Inhibition of dimethylarginine dimethylaminohydrolase (DDAH) and arginine deiminase (ADI) by pentafluorophenyl (PFP) sulfonates

Abstract

A range of pentafluorophenyl (PFP) sulfonate esters derived from the reaction of PFP vinyl sulfonate and various nitrones are shown to have significant inhibitory activity against the bacterial enzymes DDAH and ADI.Nitric oxide (NO) is an important mediator of intracellular signaling and has attracted interest as a target for therapeutic intervention, as it is widely acknowledged that there are a variety of disease states for which NO is implicated. 1 One of the most significant problems associated with the design of inhibitors, is to target the pathological excess NO production without disrupting essential NO-mediated processes, often by seeking selectivity for a particular NOS isoform. One method for potentially circumventing these problems is the indirect modulation of NO levels by inhibition of the enzyme DDAH which is responsible for controlling levels of N G -methyl-L-arginine (MMA) and N G , N G , dimethyl-L-arginine (ADMA) which are endogeneous inhibitors of NOS. 2,3 Inhibition of bacterial DDAH 4 is also of interest as it offers opportunities for the development of new anti-bacterial agents. The structurally related enzyme arginine deiminase (ADI) is also a possible antibacterial/antiprotozoal target, as various pathogenic organisms utilize ADI to generate ATP under anaerobic conditions. 5aRecently high-resolution structures of a bacterial DDAH 6 and ADI 5 have been disclosed and it has been shown in both enzymes that the active site comprises a catalytic triad containing an acidic residue (Glu / Asp), a basic residue (His) and a cysteine residue (Cys) (Figure 1). Both enzymes are known to catalyze the conversion of the substrate(s) MMA and ADMA to citrulline as shown in figure 1. In a recent communication Knipp et al. described the cysteine modification of DDAH by HcyNO and proposed this as a lead for the possible development of covalent inhibitors of DDAH and ADI. 7 In that work it was proposed that it should be possible to rationally design covalent inhibitors of DDAH based on those findings. That work has prompted us to disclose our own studies, which identify novel small molecule inhibitors of DDAH and ADI. Whilst the development of small molecule inhibitors of both DDAH and ADI is appealing, it is notable that there is only one known inhibitor of bacterial DDAH, which has modest affinity and is an arginine homologue, 8 and there are no known inhibitors of ADI. Herein we disclose our preliminary studies on the use of pentafluorophenyl (PFP) sulfonates as an unprecedented new class of enzyme inhibitors. The biological activity of the PFP-sulfonate group is completely unexplored and is highlighted here by the development of inhibitors of DDAH and ADI As had previously been noted by one of us in the disclosure of the crystal structure of DDAH, the active site resembles that of a cysteine protease with a catalytic triad (Fig 1). 5 The work of Roush et al. on the use of sulfonates and sulfonamides as inhibitors of cysteine proteases, 9 stimulated us to speculate that it may be possible to generate inh...

Arginine analogues incorporating carboxylate bioisosteric functions are micromolar inhibitors of human recombinant DDAH-1

“…28,29 DDAH-1 is primarily associated with the metabolism of ADMA and NMMA, whereas the biochemical role of DDAH-2 remains unclear. 31 Many exhibit moderately good inhibitory potential with selectivity primarily towards the bacterial isoenzyme PaDDAH, such as SR445 and pentafluorophenyl (PFP) sulphonates, 32,33 whereas others are effective on the human DDAH (hDDAH), in particular L-VNIO, L-257 and the corresponding methyl ester, L-291 (Fig. 30 Since DDAH plays a key role in the regulation of NO concentrations in vivo, the modulation of its activity may represent a potential therapeutic approach in the treatment of pathological states associated with excessive NO production.…”

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

“…11, 17 Pentafluorophenyl sulfonates ( 6 ) were reported as inhibitors of P. aeruginosa DDAH and may represent a promising scaffold, but tests with human DDAH-1 have not been reported, and it is unclear which aspects of their structures are important for affinity to the enzyme. 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.…”

Synthesis of isomeric 2,3,5‐trisubstituted perhydropyrrolo[3,4‐d]‐isoxazole‐4,6‐diones via 1,3‐dipolar cycloaddition reactions

“…In recent years, focus has been shifted toward asymmetric nitrone cycloaddition reactions; enantioselective [21], catalytic enantioselective [22], and diastereoselective [23] synthetic methodologies, as well as metal‐assisted stereocontrol [24] have been reported. Isoxazolidines have been found to exhibit antimicrobial activity [25–28] and have been used as enzyme inhibitors [29–31]. Isoxazolidine nucleoside analogues, in which a furanose ring has been replaced by an N,O‐heterocyclic system, are a particularly interesting group of compounds due to their potential antiviral activity [32–36].…”