Glutamate excitotoxicity is the predominant mechanism implicated in neuronal cell death associated with neurological disorders such as stroke, epilepsy, traumatic brain injury and ALS. Excessive stimulation of NMDA subtypes of glutamate receptors leads to protracted intracellular calcium elevations triggering calcium mediated neurotoxic mechanisms culminating in delayed neuronal cell death. In addition, glutamate excitotoxicity induces a NMDA dependent extended neuronal depolarization mediated by continuous calcium influx that correlates with delayed neuronal death. Attempts to prevent neuronal death by blocking calcium entry into the neurons using calcium channel blockers or NMDA receptor antagonists have failed to provide any beneficial effects in clinical trials. Thus, calcium continues to enter the neurons despite the presence of calcium entry blockers. This phenomenon is known as the "calcium paradox of stroke" and represents a major problem in developing effective therapies for treatment of stroke. Here employing a combination of patch clamp recordings, fluorescent calcium imaging and neuronal cell death assays in well-characterized in vivo and in vitro models of glutamate excitotoxicity, we report the discovery of a novel calcium permeable ion channel that is activated by excitotoxic glutamate injury and mediates a calcium current that is an early initiating step in causing neuronal death. Blocking this calcium permeable channel with high concentrations of Zn2+ or Gd3+ by removing extracellular calcium for a significant time period after the initial injury is effective in preventing calcium entry, apoptosis and neuronal death, thus accounting for the calcium paradox. This injury induced-calcium permeable channel provides a unique mechanism for calcium entry following stroke and offers a new target for extending the therapeutic window for preventing neuronal death after the initial excitotoxic (stroke) injury.

Alcohol abuse and dependence are complex disorders that are influenced by many genetic and environmental factors. Acute behavioral responses to ethanol have predictive value for determining an individual’s long-term susceptibility to alcohol abuse and dependence. These behavioral responses are strongly influenced by genetics. Here, we have explored the role of genetic influences on acute behavioral responses to ethanol using the nematode worm, Caenorhabditis elegans. First, we explored the role of ethanol metabolism in acute behavior responses to ethanol. Natural variation in human ethanol metabolism machinery is one of the most reported and reproducible associations found to alter drinking behavior. Ethanol metabolism is conserved across phyla and alteration in this pathway alters acute behavioral responses to ethanol in humans, mice, rats, and flies. We have extended these findings to the worm and have shown that loss of either alcohol dehydrogenase or aldehyde dehydrogenase results in an increase in sensitivity to the acute effects of ethanol. Second, we explored the influence of differences in basal and ethanol-induced gene expression in ethanol responsive behaviors. We identified a set of candidate genes using the basal gene expression differences in npr-1(ky13) mutant animals to enrich for genes involved in AFT. This analysis revealed ethanol changes to the expression of genes involved in a variety of biological processes including lipid metabolism. We focused on a gene involved in the metabolism of fatty acids, acs-2. acs-2 encodes an acyl-CoA synthetase that activates fatty acids for mitochondrial beta-oxidation. Animals carrying mutant acs-2 have significantly reduced AFT and we explored the role of genes in the mitochondria beta-oxidation pathway for alterations in ethanol responsive behaviors. We have shown that knockdown of ech-6, an enoyl-CoA hydratase, enhances the development of AFT. This work has uncovered a role for fatty acid utilization pathways in acute ethanol responses and we suggest that natural variation in these pathways in humans may impact the acute alcohol responses to alcohol that in turn influence susceptibility to alcohol abuse and dependence.

Using a semi-PBPK modeling/quantitative meta-analysis approach, this project investigated what factors affect pulmonary and systemic exposures of Budesonide (BUD), Tobramycin (TOB), and Ciprofloxacin (CIP) after inhalation: Three structurally different pulmonary disposition models were developed for each drug, including pulmonary absorption (all three), excretion (TOB and CIP) and sequestration (TOB) in a peripheral and central lung compartment. Systemic disposition parameters were estimated using available human mean plasma (cp(t)) and sputum (cs(t)) concentration profiles after IV administration, and GI absorption parameters were estimated from these profiles after oral administration. Pulmonary disposition parameters were estimated from cp(t) and cs(t) profiles after inhalation using various devices along with their published pulmonary deposition characteristics. Appropriate covariate models accounted for effects of Cystic Fibrosis on the systemic disposition/GI absorption for TOB and CIP. Monte Carlo Simulations (MCS) were used to optimize parameters and validate the final models and parameter spaces against published data. Despite limited available data, especially cs(t) for BUD and CIP (after IV administration), the point estimates for the final model parameters were mechanistically plausible for all three drugs and consistent with their known differences in physicochemical and ADME properties. Model predictions adequately described the observed cp(t) and cs(t) profiles as well as exposure metrics across studies. As the most lipophilic drug, BUD showed the fastest pulmonary absorption rates and highest Fpul (83%). TOB, a very hydrophilic drug, exhibited (intracellular) pulmonary sequestration, resulting in slow pulmonary absorption and excretion and low Fpul (10%). CIP - as zwitterion - showed relatively slow pulmonary absorption and excretion, leading to low Fpul (8%); pulmonary excretion accounted for 27% of CIP overall elimination. Results of a formal parameter sensitivity analysis demonstrated that, for all three drugs, after inhalation, (1) their systemic exposures (cp(t)) depend primarily on CLtot along with Fpul/sequestration combined with Foral; (2) increasing pulmonary exposures (cs(t)) can be accomplished by slowing down pulmonary absorption rates (kca) and/or slowing down mucociliary clearance from the lungs into the GI tract (kcm) – affirming the overall hypothesis guiding the project.

