Metal-Dependent Coordination Geometry Controls Autolytic Amide Bond Cleavage to Enable Prodrug Activation

Abstract We introduce a peptide prodrug activation strategy that employs the trivalent Lewis-acidic metal ions Ga3+, Fe3+, In3+, and Co3+, chelated by triazacyclononanediacetic acid monoamides. The metal chelates trigger rate-tunable, metal-mediated, autolytic amide bond cleavage (TMAC) adjacent to N-methylated and canonical amino acids. Our studies reveal that the four TMAC-compatible metal ions induce amide bond hydrolysis with divergent pH dependence and mechanistic pathways. We subsequently linked Ga3+, Fe3+, In3+, and Co3+ complexes to the N-terminus of a potent cytotoxic cargo, monomethyl auristatin E (MMAE), to produce the proof-of-concept prodrug PD1. In vitro toxicity studies establish IC50 values in HeLa and PC-3 cells that broadly correlate with complex cleavage rates; the metal ion-free PD1 conjugate, the Zn-PD1 complex, as well as the Ga3+, Fe3+, In3+, and Co3+ chelate hydrolysis products alone confer no toxicity. Attachment of a cancer-targeting peptide to the metal chelate, furnishing PD2, enables targeted delivery of the therapeutic payload in vivo, compatible with noninvasive nuclear imaging using positron emission tomography with 68Ga or single-photon computed tomography with 111In. The corresponding natGa-PD2 complex selectively suppressed tumor growth in vivo with minimal off-target toxicity. These studies establish metal identity as a general design parameter for programming amide bond cleavage and introduce TMAC as a versatile platform to engender peptide prodrugs with tunable release kinetics.