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. Ethylenediamine-tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine sidechain of lisinopril by EDC/NHS 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 pre-incubation with metal-chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal-chelate-lisinopril complexes revealed IC50 values ranging from 44 nM to 4,500 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−1min−1 (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primary from sidechain 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.

A new paradigm for drug activity is presented, which includes both recognition and subsequent irreversible inactivation of therapeutic targets. Application to both RNA and enzyme biomolecules has been demonstrated. In contrast to RNA targets that are subject to strand scission chemistry mediated by ribose H-atom abstraction, proteins appear to be inactivated through oxidative damage to amino acid side chains around the enzyme active site.

Catalytic metallopeptides that target the membrane-associated sortase A transpeptidase have been developed and evaluated as irreversible inactivators of SrtA∆N59 (sortase A, lacking the initial membrane-binding domain). The copper-binding GGH tripeptide ATCUN motif was linked to amidated forms of the cell wall sorting signal, LPET and LPETG, as sortase-targeting moieties. The resulting metallopeptides were used to determine half maximal inhibitory concentrations (IC₅₀) and rate constants for time-dependent sortase A inactivation. Michaelis-Menten behavior was observed for the catalytic metallopeptides, and k(cat), K(M) and k(cat)/K(M) parameters were obtained as 0.080 ± 0.002 min⁻¹, 23 ± 2 μM and 0.0035 ± 0.0003 μM⁻¹ min⁻¹, respectively. Concentration-dependent inhibition of SrtA∆N59 by the metallopeptides revealed IC₅₀ values ranging from 570 to 700 µM, while Cu-GGH, which lacked a targeting motif, had no measurable IC₅₀ value (>2,000 µM). Time-dependent inactivation of SrtA revealed a range of catalytic activities, with Cu-GGHGLPETG-NH2 demonstrating the fastest rate of inactivation in the presence of ascorbate and hydrogen peroxide coreactants. The active site of the enzyme comprises residues Cys-184, Arg-197 and His-120. LC-MS/MS analysis of the reaction products demonstrated modification of Cys-184 to cysteine sulfonic acid (+48 amu). Results obtained from a DTNB assay support oxidation of the Cys-184 residue. LC-MS/MS also suggested oxidation of the Arg-197 containing peptide. 2D NMR analysis was performed to assess the possible oxidation of His-120, however, none was observed. These compounds possess the potential for irreversible inactivation of SrtA through oxidative modification of essential residues required for substrate binding.

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 were tested for both reversible binding and irreversible catalytic inactivation 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 the 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 orientation factors (double-filter effect).

Amphiphilic chitosan, bPalm-CS-HTAP, having N -(2-((2,3-bis(palmitoyloxy)propyl)amino)-2-oxoethyl) (bPalm) groups as double hydrophobic tails and O -[(2-hydroxyl-3-trimethylammonium)] propyl (HTAP) groups as hydrophilic heads was synthesized and evaluated for its self-assembly properties and potential as a gene carrier. The degree of bis-palmitoyl group substitution ( DS bPalm ) and the degree of quaternization ( DQ ) were approximately 2 and 56%, respectively. bPalm-CS-HTAP was found to assemble into nanosized spherical particles with a hydrodynamic diameter ( D H ) of 265.5 ± 7.40 nm (PDI = 0.5) and a surface charge potential of 40.1 ± 0.04 mV. bPalm-CS-HTAP condensed the plasmid pVAX1.CoV2RBDme completely at a bPalm-CS-HTAP:pDNA ratio of 2:1. The self-assembled bPalm-CS-HTAP/pDNA complexes could enter HEK 293A and CHO cells and enabled gene expression at negligible cytotoxicity compared to commercial PEI (20 kDa). These results suggested that bPalm-CS-HTAP can be used as a promising nonviral gene carrier.