Kcat inhibitors

Mechanism-based inhibitors (also known as suicide inhibitors or as kcat inhibitors) are actually substrates for their target enzymes. A reactive group is only revealed by enzyme action it is therefore not subject to hydrolysis until it has been revealed in the vicinity of the enzyme. The ability of the inhibitor then to inactivate the enzyme will depend upon relative rates of (a) covalent bond formation with the enzyme, (b) diffusion of the reactive entity away from the enzyme, and (c) hydrolysis. 

Figure 11. Acetylenic secosteroids as kcat inhibitors of P. testosteroni...

As these inhibitors owe their activity to the kcat term (i.e., the enzyme s usual mode of action) they have been designated "kcat inhibitors" by Rando (1), while Abeles and Maycock (2 ) have used the term "Suicide Enzyme Inactivators" because the enzyme, in accepting such a "booby-trapped" substrate commits suicide by its own mechanism of action. 

The most useful, and thus far successful, examples have involved irreversible reactions of nucleophilic functions of an enzyme s reactive site with an enzymatically activated Kcat inhibitor of a Michael-type addition reaction. The activation invariably requires participation of the enzyme s prosthetic group (e.g., flavin of monoamine oxidase) or coenzymes such as pyridoxal (vitamin B) as its phosphate, which is associated with several enzymes (e.g., threonine dehydrase, ornithine decarboxylase, a-ketoglutarate transaminase). 

To function successfully, therefore, a Kcat inhibitor must be able to bind to the enzyme s reactive site with high affinity and carry a latent group readily convertible by the enzyme to a function capable in turn of alkylating (or acylating) the site covalently, thus inactivating it permanently. Examples will illustrate this type of potential drug. 

Suicide Substrates. Much like affinity labels, suicide inhibitors first form a reversible complex with the target enzyme due to the structural similarity between inhibitor and substrate. In a subsequent time dependent step, an irreversible complex (usually covalent) is formed with an appropriately positioned amino acid side chain. Unlike the affinity label, suicide substrates (65) are not inherently reactive and must undergo activation by the target enzyme before the irreversible complex is formed. Therefore, these inhibitors are generally more selective than affinity labels. Since the enzyine catalyzes its own inactivation, these inhibitors are also known as kcat inhibitors (66), enzyme-activated, irreversible inhibitors (54) and Trojan horse reagents (6. ... 

The results were indicative of the crucial role of the electric dipole moment of ligands and its interaction with the negatively charged residue Glu225 and, moreover, the dipole proved to be an useful observable for discriminating between substrate and inhibitor (Fig. 2.5). A striking correlation was found between the energetics associated with this interaction and the kcat values... 

In addition to the TDI experiment, the partition ratio measures the TDI efficiency. Specifically, the partition ratio is the number of inactivation kinetic events (k nact) versus the number of substrate turnover events per unit enzyme (kcat) [161], Thus, the most potent partition ratio is zero. The most common experimental setup for determining the partition ratio is the titration method that increases the inhibitor concentration relative to a known amount of enzyme. After the incubations, a secondary incubation containing a probe substrate similar to the TDI experiment is used to define the remaining activity. For accurate determination of the partition ratio from the titration method, it is assumed that the inhibitor is 100% metabolized ... 

The kinetic parameters kcat and Km were determined without inhibitor and at several inhibitor concentrations. The inhibition constants K,e and Kies were calculated from plots of KJkcat and /kcat as a function of the concentration of inhibitor. 

Antibody 15C5 was able to catalyse the hydrolysis of the triester [105] with cat 2.65 x 10 3 min 1 whilst a second antibody from the same immunization programme was later found to hydrolyse the acetylcholinesterase inhibitor Paraoxon [106] with kcat = 1.95 x 10 3min-1 at 25°C (Appendix entry 6.2) (Lavey and Janda, 1996b). Antibody 3H5 showed Michaelis-Menten kinetics and was strongly inhibited by the hapten [104]. It exhibited a linear dependence of the rate of hydrolysis on hydroxide ion concentration, suggesting that 3H5 effects catalysis by transition state stabilization rather than by general acid/base catalysis. 

