Metabolite mimicry identifies butyrate analogs with select protective functions in the intestinal mucosa

Microbial-derived short-chain fatty acids regulate a variety of pathways in the healthy colonic mucosa. In particular, butyrate serves as the primary energy source for colonocytes and regulates gene transcription by stabilizing the transcription factor hypoxia-inducible-factors (HIF) and functioning as a histone deacetylase (HDAC) inhibitor. A limitation of butyrate as a therapeutic is its rapid metabolism in differentiated colonocytes. Furthermore, intestinal stem cells (ISCs) respond differently to butyrate, preferentially using glucose for energy procurement. To address these limitations, we explored metabolite-mimicry to discover compounds with potent or selective biological responses within the butyrate pathway(s). We discovered an analog, 3-chlorobutyrate (3-Cl BA), that significantly enhances epithelial barrier formation and wound healing in vitro. Mechanistically, we revealed that 3-Cl BA is a potent HDAC inhibitor. Furthermore, unlike butyrate, 3-Cl BA does not stabilize HIF and it is not used as metabolic fuel. In vivo studies in a DSS-colitis model revealed that contrary to butyrate, 3-Cl BA is protective. Studies in stem-like colonoids demonstrated that only butyrate inhibits ISC proliferation and differentiation. Furthermore, it was recently reported that HIF stabilization inhibits ISCs activity. Given the fact that butyrate but not 3-Cl BA stabilizes HIF, we surmised that 3-Cl BA would circumvent these detrimental functional consequences. We demonstrate here that pharmacologic HIF stabilization inhibits colonoid differentiation and that genetic loss of HIF significantly promotes ISC differentiation. This study reveals a promising butyrate analog protective in colitis and demonstrates the advantages of metabolite-mimicry to dissect selective biological functions from major metabolites in the gut. Significance statementButyrate is a well-studied microbial short-chain fatty acid that regulates a number of mucosal pathways and is paramount in maintaining intestinal integrity. In health, it is a major source of energy for colonocytes and regulates gene transcription. The role of butyrate in disease is still controversial and not well understood. When butyrate is not metabolized or well-utilized (e.g. disease), it accumulates in intestinal stem cells leading to reduced cell proliferation and differentiation, thereby hampering intestinal barrier recovery. In this study, we describe a butyrate analog that enhances epithelial barrier formation and wound healing. Furthermore, as opposed to native butyrate, this butyrate analog is protective in a colitis mouse model and does not exhibit detrimental influences on intestinal stem cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697087v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1dd0f0borg.highwire.dtl.DTLVardef@6d27forg.highwire.dtl.DTLVardef@1e5d07corg.highwire.dtl.DTLVardef@dab74f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Microbially derived butyrate plays a key role in intestinal homeostasis. It is the primary source of energy for colonocytes, contributing to a metabolic and oxygen gradient as it is metabolized by differentiated cells along the intestinal crypt. Through the regulation of transcription factors such as HIF and the inhibition of HDAC, it regulates barrier formation and wound healing promoting a strong tight junction profile. Furthermore, well oxygenated ISCs at the bottom of the crypt are unaccustomed to the effects of butyrate, including HIF stabilization (left). In disease, loss of intestinal architecture leads to a disrupted metabolic/oxygen gradient where butyrate accumulates in stem cells leading to decreased proliferation, differentiation, and increases in apoptosis. 3-Cl BA selectively acts as an HDACi and does not stabilize HIF, exhibiting no significant detrimental effects on ISCs (right). Created in BioRender. Ornelas, A. (2026) https://BioRender.com/x9cy8mw C_FIG