Phase II Metabolite-Inspired Tripeptide Editing Generates Anti-Inflammatory Candidates for Treatment-Refractory...

Ulcerative colitis remains a major clinical challenge due to limited availability of safe, orally active anti-inflammatory drugs. Although phase II metabolism is traditionally viewed as a detoxification process, certain metabolites retain or exhibit enhanced pharmacological activity, offering an underexplored drug discovery opportunity. Here, we investigated phase II metabolite-inspired tripeptide editing using M13, a glutathione-conjugated metabolite of the anti-inflammatory natural product 6-shogaol, as a lead scaffold. A focused library of 38 analogs was generated by systematic tripeptide editing while preserving the central cysteine-containing 6-shogaol-derived scaffold. Phenotypic nuclear factor-κB (NF-κB) reporter screening identified 29 of 38 analogs with greater inhibitory activity than M13 at the screening concentration. Two selected leads, MLY2 and MLY8, demonstrated enhanced anti-inflammatory activity in Dextran Sulfate Sodium-induced experimental colitis, including restoration of colon length and reductions in fecal lipocalin-2 and pro-inflammatory cytokines. Both compounds also suppressed inflammatory cytokine production more effectively than M13 in ex vivo colonic biopsies from patients with treatment-refractory ulcerative colitis. In parallel, MLY2 and MLY8 showed no detectable mutagenicity under the Ames assay conditions and were well tolerated in a preliminary single-dose maximum tolerated dose study. Collectively, these findings establish phase II metabolite-inspired tripeptide editing as a productive lead-discovery strategy and identify MLY2 and MLY8 as anti-inflammatory leads warranting further preclinical characterization.