Relative Selectivity of Covalent Inhibitors Requires Assessment of Inactivation Kinetics and Cellular Occupancy: A Case Study of Ibrutinib and Acalabrutinib

Abstract

Kinases form an attractive class of targets for small molecule inhibitors, but similarity among their adenosine triphosphate binding sites presents difficulties for developing selective drugs. Standard methods of evaluating selectivity of most reversibly bound drugs account for binding affinity but not the two-step process, affinity and inactivation, occurring during covalent inhibition. To illustrate this concept, we assessed the selectivity of Bruton's tyrosine kinase (BTK) over TEC kinases by two novel therapeutics: ibrutinib and acalabrutinib. The two-step process and time-dependent inhibition unique to covalent inhibitors were evaluated with two biochemical assays measuring enzymatic function and inhibition kinetics. The selectivity for BTK over TEC found in these biochemical analyses was 1-1.5 for ibrutinib and 3.0-4.2 for acalabrutinib. To further assess drug selectivity in a more physiologically relevant context, we developed cellbased occupancy assays that quantify the percentage of druginactivated kinases. Cellular selectivity of BTK over TEC was determined after MWCL-1 cells, and samples from patients with chronic lymphocytic leukemia (CLL) were treated for durations and concentrations based on human pharmacokinetics of each drug. In MWCL-1 cells, BTK/TEC selectivities measured at 0.5, 1, and 3 hours were 2.53, 1.05, and 1.51 for ibrutinib and 0.97, 1.13, and 2.56 for acalabrutinib, respectively. The equivalent selectivity measured in samples from patients with CLL were 1.31 6 0.27 and 1.09 6 0.11 for ibrutinib and acalabrutinib, respectively. Collectively, our data show that when properly accounting for time-dependent factors and relevant cellular context, ibrutinib and acalabrutinib demonstrate similar selectivity for BTK over TEC. SIGNIFICANCE STATEMENT This study shows relative selectivity of covalent inhibitors toward different kinase targets should be assessed with both affinity and inactivation kinetics to accurately account for time-dependent effects of covalent binding and assessed in a cellular matrix to reproduce the physiologic context of target inhibition. This is illustrated with a case study of ibrutinib and acalabrutinib for which selectivity assessment with appropriate assays, as opposed to measuring binding affinity with KINOMEscan alone, corroborate emerging clinical data demonstrating similar safety profiles between the therapies.

A steady-state algebraic model for the time course of covalent enzyme inhibition

“…The results of fit are shown graphically in Figure 4. Note that the residuals of fit in the bottom panel are distributed randomly, which means that the single-exponential Eqn (25) is an adequate fitting model for this data, even though the data were simulated on the basis of a doubleexponential Eqn (5). The best-fit values of the apparent first-order rate constant k obs obtained at each inhibitor concentration are collected in Table 2.…”

A Steady-State Algebraic Model for the Time Course of Covalent Enzyme Inhibition

“…Each of the simulated progress curves were fit individually and separately to the standard algebraic model [1, sect. 9.1] for the time course of covalent inhibition, represented by the exponential Eqn (22), where v i is the initial reaction rate in instrument units and k obs is the apparent first-order rate constant corresponding to each inhibitor concentration.…”

“…The best-fit values of the apparent first-order rate constant k obs obtained at each inhibitor concentration are collected in Table 2. Table 2: Best-fit values of the apparent first-order rate constant k obs obtained by fitting the progress curves shown in Figure 4 to the exponential Eqn (22).…”

“…In the second category of mathematical models are algebraic equations, such as Eqn (22) originally derived by Kitz & Wilson [10], where the definition of k obs is given by Eqns (23)-(24). The Kitz-Wilson equation Eqn (22) applies only to covalent inhibition assays where the uninhibited positive control reaction proceeds at a strictly constant rate, meaning that the plot of product concentration over time is linear.…”

“…In the specific case of the Kitz-Wilson single-exponential model represented by Eqn (22), Cornish-Bowden [21, sec. 7.2.2] pointed out that "if k 2 is not small enough to allow formation of E•I to be treated as an equilibrium [...] the loss of activity does not follow simple first-order kinetics: there is no exact analytical solution, but the kinetics may still be analyzed by numerical methods."…”