Cysteine protease inhibitor(Boehringer Ingelheim)

Ascaris lumbricoides cystatin (Al‐CPI), a cysteine protease inhibitor, has shown anti‐inflammatory effects in models of house dust mite‐induced respiratory allergy and experimental colitis. Since monocytes are primary targets of cystatins, and helminth‐derived products can induce immunoregulatory monocytes that mitigate autoimmune and inflammatory diseases in murine models, we aimed to investigate the immunoregulatory effects of Al‐CPI on human monocytes and its mechanisms. Monocytes were isolated from healthy donors using CD14+ magnetic beads and treated with recombinant Al‐CPI (rAl‐CPI) before activation with LPS. Surface marker expression and caspase‐1 activity were analysed by flow cytometry, while cytokine levels were quantified using bead‐based assays. Caspase‐8 inhibition was measured by fluorescence, and pro‐IL‐1β expression was evaluated by Western blot. RNA‐seq and differential gene expression analyses were performed using DESeq2 and GSEA. rAl‐CPI reduced cell surface expression of HLA‐DR and CD14 as well as IL‐1β levels in cell culture supernatants. Transcriptomic analysis identified 30 differentially expressed genes (12 upregulated and 18 downregulated; padj < 0.05 and fold change [FC] < |1.5|), including the downregulation of CD14 and SLAMF1 , both involved in LPS‐mediated signalling through the TLR4‐TRIF pathway. GSEA analysis showed that rAl‐CPI plus LPS induced an enrichment of the IFN signalling pathway compared with LPS (NES = 1.88; p adj < 0.0001). Further analysis of IL‐1β production and maturation showed that rAl‐CPI did not alter pro‐IL‐1β levels, but reduced caspase‐1 activation. In conclusion, rAl‐CPI may impair IL‐1β maturation and release by modulating the alternative inflammasome activation, potentially through downregulation of CD14 and other key regulatory molecules involved in this pathway.

E-64 is an irreversible cysteine protease inhibitor prominently used in chemical biology and drug discovery. Here we uncover a nonribosomal peptide synthetase-independent biosynthetic pathway for E-64, which is widely conserved in fungi. The pathway starts with epoxidation of fumaric acid to the warhead (2 S ,3 S )- trans -epoxysuccinic acid with an Fe(II)/α-ketoglutarate-dependent oxygenase, followed by successive condensation with an l -amino acid by an adenosine triphosphate grasp enzyme and with an amine by the fungal example of amide bond synthetase. Both amide bond-forming enzymes display notable biocatalytic potential, including scalability, stereoselectivity toward the warhead and broader substrate scopes in forming the amide bonds. Biocatalytic cascade with these amide bond-forming enzymes generated a library of cysteine protease inhibitors, leading to more potent cathepsin inhibitors. Additionally, one-pot reactions enabled the preparative synthesis of clinically relevant inhibitors. Our work highlights the importance of biosynthetic investigation for enzyme discovery and the potential of amide bond-forming enzymes in synthesizing small-molecule libraries.