Tgrx-678, a Novel Allosteric Inhibitor of BCR-ABL1, Demonstrates Preclinical Anti-Leukemia Activity, High Oral Bioavailability and Synergism with Ponatinib to Suppress the Highly Resistant Compound Mutations

Introduction Clinical resistance to the treatment of BCR-ABL1-positive chronic myeloid leukemia (CML) patients is often conferred by mutation(s) in the ATP-binding site of ABL1 kinase domain. ABL1 is subject to auto-inhibition mediated by myristoylation-triggered conformational change, which can be exploited to overcome resistance, as validated by allosteric inhibitors such as GNF2, GNF5 and asciminib. TGRX-678, a novel allosteric inhibitor designed to target the myristoyl pocket of ABL1, is currently being investigated in a phase I clinical trial (NCT05434312). Herein we report the selectivity, potency, anti-leukemia activity and in vivo pharmacokinetic (PK)/pharmacodynamic(PD) characteristics of TGRX-678. Methods For in vitro study, ABL1b 65-534 fragments were used following the protocol for Z'-LYTE kinase assay kit (Thermo Fisher). For cellular assays, CML cell lines or engineered Ba/F3 cells were incubated with titrating TGRX-678 for 72 hrs, then CellTiter Glo reagent (Promega) was added and the chemiluminescence was measured. For in vivo studies, SD rats received TGRX-678 or asciminib and plasma samples were collected for analysis. NOD-SCID mice were inoculated with CML or Ba/F3-BCR-ABL1 T315I cells and received oral TGRX-678. Tumor volumes and body weights were recorded for 3 to 5 weeks. Results TGRX-678 exhibited minimum off-target inhibition at 1 μM in a panel of 298 kinases, but inhibited the enzymatic activity of both ABL1 and the T315I mutant with IC50 values of 2.96 nM and 1.15 nM, respectively. The anti-proliferative activity of TGRX-678 was determined in CML cell lines including K562, KU812, KCL22-S, KCL22-R, LAMA-84 and MEG-01 with IC50 values ranging from 1.1 nM to 6.56 nM. TGRX-678 had no cytotoxicity in 29 cell lines representing other hematological malignancies and solid tumors. Using engineered Ba/F3 cell lines, TGRX-678 was active against the native BCR-ABL1 (IC50 = 4.11 nM) and the clinically prominent mutants including G250E (IC50 = 2.49 nM), Q252H (IC50 = 2.38 nM), Y253H (IC50 = 5.6 nM), E255K (IC50 = 1.01 nM), E255V (IC50 = 2.73 nM) and T315I (IC50 = 66.1 nM). The cellular activity of TGRX-678 was attenuated by mutations in the myristoyl pocket such as A337V, P465S, V468F and I502L or mutations in the SH3-kinase domain interface including P223S and K294E, underpinning its allosteric mode of action. In SD rats, oral administration of TGRX-678 at 20 mg/kg achieved a maximum plasma concentration (C max) of 2369 ng/mL, which was 1.3-fo