Competitive inhibitors of the salvage pathway enzyme purine-nucleoside phosphorylase (purinenucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have been designed by using the three-dimensional strucre of the enzyme as determined by x-ray crystallography.

1-Aminocyclopropanephosphonate (ACPP) was synthesized, and its effects on the pyridoxal 5'-phosphate linked enzymes 1-aminocyclopropanecarboxylate (ACPC) deaminase from Pseudomonas sp. ACPC and alanine racemase from Bacillus stearothermophilus were studied. ACPP was found to be a potent inhibitor of both enzymes with Km/Ki ratios of 500 and 2000, respectively. Inhibition for both enzymes was characterized by slow-binding (second-order rate constants less than 150 M-1 s-1) slow-dissociating behavior. Analysis of the pre-steady-state kinetics revealed a kinetically detectable intermediate E.I complex in the inhibition mechanism for the racemase but not for the deaminase. The one-step deaminase inhibition (Formula: see text) mechanism had an association rate constant (k1) of 100 M-1 s-1, a value 10(6)-fold slower than diffusion, suggesting either a slow alignment of the inhibitor at the enzyme active site or, more likely, the same mechanism as followed by racemase but with an E.I to E.I conversion rate (k3) that is sufficiently fast on the steady-state time scale so as to hinder detection of the initial weakly associated E.I intermediate. The E to E.I transition for the deaminase was further monitored by ultraviolet-visible and circular dichroism (CD) spectroscopies and found to exhibit a time-dependent shift in the visible absorption spectrum lambda max from 418 nm for the native enzyme to 333 nm at steady state, again consistent with a rapid E to E.I and slow E.I to E.I behavior. A rate constant for the absorbance shift of 150 M-1 s-1 was consistent with the k1 calculated in the inhibition studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of the zinc protease neutral endopeptidase (NEP, EC 3.4.24.11) offer significant therapeutic interest as antihypertensives due to their ability to potentiate the biological action of the circulating natriuretic hormone ANF (atrial natriuretic factor). N-Phosphonomethyl dipeptides bearing a central (4-phenyl)phenylalanine residue have been designed to exert potent and selective NEP inhibition. In particular, (S)-3-[N-[2- [(phosphonomethyl)amino]-3-(4-biphenylyl)propionyl]amino]propionic acid (10a) (CGS 24592) displayed high inhibitory potency in vitro (IC50 = 1.9 +/- 0.1 nM) and a long plasma half-life in rats but lacked oral bioavailability. This drawback was overcome by using esterase-sensitive (acyloxy)alkyl phosphonates. More remarkable, several diaryl phosphonate derivatives of 10a also performed as effective prodrugs. Specifically, the structurally simple diphenyl phosphonate 18 (CGS 25462) induced potent inhibition of NEP ex vivo for at least 8 h after oral administration to rats (30 mg/kg). Its antihypertensive effect was demonstrated in DOCA-salt rats. At 30 mg/kg orally, 18 caused a significant reduction in mean arterial pressure measuring -35 +/- 7 mmHg at 5-h postdosing. The alpha-aminomethyl phosphonate 18 represents a new generation of selective NEP inhibitors that combine high potency, long duration of action, and oral bioavailability. Therefore, it holds promise as a novel therapeutic agent for the treatment of human hypertension and congestive heart failure.

Background and purpose: Thyroid hormone receptor (TR) agonists are in clinical trials for the treatment of hypercholesterolaemia. As statins are the standard of clinical care, any new therapies must have adjunctive activity, when given in combination with statins. As already known for the statins, the cholesterol lowering effect of TR activation involves increased expression of the low-density lipoprotein receptor. Using animal models, we tested whether TR activation would have additive cholesterol lowering activity in the presence of effective doses of a statin. Experimental approach: We evaluated the activity of a liver-targeted prodrug, MB07811, of a novel TH receptor b agonist, MB07344, as monotherapy and in combination with atorvastatin in rabbits, dogs and monkeys. Key results: In rabbits, MB07344 (i.v.) decreased total plasma cholesterol (TPC) comparable to that achieved with a maximally effective dose of atorvastatin (p.o.). The addition of MB07344 to atorvastatin resulted in a further decrease in TPC. Similarly, the addition of MB07811 (p.o.) to atorvastatin treatment decreased TPC beyond the level achieved with either agent as monotherapy. In dogs and monkeys, atorvastatin and MB07811 were administered as monotherapy or in combination. Consistent with the rabbit studies, the combination treatment caused a greater decrease in TPC than either MB07811 or atorvastatin administered as monotherapy. Conclusions and implications:We conclude that the effects of MB07811 and atorvastatin in lowering cholesterol are additive in animals. These results would encourage and support the demonstration of similarly improved efficacy of combination versus monotherapy with such agents in the clinic.