Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor

Abstract

Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell-driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell-driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.

Graphical abstract

Figure 1.

Pirtobrutinib structure, binding mode, and effects on BTK stability and activity. (A) Chemical structure of pirtobrutinib. (B) Superposition of the crystal structures of BTK and BTK C481S in complex with pirtobrutinib (magenta and light gray cartoon/stick representation, respectively; selected water molecules are shown as red spheres, and hydrogen bond interactions are illustrated as dashed lines). (C) BTK in complex with pirtobrutinib (magenta cartoon/stick representation) overlayed with BTK covalently bound to ibrutinib (cyan cartoon/stick representation; PDB ID 5P9J). (D) Dose-response curves showing inhibition of BTK and BTK C481S kinase activity by pirtobrutinib (n = 10) (mean and SD are graphed). (E) Tm graphs showing pirtobrutinib- and cBTKi-induced increases in full-length and SH3-SH2-KD BTK Tm. Light and dark blue bars represent the first and second pirtobrutinib-induced melting events, respectively; ∗P < .05 (compared with Apo Tm); #P < .05 (compared with each cBTKi’s Tm). (F) Dose-response curves showing inhibition of cellular Y223 BTK phosphorylation in HEK293 cells stably expressing BTK by pirtobrutinib, ibrutinib, zanubrutinib, and acalabrutinib (n = 3) (mean and SD are graphed). FL, full-length; SD, standard deviation.

Figure 2.

Differential impact of pirtobrutinib and cBTKis on BTK phosphorylation in Ramos RA1 cells and human CLL cells. Autophosphorylation at BTK Y223 and phosphorylation at BTK Y551 were measured by Simple Western in Ramos RA1 cells (A) and human CLL cells (B and C). (A) Ramos RA1 cells were treated with pirtobrutinib, ibrutinib, acalabrutinib, or zanubrutinib for 2 hours before stimulation with anti-IgM for 10 minutes. A representative Simple Western image shows the differential effect of pirtobrutinib vs the cBTKis on BTK Y551 phosphorylation in these cells. (B) PBMCs from 4 treatment-naive donors with CLL were treated with ascending doses of pirtobrutinib. Simple Western images show the potent inhibition of pirtobrutinib on BTK Y223 phosphorylation, with corresponding dose-response curves and calculated IC₅₀ values shown below. Donor 4 was tested in 2 independent experiments with mean ± SD shown. (C) PBMCs from the 4 donors with CLL were treated with pirtobrutinib or ibrutinib at 10 or 100 nM. Simple Western images and a corresponding bar chart show the differential effect of pirtobrutinib and ibrutinib on Y551 phosphorylation in the PBMCs from the 4 donors with CLL (mean and SD are graphed). DMSO is measured in percentage for all blots. pY223, phospho-BTK Y223; pY551, phospho-BTK Y551; tBTK, total BTK.

Figure 3.

Pirtobrutinib inhibits cellular proliferation, B-cell activation, and calcium mobilization. (A) Activity of pirtobrutinib, ibrutinib, acalabrutinib, and zanubrutinib on cellular proliferation of TMD8 cells and (B) TMD8 cells expressing BTK C481S (mean and SD are graphed). (C) Pirtobrutinib inhibited anti-IgM–stimulated calcium flux in TMD8 and (D) REC-1 cells. Representative traces of 3 independent experiments are shown. (E) Human PBMCs from healthy donors were treated with pirtobrutinib, and B-cell activation was measured by upregulation of the CD69 activation marker after IgM stimulation. Representative flow cytometry density plots showing a reduction in the percentage of CD19⁺ B cells expressing CD69 with increasing doses of pirtobrutinib. (F) Dose-response curve of pirtobrutinib inhibition of B-cell activation in PBMCs from 4 separate healthy donors. Data are mean ± SD.

Figure 4.

In vivo efficacy of pirtobrutinib in BTK and BTK C481S mutant lymphoma xenograft models. Tumor-bearing mice were treated with pirtobrutinib with the indicated doses, for the indicated time. Means and standard error of the mean were plotted for each treatment group vs days of treatment (A) or days after randomization (B-D). For OCI-LY10 xenografts (A), tumor volumes were measured 3 times per week, and the tumor volumes were measured for an additional 35 days after the last dose. For TMD8 xenografts (B), tumor volumes were measured 3 times per week. The experiment was stopped on day 14 after randomization for the vehicle control group, and on day 18 after randomization for the other groups. For REC-1 xenografts (C), tumor volumes were measured biweekly. For TMD8 BTK C481S xenografts (D), tumor volumes were measured 2 or 3 times per week.

Figure 5.

Hypothesized binding models for pirtobrutinib and covalent BTK inhibitor. (A) We hypothesize that pirtobrutinib stabilizes BTK in a closed, inactive conformation, whereas (B) cBTKi binding destabilizes the closed conformation, providing upstream kinase access to phosphorylate Y551. (C) A cartoon representation showing the domain organization of BTK.