• Inhibiting the Iron-regulated Heme Oxygenase (HemO) of Pseudomonas aeruginosa via Competitive and Non-competitive Mechanisms

      Heinzl, Geoffrey Addison; Wilks, Angela; Xue, Fengtian; 0000-0001-5291-5999 (2016)
      The discovery and development of new antimicrobials has become a top priority as resistance to known therapeutics continues to grow. While most antimicrobials target essential functions, some in the field question this historical approach and instead propose targeting virulence factors, rendering pathogens non-pathogenic. In most Gram-negative bacteria, virulence is globally regulated by iron via the ferric uptake regulator (Fur). Recent studies show that in the host, iron is preferentially acquired via heme uptake and utilization. Pseudomonas aeruginosa encodes two heme uptake systems, both of which terminate in the oxidative cleavage of heme by the iron-regulated heme oxygenase (HemO). HemO is required for the efficient utilization of heme as an iron source in P. aeruginosa. Thus, inhibiting HemO will globally reduce virulence via disrupting the utilization of heme as an iron source. Previous work identified small-molecule inhibitors of HemO via computer-aided drug design techniques, which were validated in vitro and in vivo. Several of those compounds were further explored for optimization using medicinal chemistry, biochemistry, and microbiology techniques. Compounds were synthesized, characterized, and assessed for binding, inhibitory activity in cellulo, and antimicrobial activity. Binding was analyzed by fluorescence quenching, saturation transfer difference (STD)-NMR, heteronuclear single quantum coherence (HSQC) NMR, molecular dynamics simulations, and hydrogen-deuterium exchange mass spectrometry (HXMS). Two lead compounds were shown to bind in the heme-binding site of HemO with low micromolar affinity. Another lead compound was shown to bind to a previously unidentified back site of HemO, which was identified in silico and verified with HXMS. To analyze the mechanism of back side inhibition of HemO, site-directed mutagenesis eliminated a salt bridge (D99-R188) adjacent to the back site. These mutations disrupted the essential hydrogen-bonding network in the distal pocket, as evidenced by poor stability of intermediates and altered structural dynamics. Together, these data show that inhibiting HemO with small-molecules can be achieved on two sites of the enzyme, both the heme-binding site and the newly discovered back site. Future work includes improvement of heme-binding site inhibitors, development of novel inhibitors, and confirming the antivirulent activity of HemO inhibitors in an infection model.
    • Studies on Extracellular Signal-Regulated Kinase-1/2 (ERK1/2) Function and Identification of Small Molecule Inhibitors that Selectively Target ERK1/2 Regulation of Activator Protein-1 (AP-1) Function in Cancer Cell Proliferation

      Zhang, Jun; Shapiro, Paul, Ph.D. (2014)
      Excessive activation of the Raf/MEK/ERK pathway participates in the pathogenesis of numerous human tumors. In this pathway, extracellular signal regulated kinase 1/2 (ERK1/2) occupy a unique position on which signals from various membrane receptors converge. Therefore, ERK1/2 have been appealing targets for the development of anticancer drugs. In the recent years, careful examination of ATP binding site of protein kinases coupled with structure-based drug design methods has largely facilitated development of specific protein kinase inhibitors. The crystal structures of ERK2 with ATP complex refined to 2.3 Å have been reported previously. However, the details of interactions between ERK2 and ATP binding pocket were not certain because of low resolution. We have obtained high resolution crystals and refined the structures of the apoenzyme, ATP-bound ERK2, ADP-bound ERK2 to 1.8, 1.7 and 1.8 Å respectively. We then described the identification of a novel lead compound with a thienyl benzenesulfonate scaffold that targets the F-domain Recruitment site (FRS) of ERK1/2. Our crystal structure data suggested that this compound interacted with ERK2 in the vicinity of the FRS. Biological analyses further showed that this compound, along with a few structurally similar analogs derived from computational methods based on information provided by the crystal structure, preferentially inhibited F-site containing substrates that form the activator protein-1 (AP-1) transcription factor complex including c-Fos, Fra1, and FosB. Cell viability analysis revealed that melanoma cell lines that harbor constitutively activated ERK1/2 driven by activating B-Raf mutants were more sensitive to growth inhibition by these compounds. Lastly, we discovered a novel mechanism by which ERK1/2 activation regulates the phosphorylation of protein kinase C (PKC) isoforms PKCβII/δ. Our studies demonstrated that growth factor mediated ERK1/2 activation regulated PKCβII/δ activity as inhibition of ERK1/2 affected PKC substrate phosphorylation. Moreover, we provide evidence that inhibition of ERK1/2 reduced cell migration through a mechanism involving regulation of PKCβII. Thus, these data indicate that ERK1/2 proteins act as upstream regulators of PKC proteins and that inhibition of this regulation may have utility in preventing cancer cell migration and metastasis.
    • 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.