Cellular senescence a specialized form of growth arrest that contributes to the pathogenesis of several aging-related disorders including cancer. While by definition tumor cells are considered immortalized, they can undergo senescence when exposed to conventional and targeted cancer therapy. Therapy-Induced Senescence (TIS) represents a fundamental response to therapy and impacts its outcomes. However, TIS has been considered a positive therapeutic goal since senescent tumor cells are expected to enter a state of permanent growth abrogation. In this work we examined the hypothesis that a subpopulation of senescent cells can re-acquire proliferative potential after a state of senescent dormancy, indicating that senescence is not obligatorily an irreversible process. Our observations indicate that H460 non-small cell lung cancer cells induced into senescence by exposure to etoposide, and enriched based on β-galactosidase staining and size, were shown to recover reproductive capacity, which was accompanied by resolution of the DNA-damage-response (downregulation of p53 and p21Cip1 induction), attenuation of the Senescence-associated Secretory Phenotype (SASP). To overcome the reservation that the newly dividing cells may not have been derived from the senescent population and in an effort to establish that escape from TIS is feasible, tumor cells induced into senescence by chemotherapy were enriched for senescence by flow cytometry; the subsequent division of senescent cells was demonstrable utilizing both real-time, live microscopy and High Speed Live Cell Interferometry (HSLCI). Furthermore, sorted senescent cells were observed to form tumors when implanted in immune deficient mice and with a significant delay in immunecompetent mice. As chemotherapy induced senescent cells have been identified in patient tumors, it is reasonable to propose that tumor cells that escape from senescence could contribute to disease recurrence. In addition, therapy-induced senescence could prove to reflect one form of tumor dormancy. Recently, ABT263 has been used as a senolytic drug, effectively eliminating senescent cells from aging-related animal models. Here, we utilize ABT263 in a two-hit approach to eliminate senescent tumor cells that persistent after exposure to chemotherapy. ABT263 results in the killing of senescent tumor cells in a concentration-dependent manner and shifts the response towards apoptotic cell death. Furthermore, sequential administration of ABT263 interferes with the ability of senescent tumor cells to recover growth potential. These results indicate that senescent tumor cells can contribute to cancer relapse by acquiring proliferative properties and that senolytic therapy allows for the clearance of dormant senescent tumor cells and will potentially decrease cancer recurrence rates.

It has been shown that the amino terminus and second extracellular loop (EC2) of CXCR2 are crucial for ligand binding and receptor activation. The lack of an ionic lock motif in the third intracellular loop of CXCR2 focuses an investigation of the mechanism by which these two extracellular regions contribute to receptor recognition and activation. The first objective of this investigation was to predict the structure of CXCR2 based on known structures of crystallized GPCRs. Rhodopsin, β2-adrenergic receptor, CXCR4 were used for homology modeling of CXCR2 structure. Highly conserved motifs found in sequence alignments of the template GPCRs were helpful to generate CXCR2 models. We also studied solvent accessibility of residues in the EC2 of CXCR2 in the inactive state. Most of the residues in the EC2 were found to be solvent accessible in the inactive state, suggesting the residues might be involved in ligand recognition. Second, we studied the role of charged residues in the EC2 of CXCR2 in ligand binding and receptor activation using constitutively active mutants (CAM) of CXCR2, D9K and D9R. Combinatorial mutations consisting of the CAM in the amino terminus and single mutations of charged residues in the EC2 were generated to study two concepts including “attraction” and “repulsion” models. The mutant receptors were used to test their effects on cell surface expression, ligand binding, receptor activation through PLC-β3, and cellular transformation. All the mutations in the repulsion model result in CXCR2 receptors that are unable to bind ligand, suggesting that each of the Arg residues in the EC2 are important for ligand recognition. Interestingly, mutations in the attraction model partially inhibited receptor activation by the CAM D9K, suggesting that Glu198 and Asp199 residues in the EC2 are associated with receptor activation. Furthermore, a novel CAM, E198A/D199A, was identified in this study. These negatively charged residues are very close to a conserved disulfide bond linking the EC2 and the third transmembrane. In this sense, these current discoveries concerning the structural basis of CXCR2 and interdisciplinary approaches would provide new insights to investigate unknown mechanisms of interaction with its cognate ligands and receptor activation.

