Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.
We describe our work to establish
structure- and fragment-based
drug discovery to identify small molecules that inhibit the anti-apoptotic
activity of the proteins Mcl-1 and Bcl-2. This identified hit series
of compounds, some of which were subsequently optimized to clinical
candidates in trials for treating various cancers. Many protein constructs
were designed to identify protein with suitable properties for different
biophysical assays and structural methods. Fragment screening using
ligand-observed NMR experiments identified several series of compounds
for each protein. The series were assessed for their potential for
subsequent optimization using
1
H and
15
N heteronuclear
single-quantum correlation NMR, surface plasmon resonance, and isothermal
titration calorimetry measurements to characterize and validate binding.
Crystal structures could not be determined for the early hits, so
NMR methods were developed to provide models of compound binding to
guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine
series was optimized to a
K
d
of 40 μM
before a thienopyrimidine hit series was identified which subsequently
led to the lead series from which the clinical candidate S 64315 (MIK
665) was identified. For Bcl-2, the fragment-derived series were difficult
to progress, and a compound derived from a published tetrahydroquinone
compound was taken forward as the hit from which the clinical candidate
(S 55746) was obtained. For both the proteins, the work to establish
a portfolio of assays gave confidence for identification of compounds
suitable for optimization.
Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, k) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.
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