Time dependent inhibitors

Coumarincarboxylate derivatives are versatile, efficient, low molecular weight, nonpeptidic protease inhibitors. Both esters and amides behave as time-dependent inhibitors of a-chymotrypsin but the esters are clearly more efficient than the corresponding amides. The criteria for a suicide mechanism are met. The presence of a latent alkylating function at the 6-position (chloromethyl group) is required to produce to inactivation by a suicide mechanism (Scheme 11.3, pathway a). Aryl esters, in particular the meta-substituted phenyl esters are the best inhibitors. Thus, m-chlorophenyl 6-(chloromethyl)-2-oxo-27/-l-benzopyran-3-carboxylate is one of the well-known inactivator of a-chymotrypsin (kJK, = 76(),000M s 1 at pH 7.5 and 25 °C, Table 11.1). 

A number of 2,3-methanophenylalanine derivatives are efficient inhibitors of DOPA carboxylase [64]. For instance, 2-(3,4-dihydroxyphenyl) ACC 57, due to its structural analogy with a-methyl DOPA 58, is a reversible time-dependent inhibitor of DOPA carboxylase and of tyrosine amino transferase, Eq. (22) [65]. 

The first (3-lactams LE inhibitors were naturally occurring bicyclic compounds, such as clavams and cephalosporins [338], but more recently, synthetic monocyclic (3-lactams have been developed. Time-dependent inhibitors of enzyme HLE, based on the cephem nucleus, have been reported. A series of cephalosporin tert-butyl esters have been examined, and the activity of these compounds has been found to be very sensitive to the C-7 substituents, the greatest activity being showed by small, a-oriented, and electron-withdrawing groups. Additionally, the oxidation... 

All three assumptions can be violated in the case of CYP enzymes, depending on the design of the in vitro CYP inhibition study. The first assumption can be potentially violated if the drug being tested is a time-dependent inhibitor (e.g., one with a slow on rate see below). The potency of some inhibitors (e.g., the CYP3A inhibitors ketoconazole and clotrimazole) is such that the free concentration of the inhibitor tends to approach the concentration of the enzyme (40), a violation of the second assumption. In the case of such tight-binding inhibition, an apparent A) value (A i a ) )) can be estimated, as follows ... 

All of the azides investigated were time-dependent inhibitors at millimolar concentrations and the inhibition was reversible in each case, with hepatic glutathione 5-transferase proving the most sensitive enzyme. Inhibitor potency appears to depend upon the substrate employed, -heptyl and allyl azides (60) and (62) being the most potent with NBC, and -butyl and -hexyl azide (57) and (59) when DNCB was included in the assay. Kinetic studies, where the GSH and DNCB concentrations were independently varied, indicated that compounds (61),(63) and (64) were noncompetitive inhibitors, while allyl azide (62) and the n-alkyl azides (56)-(60) inhibited the enzyme in a competitive manner. From these observations, the authors speculate that, in a process reminiscent of that known to occur with alkyl and aryl halides, glutathione 5-transferase may catalyse the conjugation of azides with GSH in vivo. 

Most P450 inhibitors act via reversible (competitive or noncompetitive mechanisms) with which their inhibitory potential can be estimated from their ICS0 or Ki values. Some inhibitors are "mechanism-based" or "time-dependent" inhibitors, which can cause irreversible inhibition due to the formation of reactive metabolites by the CYP isoform, leading to covalent binding to the active site and thereby causing irreversible inhibition of the affected enzyme molecule.31 Irreversible inhibitors therefore will have prolonged inhibition of the enzyme even after clearance of the drug in question. fCinact is a measurement of the potency of such "mechanism-based" inhibitors. 

Time-dependent inhibition observed The inhibitor is a time-dependent inhibitor. In vivo studies will need to be performed to further define its drug-drug interaction potential. 

Additional safety concern A time-dependent inhibitor may need to be further studied to define its hepatotoxic potential, as a number of time-dependent P450 inhibitors are found to cause idiosyncratic hepatotoxicity. 

The monoester 2 was converted by standard methods into the cyclopropene equivalent of aminocyclopropane carboxylic acid." The cyclopropene is a time-dependent inhibitor of ethenc biosynthesis and an extremely poor substrate for acetylene produetion and a preventative of senescence. 

The strict specificity of PFL for pyruvate and formate as substrates places some restrictions on the structure of potential active site-directed inhibitors. Therefore, acetyl phosphinate (207) is a natural candidate for a PFL inhibitor, as it is not only a pyruvate analog, but is also a derivative of hypophosphite, a known mechanism-based PFL inactivator. In the absence of CoA, 100 mM acetyl phosphinate is an effective time-dependent inhibitor of PFL, although the inactivation reaction does not appear to be first order. If 55 mM CoA is included in the inactivation reaction mixture, first-order kinetics are now observed, and with 10 mM acetyl phosphinate the half-life of the inactivation reaction is 3 min. In the presence of 5 mM pyruvate (no CoA), PFL is completely protected from inactivation by 100 mM acetyl phosphinate. 

