I am used to pre-incubate the test compounds with enzyme for durations ranging from 15 min to 45 min before adding the substrate and initiating the reactions. However, in process of designing a high-throughput method, that seems to be a lagging factor.
Hence the question.
Whether the pre-incubation step is necessary may depend on the mode of inhibition.
if you are looking for simple reversible inhibitors you do not need to pre-incubate. Clearly the inhibitor has to be added before substrate regardless, but a lengthy pre-incubation is not needed.
if you are using a pure enzyme target, the only reason why you might need a preincubation step is if the inhibitor covalently modifies the active site. This will be time dependent and the assay will be more likely to produce ‘hits’ the longer you allow.
The only other reason I can think of, which would apply to crude extracts containing your enzyme target, is if you are using pro-drugs and need the removal of protecting groups by esterases, for example, in the crude extract.
I agree generally with Nick Gee and would like to make a few more points.
Typically when one is screening a generic compound library for noncovalent inhibitors, the potency of the inhibitors is expected to be pretty weak. Such weak inhibitors (assuming they are specific inhibitors, see below) are expected to be in rapid equilibrium with the target enzyme, so there is no need to preincubate. An exception would be if you are screening a set of compounds that are expected to contain slow-binding inhibitors, which might be the case if the compounds in the screen are related to known slow-binding inhibitors of the enzyme. (A second exception would be if you are screening a set of compounds that are supposed to be covalent inhibitors, since their potency is time-dependent, as Nick Gee said.)
Time-dependence of inhibition is one typical characteristic of non-specific inhibitors such as aggregators or reactive contaminants of compound samples. Therefore, a test for such undesirable modes of inhibition is to compare the potency of the inhibitor sample at different times of the reaction, or with and without preincubation followed by a short reaction time. This test would, of course, not be done during the initial screen but could constitute part of the hit follow-up.
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