The enzyme DDAH metabolizes methylarginines that are inhibitors of nitric oxide synthase (NOS). Substrate-based inhibitors of mammalian DDAH have been synthesized, with optimization to give selective inhibition of DDAH with no significant direct effect on NOSs. These are the first examples of reversible DDAH inhibitors with significant activity and selectivity. In vivo administration increases plasma ADMA levels, giving proof of concept that these inhibitors can be used to probe the physiological effects of DDAH inhibition, with potential for pharmaceutical use of DDAH inhibitors in diseases where excess NO production is implicated.

In patients with chronic kidney disease, high plasma levels of the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine, are thought to contribute to decline in renal function. Here we took a candidate gene approach to determine any causal role of asymmetric dimethylarginine in the progression of chronic kidney disease. The impact of single-nucleotide polymorphisms in the genes encoding the two isoforms of the asymmetric dimethylarginine-degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1 and DDAH2), on enzyme expression, plasma asymmetric dimethylarginine levels, and longitudinal changes in estimated glomerular filtration rate were determined in various patient groups. There was evidence suggesting that the rs17384213 DDAH1 GG genotype was associated with increased expression of its mRNA in kidney allografts. Healthy subjects carrying the rs17384213 G allele had lower plasma asymmetric dimethylarginine, and a similar borderline association was found in patients with chronic kidney disease. This allele, however, was independently associated with a steeper decline in renal function in two separate cohorts of patients with chronic kidney disease. We conclude that polymorphisms in DDAH1 alter the rate of decline of glomerular filtration rate in subjects with chronic kidney disease. Our findings show that DDAH1 modulates plasma asymmetric dimethylarginine and contributes to the decline in renal function. However, it appears that increases in circulating methylarginine did not mediate progressive kidney injury.

An efficient process for the discovery of inhibitors of DDAH enzymes, without the requirement for high throughput screening, is described. Physicochemical filtering of a 308,000-compound library according to drug likeness followed by reciprocal nearest neighbour selection produced a representative subset of 35,000 compounds. Virtual screening on a dual processor PC using FlexX, followed by biological screening, identified two hit series. Similarity searches of commercial databases and chemical re-synthesis of pure compounds resulted in SR445 as an inhibitor of Pseudomonas aeruginosa DDAH at 2 microM.

Three cases of mild metabolic hyperoxaluria (with glycollaturia) are described. They showed different types of response to pyridoxine. One responded to low dose, one responded at first to low dose but became resistant, and the third showed temporary response to high dose. One case also had primary hyperparathyroidism and one had medullary sponge kidneys and hypercalciuria. It is important to measure urinary oxalate (and glycoUate) in all cases of calcium oxalate urolithiasis.

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...

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...

No abstract

24 h urine compositions of male stone formers with idiopathic hypercalciuria prior to treatment were compared with those of male general practitioners without urolithiasis. Urinary urate was slightly higher in the stone formers than in the normals but this was not statistically significant. Furthermore, when results were corrected for the higher creatinine excretions of the stone formers then the reverse was true and statistically significant. All subjects with urinary urate over 7.0 mmol/24 h were separately studied. In these groups the normals had higher urate and creatinine excretions than the stone formers but when results were corrected for creatinine the difference in the urate excretions disappeared. In long term follow up studies urinary calcium was lowered by diet and more so by diet supplemented with either Bendrofluazide or cellulose phosphate. Each drug raised urinary oxalate slightly and this was statistically significant, while both drugs together caused an even bigger rise in oxalate excretion. An unexpected finding was a rise in urinary urate with cellulose phosphate.