Substrate concentration is an important factor when studying competitive enzyme inhibition. As the concentration of substrate increases, Michaelis-Mentin kinetics states that the efficacy of an inhibitor will decrease. This is because the competition for the inhibitor will be higher, due to there being more substrate molecules present. To obtain the same inhibitory effect, the concentration of inhibitor must be increased as substrate concentration increases. However, this may not be possible physiologically, as off-target effects will increase with potential side effects.
Substrate concentration does not alter the efficacy of non-competitive inhibitors (allosteric modulators), as these inhibitors do not directly interact with the active (orthosteric) site.
Enzyme affinity is important when selecting or designing an inhibitor. If the inhibitor does not bind, or binds poorly, to the target, it is unlikely to work well.
IC50 (inhibitory concentration) is the concentration of inhibitor at which 50% of the enzymes are bound by the inhibitor. This means that the Vmax of the enzyme-catalysed reaction is halved. This can be a useful indicator when determining the relative potency of an inhibitor (many drugs are inhibitors).