The total cDNA construct of NPN43C9 was expressed efficiently in E. coli cells and protein purified, and its catalytic properties were assessed in both the monoclonal antibody and the single-chain antibody (scFv) 7A4-1/212 for the hydrolysis of p-nitroanilide [50] and the related p-chlorophenyl ester [51a] (Fig. 19). Virtually identical kcat and Km values were obtained for both 7A4-1/212 and NPN43C9. This activity was decreased in both cases by the addition of the inhibitor /n-nitroanilide [52], which gave = 800 p,M and 400 p,M for the NPN43C9 and 7A4-1/212, respectively. 

A more stringent criterion emanates from the thermodynamic cycle already encountered in Chapter 2 (Figure 2.2). For an inhibitor, which necessarily is an imperfect mimic of the transition state, and thus for which clearly K XT, the relationship kcat = kuncat (XS/XT) [the Kurz equation Chapter 2, Eq. (2.8)], has to be modified to Eq. (9.12), reflecting the proportionality of X, and XT, and setting Xs = XM. 

This approach has been applied extensively to peptidases, as the substrate or inhibitor structure can be varied most liberally. Correlations between inhibitor K and substrate Ku/kcat have been demonstrated for the phosphorus-containing peptide inhibitors of thermolysin (Figure 9.3) (Bartlett, 1983 Morgan, 1991), carbox-ypeptidase A (Hanson, 1989), and pepsin (Bartlett, 1996). Each series of inhibitors shows a slope close to unity in the log K vs. log (KM/kcat) plot importantly, the structural variations at the P2 residue should not affect kuncat significantly. 

Two types of inhibitors, pyrazoles and imidazoles (with E-NAD+) and iso-butyramide (with E-NADH), form tight ternary complexes with E-coenzyme, allowing single turnover to be observed (through photometry at 290 nm or fluorescence caused by NADH) and thus titration of the active sites (see Section 9.2.3.). Pyrazole and isobutyramide are kinetically competitive with ethanol and acetaldehyde, respectively. If the reaction E + NADH + aldehyde is run in the presence of a high concentration of pyrazole, the complex E-NAD+ formed by dissociation of alcohol immediately binds pyrazole for a single turnover only. Under favorable conditions, a single NADH oxidation can be observed by stopped-flow techniques to find a kcat of about 150 s 1 and a deuterium isotope effect kD 4 as expected (see Section 9.2.5). 

Uncompetitive inhibition occurs when the inhibitor binds with ES but not with E (1 /Ki = 0). The manifestation of this type of inhibition is an Eadie-Hofstee graph with lines intersecting on the rate/[S] axis (Fig. 11.13B). The general case where the parameters of Scheme 11.20 are finite and Km and kcat k cat is called mixed inhibition, and the lines of the Eadie-Hofstee plots do not intersect on either axis. 

Suicide inhibitors, alternatively known as Kcat or irreversible mechanism based inhibitors (IMBIs), are irreversible inhibitors that are often analogues of the normal substrate of the enzyme. The inhibitor binds to the active site, where it is modified by the enzyme to produce a reactive group, which reacts irreversibly to form a stable inhibitor-enzyme complex. This subsequent reaction may or may not involve functional groups at the active site. This means that suicide inhibitors are likely to be specific in their action, since they can only be activated by a particular enzyme. This specificity means that drugs designed as suicide inhibitors could exhibit a lower degree of toxicity. 

The value of kcat and Ki can also be estimated by quantifying the rate of metabolite formation from the inhibitor either simultaneously with the decline in enzyme activity or under the same incubation conditions. The rate of change of metabolite, dM( )/dt, is given by... 

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