Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor.

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1, N5-(1-imino-2-chloroethyl)-l-ornithine ( KI = 1.3 µM, kinact = 0.34 min-1), was conceptually dissected into two fragments and each characterized separately: l-norvaline ( Ki = 470 µM) and 2-chloroacetamidine ( KI = 310 µM, kinact = 4.0 min-1). This analysis suggested that the two fragments were not linked in a manner that allows either to reach full affinity or reactivity, prompting the synthesis and characterization of three analogues: two that mimic the dimethylation status of the substrate, N5-(1-imino-2-chloroisopropyl)-l-ornithine ( kinact /KI = 208 M-1 s-1) and N5-(1-imino-2-chlorisopropyl)-l-lysine ( kinact /KI = 440 M-1 s-1), and one that lengthens the linker beyond that found in the substrate, N5-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, KI = 0.19 µM, kinact = 0.22 min-1). Cl-NIL is one of the most potent inhibitors reported for DDAH1, inactivates with a second order rate constant (1.9 × 104 M-1 s-1) larger than the catalytic efficiency of DDAH1 for its endogenous substrate (1.6 × 102 M-1 s-1), and has a partition ratio of 1 with a >100 000-fold selectivity for DDAH1 over arginase. An activity-based protein-profiling probe is used to show inhibition of DDAH1 within cultured HEK293T cells (IC50 = 10 µM) with cytotoxicity appearing only at higher concentrations (ED50 = 118 µM). A 1.91 Å resolution X-ray crystal structure reveals specific interactions made with DDAH1 upon covalent inactivation by Cl-NIL. Dissecting a covalent inactivator and analysis of its constituent fragments proved useful for the design and optimization of this potent and effective DDAH1 inhibitor.

Selective Covalent Protein Modification by 4-Halopyridines through Catalysis.

We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4-chloropyridine with amino acids and thiols was ranked with respect to common covalent protein-modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment-sized 4-halopyridines inactivated human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off-pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrated that the pKa and kinact /KI values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.

Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma.

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. In this study, N(5) -(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) is shown to be a potent time- and concentration-dependent inhibitor of purified human DDAH-1 (KI =1.3±0.6 µM; kinact =0.34±0.07 min(-1) ), with >500-fold selectivity against two arginine-handling enzymes in the same pathway. An activity probe is used to measure the "in cell" IC50 value (6.6±0.2 µM) for Cl-NIO inhibition of DDAH-1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50/64 (78 %) of melanoma lines tested showed increased levels of DDAH-1 relative to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl-NIO shows a subsequent decrease in cellular nitric oxide production. Cl-NIO is a promising tool for the study of methylarginine-mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production, such as septic shock and idiopathic pulmonary fibrosis.

Rational design of a transition state analogue with picomolar affinity for Pseudomonas aeruginosa PvdQ, a siderophore biosynthetic enzyme.

The Pseudomonas aeruginosa enzyme PvdQ can process different substrates involved in quorum-sensing or in siderophore biosynthesis. Substrate selectivity was evaluated using steady-state kinetic constants for hydrolysis of N-acyl-homoserine lactones (HSLs) and p-nitrophenyl fatty acid esters. PvdQ prefers substrates with alkyl chains between 12 and 14 carbons long that do not bear a 3-oxo substitution and is revealed here to have a relatively high specificity constant for selected N-acyl-HSLs (kcat/KM = 10(5) to 10(6) M(-1) s(-1)). However, endogenous P. aeruginosa N-acyl-HSLs are ≥100-fold disfavored, supporting the conclusion that PvdQ was not primarily evolved to regulate endogenous quorum-sensing. PvdQ plays an essential biosynthetic role for the siderophore pyoverdine, on which P. aeruginosa depends for growth in iron-limited environments. A series of alkylboronate inhibitors was found to be reversible, competitive, and extremely potent (Ki ≥ 190 pM). A 1.8 Å X-ray structure shows that 1-tridecylboronic acid forms a monocovalent bond with the N-terminal ß-chain Ser residue in the PvdQ heterodimer, mimicking a reaction transition state. This boronic acid inhibits growth of P. aeruginosa in iron-limited media, reproducing the phenotype of a genetic pvdQ disruption, although co-administration of an efflux pump inhibitor is required to maintain growth inhibition. These findings support the strategy of designing boron-based inhibitors of siderophore biosynthetic enzymes to control P. aeruginosa infections.

Quinobis(imidazolylidene): synthesis and study of an electron-configurable bis(N-heterocyclic carbene) and its bimetallic complexes.

Reaction of bromanil with N,N'-dimesitylformamidine followed by deprotonation with NaN(SiMe(3))(2) afforded 1,1',3,3'-tetramesitylquinobis(imidazolylidene) (1), a bis(N-heterocyclic carbene) (NHC) with two NHC moieties connected by a redox active p-quinone residue, in 72 % yield of isolated compound. Bimetallic complexes of 1 were prepared by coupling to FcN(3) (2) or FcNCS (3; Fc=ferrocenyl) or coordination to [M(cod)Cl] (4 a or 4 b, where M=Rh or Ir, respectively; cod=1,5-cyclooctadiene). Treatment of 4 a and 4 b with excess CO(g) afforded the corresponding [M(CO)(2)Cl] complexes 5 a and 5 b, respectively. Analysis of 2-5 by NMR spectroscopy and X-ray diffraction indicated that the electron-deficient quinone did not significantly affect the inherent spectral properties or coordination chemistry of the flanking imidazolylidene units, as compared to analogous NHCs. Infrared spectroscopy and cyclic voltammetry revealed that decreasing the electron density at ML(n) afforded an increase in the stretching energy and a decrease in the reduction potential of the quinone, indicative of metal-quinone electronic interaction. Differential pulse voltammetry and chronoamperometry of the metal-centered oxidations in 2-4 revealed two single, one-electron peaks. Thus, the metal atoms bound to 1 are oxidized at indistinguishable potentials and do not appear electronically coupled. However, the metal-quinone interaction was used to increase the electron density at coordinated metal atoms. Infrared spectroelectrochemistry revealed that the average nu(CO) values for 5 a and 5 b decreased by 14 and 15 cm(-1), respectively, upon reduction of the quinone embedded within 1. These shifts correspond to 10 and 12 cm(-1) decreases in the Tolman electronic parameter of this ditopic ligand.