# Inhibitor trapping

Inhibitor trapping, also known as ligand trapping or ligand entrapment, pertains to an inhibition mechanism in which a small molecule ligand becomes enclosed and locked within the structure of the protein target. During this process, the protein molecule acts as a trap, effectively immobilizing the small molecule within it. Simultaneously, the ligand confines the protein in a specific inhibited conformation by significantly reducing its dynamic movements. Inhibitor trapping leads to a substantial reduction in the dissociation rate of the ligand, resulting in increased binding affinity and activity.
The inhibitor trapping is contingent on the existence of open and closed protein conformations. The inhibitor gains entry in the open conformation and becomes entrapped during the formation of the closed form by preventing the transition to the open conformation. For example, the enzyme hexokinase that phosphorylates glucose in the first step of glycolysis can adopt open and closed conformation. The substrate glucose binds to the open form and induces the formation of a closed form where the phosphorylation reaction takes place. When the monosaccharide xylose is used instead of glucose, the hexokinase adopts the closed form, but it cannot reopen, leading to potent inhibition of the enzyme.
Most frequently, the opening and closing of the protein occurs through specialized loop regions that have evolved to adopt an open and closed conformation. In N-myristoyltransferases, such function performs the Ab-loop, in kinases the P-loop, and in streptavidin- loop 3-4. After ligand binding, these loop regions adopt a closed conformation and function as a ‘lid’ over the binding pocket, sterically inhibiting the dissociation of the small molecule. Interactions between the ligand and the protein within the binding site prevent the reopening of the loop regions, thus trapping the small molecule inside the protein structure.
Inhibitor trapping has a profound effect on binding affinity. For example, the entrapment of biotin increases its binding affinity over 3.7 million-fold, of nilotinib over 12,000-fold, of imatinib over 1700-fold, and of the NMT inhibitor—IMP-1088 over 5200-fold. This effect is mainly due to a dramatic decrease in the dissociation rate constant koff, respectively, an increase in the inhibitor residence time (1/koff). The values of the residence time indicate how many times the binding affinity increases as a result of the entrapment of the ligand.
Drugs exhibiting a high degree of entrapment may possess desirable properties for clinical use. For example, the entrapment of imatinib in the Abl kinase could be pivotal to its success in treating chronic myeloid leukemia (CML). Nilotinib, which shows an even higher degree of entrapment, is effective against the resistant mutations observed in the P-loop of the Abl kinase domain in patients treated with imatinib]. The clinical success of drugs that exhibit high entrapment may be attributed to their higher selectivity. For instance, while imatinib can inhibit c-Kit and platelet-derived growth factor receptor (PDGFR) in addition to Abl, it demonstrates a high level of selectivity within the 500-member protein kinase family, and lapatinib inhibits only the kinases of the EGFR subfamily. In contrast, staurosporine shows a low degree of entrapment and displays almost no selectivity against the different protein kinases. As a result, staurosporine exhibits significant off-target toxicity, rendering it unsuitable for clinical use.
In the context of drug design, inhibitor entrapment produces outcomes that are difficult to explain through conventional models, such as a lock and key, induced fit, and conformational selection. Examples of these include the observations that in some protein-ligand complexes, the direct interactions between the ligand and the protein are not correlated with the binding affinity and activity of the ligands, that some bonds within the protein-ligand complexes are “special” and contribute dramatically by hundreds to thousands of folds to binding affinity instead of the expected difference of several folds. In the literature, numerous examples can be found where the addition of a single methyl group dramatically increases binding affinity and potency, often by hundreds or even thousands of folds. Due to the highly unanticipated effect on the methyl group, this phenomenon has become known as a “magic” methyl effect and has been observed in diverse proteins besides the kinase family, including other enzymes and G-protein-coupled receptors. The role of the methyl group in these examples could be to enhance the entrapment of the inhibitors. However, this phenomenon is not unique to the methyl group, and a similar disproportionately high contribution to affinity has been seen for other bonds, including salt bridges, hydrogen bonds, halogen bonds, and stacking interactions.
The inhibitor trapping requires the formation of a closed protein conformation, wherein the small molecule is captured inside. The existence of this closed, inhibited state is contingent on low protein dynamics, as increased dynamics could lead to the opening of the structure and dissociation of the ligand. Therefore, potent inhibition arises as a result of imposing a conformational “freeze” on the protein structure, effectively trapping it in a specific conformation. Hence, the inhibitor-trapping mechanism exhibits dual effects: as the inhibitor becomes entrapped within the protein structure, the protein structure itself becomes “locked” into a specific conformational state.
Ligand entrapment distinguishes itself from other models of inhibition, where the binding affinity is often evaluated by estimating the change in free binding energy caused by the transfer of the ligand from the solution to the protein's binding site. In contrast, the inhibitor trapping model suggests that the affinity of the inhibitor is influences by the protein's conformational dynamics, which determine whether the ligand will remain bound within its binding site