E-64 (1) is a fungal natural product that has prominent roles in drug discovery and chemical biology (Fig. 1a). Isolated from Aspergillus japonicus TPR-64 in 1978, 1 is a classic trans-epoxysuccinic acid (t-ES)-based irreversible, potent and selective inhibitor against cysteine proteases such as papain, calpain and cysteine cathepsins (Fig. 1a). Cysteine proteases are ubiquitously conserved in all kingdoms of life, have multifaceted physiological roles and are potential drug targets for multiple diseases. Upon binding to a cysteine protease, the electrophilic t-ES warhead in 1 is covalently captured by the thiolate of the catalytic cysteine (Fig. 1a), a feature that led to the development of probes based on 1 for activity-based protein profiling (ABPP). Numerous biosynthetic variants of 1 have been isolated from fungi (Supplementary Fig. 1). Extensive synthetic efforts have resulted in a variety of analogs such as CA-074, CLIK-148 (3) and NYC-488 that show selectivity toward cathepsin B, cathepsin L and calpain, respectively (Fig. 1b and Supplementary Fig. 1). E-64d (loxistatin), a prodrug for E-64c (2; loxistatin acid), was in phase 3 trials for the treatment of muscular dystrophy (Fig. 1b) and was also repurposed for treating viral infections such as coronavirus disease. Despite such a decorated track record of 1, the enzymes responsible for the formation of 1 have remained elusive.
a, Structure, mode of action and retrobiosynthesis of 1. E-64 contains the t-ES warhead that is the site of covalent inhibition of cysteine proteases. E-64 is proposed to form from the condensation of (2 S,3 S)-t- ES, l-Leu and agmatine. b, Structures of synthetic cysteine protease inhibitors based on 1. c, Structure of dapdiamide E. d, Mechanism and application of bacterial ABSs such as McbA from the marinacarboline biosynthetic pathway. e, Mechanism and applications of bacterial ATP-grasp enzymes such as TabS from the tabtoxin biosynthetic pathway. f, Stepwise combination of two bacterial amide bond-forming enzymes CysC and CysD to synthesize cystargolide analogs. g, In this work, we discovered and biochemically characterized the fungal ATP-grasp enzyme and ABS from the biosynthesis of 1 and used the enzymes in the synthesis of diverse E-64 analogs.
Biosynthetically, 1 is formed from the stepwise condensation of three distinct building blocks, the warhead (2 S,3 S)-t-ES (a dicarboxylic acid), l-Leu (an amino acid) and agmatine (an amine) (Fig. 1a,b), using amide bond-forming enzyme(s) (Fig. 1a). Amide bond-forming enzymes have found widespread interest as biocatalysts for the synthesis of pharmaceuticals because of the ubiquity of the amide functionality (Extended Data Fig. 1a,b). Enzymatic synthesis of amides can obviate the protection–deprotection steps associated with functionally rich building blocks and can achieve chemoselectivity and regioselectivity with atom economy. Two particular classes of enzymes that are not associated with nonribosomal peptide synthetases (NRPSs) (Extended Data Fig. 1c,d and Supplementary Fig. 2), adenosine triphosphate (ATP) grasp enzymes and amide bond synthetases (ABSs), have attracted attention because of their direct activation and amidation of carboxylic acids without the need for partnering enzymes.
