Onset of inhibition

Some inhibitors bind to, or dissociate from, their target enzymes slowly, thus leading to a time dependence for the onset of inhibition. 

DETERMINING kobs THE RATE CONSTANT FOR ONSET OF INHIBITION... 

To distinguish between simple, reversible slow binding (scheme B) and an enzyme isomerization mechanism (scheme C), one can examine the dependence of kobs on inhibitor concentration. If the slow onset of inhibition merely reflects inherently slow binding and/or dissociation, then the term kobs in Equations (6.1) and (6.2) will depend only on the association and dissociation rate constants k3 and k4 as follows ... 

Interestingly, although many transition state analogs bind noncovalently to the target enzyme s active site via a one-step kinetic mechanism (Scheme la) and would therefore be expected to exhibit no time-dependent properties of inhibition, inhibitors with Kj values of < 10 10 M (like coformy-cin) usually have a slow onset of inhibition kobserved < 10 2 s 1 (i.e., an approach to equilibrium inhibition of > 1 min).161 This is merely an assay artifact due to... 

Figure 3. Slow-onset of inhibition by 1 -deoxynojirimycin may be due to slow protonation of the inhibitor.

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). 

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. 

Fig. 4.8 Typical generation rate curves of coli at 37 °C in the absence (+) and presence of 2 LI M of various rifampicin derivatives possessing different lipophilic properties (log k,). The variation in lag phase (onset of inhibition) is clearly shown. (Reprinted from Fig. 1 of ref. 105.)...

Note the concentration-independent onset of inhibition on the right-hand fig. (Reprinted from Fig. 2 of ref. 105.)... 

The fact that the onset of inhibition of manganese transport and retention by iron is faster than its disappearance may also be due to a higher affinity of the carrier binding sites for iron than for manganese. Once filled up with iron these sites will resist iron deficiency for a longer time than they would need to get... 

To observe the slow onset of inhibition and the E. I complex, iiTj must be smaller than and smaller than 4. However, if is considerably smaller than 4, then the formation of the E. I complex will be effectively irreversible (i.e., the inhibitor is of the slow-tight-binding variety). Under those circumstances it will again be necessary to take depletion of free enzyme and free inhibitor into account when determining iiTi and K (78). 

Differences in rates of onset of inhibition between compounds can suggest differences in molecular mechanism. Compounds that bind to rare forms of the kinase have slower on rates than those that bind abundant forms. This is seen with both DFG-out (p38 and BRAF)13 and C-helix-out (EGFR) conformations.15 Compounds with slow on rates must have slow off rates relative to equipotent fast binders. These slow off rates give extended drug-target residence times, which can have clinical implications (see Section 4.4.2.4). 

A comparison of IV injection of pantoprazole to IV injection of famotidine in pentagastrin-stimulated patients showing the equally rapid onset of inhibition and the long duration of action of the PPL Probably, current therapy indicates a bolus of 80 mg IV pantoprazole. 

Some observations from the progress curve are characteristic of this lead. The LD50 is 2ppm in minimal medium, but tiie initial phase almost tracks like the untreated sanq>le where onset of inhibition seems to be somewhat delayed. The degree of inhibition is also sensitive to die conqiosition of the medium. In the... 

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