The Taxonomy of Covalent Inhibitors

Abstract

Covalent enzyme inhibitors are widely applied as biochemical tools and therapeutic agents. As a complement to categorization of these inhibitors by reactive group or modification site, we present a categorization by mechanism, which highlights common advantages and disadvantages inherent to each approach. Established categories for reversible and irreversible covalent inhibition are reviewed with representative examples given for each class, including covalent reversible inhibitors, slow substrates, residue-specific reagents, affinity labels (classical, quiescent, and photoaffinity), and mechanism-based inactivators. The relationships of these categories to proteomic profiling probes (activity-based and reactivity-based) as well as complementary approaches such as prodrug and soft drug design are also discussed. A wide variety of strategies are used to balance reactivity and selectivity in the design of covalent enzyme inhibitors. Use of a shared terminology is encouraged to clearly convey these mechanisms, to relate them to prior use of covalent inhibitors in enzymology, and to facilitate the development of more effective covalent inhibitors.

Figure 1

The Taxonomy of Covalent Inhibitors. Classification of covalent enzyme inhibitors by mechanism reveals different strategies used to balance reactivity and selectivity.

Figure 2

Reversible Covalent Inhibitor. A non-catalytic Cys of RSK2 is targeted by an acrylonitrile and reacts reversibly, presumably with the small fraction of ionized thiolate present at the reaction pH. In this figure and the following figures, the enzyme active site is depicted schematically in green, and for clarity, arrows are shown only for the initial attack leading to covalent bond formation.

Figure 3

Slow Substrate As a Covalent Inhibitor. The catalytic Cys thiolate⁽³⁵⁾ of Pseudomonas aeruginosa arginine deiminase (ADI) attacks L-canavanine, resulting in a long-lasting covalent intermediate that causes the observed inhibition.

Figure 4

Residue-Specific Reagent As a Covalent Inhibitor. Methyl methanethiosulfonate reaction with multiple Cys residues results in inhibition of soluble guanylate cyclase activation. The figure does not reflect labeling stoichiometry of this particular experiment.

Figure 5

Classical Affinity Label As a Covalent Inhibitor. The high effective concentration of the acrylamide in afatinib enables selective reaction with a non-catalytic Cys (C797) in EGFR, presumably with the small fraction of ionized thiolate present at the reaction pH.

Figure 6

Quiescent Affinity Label As a Covalent Inhibitor. The reaction of 2-methyl-4-chloropyridine with the active-site Cys thiolate of dimethylarginine dimethylaminohydrolase (DDAH) is catalyzed by a neighboring Asp residue.

Figure 7

Photoaffinity Label As a Covalent Inhibitor. Non-covalent binding provides a high effective molarity of an imine nitrile, formed in situ through irradiation with light, that facilitates selective reaction with a particular Glu side chain.

Figure 8

Mechanism-Based Enzyme Inactivator As a Covalent Inhibitor. The unreactive 2-vinyl-D-isocitrate is processed by the normal catalytic reaction of isocitrate lyase to produce a reactive species that covalently modifies Cys191.

Figure 9

Activity-Based Proteomic Profiling Probe As a Covalent Inhibitor. A classical affinity label (a fluorophosphonate) tethered to a purification/detection tag (biotin) broadly targets the active-site Ser of serine proteases for covalent modification.

Figure 10

Reactivity-Based Protein Profiling Probe As a Covalent Inhibitor. Proteome-wide, residue selective modification by activated sulfotetrafluorophenyl esters produces Lys residues labeled with a latent purification/detection tag. Hyper-reactive Lys residues (e.g. Lys residues with enhanced nucleophilicity due to lowered pK_a values) can be preferentially modified under certain conditions (depicted above).

Figure 11

Prodrug Form of a Covalent Inhibitor. In an acidic cell compartment, the prodrug omeprazole is converted into a classical affinity label containing a sulfenamide group that reacts with non-catalytic Cys residues, presumably with the small fraction of ionized thiolate.

Figure 12

Soft Drug Form of a Covalent Inhibitor. This classical affinity label bearing a fumarate ester reactive group covalently modifies a Cys in Bruton’s tyrosine kinase (BTK), but hydrolysis of the ester by human carboxylesterase-1 (CES1) results in rapid deactivation of the reactive group, improving selectivity.