Developing dual and specific inhibitors of dimethylarginine dimethylaminohydrolase-1 and nitric oxide synthase: toward a targeted polypharmacology to control nitric oxide

Abstract

Molecules that block nitric oxide's (NO) biosynthesis are of significant interest. For example, nitric oxide synthase (NOS) inhibitors have been suggested as antitumor therapeutics, as have inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes endogenous NOS inhibitors. Dual-targeted inhibitors hold promise as more effective reagents to block NO biosynthesis than single-targeted compounds. In this study, a small set of known NOS inhibitors are surveyed as inhibitors of recombinant human DDAH-1. From these, an alkylamidine scaffold is selected for homologation. Stepwise lengthening of one substituent converts an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor and then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N(5)-(1-iminohexyl)-l-ornithine for neuronal NOS (1.7, 3, 20, >1,900 microM, respectively) and DDAH-1 (990, 52, 7.5, 110 microM, respectively). A 1.9 A X-ray crystal structure of the N5-(1-iminopropyl)-L-ornithine:DDAH-1 complex indicates covalent bond formation between the inhibitor's amidino carbon and the active-site Cys274, and solution studies show reversible competitive inhibition, consistent with a reversible covalent mode of DDAH inhibition by alkylamidine inhibitors. These represent a versatile scaffold for the development of a targeted polypharmacological approach to control NO biosynthesis.

Figure 1

Nitric oxide biosynthesis is promoted by the enzymic activities of both NO synthase (NOS) and DDAH. DDAH catabolizes endogenous methylarginines and relieves their inhibition of NOS.

Figure 2

Diagram of selected substrates and inhibitors of NOS and DDAH-1.

Figure 3

Analytical sedimentation equilibrium ultracentrifugation of DDAH-1. Symbols represent an overlay of data collected during the last nine scans, indicative that equilibrium had been reached. The solid line represents the global simultaneous fit for a single ideal species model using Ultrascan. The fitted M_w is 34,530 Da (theoretical monomer M_w = 33,558 Da), variance = 8.7e^-5. Conditions: DDAH-1 (1 mg/ mL), NaH₂PO₄ (20 mM), NaCl (100 mM), pH 7.0 at 25 °C with a rotor speed of 20,000 rpm.

Figure 4

The active site of DDAH-1 in complex with L-IPO (13) and omit map density. Atoms colored in cyan, blue, red and yellow for carbon, nitrogen, oxygen and sulfur, respectively. Active-site residues and L-IPO are labeled. The F_o-F_c electron density map, with L-IPO and the C274 Sγ atom not included in the calculated structure factors, is shown at 3 σ in blue.

Figure 5

Reversible covalent inhibition of DDAH-1 by L-IPO (13). The the non-covalent complex (20) is expected to be in rapid equilibrium with the (R)-tetrahedral complex (21).

Figure 6

Lineweaver-Burk plot of DDAH-1 inhibition by L-IPO (13). L-IPO is included in assay mixtures at 0 (●), 62.5 (▼), 125 (▲), 250 (□) and 500 (■) μM at pH 7.27, 25 °C. Intersecting fits at 1/V_max indicates competitive inhibition against the SMTC substrate. A K_i value of 50 ± 4 μM is determined as described in Experimental Procedures.

Figure 7

Alternative loop conformations observed in DDAH-1. Structural variance in residues 167 – 173 is observed depending on the bound inhibitor. Protein backbone is shown as a ribbon representation, bound inhibitors as ball and stick representations, and selected residues (Arg 145, Leu172, His 173, Cys 274) in stick form. Apo protein is in white, and the l-citrulline (4), L-IPO (13) and L-257 (16) complexed structures in green, light blue and orange, respectively. The figure is constructed using coordinates from this work and from protein data bank accession codes 2JAI and 2JAJ (38).

Figure 8

Comparison of DDAH-1 and NOS active sites. The left panel shows a superimposition of human DDAH-1 with bound L-IPO (13) (green) and L-257 (16) (pink). The right panel shows a superimposition of bovine eNOS bound by L-NIO (12) (green) and rat nNOS bound by N^ω-propyl-l-arginine (pink), a compound of comparable length to N⁵-(1-iminopentyl)-l-ornithine (14). Inhibitors are shown in ball and stick format and colored by heteroatom as described earlier. The heme groups of NOS are shown in stick models. Surface features (light blue) are shown for the L-257-DDAH-1 complex and the N^ω-propyl-l-arginine-nNOS complex to highlight the active-site cavity shape. Figures are constructed using coordinates from this work and protein data bank accession codes 2JAJ, 1ED6 and 1MMV (23, 38, 82).