Promising New Antifungal Treatment Targeting Chorismate Synthase from Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides, is a systemic mycosis with granulomatous character and a restricted therapeutic arsenal. The aim of this work was to search for new alternatives to treat largely neglected tropical mycosis, such as PCM. In this context, the enzymes of the shikimate pathway constitute excellent drug targets for conferring selective toxicity because this pathway is absent in humans but essential for the fungus. In this work, we have used a homology model of the chorismate synthase (EC 4.2.3.5) from Paracoccidioides brasiliensis (PbCS) and performed a combination of virtual screening and molecular dynamics testing to identify new potential inhibitors. The best hit, CP1, successfully adhered to pharmacological criteria (adsorption, distribution, metabolism, excretion, and toxicity) and was therefore used in in vitro experiments. Here we demonstrate that CP1 binds with a dissociation constant of 64 ± 1 µM to recombinant chorismate synthase from P. brasiliensis and inhibits enzymatic activity, with a 50% inhibitory concentration (IC50) of 47 ± 5 µM. As expected, CP1 showed no toxicity in three cell lines. On the other hand, CP1 reduced the fungal burden in lungs from treated mice, similar to itraconazole. In addition, histopathological analysis showed that animals treated with CP1 displayed less lung tissue infiltration, fewer yeast cells, and large areas with preserved architecture. Therefore, CP1 was able to control PCM in mice with a lower inflammatory response and is thus a promising candidate and lead structure for the development of drugs useful in PCM treatment.

Systematic analysis and integrative discovery of active-site subpocket-specific dehydroquinate synthase inhibitors combating antibiotic-resistant Staphylococcus aureus infection.

Shikimate pathway plays an essential role in the biosynthesis of aromatic amino acids in various plants and bacteria, which consists of seven key enzymes and they are all attractive targets for antibacterial agent development due to their absence in humans. The Staphylococcus aureus dehydroquinate synthase (SaDHQS) is involved in the second step of shikimate pathway, which catalyzes the NAD + -dependent conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate via multiple steps. The enzyme active site can be characterized by two spatially separated subpockets 1 and 2, which represent the reaction center of substrate adduct with NAD + nicotinamide moiety and the assistant binding site of NAD + adenine moiety, respectively. In silico virtual screening is performed against a biogenic compound library to discover SaDHQS subpocket-specific inhibitors, which were then tested against both antibiotic-sensitive and antibiotic-resistant S. aureus strains by using in vitro susceptibility test. The activity profile of hit compounds has no considerable difference between the antibiotic-sensitive and -resistant strains. The subpocket 1-specific inhibitors exhibit a generally higher activity than subpocket 2-specific inhibitors, and they also hold a strong selectivity between their cognate and noncognate subpockets. Dynamics and energetics analyses reveal that the SaDHQS active site prefers to interact with amphipathic and polar inhibitors by forming multiple hydrogen bonds and van der Waals packing at the complex interfaces of the two subpockets with their cognate inhibitors.

Nonmevalonate terpene biosynthesis enzymes as antiinfective drug targets: substrate synthesis and high-throughput screening methods.

The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.