A-249 Development of non-lethal mechanism-based small molecule inhibitors of the gut microbial phenylacetylglutamine pathway in cardiovascular disease

Background Phenylacetylglutamine (PAG) is a compound found in human circulation derived from the gut microbial metabolite phenylacetic acid. This compound has recently been identified through a collection of metabolomic studies and animal models to be both clinically and mechanistically linked to the development of cardiovascular disease. The multistep, metaorganismal production of PAG begins with dietary protein derived phenylalanine, which when taken up into gut microbes is converted to phenylpyruvic acid by multiple catalytic strategies. Phenylpyruvic acid is further metabolized through at least two distinctive microbial pathways. One pathway involves an oxidative decarboxylation by microbial phenylpyruvate ferredoxin:oxidoreductase (PPFOR), producing phenylacetyl-CoA, which is further hydrolyzed to phenylacetic acid. Alternatively, a non-oxidative decarboxylation reaction is catalyzed by phenylpyruvate decarboxylase (PPDC), producing phenylacetaldehyde, which is further oxidized to phenylacetic acid. Ultimately, phenylacetic acid, when taken up by the human host, is rapidly conjugated to glutamine to form phenylacetylglutamine by a phase II detoxification reaction for improved urinary excretion. Towards the aim of prevention and treatment of PAG-linked diseases we have developed gut microbe targeting, small molecule inhibitors designed to inhibit microbial phenylacetic acid production in order to suppress host PAG levels. Methods We have synthesized a series of small molecule inhibitors designed to selectively target PPFOR and PPDC, taking advantage of the fact that while both enzymes perform distinct reactions they both share a common point by requiring thiamine pyrophosphate (TPP) as a cofactor for catalysis. These compounds are designed as mechanism-based inhibitors: substrate analogues, which upon catalysis, produce reactive electrophilic species capable of reacting with nucleophilic active site residues, irreversibly inactivating the target enzymes. This strategy seeks to create potent, highly selective inhibitors, due to dependency on the specific enzymatic reaction for inhibitor activation. To assay in vitro inhibitor efficacy, several distinct assays of microbial phenylacetic acid production were established using HPLC-MS/MS and spectrophotometric techniques (e.g. recombinant microbial enzymes cloned and expressed using bacterial expression vectors, and systems of monocultures or polymicrobial communities). For example, assays of recombinant PPDC were developed by cloning PPDC from the aerotolerant microbe P. mirabilis and quantifying the conversion of phenylpyruvic acid into phenylacetaldehyde by HPLC-MS/MS; assays of recombinant PPFOR were also developed (requiring strict anaerobic conditions) using recombinant PPFOR from the anaerobe B. thetaiotaomicron, and monitoring the CoA dependent oxidative decarboxylation of phenylpyruvic acid by spectrophotometric detection of the coupled reduction of methyl viologen. Results When tested in our assays, several of these compounds have shown potent inhibition (EC50 in the sub-micromolar range) of both recombinant PPFOR and PPDC as well as inhibition of phenylacetic acid production in polymicrobial cultures. Our ongoing studies are aimed at further refining structure-activity relationships (SAR) for lead candidates to optimize pre-specified PK-PD requirements, with the long term goal of utilizing these inhibitors in preclinical animal models of disease. Conclusion Our results thus far demonstrate feasibility in creating single non-lethal high potency small molecule inhibitors for the potential therapeutic targeting of a gut-microbiome pathway that is clinically and mechanistically linked to the development and progression of cardiovascular disease – the PAG pathway.