Characterization of C‐Alkyl Amidines as Bioavailable Covalent Reversible Inhibitors of Human DDAH‐1

Abstract

C-alkyl amidine analogs of asymmetric Nω,Nω -dimethyl-L-arginine are dual-targeted inhibitors of both human DDAH-1 and nitric oxide (NO) synthase, and provide a promising scaffold for developing therapeutics to control NO overproduction in a variety of pathologies including septic shock and some cancers. Using a two-part clickchemistry-mediated activity probe, a homologated series of C-alkyl amidines are ranked for their ability to inhibit DDAH-1 within cultured HEK 293T cells. N5-(1-iminopentyl)-L-ornithine was determined to be the most potent compound in vitro (Kd = 7 μM) as well as in cultured cells, and the binding conformation and covalent reversible mode of inhibition was investigated by comparison of interactions made with DDAH-1 and a catalytically inactive C274S variant as gauged by Xray crystallography and isothermal titration calorimetry. By interrupting the ability of the inhibitor to form a covalent bond, the contribution of this interaction can be estimated. These results suggest further stabilization of the covalent adduct as a promising strategy for lead optimization in the design of effective reagents to block NO synthesis.

Developing an Irreversible Inhibitor of Human DDAH‐1, an Enzyme Upregulated in Melanoma

“…Therefore, the selectivity of DDAH-1 inhibitors against arginase and nitric oxide synthase is an important consideration that has been noted previously. [7a, 11] Cl-NIO does not inhibit endothelial NOS at concentrations up to 500 μM (Figure 3), giving a conservative selectivity ratio of approximately ( K i, eNOS : K I, DDAH-1 ) ≥ 500 : 1. NOS isoforms have conserved active sites and do not typically show marked differences in selectivity between arginine-like inhibitors, [12] so only the endothelial isoform was tested here.…”

“…[7a] Briefly, N α-BOC-L-ornithine (250 mg, 1.08 mmol) was dissolved in water (4 mL) in an ice bath and treated with NaOH (2.5 M) to adjust the solution pH to 10. 2-Chloro-1-ethoxy-ethanimine (462 mg, 2.94 mmol) was slowly added in portions while maintaining the mixture at pH 10.…”

“…[7b, 14] Cultured HEK293T cells were seeded in a 12-well polystyrene plate using complete growth medium containing DMEM with 10% FBS (Invitrogen, Carlsbad, CA) and grown to 80 % confluency. The pEF6a-hDDAH-1 plasmid [14] was transiently transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).…”

“…IPO, K i = 52 μM) (Figure 1). [7] We also found that the fragment-sized compound 2-chloroacetamidine (CAA) is a covalent irreversible inhibitor of P. aeruginosa DDAH that selectively modifies the active-site Cys residue, albeit with weak potency ( K I = 3.1 mM; k inact = 1.2 min −1 ). Other groups have demonstrated the utility of 2-haloacetamidines in the design of selective inhibitors for other enzyme targets and these ‘warheads’ were used in both cell culture and animals, providing precedence for their wider application.…”

Designing modulators of dimethylarginine dimethylaminohydrolase (DDAH): A focus on selectivity over arginase

“…However, most of these l-arginine analogues additionally affect NOSs activity 7,[10][11][12] . In fact, it is still a matter of debate whether selectivity over NOSs is essentially needed, considering that inhibition of both NOSs and DDAH represents a dual mode of action and should increase potency of .…”