Slow binding inhibitors examples

SOME EXAMPLES OF PHARMACOLOGICALLY INTERESTING SLOW BINDING INHIBITORS... 

Some Examples of Pharmacologically Interesting Slow Binding Inhibitors 157... 

Equation 16. Figure 13 gives an example of a progress curve for a slow-binding inhibitor. For each curve with inhibitor present, there is an initial burst followed by a slower steady-state rate. 

As in the peptide substrate or electrophilic carbonyl-based inhibitor series, the addition of a peptide chain to take advantage of binding interactions in the 8,-85 subsites of elastase results in increased potency and selectivity, for example, (10-1) vs. (10-2) Table 2.10) [154]. In contrast to the above two series, boronic acid (10-5) whose P,-Phe residue was chosen for it to be a specific a-chymotrypsin inhibitor (final A, = 0.16 nM vs. chymotrypsin), retained good activity against both HLE and PPE. Also each of the peptide boronic acids corresponding to the better substrates for a-chymotrypsin, PPE, and HLE (for example, (10-6, -7, and -8), respectively) were slow-binding inhibitors of those enzymes [155]. 

The usefulness of knowing that the reactions of individual species are kineti-cally linked is illustrated by studies on the time dependence of enzyme inactivation in the presence of a slow-binding inhibitor or inactivator, for example. The reaction can be considered as... 

Table 6.1 Some examples of slow binding enzyme inhibitors...

On the other hand, when K Kf, the concentration of inhibitor required to observe slow binding inhibition would be much less than the value of K, for the inhibitor encounter complex. When, for example, the inhibitor concentration is limited, due to solubility or other factors, and therefore cannot be titrated above the value of Kif the steady state concentration of the El encounter complex will be kinet-ically insignificant. Under these conditions it can be shown (see Copeland, 2000) that Equation (6.6) reduces to... 

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. 

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. 

Potent HPPD inhibitors have a common structural feature in the form of a 1,3-diketone moiety (9, 10, II). Kinetics experiments show that these potent inhibitors exhibit the characteristics of slow-tight binding inhibitors (12, 13, 14, IS). Figure 2 contains examples of various structural classes of inhibitors all with the 1,3-diketone motif. Mesotrione and the diketonitrile form of... 

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