A Continuous, Fluorescent, High-Throughput Assay for Human Dimethylarginine Dimethylaminohydrolase-1

Abstract

Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possesses adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4,446 compounds and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date, and enables screening of compound libraries using [S] =KM conditions, while displaying Z′ factors from 0.6 – 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.

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

“…The HTS assay performance has been documented elsewhere. 19, 20 To discover novel scaffolds suitable for inhibitor development, we chose a more conservative fragment-based approach in which smaller, less potent compounds are identified as starting points for drug development. The benefits of this approach are reviewed elsewhere, and include the ability to identify compact, easily modified core structures in which most of the atoms contribute directly to binding affinity.…”

“…31 We previously screened this library for inhibitors of human DDAH-1 and reported only on the assay’s performance. 19 The primary screening hits from that assay are now reported here, and are combined with additional primary screening hits from a second HTS assay of the same library completed by using the P. aeruginosa DDAH isoform. We then designed a rigorous series of validation tests that were applied to these pooled primary hits.…”

“…32 This thiol is then detected by a chromogenic or fluorogenic reagent and the resulting increase in signal is compared to control wells to observe any apparent inhibition by library compounds. 19, 20 Each assay is done in the presence of a non-ionic detergent to reduce non-selective inhibition by “aggregators.” 33, 34 Primary screens of the 4000-member fragment library in duplicate at 100 µM of each compound for each DDAH isoform identified 44 compounds as possible inhibitors of P. aeruginosa DDAH and 79 compounds as possible inhibitors of human DDAH-1, reflecting a 1 % and 2 % primary hit rate, respectively (Figure 3). This primary hit rate is much higher than is typically seen when screening diverse libraries of drug-like molecules, but is typical for libraries of fragment-sized molecules.…”

“…The duration of the enzyme and inhibitor preincubation step in the P. aeruginosa DDAH screen (≤ 1 h) was longer than in the human DDAH-1 screen (10 min). 19, 20 Since inhibitors 10 and 11 are time-dependent inactivators, an increase in the duration of this step results in a lower apparent IC 50 value and, therefore, a more easily detectable “hit.” Future HTS protocols that lengthen the duration of this step would have an enhanced ability to detect other time-dependent inactivators.…”

“…18 Through a high-throughput screening (HTS) approach, we identified ebselen ( 7 ) as an inhibitor of human DDAH-1, but the polypharmacology of this compound complicates its use. 19, 20 Recently, HTS of a 130,000 member diverse library using saturating concentrations of substrate ([S] > K M ), revealed a number of human DDAH-1 inhibitors, but their structures suggest that the hits are mostly reactive electrophiles. 21 Two promising inhibitors were identified ( 8 , 9 ) 21 , but the mode of inhibition by 8 (Hill coefficient = 1.8) was not reported, and compound 9 did not inhibit human DDAH-1 in our hands (Sigma-Aldrich, ( m/z ) M + H + calc’d for 231.10, found 231.10; ( m/z ) M + Na + calc’d for 253.08, found 253.08; Gayle Burstein;T.W.L; W.F; University of Texas, Austin, unpublished observations).…”