• Design and Discovery of Novel Small Molecule Inhibitors targeting Heme Oxygenase (HemO) Dependent Iron Acquisition and Heme Signaling in Pseudomonas aeruginosa.

      Robinson, Elizabeth; Wilks, Angela; Xue, Fengtian, Ph.D.; 0000-0002-7770-4060 (2021)
      The recent rise in antibiotic resistance particularly those pertaining to hospital acquired infections has highlighted the need for alternative therapeutic approaches. Alternative approaches include anti-virulence strategies that differ from current treatments targeting essential pathways in pathogens and instead target systems and factors required for virulence and infection. Gram-negative opportunistic multi-drug resistant (MDR) pathogens like Pseudomonas aeruginosa are ranked at a serious threat level by the CDC and are infamous for causing life threatening infections in immunocompromised populations such as patients with ventilator-assisted pneumonia, open surgical wounds, and cystic fibrosis. Pathogenic bacteria including P. aeruginosa, require the essential micronutrient iron for their survival and virulence. It has been reported that during acute and chronic infections P. aeruginosa preferentially utilizes heme as its iron source over siderophore mechanisms. P. aeruginosa encodes two non-redundant heme uptake systems that both utilize the iron regulated heme oxygenase enzyme (HemO) to release iron and the biliverdin (BVIX) metabolites BVIXβ and -δ. It has been shown that HemO catalytic activity is required to drive heme uptake into the cell. Furthermore, the products of extracellular heme metabolism BVIXβ and -δ function as signaling and regulatory molecules in several virulence traits. Therefore, HemO represents an ideal therapeutic target due to its dual function in limiting both iron and the heme metabolites that regulate several virulence traits. We hypothesize such a dual function strategy will also increase the barrier to resistance. The work herein applies a structure-based design and high-throughput screening approach followed by in vitro and in cell characterization of lead compounds. Further computer-aided drug design (CADD) and guided chemical synthesis optimization were employed to design and discover novel small molecule scaffolds and inhibitors of HemO. The approaches resulted in lead compounds with nanomolar binding affinity and inhibition of HemO enzyme activity both in vitro and in vivo. Additionally, I developed a new method for the production and purification of BVIXβ and -δ, in > 500-fold increased yields, to further study their role in P. aeruginosa virulence and infection.
    • Discovery and Evaluation of Small Molecule Inhibitors of the S100B-p53 Interaction

      McKnight, Laura Emily; Weber, David J., Ph.D. (2012)
      S100B is a calcium-binding protein that is highly upregulated in malignant melanoma and is currently used as a prognostic indicator for the disease. S100B has been shown to bind to p53, decreasing p53 protein levels and inhibiting its function. Small molecule inhibitors are being investigated which use the S100B-p53 interaction as a therapeutic target, and the drug pentamidine was found to bind S100B; pentamidine-derived compounds were then designed, synthesized, and analyzed. Molecular Dynamics simulations of the pentamidine-S100B complex were performed in an effort to determine what properties would be desirable in a pentamidine-derived compound as an inhibitor for S100B. These simulations predicted that increasing the linker length of the compound would allow a single molecule to span both pentamidine binding sites on the protein. The resulting compound, SBi4211 or heptamidine, was synthesized and experiments to study its inhibition of S100B were performed. The 1.65 Å X-ray crystal structure was determined for Ca2+-loaded S100B bound to heptamidine and gives high-resolution information about key contacts that facilitate the interaction between heptamidine and S100B. Additionally, NMR HSQC experiments with both compounds show that SBi4211 causes perturbations in the chemical shifts of the same residues of S100B as pentamidine. SBi4211 is able to selectively kill melanoma cells with S100B over those without S100B, indicating that its binding to S100B has an inhibitory effect and that this compound may be useful in designing higher-affinity S100B inhibitors as a treatment for melanoma and other S100B-related cancers.
    • 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.
    • Inhibition of TLR2 Signaling by Small Molecule Inhibitors Targeting a Pocket Within the TLR2 TIR Domain

      Mistry, Pragnesh Dilipkumar; Vogel, Stefanie N. (2015)
      Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll-Interleukin-1 receptor resistance (TIR) domains. For all TLRs, except TLR3, recruitment of the adapter, MyD88, to the TIR domains results in downstream signaling that culminates in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate diseases caused by TLR-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a "pocket" adjacent to the highly conserved P681 and G682 residues of the BB loop. Using "Computer-Aided Drug Design" (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this "BB loop pocket" and, potentially, disrupt TLR2 signaling. In silico screening identified 149 lead compounds and 20 FDA-approved drugs based on their predicted ability to bind in the BB loop pocket. These were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. In this screen, C16H15NO4 ("C29") was identified as a potential TLR2 inhibitor. C29, and a derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages induced by synthetic and bacterial TLR2 agonists. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6 signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle for this novel CADD approach for the identification of inhibitors of TLR signaling.
    • Novel Cholinergics for Treatment of Central Nervous System Disorders

