Strategies for discovering and derisking covalent, irreversible enzyme inhibitors

Abstract

This article presents several covalent inhibitors, including examples of successful drugs, as well as highly selective, irreversible inhibitors of emerging therapeutic targets, such as fatty acid amide hydolase. Covalent inhibitors have many desirable features, including increased biochemical efficiency of target disruption, less sensitivity toward pharmacokinetic parameters and increased duration of action that outlasts the pharmacokinetics of the compound. Safety concerns that must be mitigated include lack of specificity and the potential immunogenicity of protein-inhibitor adduct(s). Particular attention will be given to recent technologies, such as activity-based protein profiling, which allow one to define the proteome-wide selectivity patterns for covalent inhibitors in vitro and in vivo. For instance, any covalent inhibitor can, in principle, be modified with a 'clickable' tag to generate an activity probe that is almost indistinguishable from the original agent. These probes can be applied to any living system across a broad dose range to fully inventory their on and off targets. The substantial number of drugs on the market today that act by a covalent mechanism belies historical prejudices against the development of irreversibly acting therapeutic small molecules. Emerging proteomic technologies offer a means to systematically discriminate safe (selective) versus deleterious (nonselective) covalent inhibitors and thus should inspire their future design and development.

Figure 1

Mechanism-based covalent inhibition via disulfide adduct formation.

Figure 2

Examples of covalent inhibitors including their protein target(s) with active-site nucleophile. The arrow indicates the position of attack by the nucleophile on the drug resulting in covalent modification of the target.

Figure 3. Assessment of global selectivity of covalent inhibitors by activity-based protein profiling (ABPP)

(A) Representative structure of an activity-based probe (ABP), which contains a reactive group, a linker or binding group and a reporter tag. (B) Competitive ABPP to determine the selectivity of an inhibitor against an enzyme family that is targeted by a particular ABP (with fluorescent reporter tag in this example). Probe-labeled proteins are analyzed by SDS-PAGE (in-gel fluorescence) and those that show significant reductions in fluorescent intensity in the presence of inhibitor are scored as targets of the inhibitor. (C) Click chemistry ABPP profiling to characterize the selectivity of covalent inhibitors in vivo. Covalent inhibitors are converted to activity-based probes via incorporation of an alkyne handle and these probes are administered to living systems (cells or animals). Probe-labeled proteins are conjugated to rhodamine-azide using click chemistry and analyzed by SDS-PAGE (in-gel fluorescence).

Figure 4. Representative activity-based probes for individual enzyme families or subfamilies

Reactive groups are highlighted. Tag: Biotin, rhodamine, TAMRA, BODIPY or HA.

Figure 5. Representative photoreactive activity-based probes that achieve target selectivity through binding affinity and covalent labeling is accomplished by exposure to UV light

Tag: Biotin, rhodamine or TAMRA.

Figure 6

Covalent fatty acid amide hydrolase inhibitors; OL-135 is reversible.

Figure 7

Covalent inhibitors and clickable covalent probes emerging from chemoproteomic endeavors.