10 years into the resurgence of covalent drugs

Abstract

In the first decade of targeted covalent inhibition, scientists have successfully reversed the previous trend that had impeded the use of covalent inhibition in drug development. Successes in the clinic, mainly in the field of kinase inhibitors, are existing proof that safe covalent inhibitors can be designed and employed to develop effective treatments. The case of KRAS_G12C covalent inhibitors entering clinical trials in 2019 has been among the hottest topics discussed in drug discovery, raising expectations for the future of the field. In this perspective, an overview of the milestones hit with targeted covalent inhibitors, as well as the promise and the needs of current research, are presented. While recent results have confirmed the potential that was foreseen, many questions remain unexplored in this branch of precision medicine.

Keywords: FDA-approved drugs; covalent warhead; drug discovery; targeted covalent inhibitors.

Figure 1.. Targeted covalent inhibition.

(A) Structural features of a targeted covalent inhibitor. (B) Targeted covalent inhibition entails a two-step mechanism: affinity encounter complex formation (determined by K_d = k_–1/k₁) and covalent bond formation (determined by rate constant k₂). (C) Bar graph illustrating the timeline for the use of the terms ‘irreversible inhibition/inhibitors’ and ‘covalent inhibition/inhibitors’ in scientific literature. Data source: Pubmed, collected on 31 May 2020. The displayed data sets were obtained with the following search strings: Set A: ‘irreversible inhibitor’ OR ‘irreversible inhibition’; Set B: ‘covalent inhibitor’ OR ‘covalent inhibition’.

Figure 2.. Recent development of TCIs in the clinic.

(A) Chemical structures of selinexor (left, FDA approved in 2019) and leptomycin B (right); both compounds bind covalently to Cys528 of XPO-1. (B) Ras activation cascade: upon detection of extracellular signal (e.g., EGF), tyrosine kinase receptors (e.g., EGFR) dimerize and cross-phosphorylate. Phosphate groups are recognized by an adaptor protein (e.g., GRB2), which activates a GEF protein (e.g., SOS1). SOS1 binds to GDP-RAS and induces nucleotide release. Cytoplasmic abundant GTP binds to RAS, resulting in the active conformation for signal transduction. Effector binding (e.g., Raf) occurs and the signal is further propagated to the cytoplasm, ultimately leading to regulation of specific gene expression. (C) Optimized covalent probes for KRAS_G12C. (D) Clinical candidates against KRAS_G12C for which the chemical structure is reported in the literature.

Figure 3.. FDA-approved covalent inhibitors.

(A) Types of warheads found in FDA-approved drugs. In prodrugs, the warhead is masked. (B) Target moieties covalently bound by FDA-approved drugs. DNA includes all nonspecific alkylating agents (the most commonly modified site is N7 in purines). Cofactors include PLP, heme groups, FAD and NADH. Data source: Supplementary Table 1 (n = 88). Sec: Selenocysteine.

Figure 4.. Nonexhaustive collection of warheads used for TCI design.

The warheads are grouped in panels by target residues (Cys, pink; catalytic Ser/Thr, cyan; Lys, green; Tyr/His/Ser, purple; Asp/Glu, blue; His/Ser, orange). Some warheads may be used to target multiple residues.

Figure 5.. Overview of available screening platforms for the discovery of novel TCIs.

(A) Intact protein mass spectrometry: binding is shown by a shift in the measured protein MW. (B) Fragment tethering: hits are selected by favorable affinity under thermodynamic equilibrium conditions. (C) Proteomic profiling: targeted proteins are identified by the decrease in signal detected upon pull-down with broad spectrum covalent probes. (D) qIT assay: a fluorogenic substrate (CPM) is used to monitor the labeling reaction. (E) Covalent docking: hits are selected by in silico simulations. KRAS_G12C with ARS1620 in Molecular Operating Environment (MOE, Chemical Computing Group, Montreal, Canada) was used as an example to show the covalent docking interface (PDB: 5V9U). (F) X-ray crystallography: structural information of covalent fragment binding is generated. The structure shown derives from the Nir London lab’s recent effort for the COVID Moonshot campaign (fragment 1351 binding to COVID-19 M^pro catalytic Cys).