N- versus C‑Domain Selectivity of Catalytic Inactivation of Human Angiotensin Converting Enzyme by Lisinopril-Coupled Transition Metal Chelates

The N- and C-terminal domains of human somatic angiotensin I converting enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates was tested for both reversible binding and irreversible catalytic inactivation of each domain of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of M–chelate–lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and catalytic factors (double-filter effect)

Targeted Catalytic Inactivation of Angiotensin Converting Enzyme by Lisinopril-Coupled Transition-Metal Chelates

A series of compounds that target reactive transition-metal chelates to somatic angiotensin converting enzyme (sACE-1) have been synthesized. Half-maximal inhibitory concentrations (IC₅₀) and rate constants for both inactivation and cleavage of full-length sACE-1 have been determined and evaluated in terms of metal chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine side chain of lisinopril by 1-ethyl-3-[3-(dimethylamino)­propyl]­carbodiimide hydrochloride/<i>N</i>-hydroxysuccinimide coupling. The resulting amide-linked chelate–lisinopril (EDTA–lisinopril, NTA–lisinopril, DOTA–lisinopril, and GGH–lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel, and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK­(Dnp)-OH, a derivative of bradykinin, following preincubation with metal chelate–lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal chelate–lisinopril complexes revealed IC₅₀ values ranging from 44 to 4500 nM for Ni–NTA–lisinopril and Ni–DOTA–lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal chelate–lisinopril complexes revealed a remarkable range of catalytic activities, with second-order rate constants as high as 150 000 M^–1 min^–1 (Cu–GGH–lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primarily from side chain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal chelate–lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1