STRUCTURAL MODIFICATIONS OF BUTYRATE REVEAL TEMPLATES FOR INTESTINAL EPITHELIAL HIF STABILIZERS

Abstract

Abstract Microbiota-derived short chain fatty acids, particularly butyrate (BA), show multiple beneficial influences on health. In the colon, BA ranges from 10-20 mM and up to 99% is utilized as a metabolic fuel by the mucosa. BA plays a key role in epithelial barrier regulation, reduces inflammation, and regulates cell growth and differentiation. There are multiple mechanisms by which BA contributes to gut health, many due to its regulatory capacity for gene expression. Our group reported (PMID: 34190032) a direct influence of BA on the stabilization of the transcription factor hypoxia-inducible factor (HIF). It is known that HIF stabilization is essential for appropriate mucosal barrier regulation and the coordination of regenerative capacity in the intestine. However, BA is constantly metabolized limiting its HIF stabilization effect. This observation led to the design and investigation of BA-mimicking compounds that stabilize HIF, but may not be involved in metabolism. We hypothesize that structural modifications of BA yield analogues that stabilize HIF with better efficacy and a longer biological half-life. A library of BA derivatives was screened in search of a non-endogenous analogue with higher potency and/or longer half-life for HIF stabilization. In vitro screenings at physiologically-relevant concentrations (5 mM) were performed using intestinal epithelial cell lines (T84 and CaCO2) in normoxia to determine HIF-1a protein abundance. Various analogues stabilized HIF, with 4-mercapto-butyrate (MBA) being the most promising candidate. The best analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CAIX by q-PCR. MBA exhibited higher induction of these targets compared to BA. Time-course studies revealed that MBA exhibits significantly longer half-life as observed in the stabilization of HIF and induction of gene targets for up to 72 h compared to BA (24 h). Loss of gene induction was observed in MBA treated cells expressing lentiviral shRNA against HIF1b (HIF1b KD), supporting a HIF-dependent transcription. Furthermore, it’s been widely reported that BA enhances epithelial barrier, primarily through HIF coordinated barrier protection. Among several other BA derivatives, MBA enhanced and prolonged epithelial barrier function in intestinal epithelial cells. In vivo studies in C57BL/6 mice explored the induction of HIF targets in tissue and specifically erythropoietin (EPO) in circulation following administration of BA and MBA at similar doses. These studies revealed that induction of EPO with a single dose of MBA exceeded that of native BA. In colonic and kidney tissue, GLUT1 and BNIP3 were moderately induced in vivo. Thus, a non-endogenous BA derivative (MBA) stabilizes HIF at higher potency and longer half-life than native BA and will be used as a template for the development of HIF stabilizing agents.