Search

CAS 1609 (compound 1) and CHF 2363 (compound 2) are two furoxan derivatives able to release nitric oxide (NO) under physiological conditions, and display typical NO-dependent vasodilator activity. The potential genotoxic effects of compound 1 and of the water-soluble analogue of CHF 2363 (compound 2a) were investigated. The results show that the two compounds induce genotoxic effects only at concentrations that significantly reduce cell viability. However, in the case of compound 1 this range of concentrations is one order of magnitude higher than the one leading to the beneficial effects, while in the case of compound 2a these ranges partially overlap. In both cases the release of NO plays a key role in the induction of the cytotoxic and genotoxic effects, since the non-NO-donating furazan analogues display a different toxicological profile, and since the effects were reduced in the presence of oxyhaemoglobin, a well-known NO-scavenger

The inhibition mechanism of the dimeric human placenta glutathione transferase (GST) P1-1 by calvatic acid and the reaction intermediates, i.e. the diazocyanide analogue of calvatic acid, has been investigated at pH 7.0 and 30.0 degrees C. Experiments performed at different molar ratios of inhibitor/GST P1-1 indicate that 1 mol calvatic acid inactivates 1 mol GST P1-1, containing two catalytically equivalent active sites. However, 2 mol of the diazocyanide analogue of calvatic acid inactivate 1 mol GST P1-1. Two disulfide bridges/dimer, probably between Cys47 and Cys101, have been formed during the reaction of GST P1-1 with calvatic acid and its diazocyanide analogue. The apparent second-order rate constants for GST P1-1 inactivation by calvatic acid and its diazocyanide analogue are 2.4+/-0.3 M(-1) s(-1) and (8.5+/-0.7) x 10(3) M(-1) s(-1), respectively. The reaction of calvatic acid with free L-cysteine can be described by a simple process with an apparent second-order rate constant of (5.0+/-0.4) x 10(1) M(-1) s(-1). In contrast, a transient species occurs during the reaction of the diazocyanide analogue of calvatic acid with free L-cysteine. Kinetics may be described by a second-order process [the rate constant being (8.0+/-0.5) x 10(3) M(-1) s(-1)] followed by a first-order decay [the rate constant corresponding to (1.2+/-0.1) x 10(1) s(-1)]. Calvatic acid represents an enzyme inhibitor acting much slower than its reaction intermediates (i.e. its diazocyanide analogue).

The inhibition mechanism of the dimeric human placenta glutathione transferase (GST P1-1) by the antibiotic p-carboxyphenylazoxycyanide (calvatic acid) has been investigated at pH 7.0 and 30.0 degrees C. Experiments performed at different calvatic acid/GST P1-1 molar ratios indicate that one mole of calvatic acid inactivates one mole of the homodimeric enzyme molecule, containing two catalytically equivalent active sites. The apparent second order rate constant for GST P1-1 inactivation is 2.4 +/- 0.3 M-1 s-1. The recovery of all the 5,5'-dithio-bis(2-nitro-benzoic acid)-titratable thiol groups as well as the original catalytic activity of GST P1-1 after treatment of the inhibited enzyme with dithiothreitol indicates that two disulfide bridges per dimer, likely between Cys47 and Cys101, have been formed during the reaction with calvatic acid. To the best of the authors knowledge, calvatic acid represents a unique case of enzyme inhibitor acting also throughout its reaction product(s).

The inhibitory effect of bis-, tris- and tetra-benzamidine derivatives (DAPP, TAPB and TAPP, respectively) on the catalytic properties of bovine beta-trypsin (beta-trypsin), human alpha-thrombin (alpha-thrombin) and porcine pancreatic beta-kallikrein-B (beta-kallikrein-B) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, benzamidine, DAPP, TAPB and TAPP, show the same value of the association inhibition constant (Ki, M-1) for beta-trypsin; at variance, the affinity of DAPP, TAPB and TAPP for alpha-thrombin and beta-kallikrein-B is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to beta-trypsin, alpha-thrombin and beta-kallikrein-B as well as for DAPP, TAPB and TAPP association to beta-trypsin reflects the acidic-pK shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for DAPP, TAPB and TAPP binding to alpha-thrombin and beta-kallikrein-B appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues. Considering the X-ray three dimensional structures and the computer-generated molecular models of the serine proteinase inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to beta-trypsin, alpha-thrombin and beta-kallikrein-B has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).