Discovery of HZ-L105, a Next Generation of Bcl-2 Inhibitor, Overcomes Bcl-2 Mutation and Exhibits Superior Antitumor Activity

Introduction: Bcl-2, a key regulator of apoptosis pathway, is over-expressed in many hematologic malignancies, which results in blocking of apoptosis and cell survival. Targeting Bcl-2 for the treatment of malignant hematological tumors has been well validated and demonstrated by FDA approval of the first generation Bcl-2 inhibitor Venetoclax for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and acute myeloid leukemia (AML). Although Venetoclax provides significant clinical benefits, there are reports show that after long-term monotherapy patients develop disease progression , often driven by acquisition of Bcl-2 Gly101Val (G101V), Asp103Tyr (D103Y) resistance mutations. Here, we described the discovery of a next generation Bcl-2 inhibitor HZ-L105 as with high potency against Bcl-2, Bcl-2(G101V) and Bcl-2(D103Y) for treatment of Bcl-2 dependent and Venetoclax-resistant hematological cancers. Methods:Biochemical inhibition assays were conducted towards Bcl-2/Bcl-2 G101V/Bcl-2 D103Y/Bcl-xL by time-resolved fluorescence resonance energy transfer (TR-FRET);Cellular proliferation tests of RS4;11 and RS4;11-G101V overexpressed stable line were carried out using MTS assay;Human platelet viability was measured by Cell-titer Glo assay;Cytochrome P450 inhibition assays were performed using fluorometric assay;In vivo pharmacodynamic (PD) studies were performed in RS4;11-derived subcutaneous xenograft SCID mouse model;Preclinical pharmacokinetics (PK) studies were assayed in mice and dogs and parameters were determined by LC-MS. Results: In biochemical assays, HZ-L105 potently inhibited Bcl-2 WT, Bcl-2 G101V and Bcl-2 D103Y with IC50 of 0.54 nM, 6.16 nM and 5.57 nM，while Venetoclax's IC50 of 1.62 nM, 440.6 nM and 279.8 nM, respectively. Therefore, it is expected to overcome the acquisition of Bcl-2 resistance mutations. In cellular assays, HZ-L105 suppressed cell growth not only in RS4;11 but also in RS4;11-G101V stable line (IC50=0.30 nM in RS4;11 and 243.36 nM in RS4;11-G101V), which was much more superior to Venetoclax (IC50= 2.91 nM in RS4;11 and 3360.79 nM in RS4;11-G101V). HZ-L105 was also highly selective against Bcl-2, showing >1000 folds selectivity to Bcl-xL. Furthermore, platelet viability assay in vitro showed HZ-L105 induced hypotoxicity as similarly to Venetoclax, suggesting its good potential to avoid thrombocytopenia due to Bcl-xL inhibition. In the preclinical Pharmacodynamics studies, oral administration of HZ-L105 induced rapid and robust antitumor effect in RS4;11 ALL xenografts. HZ-L105 at a dose of 5 mg/kg demonstrated significantly excellent efficacy than Venetoclax at a dose 15mg/kg in RS4;11-derived xenograft mouse model. In the preclinical pharmacokinetic studies, HZ-L105 showed excellent pharmacokinetic properties in ICR mice and beagle dogs, with long T1/2 and high dose-normalized AUC. HZ-L105 did not reduced hERG tail current (% current blocked 2.87% at 30μM), indicating the compound has a low potential to induce QT prolongation. HZ-L105 has less drug-drug interaction potential, as evidenced by no inhibitory effect on CYPs, especially in CYP2C9 (IC50 >20 μM) compared with Venetoclax (IC50=1.12 μM). Besides, in 14-days oral gavage toxicity study in mice, no compound related deaths occurred at 15, 50 and 300 mg/kg/day, and only leukopenia was observed similarly to Venetoclax. Conclusion: In summary, HZ-L105 as a next generation Bcl-2 inhibitor, showed great potency on wild type and acquisition resistance mutations of Bcl-2 in vitro, excellent oral bioavailability, superior anti-tumor activities on preclinical xenograft model, and safety profiles,. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal