Discovery of structurally-diverse inhibitor scaffolds by high-throughput screening of a fragment library with dimethylarginine dimethylaminohydrolase

Abstract

Potent and selective inhibitors of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) are useful as molecular probes to better understand cellular regulation of nitric oxide. Inhibitors are also potential therapeutic agents for treatment of pathological states associated with the inappropriate overproduction of nitric oxide, such as septic shock, selected types of cancer, and other conditions. Inhibitors with structures dissimilar to substrate may overcome limitations inherent to substrate analogs. Therefore, to identify structurally-diverse inhibitor scaffolds, high-throughput screening (HTS) of a 4000-member library of fragment-sized molecules was completed using the Pseudomonas aeruginosa DDAH and human DDAH-1 isoforms. Use of a substrate concentration equal to its KM value during the primary screen allowed for the detection of inhibitors with different modes of inhibition. A series of validation tests were designed and implemented in the identification of four inhibitors of human DDAH-1 that were unknown prior to the screen. Two inhibitors share a 4-halopyridine scaffold and act as quiescent affinity labels that selectively and covalently modify the active-site Cys residue. Two inhibitors are benzimidazole-like compounds that reversibly and competitively inhibit human DDAH-1 with Ligand Efficiency values ≥ 0.3 kcal / mol / heavy (non-hydrogen) atom, indicating their suitability for further development. Both inhibitor scaffolds have available sites to derivatize for further optimization. Therefore, use of this fragment-based HTS approach is demonstrated to successfully identify two novel scaffolds for development of DDAH-1 inhibitors.

Specific Lowering of Asymmetric Dimethylarginine by Pharmacological Dimethylarginine Dimethylaminohydrolase Improves Endothelial Function, Reduces Blood Pressure and Ischemia-Reperfusion Injury

“…Previous attempts to discover small molecule modulators of DDAH activity have not been successful (Ghebremariam et al, 2012;Linsky and Fast, 2012). Thus, at the present time, pharmacological agents that can specifically lower pathological ADMA are not available.…”

Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

“…DDAH inhibition and activation have been suggested as useful molecular probes to better understand cellular regulation of nitric oxide. Several potent DDAH inhibitors have recently been published [ 43 ] and suggested as potential therapeutic agents for treatment of pharmacological states associated with inappropriate under- and over-production of nitric oxide, such as septic shock [ 44 ]. Therefore, ADMA and especially DDAH changes in the perilesional area might offer potential new therapeutic targets in treatment of TBI and secondary brain damage development.…”

The Second Life of Methylarginines as Cardiovascular Targets

“…Most of the existing evidence suggests that therapeutic strategies aimed at upregulation of DDAH activity and subsequent ADMA lowering might be beneficial to patients with CVD. As a result, several groups performed experimental screens for modulators of DDAH activity to identify DDAH-activators [59,60,61]. These screens led to discovery of specific DDAH inhibitors [62,63], however, no specific and efficient DDAH activators were reported.…”