Competitive reversible inhibitors

We conclude the discussion of formal kinetics with a practical consideration. When two isotopomers simultaneously present in an enzyme substrate mixture compete for the same active site on the free enzyme E, one can write  

The same concentration of free enzyme enters both Equations 11.55 and 11.56. Dividing and introducing Equation 11.20 yields  

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The inactivation is normally a first-order process, provided that the inhibitor is in large excess over the enzyme and is not depleted by spontaneous or enzyme-catalyzed side-reactions. The observed rate-constant for loss of activity in the presence of inhibitor at concentration [I] follows Michaelis-Menten kinetics and is given by kj(obs) = ki(max) [I]/(Ki + [1]), where Kj is the dissociation constant of an initially formed, non-covalent, enzyme-inhibitor complex which is converted into the covalent reaction product with the rate constant kj(max). For rapidly reacting inhibitors, it may not be possible to work at inhibitor concentrations near Kj. In this case, only the second-order rate-constant kj(max)/Kj can be obtained from the experiment. Evidence for a reaction of the inhibitor at the active site can be obtained from protection experiments with substrate [S] or a reversible, competitive inhibitor [I(rev)]. In the presence of these compounds, the inactivation rate Kj(obs) should be diminished by an increase of Kj by the factor (1 + [S]/K, ) or (1 + [I(rev)]/I (rev)). From the dependence of kj(obs) on the inhibitor concentration [I] in the presence of a protecting agent, it may sometimes be possible to determine Kj for inhibitors that react too rapidly in the accessible range of concentration. ... 

Note that in some cases one may follow the time course of covalent E-A formation by equilibrium binding methods (e.g., LC/MS, HPLC, NMR, radioligand incorporation, or spectroscopic methods) rather than by activity measurements. In these cases substrate should also be able to protect the enzyme from inactivation according to Equation (8.7). Likewise a reversible competitive inhibitor should protect the enzyme from covalent modification by a mechanism-based inactivator. In this case the terms. S and Ku in Equation (8.7) would be replaced by [7r] and K respectively, where these terms refer to the concentration and dissociation constant for the reversible inhibitor. 

A method has been described by which the effects of reversible competitive monoamine oxidase inhibitors might be estimated successfully ex vivo (Green, 1984). This method relies upon the ability of a reversible competitive inhibitor (perhaps administered chronically) to protect against the effects of an irreversible inhibitor (administered as a single dose) that binds to the enzyme active site. As inhibition by an irreversible inhibitor can be measured quite easily ex vivo, the degree of irreversible inhibition in an animal coadministered a reversible competitive inhibitor would be less than that in a control animal that received only the irreversible inhibitor. The difference would provide an estimate of the degree to which enzyme was bound (protected) by the reversible inhibitor in vivo. 

Several inhibitors of AChE have been developed for use in treating Alzheimer s disease, which requires that the drugs readily enter the CNS. These inhibitors are structurally unrelated and vary in their mechanism of inhibition, although all are reversible inhibitors. Tacrine (Cognex) is a monoamine acridine. Donepezil (Aricept) is a piperidine derivative that is a relatively specific inhibitor of AChE in the brain, with little effect on pseudo-ChE in the periphery. Galanthamine (Reminyl) is a tertiary alkaloid and phenanthrene derivative extracted from daffodil bulbs that is a reversible competitive inhibitor of AChE it also acts on nicotinic receptors. 

Over the years, many reversible competitive inhibitors selective for MAO A have been developed, including many a-methylamines. In contrast, very few effective MAO B-selective reversible inhibitors have been reported to date. Accumulated experience has indicated that a-alkylamines are inhibited steiically from binding to MAO B—and thus are MAO A selective. No complementary steric inhibition selective for MAO A is available [6b]. However, it was demonstrated that, while 5-fluoro-a-methyltryptamine (5) is a selective MAO A substrate, jS-substitution, as in p-chloro-/J-methylphenylamine (6), favors MAO B selectivity [28]. 

Reversible, competitive inhibitors of COX-1 and COX-2. Inhibitors such as ibuprofen, piroxicam or mefenamic acid compete against arachidonic acid to bind at the catalytic center. 

Inhibition by drugs The statin drugs, including simvastatin, lovastatin, and mevastatin, are structural analogs of HMG CoA, and are reversible, competitive inhibitors of HMG CoA reductase (Figure 18.7). They are used to decrease plasma cholesterol levels in patients with hypercholesterolemia.1... 

A common practice is to conduct the immobilization procedure in the presence of species that occupy the active site of the enzymes, such as substrates, cofactors, reversible competitive inhibitors, or products, at concentrations preferably above their Michaelis or inhibition constants. Such a precaution is not necessary for the immobilization of CMP-Neu5Ac synthetase. 

