Manipulation of urease within the gut microbiome using small-molecule inhibitors

Abstract

Microbial urea metabolism plays a critical role in shaping gut microbial ecology and host nitrogen balance, yet its mechanistic underpinnings and therapeutic potential remain underexplored. In my thesis work, we sought to advance our understanding of urea metabolism within the gut microbiome and evaluate the therapeutic potential of small-molecule urease inhibitors to modulate host-microbe interaction. In my thesis work we sought to advance our understanding of urea metabolism within the gut microbiome and evaluate the therapeutic potential of small-molecule urease inhibitors to modulate host-microbe interactions. Using an interdisciplinary approach that integrated bioinformatics, chemical biology, organic synthesis, microbiome profiling, and in vivo physiology, we investigated microbial urease as a conserved and druggable enzyme within the gut microbiome. We identified and characterized urease homologs across human, ancient, and murine microbiomes, revealing broad phylogenetic distribution and active site conservation. We further evaluated benurestat, a hydroxamic acid-based inhibitor, through structure–activity relationship studies and demonstrated its potency, gut-restriction, and therapeutic efficacy in reducing ammonia levels and improving survival in murine models of acute liver injury. Altogether, my thesis establishes a mechanistic and translational framework for targeting microbial urease and supports the development of gut-restricted small-molecule inhibitors to modulate host-microbe interactions in disease Chapter 2 presents a comprehensive bioinformatic and structural analysis of urease-encoding genes across human, murine, and ancient microbiomes. Using a sequence-structure-function pipeline developed in collaboration with the Huttenhower lab, we identified hundreds of microbial urease homologs, characterized the structural conservation of a selection of diverse sequences, and highlighted widespread preservation of active site residues across phylogenetically diverse taxa. These findings underscore the broad evolutionary conservation of urease and support its tractability as a microbiome-targeted enzyme. This chapter also addresses common challenges in urease annotation and emphasizes the need for integrated functional validation. Chapter 3 focuses on the discovery and structure–activity relationship of hydroxamic acid-based urease inhibitors, with an emphasis on benurestat as the main scaffold. Comparative biochemical assays demonstrated that benurestat is a potent and selective inhibitor of bacterial urease, outperforming earlier compounds such as acetohydroxamic acid. Through synthetic modifications and docking-guided design, I characterized key structural determinants of inhibitor potency and selectivity. Notably, halogenated derivatives improved activity, while modifications to the α-amino acid motif reduced efficacy, pointing to structural constraints within the urease active site. These studies provide medicinal chemistry insights that guide further optimization of gut-restricted urease inhibitors. Chapter 4 establishes the in vivo therapeutic potential of benurestat, a gut-restricted small-molecule urease inhibitor, in murine models of hyperammonemia and acute liver injury. Unlike less selective or systemically absorbed analogs, benurestat significantly reduced both fecal and serum ammonia levels, preserved gut microbial structure, and conferred complete protection against lethal thioacetamide-induced liver injury. Comparative studies with other known urease inhibitors, including AHA, flurofamide, and ebselen, demonstrated benurestat’s superior potency, gut localization, and therapeutic efficacy. Control experiments with an inactive analog (KRC40) confirmed that the observed effects were driven by specific inhibition of microbial urease. Metagenomic analysis further revealed that benurestat may be modulating the gut microbiome without inducing broad dysbiosis and supporting a distinct microbial signature under hepatic stress. Together, these findings validate microbial urease as a clinically relevant and tractable target and demonstrate that gut-restricted enzyme inhibition can yield meaningful host benefits by precisely modulating microbial metabolism in disease. Collectively, this thesis defines a thorough mechanistic and translational framework for targeting gut microbial urease to modulate host-microbe interactions in disease. Through an integrated approach combining comparative genomics, structural bioinformatics, chemical biology, synthetic chemistry, and in vivo murine models, this work reveals that urease is a broadly conserved and druggable microbial enzyme across human-associated microbiomes. Detailed structure–activity relationship studies guided the identification and optimization of benurestat, a potent and gut-restricted urease inhibitor that effectively lowers systemic ammonia levels and protects against lethal liver injury in preclinical models. Rigorous experiments confirmed urease-specific mechanisms, while metagenomic profiling demonstrated that therapeutic efficacy can be achieved without inducing broad disruption of the gut microbiota. Altogether, this body of work not only establishes urease as a clinically relevant microbial target but also advances the broader concept of using small-molecule inhibitors to selectively modulate microbial metabolism with precision and translational potential.