2 research outputs found
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 (IC<sub>50</sub>) 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)Âpropyl]Âcarbodiimide 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-RPPGFSAFKÂ(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 IC<sub>50</sub> 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<sup>–1</sup> min<sup>–1</sup> (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