Presented by: Amanda Graboski
Chronic kidney disease (CKD) afflicts nearly 500 million people worldwide and is one of the fastest growing causes of mortality. A key consequence of a diseased kidney is the serum retention of toxic compounds that have a broad impact on human physiology. One of the most dangerous uremic toxins is indoxyl sulfate (IS), a metabolite produced solely from the breakdown of tryptophan by gut microbial tryptophanases (TPases). High IS levels in preclinical and clinical models have been correlated with altered mitochondrial oxidative phosphorylation, renal fibrosis, and six different phenotypes of cardiovascular disease. Recent studies showed the genetic elimination of TPase in an artificial microbiome of germfree mice prevented the formation of IS and reduced biomarkers of kidney injury, suggesting that inhibition of TPase could prevent or reduce uremic toxicity. In this study, we define the structural and functional landscape of gut microbial TPases to gauge its drugability and to guide novel inhibitor design. First, we selected a group of diverse TPases from the 184 sequences present in the Integrated Genome Catalog of human fecal metagenomes and examined their activity against tryptophan using substrate-turnover assays. We also elucidated the crystal structures of these TPases, revealing highly conserved tertiary structure and active site architecture. Using this structural information and the well-characterized catalytic mechanism, we designed and synthesized a panel of structure-based and mechanism-inspired inhibitors. A handful of these novel tryptophan-like compounds show inhibitory activity, one displaying a 20-30-fold greater affinity relative to the natural substrate. UV-vis spectral readings suggest activity as a transition state analog, and a cocrystal structure reveals many aromatic contacts with conserved active site residues. Minimal toxicity against mammalian and microbial cells was observed when dosed up to 500mM and target engagement in microbial cells and murine models is currently being evaluated. The discovery of a potent TPase inhibitor will help to unravel the molecular basis of increased serum IS levels and may serve as a therapeutic avenue for mitigating IS-induced toxicity in CKD.
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