Lysine-Targeted Covalent Inhibitors Gaining Traction

In the past decade, targeted covalent inhibitors (TCIs) has experienced a resurgence due to its sustained target engagement and improved proteome-wide selectivity. Currently, targeting cysteine residues with acrylamides and other α, β-unsaturated carbonyl compounds is the predominant strategy in TCI development. Although lysine is more prevalent than cysteine and possesses lower mutation rates, lysine has been much less frequently considered and more challenging as a residue targeted by TCIs due to its low nucleophilicity. Recently, Jack Taunton and his colleagues reveled their two related work in JACS. 

Based on the co-crystal structure of Hsp90 with a reversible inhibitor PU-H71, they designed two arylsulfonyl fluorides 1 and 2 and co-incubated them with Hsp90 protein (Figure 1a, b). Mass spectrometry detected the formation of a 1:1 covalent adduct. Mutation of Lys58 to arginine abrogated the covalent modification, indicating that the TCI selectively modified Lys58 (Figure 1c).  Moreover, the co-crystal structure provided unambiguous evidence for a covalent bond formation between Lys58 and the sulfonyl group (Figure 1e). Subsequently, they applied compound 2 with an alkyne group to examine its cellular target engagement. The pull-down experiment revealed a prominent 90-kDa band, which disappeared in the presence of compound 1 competition, indicating an on-target effect. (Figure 1f).

Figure 1. Design of lysine-targeted arylsulfonyl fluoride inhibitors of Hsp90.

To optimally position the warhead to facilitate nucleophilic attack by Lys58, they constrained the propylamine linker with a 4-, 5-, or 6-membered ring (Figure 2a). Among them, compound 5 with (S)-methylpiperidine linker exhibited the fastest modification rate (t_1/2 ∼ 5 min), while its enantiomer 6 was the slowest compound tested (t_1/2 ∼ 200 min) (Figure 2b).

Figure 2. Linker conformational constraints dramatically affect k_inact.

Similar to TCIs, compound 5 exhibited concentration- and time-dependent occupancy of endogenous cellular Hsp90, as revealed by reduced Hsp90 labeling by the competitive clickable probe 2 (Figure 3a and 3b). In addition, compound 5 still exhibited potent occupancy and inhibition after washout, while compound 6 and PU-H71 did not.

Figure 3. Covalent and noncovalent inhibition of Hsp90 in cells.

Alternatively, they identified two lysine-targeted eIF4E inhibitor hits via two rounds of virtual covalent docking screening. After treatment with 30 μM compound for 3 h, hits 8 and 9 labeled eIF4E to 68% and 41%, respectively (Figure 4a, b). The co-crystal structure of hit 9 with eIF4E confirmed the binding mode predicted by docking, in which the 4-pyridylmethyl substituent of 9 fits into the hydrophobic pocket and a covalent bond is formed between lys162 and the sulfonyl group (Figure 4c). Further modification of either adding a 2-Cl substituent to the pyridine or replacing the isoquinolone core with a quinazolinone bearing an exocyclic amine increased the potency by 50-600 folds (Figure 4e, f).

Figure 4. Structure-guided optimization of covalent eIF4E inhibitors.

Besides, compound 12 also showed high cellular covalent modification efficiency, where a dose-dependent shift of eIF4E (25 kDa) to a form with a higher molecular weight (MW) was observed in 12 treated cell lysates (Figure 5a, lysates). Consistently, the m7GTP pulldown samples revealed a dose-dependent decrease in bound eIF4E (Figure 5a, elution). Moreover, 12 induced band shift of eIF4E was not observed in K162R eIF4E cells (Figure 5b).

Figure 5. Covalent inactivation of eIF4E in cells.

Taunton’s work provided two different approaches to identify lysine-targeted covalent inhibitors and indicated arylsulfonyl fluorides is a complementary electrophile capable of efficiently targeting lysines. The Hsp90 covalent inhibitor suggests the solvent-exposed lysine could be targeted for TCI design. The eIF4E covalent inhibitor demonstrates that docking strategy is an efficient method for the discovery of covalent inhibitors, especially those lacking a ligand as a starting point. These two contributions from the Taunton group provide a lot of opportunities and guidance to use arylsulfonyl fluorides for lysine targeting.