CHARACTERIZATION AND INHIBITION OF GUT MICROBIAL TRYPTOPHANASE AND ITS IMPLICATIONS IN CHRONIC KIDNEY DISEASE

Chronic kidney disease (CKD) is defined as the progressive decline in kidney function over months to years and afflicts nearly 800 million people worldwide. 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 (TIL). 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 TIL in an artificial microbiome of germfree mice prevented the formation of IS and reduced biomarkers of kidney injury, suggesting that inhibition of TIL could prevent or reduce uremic toxicity. Here, we characterize the structural and functional landscape of TIL enzymes within the human gut microbiome. We start by mining for TIL sequences within clinically relevant gut microbiome databases, identifying 183 unique sequences. We then characterize the TIL-ome using a combination of classical biochemistry, protein crystallography, and structural metagenomics. We find that TILs are remarkably homologous in structure and function despite deriving from diverse taxa, likely attributed to narrow substrate preference. We next leverage this structural and functional data to aid in a medicinal chemistry campaign for a potent pan-inhibitor of TILs. We identified novel scaffolds through in silico screening and designed a panel of tryptophan-like analogs using our resolved atomic structures. We then synthesized these molecules using a novel biocatalytic pipeline and traditional synthetic chemistry in tandem. Nearly all in silico and structure-inspired compounds displayed significantly reduced or negligible activity leading us to pursue mechanism-based inhibitors which showed the greatest success, producing our lead compound (3S) ALG-05. UV-vis spectroscopy and x-ray crystallography validate that (3S) ALG-05 behaves as a transition state analog, stalling the quinonoid intermediate of the catalytic mechanism. In microbial culture, treatment with (3S) ALG-05 successfully reduced indole levels and no toxicity was observed across a wide range of doses against both microbial and mammalian cells. (3S) ALG-05 treatment in mice reduced cecal indole and serum indoxyl sulfate levels while displaying minimal changes in other tryptophan metabolizing pathways. Lastly, we utilize and validate existing PLP-like probes to pulldown TILs in fecal samples of healthy and CKD patients with the goal of better understanding how gut microbial dysbiosis can impact protein expression, uremic toxin generation, and metabolite accumulation in advanced disease. Together, the studies conducted in this dissertation characterize the structural and functional diversity of TILs, leverage this knowledge to safely and successfully pan-inhibit them in vivo, and begin quantifying differences in TIL abundance and species derivation in CKD.