      Johnson, Chad; Coop, Andrew; 0000-0001-7584-3000 (2019)
      Approximately 16% of Americans are diagnosed with major depressive disorder, a mental disorder thought be caused by a combination of characterized by genetic, biological, environmental, and psychological factors. It can be accompanied by low self-esteem, loss of interest in normally enjoyable activities, low energy, and diminished quality of life. Between 2-7% of adults with this disorder die by suicide. In addition, almost half of patients who are treated initially with an SSRI do not achieve complete remission, and nearly a third after four different treatment regimens (nimh.nih.gov). While counseling and antidepressant medication can be effective treatments, current selective serotonin re-uptake inhibitors (SSRI's) take weeks before therapeutic effects are observed. This "delay" period of action is not well understood and presents a significant challenge for medical professionals in the management of major depression. Mechanisms of anti-depressants have been a major focus of both current/past research in hopes of developing more effective and faster acting drugs. Directly related to this, clinical data (nimh.nih.gov) that oral and intravenous treatment with the muscarinic cholinergic antagonist scopolamine had rapid anti-depressant effects in humans--likely mediated through an antimuscarinic effect. Unfortunately, scopolamine can produce cognitive impairment including memory disturbances due to its anticholinergic properties. Since major depressive disorder is associated with deficits in cognition, this would produce an undesired additive effect that would only exacerbate the problem. It is our goal to identify a muscarinic antagonist that may be able to relieve depression and have little to no effect on memory or cognition. The 3-exo-1-azabicyclo[2.2.1]heptane, 1-azabicyclo[2.2.2]octane, 1-azabicyclo[3.2.1]octane, and N-methyltetrahydropyidine 3 (and 4)-substituted-1,2,4-oxadiazoles appear to be excellent chemical scaffolds for the generation of potent muscarinic agonists/antagonists. In order to probe the orthosteric site of the mAChRs we designed a large library of compounds and evaluated them via a battery of pharmacological assays to confirm both their antidepressant and cognitive effects. This resulted in the identification of lead compound (CJ2100) that showed potent antidepressant activity without cognitive impairment. (Supported by NIMH Grant 107499)
    • Structure-based optimization of small molecule transcription therapy targeting the BCL6 oncoprotein

      Tsourounis, Marilyn; Coop, Andrew (2009)
      BCL6, a member of the BTB/Zinc finger family of proteins is a transcriptional repressor that is constitutively expressed in over 50% of patients with Diffuse Large B Cell Lymphomas (DLBCL). BCL6 is responsible for the repression of at least 14 different genes linked by roles in B cell activation, differentiation, inflammation, and cell cycle control. Prior to cell differentiation, normal BCL6 encourages apoptosis by repressing expression of anti-apoptotic BCL6 target genes in the germinal center. Specific mutations and translocations in the 5' regulatory region of the BCL6 gene have been implicated in the constitutive expression of BCL6. This results in the aberrant replication of B cells that can escape the germinal center and lead to the formation of lymphomas. Preliminary proof of principle experiments have illustrated that an interaction between the BTB domain of BCL6 and unique regulatory sequences of its critical corepressors NCoR and SMRT are responsible for the mechanism of transcriptional repression of BCL6 at a "lateral groove" motif. Peptides that block the BCL6-SMRT interface kill lymphoma cells in vitro and in vivo, without causing toxicity to normal tissues. Small molecule inhibitors based on the 3D BCL6-SMRT interface were discovered via a target based drug design approach utilizing computer aided chemical database screening. Lead compounds displaying the desired biological activity in biochemical and functional assays were identified and selected for chemical derivation. Over 70 compounds have been developed and synthesized and are currently being evaluated for BCL6 inhibitory activity, as a part of our successive chemical optimization process to improve binding affinity, selectivity, and solubility. Preliminary in vitro studies of our first generations of indolylidene inhibitor compounds have demonstrated that an N-carboxylic acid and nature of the central heterocyclic ring are significant for activity. These results are consistent with CADD results determination of an energetically favorable interaction between the carboxylic charged moieties of lead compounds identified from primary database screens and key Arginine and Histidine residues in the putative binding site of the BCl6 BTB domain. Other CADD identified lead candidates possessing a diarylheterocyclic based scaffold were selected for further optimization to generate a chemically diverse library of molecules for SAR investigation of BTB lateral groove blockade and are presented. As continuation of this work, the development of small molecule inhibitors to target a secondary binding site of the BTB domain is described. Our structure-based strategy for introducing chemical modifications to our existing small molecule inhibitors along with proposed synthetic methodology to develop targets achieving this unique biological profile are reported.
    • The use of conformational sampling in CHARMM protein force field optimization and ligand-based drug design

      Shim, Jihyun; MacKerell, Alexander D., Jr. (2013)
      Sampling of the conformational space of biomolecules in computer simulations allows researchers to investigate atomistic details of biological phenomena such as protein folding and ligand binding. Conformational sampling based on empirical energy functions depends on the force field and is aided by enhanced simulation methods. This thesis discusses conformational sampling methods and force fields, along with application of conformational sampling to force-field optimization and ligand-based drug design. Extensive conformational sampling was performed for small peptides and drug-like molecules using temperature replica-exchange and Hamiltonian replica-exchange molecular dynamics. Obtained conformational ensembles were then used to improve peptide-backbone and side-chain parameters in the CHARMM protein force fields, thereby yielding more accurate conformational properties. Obtained ensembles were also applied to ligand-based drug design where a novel method based on the conformationally sampled pharmacophore approach was used to identify quantitative structure-activity relationships (SARs) of μ opioid receptor ligands. Based on the SARs, we proposed ligand-binding orientations related to receptor activation. The binding orientations were further investigated using simulations of selected ligands bound to the 3-dimensional -opioid receptor structures. Our studies validate ligand-based SARs and show atomistic details of ligand-receptor interactions and the mechanism of µ opioid receptor activation.