Galantamine, unlike the other anticholinesterases in clinical use, is derived from the alkaloids from the daffodil and snowdrop family. It is a reversible, competitive inhibitor of acetylcholinesterase with some inhibitory action on butyryl cholinesterase. It is also an agonist at nicotinic receptor sites. Although a clinically effective drug, galantamine frequently causes gastrointestinal side effects. 

Most therapeutic drugs are reversible competitive inhibitors, which bind at the catalytic (active site) of the enzyme. Competitive inhibitors are especially attractive as clinical modulators of enzyme activity because they offer two routes for the reversal of enzyme inhibition, by decreasing the concentration of inhibitor or by raising the concentration of substrate. 

If the dmg is an inhibitor, we are dealing with a ternary system of receptor, physiological agonist, and our inhibitory dmg. We will examine two cases Reversible competitive inhibitors (Fig. 3.2, top) and irreversible ones (Fig. 3.2, bottom). 

A series of (3-alkenylphenyldifluoromethyl)phosphonic acids (234), has been synthesized on non-crosslinked polystyrene (NCPS) support and examined for inhibition with protein tyrosine phosphatase IB. Phosphonic acid (234) was the most potent of this series of compounds being a reversible, competitive inhibitor with a K of 8.0 1,4 pM (Figure 41)." ... 

CNS. These H, receptor antagonists are reversible, competitive inhibitors of the pharmacological actions of histamine on Hj receptors. 

The rate of absorbance change for the uninhibited enzyme was linear with time (Figures 1A and B). A lower, but constant, rate was observed with trans-3-amino-cvclohexene carboxyl ate, which is characteristic of a reversible, competitive inhibitor (Figure 1A). 

In general, an irreversible mechanism of inhibition might reduce the versatility of a drug for in vivo applications. Thus, a major challenge for the ongoing pharmaceutical research is the development of potent and selective, but reversible, inhibitors of FAAH. Based on ot-ketoheterocycle protease inhibitors (Edwards et al., 1995), potent reversible competitive inhibitors were developed, combining an unsaturated acyl chain and an a-keto-A4-oxazolopyridine, with incorporation of a second weakly basic N-atom. This class of compounds... 

Exemestane (Aromasin) is a more potent, orally administered analog natural substrate androstenedione that lowers estrogen levels more effectively than formestane. In contrast to the reversible competitive inhibitors anastrozole and letrozole, exemestane irreversibly inactivates the enzyme, and, therefore is referred to as a suicide substrate. Doses of 25 mg per day inhibit aromatase activity by 98%, and lower plasma estrone and estradiol levels by about 90%. It... 

Further work on the 7a-(4 -aminophenyl)thioandrostenedione (61, 7-APTA) was undertaken. The synthesis of an unsaturated derivative of (61) has resulted in the conversion of a reversible competitive inhibitor of AR into an enzyme-activated irreversible inhibitor. 7a-(4 -Aminophenyl)thio-l,4-androstadiene-3,17-dione (62, 7-APTADD) has an apparent Ki value of 9.9 + 1.0 nM (.Sfn, for androstenedione of 52.5 + 5.9 nM) and demonstrated rapid... 

STRUCTURE-ACTIVITY RELATIONSHIP All the available Hj-receptor antagonists are reversible competitive inhibitors of the interaction of histamine with Hj receptors. Like histamine, many Hj antagonists contain a substituted ethylamine moiety. Unlike histamine, which has a primary amino group and a single aromatic ring, most Hj antagonists have a tertiary amino group linked by a two- or three-atom chain to two aromatic substituents (e.g., diphenhydramine). 

As reversible, competitive inhibitors of enzymes, porphyrins can be used for identification and quantification of a substrate or other competitive inhibitors of enzymes. This approach has been successful in the development of cholinesterase and organophosphorus hydrolase bearing glass surfaces for the detection of substrates and inhibitors, including organophosphate compounds. The technique has demonstrated detection limits for sarin (GB) below the Immediately Dangerous to Life or Health levels mdicated by CDC/NIOSH. 

The COX-2-selective diarylheterocycHc inhibitors have been reported to be a reversible competitive inhibitor of COX-1 while demonstrating time-dependent irreversible inhibition of COX-2 which accounts for the potency and selectivity demonstrated by members of this structural class [229]. The phenylsulphonamide moiety of the diarylheterocycles associate within a side pocket present in the active site of COX-2 and this pocket is more accessible in COX-2 than in COX-1, which is the result of the substitution of a vahne for an isoleucine foimd at position 523 in COX-1 [235]. 

We evaluated the four diastereomers of 17, as well as the flexible macrocyclic ether 18 and the comparison compounds 19 and 20, as reversible, competitive inhibitors of thermolysin, and determined the inhibition constants given in Table 6. The stereoisomer of 17 that turned out to have the S,S-conHguration as designed proved to be the most potent inhibitor, with a K value significantly lower than those of the other isomers, as well as the comparison compounds. The ca. 50-fold enhancement in binding affinity of... 

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