• Antagonism of the Alpha-Helix Mediated Protein-Protein Interactions of the Bcl-2 and c-Myc Oncoprotein Families: Proteomimetic and Small-molecule Strategies

      Yap, Jeremy; Fletcher, Steven (2014)
      The Bcl-2 oncoprotein family includes both anti- and pro-apoptotic proteins that are normally localized within the mitochondrial outer membrane. The over-expression of the anti-apoptotic proteins (such as Bcl-xL, Bcl-2, and Mcl-1) is associated with cancer and chemotherapeutic resistance. Pro-apoptotic Bcl-2 proteins (such as Bak and Bim) initiate the intrinsic apoptotic pathway via oligomerization at the mitochondrial membrane. However, in the presence of over-expressed anti-apoptotic Bcl-2 proteins, pro-apoptotic Bcl-2 proteins are sequestered and the intrinsic apoptotic pathway is antagonized. Specifically, the conserved BH3 alpha-helix of the pro-apoptotic proteins engage the hydrophobic binding crevices of the anti-apoptotic proteins largely through hydrophobic (i), (i + 3/4) and (i + 7) residues on one face of the helix. Though potent inhibitors of Bcl-2 and Bcl-xL have been identified, chemically diverse pan-Bcl-2 and Mcl-1 specific inhibitors are lacking. Inspired by the recent advances in alpha-helix mimicry and fragment-based drug design, we have successfully synthesized potent (Ki ~ 150 nM) pan-Bcl-2 inhibitors based on trisbenzamide and salicylate scaffolds and validated their activities in vitro. The c-Myc oncoprotein is an intrinsically disordered (ID) transcription factor of a vast number of genes that are involved in cell proliferation and growth. Similar to anti-apoptotic Bcl-2 proteins, overexpression of c-Myc is associated with a myriad of cancers such as prostate, breast, and lung tumors. Though biologically inactive in its ID monomeric form, the transcriptional activation of c-Myc is initiated upon binding its obligatory protein partner Max. The transcriptionally active c-Myc-Max heterodimers recognize and bind the hexanucleotide sequence 5'-CACGTG-3' on dsDNA, where the transactivation domain of c-Myc recruits additional transcriptional machinery. Owing to its ID properties, in the absence of Max, c-Myc does not exhibit any secondary structure that may function as a basis for drug design. While several c-Myc specific inhibitors have been identified through high-throughput screening, few structure-activity relationship (SAR) studies have been reported. Towards developing potent c-Myc inhibitors, we conducted an SAR study on the c-Myc inhibitor 10074-G5 (IC50 = 146 uM), which resulted in the discovery of an improved inhibitor, JY-3-094 (IC50 = 33 uM) whose ester prodrugs exhibited potent cell activities (IC50 < 10 uM).
    • Inhibition of Anti-Apoptotic Protein MCL-1 and Epigenetic Reader BRD4 with Small Molecule Inhibitors: Structure-Based Drug Design and Polypharmacology

      Chen, Lijia; Fletcher, Steven; 0000-0002-5721-5091 (2016)
      One of the hallmarks of cancer is the evasion of programmed cell death. Programmed cell death is strictly regulated by the intrinsic apoptosis pathway, in which BCL-2 anti/pro-apoptotic proteins play vital roles. In normal cells, the delicate balance between anti-apoptotic BCL-2 proteins and their functional sequester: pro-apoptotic proteins, is precisely maintained to ensure the execution of cell death or cell proliferation. However, the apoptotic pathway is often dysregulated in cancer cells, demonstrated by the frequent observed over-expression of anti-apoptotic protein(s), such as BCL-2, BCL-xL and MCL-1. Therefore, targeted inhibition of BCL-2 anti-apoptotic proteins using small molecule inhibitors has been extensively researched to restore apoptosis and kill cancer cells. A decade of research is rewarded by the recent approval of the BCL-2 selective inhibitor Venetoclax as a second line therapy for chronic lymphocytic leukemia (CLL) patients. Nonetheless, due to the heterogenetic nature of cancers, inhibitors targeting other anti-apoptotic BCL-2 proteins should also be developed not only for different cancer types, but also for the potential resistance generated by MCL-1 up-regulation after BCL-2 inhibition. To date, numerous studies are currently ongoing to discover small molecule inhibitors targeting different anti-apoptotic BCL-2 proteins, primarily MCL-1 and/or BCL-xL, in order to provide insight to dissect these proteins' biological functions, and potentially yield various tool compounds as clinical candidates. Utilizing structure-based drug design strategy, we have designed and synthesized two distinct scaffolds: tetrahydroquinoline (THQ) and salicylate as MCL-1 inhibitors. SAR analysis has been conducted based on their biochemical binding affinities to facilitate the understanding of binding pocket properties. In vitro cell growth inhibition was also evaluated to estimate their potency at cellular level. Another survival mechanism of cancer is through the dysregulation of post-translational modifications, such as aberrant acetylation of histone lysine residues, which abnormally turn on the transcription and expression of oncogenes. Particularly, the bromodomain BRD4 protein is heavily involved in this process as an epigenetic reader to recognize the acetylated lysine residues on histone and regulate the transcription of several oncogenes including c-MYC and BCL-2. Therefore, inhibition of BRD4 may serve as an indirect method to modulate the level of c-MYC and BCL-2 oncoprotein in cancers. Recently, a PLK1 inhibitor BI-2536 was reported as a potent BRD4 inhibitor. The dual inhibition by a single drug molecule might provide the platform for a polypharmacological strategy to treat various cancers. With the guidance of structure-based drug design, we have synthesized and evaluated an SAR library of BI-2536 to dissect their PLK1 and BRD4 inhibition profiles. The fine-tuning modifications of functional groups on BI-2536 furnished both selective BRD4 inhibitors and stronger dual PLK1/BRD4 inhibitors.
    • Targeting Aberrant alpha-Helix Mediated Protein-Protein Interactions with Densely Functionalized Heterocycles

