The Design and Development of Dual MCL-1/BCL-2 and HDM2/Bcl-2 Protein Family Inhibitors Using a Polypharmacology Approach

Abstract

Apoptosis, a cellular process that leads to cell death, is a vital signaling pathway for maintaining homeostasis. Intracellular-activated apoptosis is regulated by the B-cell lymphoma 2 (BCL-2) family of proteins, which encompasses two classes of proteins: the pro-apoptotic and anti-apoptotic members. Apoptosis is controlled by a protein-protein interaction (PPI) between the two members. Specifically, the anti-apoptotic proteins’ surface hydrophobic binding groove binds to the α-helical Bcl-2 homology 3 (BH3) domain of the pro-apoptotic proteins, thus inhibiting apoptosis. During apoptotic conditions, BH3 activator proteins are expressed and disrupt the PPI, initiating apoptosis. During tumorigenesis, the anti-apoptotic proteins are overexpressed and capture the activator proteins before they can act, progressing tumor development. A strategy developed to overcome this oncogenic transformation is BH3 mimicry, the design of small molecules that behave like BH3 activators to free the pro-apoptotic proteins. Though potent BH3 mimetics have been synthesized, cytotoxic and resistance issues have arisen. Specifically, BCL-XL inhibition causes thrombocytopenia within patients and BCL-2 inhibition causes resistance mechanisms to emerge that involve the upregulation of MCL-1. Presently, there are no potent dual inhibitors of BCL-2 and MCL-1 to overcome these issues. Additionally, p53 has been shown to regulate apoptosis through the Bcl-2 family by either direct interactions or increasing their expression. P53 is rapidly degraded due to the overexpression of HDM2, a ubiquitin ligase, within cancer cells. The PPI between p53 and HDM2 resembles the PPI between the members of the Bcl-2 family. Also, Venetoclax (BCL-2 inhibitor) and idasanutlin (HDM2 inhibitor) act synergistically in combination therapies. Thus, we followed a polypharmacology approach to synthesize dual BCL-2/MCL-1 and dual HDM2/Bcl-2 family inhibitors. We were able to create potent dual MCL-1/BCL-2 indazole inhibitors (Ki MCL-1 < 1.50 µM, BCL-2 < 0.050 µM, BCL-XL > 10.00 µM), dual HDM2/Bcl-2 family pyrazole and imidazole inhibitors (Ki MCL-1 < 0.050 µM, HDM2 < 25.00 µM), HSQC-confirmed nicotinate-based MCL-1 inhibitors (Ki MCL-1 < 3.00 µM) and a new alpha-helix mimetic scaffold for disrupting PPIs. Further optimization of these inhibitors is planned, along with cell viability studies. Overall, these inhibitors can serve as starting points for future experiments and polypharmacology designs.