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:

1. 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);

2. Cellular proliferation tests of RS4;11 and RS4;11-G101V overexpressed stable line were carried out using MTS assay;

3. Human platelet viability was measured by Cell-titer Glo assay;

4. Cytochrome P450 inhibition assays were performed using fluorometric assay;

5. In vivo pharmacodynamic (PD) studies were performed in RS4;11-derived subcutaneous xenograft SCID mouse model;

6. 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 T_1/2 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.

No relevant conflicts of interest to declare.