Burkhart, J.P, N.P Peet, C.L. Wright, and J.O. Johnston (1991). Novel time-dependent inhibitors of human placental aromatase. J. Med. Chem. 34, 1748-1750. 

Reversible inhibitors usually compete with substrate for binding at the active site. Ketoconazole (Figure 10.2) is one example of a reversible inhibitor of CYP3A4. Because a rapid equilibrium is involved, the potency observed for such an inhibitor will be independent of incubation time. This isn t true for the other two types of CYP inhibitors, which are therefore known as time-dependent inhibitors (TDIs). 

Quick primary screens for CYP inhibition can fail to identify time-dependent inhibitors (TDIs) as noted in Box 10.2. These are not at all uncommon. To identify TDIs one sample of the test compound is incubated with microsomes or an rCYP without NADPH, while a second one otherwise identical, but containing this necessary CYP co-factor, is prepared. After 30 min the substrate and NADPH (now needed for metabolism to proceed) are added to each, and the amount of metabolite formed is determined for each sample. Time-dependent inhibitors will... 

Many of the inhibitors that react with cyclooxygenase in a time-dependent irreversible manner do so by reversibly forming an El complex at the substrate site which then leads irreversibly by a first-order process to an inactive form of the enzyme, E+. The overall scheme of such a competitive, time-dependent inhibitor is then ... 

If the CYP in the gut eontributes to the metabolism of a drug and is inhibited by a time-dependent inhibitor, then a change of bioavailability of the drug due to decreased gut metabolism should also be taken into account. The ratio of to T g ctr (the intestinal wall bioavailability in the presence and absence of inhibitor, respectively) can be incorporated into the model [Eq. (4.22)]. The ratio of to Fg u can be estimated from the relative change in CLint,j... 

Hamilton CJ, Saravanamuthu A, Poupat C, Fairlamb AH, Eggleston IM (2006) Time Dependent Inhibitors of Trypanothione Reductase Analogues of the Spermidine Alkaloid Lunarine and Related Natural Products. Bioorg Med Chem 14 2266... 

Recently Withers et al. tested chalcones carrying sulfonamide groups as potential inhibitors of TcTS [65]. Impressive IC50 data could be measured - e.g., IC50 = 0.6 pM for compound 108 however, with these structural elements it could not be excluded that covalent modifications of the enzyme may occur due to the Michael acceptor properties of these derivatives. The authors did address this question, and a covalent enzyme modification was excluded since the compounds did not show a time-dependent inhibitor activity [65]. 

Fluoroadenosine has been prepared in unprotected form for the first time, using chemistry similar to that employed by Moffatt and co-workers for the synthesis of nucleocidin (Vol.lO, p.l60), but with some improvements in protecting groups and reagents. The compound was found to be a time-dependent inhibitor of adenosylhomosyteine hydrolase. ... 

The inhibition mechanism of acetylphosphinic acid analog of pymvate 3 against E. coli PDHc was further studied by Laber et al. in 1990 [47]. The results showed that 3 was a time-dependent inhibitor. At initial reaction, a reversible enzyme-inhibitor complex El was rapidly formed and then closely integrated into the complex El. All of the experiments showed that the product formed by the interaction of 3 with PDHc El was similar to these intermediates formed by a normal reaction of substrate pymvate and TPP. 

Previously, Kluger et al. s work showed that 1-1 was a good inhibitor against E. coli PDHc. 1-1 was also found to be a good competitive inhibitor of pea PDHc by Baillie et al. later [2]. According to BaiUie et al. s work, 1-2 caused time-dependent inhibition with a half-Ufe of inactivation ty-2) of approximately 12 min at 10 pM. 1-3 was an exceptional good time-dependent inhibitor with ty in the region of 3 min at 0.3 pM. 1-6 was much less active, whereas 1-5 with Me as R and H as R" were inactive at all. 

The above results suggested that substituents R and R" on the skeleton of the monosodium of acetylphosphinic acid or acetylphosphonic acid 1 would gready affect the enzyme inhibitory ability. It is worth noting that when Me as R" stayed the same, a smaller, weaker electronegativity R on the phosphorus, there are noticeable increasing inhibition activity. Baillie et al. s work showed that both 1-2 and 1-3, which resemble pyruvate in strucmre, were good PDHc inhibitors. Compared with 1-2 (R = R" = Me), 1-3 (R = H, R" = Me) is a much better time-dependent inhibitor [2]. 

---