Very slow binding inhibitors

Figure 6.2 Effect of preincubation time with inhibitor on the steady state velocity of an enzymatic reaction for a very slow binding inhibitor. (A) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the single-step binding mechanism of scheme B of Figure 6.3. (B) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the two-step binding mechanism of scheme C of Figure 6.3. Note that in panel B both the initial velocity (y-intercept values) and steady state velocity are affected by the presence of inhibitor in a concentration-dependent fashion.

Note that for very slow binding inhibitors that are studied by varying preincubation time, the fits of the exponential decay curves to Equation (6.4) provide values for both V, and kohs for each inhibitor concentration. The values of v, at each inhibitor concentration represent the v-intercepts of the best fit to Equation (6.4), and these can be used in conjunction with Equation (6.8) to obtain an independent estimate of... 

Fig. 3.14 Simulated ALIS-based dissociation rate measurements. See text for details. (A) Quench experiments modeled at varying inhibitor association rates. Even with a very slow-binding inhibitor, the decay curve resembles pure first-order dissociation kinetics. (B) Data in (A), shown on a log axis. (C) Simulated ALIS quench experiment with varying protein-ligand dissociation rates,...

The first mechanism-based inhibitor (11) of 3-deoxy-D-arahino heptulosonate 7-phosphate (DAH7P synthase) has been synthesized in 12 steps from D-arabinose and has been found to be a very slow binding inhibitor against E. coli DAH7P synthase/ ... 

Until now our discussions of enzyme inhibition have dealt with compounds that interact with binding pockets on the enzyme molecule through reversible forces. Hence inhibition by these compounds is always reversed by dissociation of the inhibitor from the binary enzyme-inhibitor complex. Even for very tight binding inhibitors, the interactions that stabilize the enzyme-inhibitor complex are mediated by reversible forces, and therefore the El complex has some, nonzero rate of dissociation—even if this rate is too slow to be experimentally measured. In this chapter we turn our attention to compounds that interact with an enzyme molecule in such a way as to permanendy ablate enzyme function. We refer to such compounds as enzyme inactivators to stress the mechanistic distinctions between these molecules and reversible enzyme inhibitors. 

Some inhibitors interact very slowly with the enzyme protein, and onset of inhibition thus exhibits time-dependence. These inhibitors are generally referred to as slow-binding inhibitors, and as slow tight-binding inhihitors if the potency of inhibition is extremely high. Analysis of these inhibitory mechanisms is complex because binding and dissociation rate constants may be determined in addition to values. Indeed, a complete analysis may require extensive use of specialized computer software, and the complexities of such analyses preclude their discussion in this chapter. However, the reader is directed to several publications from Morrison s laboratory if a slow-binding mechanism is suspected for an inhibitor of interest (Morrison, 1982 Morrison and Stone, 1985 Sculley and Morrison, 1986 Morrison and Walsh, 1988). 

If the slow-binding inhibitor described by Equation 17.26 also binds very tightly, it is referred to as a slow-tight-binding inhibitor. For inhibitors of this type, is given by Equa-... 

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent inhibitors provide a means of mapping the enzyme s active site. In contrast, reversible inhibition is characterized by a rapid equilibrium between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. In noncompetitive inhibition, the inhibitor decreases the turnover number. Competitive inhibition can be distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the substrate concentration. 

On addition of enzyme to a mixture of substrate and slow-binding inhibitor, one sees an exponential approach to a steady-state rate (if an increase in absorbance is followed, the analytical form of the curve is the same as for a presteady-state burst - see Figure 5.38). If K (fast) is the X calculated from the very initial rates and (slow) from the steady-state rate, then the relation between the two constants is given by eqn. (5.24). The exponential describing the approach to the steady state depends in a complex way on the concentration... 

Often high-affinity, or tight binding, interactions with enzymes is the result of a very slow dissociation rate of the enzyme-inhibitor binary complex. 

The very slow dissociation rates for tight binding inhibitors offer some potential clinical advantages for such compounds, as described in detail in Chapter 6. Experimental determination of the value of k, can be quite challenging for these inhibitors. We have detailed in Chapters 5 and 6 several kinetic methods for estimating the value of the dissociation rate constant. When the value of kofS is extremely low, however, alternative methods may be required to estimate this kinetic constant. For example, equilibrium dialysis over the course of hours, or even days, may be required to achieve sufficient inhibitor release from the El complex for measurement. A significant issue with approaches like this is that the enzyme may not remain stable over the extended time course of such experiments. In some cases of extremely slow inhibitor dissociation, the limits of enzyme stability will preclude accurate determination of koff the best that one can do in these cases is to provide an upper limit on the value of this rate constant. 

Except for very simple systems, initial rate experiments of enzyme-catalyzed reactions are typically run in which the initial velocity is measured at a number of substrate concentrations while keeping all of the other components of the reaction mixture constant. The set of experiments is run again a number of times (typically, at least five) in which the concentration of one of those other components of the reaction mixture has been changed. When the initial rate data is plotted in a linear format (for example, in a double-reciprocal plot, 1/v vx. 1/[S]), a series of lines are obtained, each associated with a different concentration of the other component (for example, another substrate in a multisubstrate reaction, one of the products, an inhibitor or other effector, etc.). The slopes of each of these lines are replotted as a function of the concentration of the other component (e.g., slope vx. [other substrate] in a multisubstrate reaction slope vx. 1/[inhibitor] in an inhibition study etc.). Similar replots may be made with the vertical intercepts of the primary plots. The new slopes, vertical intercepts, and horizontal intercepts of these replots can provide estimates of the kinetic parameters for the system under study. In addition, linearity (or lack of) is a good check on whether the experimental protocols have valid steady-state conditions. Nonlinearity in replot data can often indicate cooperative events, slow binding steps, multiple binding, etc. 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

Many inhibitors with very low dissociation constants appear to have a slow onset of inhibition when they are added to a reaction mixture of enzyme and substrate. This was once interpreted as the inhibitors having to induce a slow conformational change in the enzyme from a weak binding to a tight binding state. But in most cases, the slow binding is an inevitable consequence of the low concentrations of inhibitor used to determine its Ki. For example, consider the inhibition of trypsin by the basic pancreatic trypsin inhibitor. Kx is 6 X 10-14 M and the association rate constant is 1.1 X 106 s-1 M-1 (Table 4.1). To determine the value of Ki, inhibitor concentrations should be in the range of K1, where the observed first-order rate constant for association is (6 X Q U M) X (1.1 X 106 s-1 M-1) that is, 6.6 X 10-8 s 1. The half-life is (0.6931/6.6) X 108 s, which is more than 17 weeks. 

Sulindac sulfide, the bioactive metabolite of sulindac, is struchirally very similar to INDO and is a slow, tight-binding inhibitor of COX (Fig. 2b) (12, 13). As with INDO, removal of the methyl group from sulindac sulfide results in loss of COX-1 and COX-2 inhibition (14). However, it should be noted that the benzylidine double bond of rfei-methyl sulindac sulfide (DM-SS) exists in the F-conformation, whereas sulindac sulfide exists in the Z-conformer. 

Figure 2 Standard mechanism of protein serine protease inhibitors bind in a substrate-like manner that completely spans the active site, and act as substrates with a very slow kcat. They interact with both the substrate binding sites (shallow indentation) and the catalytic residues (rectangle) of the serine protease.

For slow-tight-binding inhibitors, is very small and formation of the E. I complex is essentially irreversible. Use of Equation 17.28 ensures that depletion of free enzyme and free inhibitor by formation of the E. I complex is taken into account. 

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