Covalent targeting of non-cysteine residues in PI4KIIIβ

Abstract

The synthesis and characterisation of fluorosulfate covalent inhibitors of the lipid kinase PI4KIIIβ is described. The conserved lysine residue located within the ATP binding site was targeted, and optimised compounds based upon reversible inhibitors with good activity and physicochemical profile showed strong reversible interactions and slow onset times for the covalent inhibition, resulting in an excellent selectivity profile for the lipid kinase target. X-Ray crystallography demonstrated a distal tyrosine residue could also be targeted using a fluorosulfate strategy. Combination of this knowledge showed that a dual covalent inhibitor could be developed which reveals potential in addressing the challenges associated with drug resistant mutations.

Fig. 1. Comparison of cysteine and lysine residues as targets in covalent drug discovery.

Fig. 2. Design strategy for the development of a covalent inhibitor for PI4KIIIβ.

Fig. 3. Lipid kinase pIC₅₀ screening data for 4.

Fig. 4. Time course of compound 4 reacting with recombinant PI4KIIIβ.

Fig. 5. Time course of Wortmannin reacting with recombinant PI4KIIIβ.

Fig. 6. Stability of the covalent adduct between 4 (5 μM) and PI4KIIIβ in the presence of ATP (10 mM).

Fig. 7. Time course for the reaction of 3 with recombinant PI4KIII.

Fig. 8. The o-methoxy group may increase the rate of covalent modification.

Fig. 9. Crystal structure of PI4KIIIβ covalently modified on Lys₅₄₉ with 4 and a corresponding F_o − F_c difference “omit” map contoured at 3σ (grey mesh) [PyMOL generated figure] (PDB: 8Q6F). The compound is present in the ATP binding site and makes key H-bonding interactions with Tyr₃₈₅ and hinge residue Val₅₉₈.

Fig. 10. Reaction of 8 with recombinant PI4KIIIβ.

Fig. 11. Crystal structure of PI4KIIIβ covalently modified on Tyr₃₈₅ with 8 and a corresponding F_o − F_c difference “omit” map contoured at 3σ (grey mesh) [PyMOL generated figure] (PDB: 8Q6G). The compound is present in the ATP binding site and makes key H-bonding interactions with Lys₅₄₉ and hinge residue Val₅₉₈.

Fig. 12. Reaction of 11 with recombinant PI4KIIIβ.

Fig. 13. Crystal structure of 11 engaged with Lys₅₄₉ and Tyr₃₈₅ in PI4KIIIβ and a corresponding F_o − F_c difference “omit” map contoured at 3σ (grey mesh) [PyMOL generated figure] (PDB: 8Q6H). The compound is present in the ATP binding site and makes key H-bonding interactions with hinge residue Val₅₉₈.

Fig. 14. Use of COware to generate fluorosulfate compounds.

Scheme 1. Synthesis of compound 3.

Scheme 2. Synthesis of compounds 4, 5 and 6.

Scheme 3. Synthesis of compounds 7, 8 and 9.

Scheme 4. Synthesis of compounds 10 and 11.