Liu Mengting, Zang Xin, Vlahakis Niko W, Rodriguez Jose A, Ohashi Masao, Tang Yi
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA, USA.
Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, STROBE, NSF Science and Technology Center, University of California, Los Angeles, Los Angeles, CA, USA.
Nat Chem Biol. 2025 May 9. doi: 10.1038/s41589-025-01907-2.
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 (2S,3S)-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.
E-64是一种不可逆的半胱氨酸蛋白酶抑制剂，在化学生物学和药物发现中被广泛应用。在此，我们揭示了一种E-64的非核糖体肽合成酶非依赖型生物合成途径，该途径在真菌中广泛保守。该途径始于富马酸被铁（II）/α-酮戊二酸依赖性加氧酶环氧化为弹头（2S,3S）-反式环氧琥珀酸，随后依次通过三磷酸腺苷抓握酶与L-氨基酸以及通过酰胺键合成酶的真菌实例与胺进行缩合。这两种形成酰胺键的酶均显示出显著的生物催化潜力，包括可扩展性、对弹头的立体选择性以及在形成酰胺键时更广泛的底物范围。与这些形成酰胺键的酶进行生物催化级联反应产生了一个半胱氨酸蛋白酶抑制剂文库，从而得到了更有效的组织蛋白酶抑制剂。此外，一锅法反应实现了临床相关抑制剂的制备合成。我们的工作突出了生物合成研究对于酶发现的重要性以及形成酰胺键的酶在合成小分子文库方面的潜力。