      Conlon, Ivie; Fletcher, Steven; 0000-0002-8269-299X (2020)
      Protein-protein interactions (PPIs) play crucial roles in cell proliferation, differentiation, and apoptosis. Apoptosis is a highly regulated process of cell death and its dysregulation can lead to a multitude of different pathophysiologies, such as cancer. In particular, the overexpression of pro-life Bcl-2 proteins, such as Bcl-2, Bfl-1, and Mcl-1, has been linked to cancer progression and tumorigenesis, as well as chemoresistance to a number of different chemotherapeutics. The binding counterparts of these proteins, pro-death Bcl-2 proteins such as Bim, and p53 transactivation domain (TAD), exert their effects through α-helix mediated PPIs with key residues i, i+ 3/4, and i+ 7 oriented on one side of the helix. In addition, HDM2, the E3 ubiquitin protein ligase responsible for the degradation of p53, is upregulated in numerous cancers, and given the similarities of the recognition profiles of Bim-BH3 and p53TAD, we have designed α-helix mimetic inhibitors that target Mcl-1 and HDM2. The first generation of compounds included various heterocyclic scaffolds, including isoxazoles, pyrazoles, and thiazoles, that project functional groups in a similar manner to the native α-helices. In addition, bicyclic scaffolds have been utilized in Mcl-1 selective inhibition. Therefore, we developed a second generation of compounds of isoxazoles, pyrazoles, and functionalized indoles to further explore the binding interface of Mcl-1. The recent resurgence of covalent inhibition and targeted protein degradation has led to the development of successful Bcl-2 family inhibitors. We have designed two tris-aryl α-helix mimetic scaffolds targeting the Bfl-1 pro-life protein. A unique surface-accessible cysteine within the BH3 domain allows for the development of reversible and irreversible small molecule covalent inhibitors. In addition, we have also designed a venetoclax-based PROTAC targeting Bcl-2.
    • The Design and Development of Dual MCL-1/BCL-2 and HDM2/Bcl-2 Protein Family Inhibitors Using a Polypharmacology Approach

      Drennen, Brandon; Fletcher, Steven (2019)
      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.
    • Towards Targeted Anti-Neoplastics: The Disruption of Aberrant Protein-Protein Interactions with Low Molecular-Weight Proteomimetics

      Lanning, Maryanna Elizabeth; Fletcher, Steven; 0000-0002-9511-4435 (2017)
      Protein-protein interactions (PPIs) play pivotal roles in a range of cellular processes including proliferation, differentiation, metabolism and apoptosis. Dysregulations of certain PPIs can lead to the development and progression of human cancers. In particular, the overexpression of the anti-apoptotic BCL-2 family members, specifically Mcl-1, have been linked to pancreatic, colorectal and lung cancers as well as leukemia and lymphoma. When over-expressed, MCL-1 prevents cell death by binding and sequestering the BH3 "death" domain of its pro-apoptotic counterpart, such as Bim. Mcl-1 has become an important target for the development of novel antineoplastics. As with many, helix-mediated PPIs, several key residues are hydrophobic and located on one face of the BH3 α-helix, specifically at the i, i + 3/4, i + 7 residues. In addition to exhibiting a hydrophobic face, Asp67 on the "other" face of the Bim-BH3 helix forms a salt bridge with the protein (Arg263). In an improved effort to develop more potent and more selective agents to disrupt the MCL-1-BH3 PPI, we used structure-based design, and developed two complementary strategies: synthetic α-helix (purine based) and BH3 mimetics (naphthoate based). Both scaffolds yielded molecules that disrupted the MCL-1-BIM PPI consistently, and subsequent studies were undertaken towards second-generation molecules. The designed molecules were subjected to biological assays and further structure-activity relationship (SAR) studies to increase affinity and potency in an effort to translate in vitro activity to on target cell activity. It is expected anticipated that the SAR developed in the present study will facilitate the development of novel therapeutics capable of inhibiting Mcl-1 will be identified, which can be advanced to preclinical evaluation.