metabolism by the Hexosamine Biosynthesis Pathway and Protein O-GlcNAcylation

OBJECTIVE: The hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization. METHODS: Isolated rat hearts were perfused with glucosamine (0-10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by (13)C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36. RESULTS: Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation. CONCLUSION/INTERPRETATION: These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism

Approach to the mechanism of action of hydroxychloroquine on SARS-CoV-2: a molecular docking study

We aimed to analyze the interactions of both hydroxychloroquine and chloroquine with SARS-CoV-2 and identify their possible role for the prevention/treatment of COVID-19 by molecular docking studies. Protein crystal structures of SARS-CoV-2 and ACE2, the compounds hydroxychloroquine and chloroquine, and other ligand structures were minimized by OPLS3 force field. Glide Standard Precision and Extra Precision docking are performed and MM-GBSA values are calculated. Molecular docking studies showed that hydroxychloroquine and chloroquine do not interact with SARS-CoV-2 proteins, but bind to the amino acids ASP350, ASP382, ALA348, PHE40 and PHE390 on the ACE2 allosteric site rather than the ACE2 active site. Our results showed that neither hydroxychloroquine and chloroquine bind to the active site of ACE2. However, both molecules prevent the binding of SARS-CoV-2 spike protein to ACE2 by interacting with the allosteric site. This result can help ACE2 inhibitor drug development studies to prevent viruses entering the cell by attaching spike protein to ACE2. Communicated by Ramaswamy H. Sarm

Synthesis, molecular docking, in silico ADME, and EGFR kinase inhibitor activity studies of some new benzimidazole derivatives bearing thiosemicarbazide, triazole, and thiadiazole

Epidermal growth factor receptor (EGFR), one of the important targets in the development of the anticancer compound, is a member of the ErbB receptor tyrosine kinase receptor family and is highly expressed in solid tumors. Inhibition of EGFR is important for cancer treatment to inhibit the progression and growth of EGFR-expressing tumor cells. Agents targeting EGFR are successful drugs involved in the treatment of various cancers, particularly colorectal, head, neck, lung, and breast cancers. In this study, the design of some novel benzimidazole compounds that can interact with EGFR kinase enzyme, synthesis and analysis of these compounds, and evaluation of their biological activities in vitro was aimed. To reach the target compounds, by reacting acid hydrazides with alkyl isothiocyanates, thiosemicarbazides were formed, then cyclization of these compounds with concentrated sulfuric acid or sodium hydroxide, thiadiazole, or triazole derivatives were obtained. As a result of the study, a total of 38 new benzimidazole derivatives was obtained, and the structures of these compounds were clarified by elemental analysis, mass, H-1, and C-13 NMR spectroscopy. Also, the structure of compound 4c was proven by X-ray crystallography. Molecular docking studies of the synthesized compounds have also been carried out, some molecules with high docking scores have been selected and EGFR kinase inhibitor properties have been tested. Among the compounds tested, it was determined that the most active compound was 12a, which inhibited 68% EGFR at a concentration of 10 mu M

In Vitro and in Silico Evaluation of Some New 1H-Benzimidazoles Bearing Thiosemicarbazide and Triazole as Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

A new class of benzimidazole derivatives bearing bis-triazole or bis-thiosemicarbazide structure was designed and synthesized as potential inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. In vitro EGFR kinase enzyme inhibition properties were determined in comparison with erlotinib and compound (6b) containing propyl side chain at the 4th position of triazole rings showed 13.8% inhibition. Molecular docking studies were performed and docking score and binding energy were established. The most active compound formed a hydrogen bond between the Asn818 and triazole NH at the 2nd position of the benzimidazole skeleton. Predicted ADME profiles of the compounds were calculated and found to be within the appropriate reference ranges

Effect of glucosamine on A) glucose; B) pyruvate; C) lactate and D) palmitate oxidation.

<p>^* P<0.05 vs. 0 mM glucosamine, one-way ANOVA with Dunnett's posthoc test. 0 mM (n = 6), 0.05 mM (n = 4), 0.1 mM (n = 5), 5 mM (n = 5), 10 mM (n = 4).</p

Effect of glucosamine on A, B) Overall cardiac O-GlcNAc levels; C) UDP-HexNAc concentrations and D) ATP concentrations.

<p>^* P<0.05 vs. 0 mM, one-way ANOVA with Dunnett's posthoc test. Western blots: 0 mM (n = 8), 0.05 mM (n = 5), 0.1 mM (n = 9), 1 mM (n = 4), 5 mM (n = 8), 10 mM (n = 7). HPLC: 0 mM (n = 4), 0.05 mM (n = 5), 0.1 mM (n = 5), 1 mM (n = 4), 5 mM (n = 3), 10 mM (n = 3). Note that equal protein loading for the O-GlcNAc immunoblots was assessed by Sypro staining and overall O-GlcNAc levels were normalized to untreated control group.</p

Effect of 0.1 mM glucosamine on A) unlabeled glycolytic lactate efflux and B) exogenous [3-¹³C]lactate uptake ^* P<0.05 vs. 0 mM, Student's t-test.

<p>0 mM (n = 4), 0.1 mM (n = 5).</p

Cardiac function of isolated rat hearts perfused with 0 (n = 8), 0.05 (n = 5), 0.1 (n = 10), 1 (n = 4), 5 (n = 8) and 10 mM (n = 7) glucosamine for 60 minutes (hearts were paced at 320 beats/min rate).

<p>Glucosamine had no effect either on cardiac or on coronary flow. (RPP: rate pressure product = left ventricular developed pressure×heart rate; dp/dt: change of pressure over time).</p

A) Immunoblots of Plasma membrane fraction for FAT/CD36 following 60 min perfusion with 0, 0.05, 0.1, 1, 5 and 10 mM; pan-cadherin included as a plasma membrane marker and protein loading control; B) Densitometric analysis of FAT/CD36 immunoblots normalized to 0 mM glucosamine; P<0.05 vs. 0 mM, one-way ANOVA with Dunnett's posthoc test; <i>n = 2 in each group</i>; C) Immunoprecipitation of FAT/CD36 from whole tissue and plasma membrane lysates, followed by O-GlcNAc and OGT immunoblots.

<p>Specificity of O-GlcNAc antibody was confirmed by co-incubation with 10 mM N-acetylglucosamine (GlcNAc).</p