• 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).
    • Design, Development, and Characterization of Gallium (III) Salophen Metallotherapeutics Targeting Heme Sensing and Iron Acquisition Pathways in Pseudomonas aeruginosa

      Centola, Garrick; Wilks, Angela; Xue, Fengtian, Ph.D.; 0000-0001-5965-9545 (2022)
      The development of new antibiotics is outpaced by the rise in multi-drug resistant (MDR) bacteria, creating a global health problem. Pseudomonas aeruginosa, one such bacterium, is labeled as a “critical priority” pathogen by the WHO for its resistance to treatment and prevalence in hospital-acquired infections and immunocompromised patients where it is often life threatening. Adding to this problem, most new discoveries are derivatives of existing antibiotic classes rather than new strategies. Newer approaches targeting bacterial pathways critical to infection but not survival outside the host are expected to exert less selective pressure and slow resistance onset. One such strategy is interfering with bacterial iron uptake and utilization, as iron is a key micronutrient with several iron-regulated virulence traits used to counter iron-sequestering defense mechanisms of the host. P. aeruginosa can shift between the acquisition of labile iron stores and the more abundant heme-bound iron at various stages of infection, so inhibitors targeting these pathways must account for this adaptability. One such approach to targeting iron utilization in several forms is the use of gallium, which mimics ferric iron in ionic size and charge but cannot undergo critical redox processes, thus causing toxicity in the bacteria that acquire it under the guise of iron. This work describes the synthesis and characterization of Gallium Salophen (GaSal) and subsequent analogs targeting heme and iron acquisition pathways in P. aeruginosa. In this characterization, GaSal binds to a hemophore, HasAp, secreted by P. aeruginosa, and inhibits an extra-cytoplasmic function (ECF) signaling cascade with the outer-membrane receptor HasR, which is critical for sensing and adapting to host heme levels. GaSal is simultaneously a substrate for uptake, independent of its effect on HasAp. Using a combination of cell-based assays as well as in vitro target characterization and finally preliminary animal infection studies, GaSal and subsequent derivatives are shown to be promising new developments targeting several points in the iron uptake and utilization pathways of P. aeruginosa. Continued developments aim to retain such activity and include several routes towards further optimization and development as a therapeutic.