Synthesis, molecular docking and antibacterial activity of an oxadiazole-based lipoteichoic acid inhibitor and its metabolites.

Amongst drug resistant Gram-positive bacteria, Staphylococcus aureus is a pathogen of great concern as it is the leading cause of life-threatening nosocomial and community acquired infections which are often associated with implanted medical devices. The biosynthesis of lipotheicoic acid (LTA) by S. aureus has been recognized as a promising antibacterial target, owing its critical role in the growth and survival of Gram-positive bacteria. Here we report for the first time the chemical synthesis and characterisation of an oxadiazole based compound (1771), previously described as an inhibitor of LTA biosynthesis by targeting Lta synthase enzyme (LtaS). To investigate its controversial mode of action, we also performed molecular docking studies, which indicated that 1771 behaves as a competitive inhibitor against LtaS. We also synthesised and evaluated the antimicrobial activity of 1771 metabolites which we have identified from its decomposition in mouse serum, proving that the biological activity was caused by intact 1771.

Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls.

Humans are commonly exposed to α,ß-unsaturated carbonyls as both environmental toxins (e.g. acrolein) and therapeutic drugs (e.g. dimethylfumarate, DMFU, a front-line drug for the treatment of multiple sclerosis and psoriasis). These compounds undergo rapid Michael addition reactions with amine, imidazole and thiol groups on biological targets, with reaction at protein Cys residues being a major reaction pathway. However, the cellular targets of these species (the 'adductome') are poorly understood due to the absence of readily identifiable tags or reporter groups (chromophores/fluorophores or antigens) on many α,ß-unsaturated carbonyls. Here we report a 'proof of concept' study in which we synthesize novel α,ß-unsaturated carbonyls containing an alkyne function introduced at remote sites on the α,ß-unsaturated carbonyl compounds (e.g. one of the methyl groups of dimethylfumarate). The presence of this tag allows 'click-chemistry' to be used to visualize, isolate, enrich and characterize the cellular targets of such compounds. The probes show similar selectivity and reactivity to the parent compounds, and compete for cellular targets, yielding long-lived (stable) adducts that can be visualized in intact cells (such as primary human coronary artery smooth muscle cells), and extracted and enriched for subsequent target analysis. It is shown using this approach that dimethylfumarate forms adducts with multiple intracellular targets including cytoskeletal, organelle and nuclear species, with these including the rate-limiting glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This approach should be amenable to use with multiple α,ß-unsaturated carbonyls and a wide variety of targets containing nucleophilic sites.

Kinetic assessment of Michael addition reactions of alpha, beta-unsaturated carbonyl compounds to amino acid and protein thiols.

Humans have extensive adverse exposure to alpha,beta-unsaturated carbonyl compounds (ABuCs) as these are major toxins in smoke and exhaust fumes, as well as products of lipid peroxidation. In contrast, another ABuC, dimethylfumarate, is used to treat psoriasis and multiple sclerosis. ABuCs undergo Michael adduction with amine, imidazole and thiol groups, with reaction at Cys residues predominating. Here we report rate constants, k2, for ABuCs (acrolein, crotonaldehyde, dimethylfumarate, cyclohex-1-en-2-one, cyclopent-1-en-2-one) with Cys residues present on N-Ac-Cys, GSH, bovine serum albumin, creatine kinase, papain, glyceraldehyde-3-phosphate dehydrogenase, and both wild-type and the C151S mutant of Keap-1. k2 values for N-Ac-Cys and GSH vary by > 250-fold, indicating a marked ABuC structure dependence, with acrolein the most reactive. There is also considerable variation in k2 between protein Cys groups, with these significantly greater than for GSH. A linear inverse correlation for acrolein with the thiol pKa indicates that the thiolate anion is the reactive species. The modest k2 for GSH rationalizes the detection of protein adducts of ABuCs in cells. The k2 values for dimethylfumarate also vary markedly, with the Cys151 residue on Keap-1 being particularly reactive, with the C151S mutant giving a much lower k2 value. The data for crotonaldehyde, dimethylfumarate, and cyclohex-1-en-2-one show little correlation with the Cys pKa values, indicating that steric/electronic interactions, rather than Cys ionization are important. These data indicate that protein Cys residues, and particularly Cys151 on Keap-1, react readily with dimethylfumarate, and this may help rationalize the use of this compound as a therapeutic agent.

Targeting GNE Myopathy: A Dual Prodrug Approach for the Delivery of N-Acetylmannosamine 6-Phosphate.

ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.