Biochemical Characterization of GS-4059 As a Potent and Selective Covalent Irreversible Inhibitor of Bruton's Tyrosine Kinase

Introduction:Bruton's tyrosine kinase (BTK) plays a crucial role in B-cell development, differentiation, and signaling in the B-cell receptor (BCR) signaling pathway, and is a validated target for aberrant B-cell activity in malignancy and autoimmune diseases. GS-4059 (ONO-4059) is a selective, once-daily, oral inhibitor of BTK with clinical activity against a number of relapsed/refractory B-cell malignancies. GS-4059 was designed to specifically form a covalent bond with a cysteine residue in the BTK active site. The studies presented here confirmed the biochemical mechanism of action of GS-4059, assessed the inactivation kinetics of BTK by GS-4059 and comparator compounds, and measured the selectivity of GS-4059 for BTK over other related kinases. These studies will help to establish pharmacokinetic-pharmacodynamic models to guide clinical studies, and the evaluation of potential for inhibition of other kinases will assist in understanding the safety of the molecule.

Methods: The binding of GS-4059 to BTK was characterized using mass spectrometry to detect covalent adduct formation. Ibrutinib, a known irreversible covalent inhibitor, and staurosporine, a known reversible noncovalent inhibitor, were used as positive controls. The covalent nature of GS-4059 was further tested by chasing with excess amount of ibrutinib. GS-4059 and other covalent, irreversible inhibitors of BTK including CC-292, acalabrutinib, and ibrutinib were studied to determine the inactivation kinetics of BTK and related kinases, such as epidermal growth factor receptor (EGFR) and IL2-inducible T-cell kinase (ITK), for these compounds. The rate of enzyme inactivation was studied as a function of inhibitor concentration using a Sox-based fluorescence assay that allows real-time measurement of enzyme activity.

Results: Mass spectrometry studies showed that GS-4059 is a covalent inhibitor of BTK, based on an observed mass shift of BTK following preincubation with GS-4059 (Figure 1). This mass shift was consistent with the molecular weight of GS-4059. The irreversibility of GS-4059 binding was further demonstrated by the observation that GS-4059 could not be displaced from BTK by chasing with ibrutinib, as measured by mass spectrometry. GS-4059 efficiently inactivated BTK with a rate constant k_inact/K_i of 2.4 ± 0.6 ´ 10⁴ M^-1s^-1, similar to that of the covalent BTK inhibitors CC-292 and acalabrutinib, but >10 times lower than that of ibrutinib (Figure 2, Figure 3, and Table 1). In addition, GS-4059 is a poor inhibitor of the related kinases EGFR and ITK, supporting the clinical observation of a favorable safety profile for GS-4059 (Table 1).

Conclusions: In summary, GS-4059 is a potent and selective covalent irreversible inhibitor of BTK with a different selectivity profile than other BTK inhibitors, and warrants further studies against B-cell malignancies and autoimmune diseases.

Figure 1

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Mass Spectrometry Analysis Showing Mass Shift of BTK upon Binding of GS-4059. The reversible noncovalent inhibitor staurosporine and the irreversible covalent inhibitor ibrutinib were used as controls.

Figure 1

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Mass Spectrometry Analysis Showing Mass Shift of BTK upon Binding of GS-4059. The reversible noncovalent inhibitor staurosporine and the irreversible covalent inhibitor ibrutinib were used as controls.

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Figure 2

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BTK Product Formation Progress Curves in the Presence of Increasing Concentrations of GS-4059, CC-292, Acalabrutinib, and Ibrutinib. Legends to the right of curves indicate compound concentrations.

Figure 2

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BTK Product Formation Progress Curves in the Presence of Increasing Concentrations of GS-4059, CC-292, Acalabrutinib, and Ibrutinib. Legends to the right of curves indicate compound concentrations.

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Figure 3

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Plots of k_obs as a Function of Inhibitor Concentration for GS-4059, CC-292, Acalabrutinib, and Ibrutinib.

Figure 3

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Plots of k_obs as a Function of Inhibitor Concentration for GS-4059, CC-292, Acalabrutinib, and Ibrutinib.

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