Apurinic/apyrimidinic DNA repair endonuclease-1 (APE1), first recognized as an important DNA excision repair enzyme, is also known as Redox Factor-1 (Ref-1) involved in the activation of many nuclear transcription factors in both redox-dependent and independent manner. It has been well-documented that the overexpression of APE/Ref-1 contributes to the development of chemo-resistance and is associated with tumor progression in many human malignancies [1]. Our previous study in melanoma demonstrated that the development of novel inhibitors targeting the redox regulation domain of APE/Ref-1 is a promising strategy for melanoma treatment. To date, limited successes have been reported in developing novel APE/Ref-1 inhibitors for cancer treatment. Utilizing a structure-based approach, our study identified and characterized small molecular inhibitors of APE/Ref-1. First, N-terminally truncated APE/Ref-1 protein lacking the first 40 amino acid residues (∆40APE-1wt) was cloned into the pGEX-6P1 vector to express the GST-∆40APE-1wtprotein. After cleavage of GST-tag, the concentrated ∆40APE-1wt protein was subjected to protein crystallization study. We have successfully diffracted ∆40APE-1wt crystals and collected data with a resolution of 1.57Å. The crystal structure was further determined by molecular replacement in Molrep using the already available human APE-1 structure (PDB: 5CFG). For the first time, we observed the dimerization of APE/Ref-1 protein formed under oxidative conditions, which may contribute to the redox regulation of APE/Ref-1. Such structural transformation of APE/Ref-1 protein under distinct redox conditions may pave the way for future drug development and optimization. The binding affinity of the candidate compounds with ∆40APE-1wt protein was also determined using Surface Plasmon Resonance (SPR), and the Ki values were analyzed. One of the potent inhibitors developed by our group by structure-based approach, exhibited promising anti-melanoma activities both in vitro and in vivo. Future studies on the structure-activity association are warranted.

Part I. The reaction between chiral acyl chlorides and trans -2-substituted-cyclohexanols proceeds diastereoselectively, i.e. produces mixtures of unequal amounts of diastereomers. We found for the first time that addition of pyridine or diisopropylethylamine accelerates the acylation, and unexpectedly for some substituents (RX) may completely invert its diastereoselectivity. These observations have been rationalized in terms of a stereoselective intramolecular assistance by the RX group to the acylation of the neighboring hydroxyl ("bait-and-hook" mechanism). A series of trans -2-substituted-cyclohexanols were synthesized and acylated with a racemic reagent in presence and absence of pyridine. The results showed that the presence of a nucleophilic group on the second carbon allowed for the preferred formation of one of the diastereomers in the absence of pyridine. However, in the presence of pyridine, the diastereoselectivity would inverse, and the reaction would favor the formation of the other diastereomer. To test the intramolecular acyl transfer hypothesis in detail a series of thioglucosides has been synthesized. Part II. The synthesized thioglucosides were tested as inhibitors of fungal glycosidases. Two compounds showed greater than 80% inhibition values in excess of the activity of β-D-glucosidases. More interestingly, the same compounds showed a marked enhancement of α-D-galactosidase activity by as much as 35%.

Antibodies have been widely used as reagents, homing devices, diagnostics and as therapeutic agents against different targets in clinic and research. Recently a number of monoclonal antibodies and their drug conjugates have been approved as therapeutic agents. While these molecules have great potential in various applications and therapeutics, extensive use of full length antibodies has been hampered by the high cost of production, large molecular weight and limited ability to penetrate tumor tissues. These limitations have led to the research for antibody alternatives with lower molecular weight, similar binding and affinity properties but without the lengthy and complicate process of generating antibodies. Some examples of these efforts include minibodies, fragment antigen binding (FAB), ScFv, and synthetic antibody mimics. Although these antibody alternatives have low molecular weight, as compared to the antibody, they are either derived from full size antibodies or by a long and tedious in vitro screening process. Therefore, a rational design of molecules that mimic antibody binding is a logical first step for the development of antibody alternatives. In this study, a novel approach to design antibody mimics without involving massive experimental screening was developed. The design was developed by mapping and identifying EGFR epitope region where Cetuximab CDR binds and modifying sequences using knob-socket computational model. The binding of antibody mimics were first analyzed by using MOE to obtain the binding energy, total and preserved interactions as compared to the interactions between EGFR and Cetuximab. Further, the designed antibody mimics were used to form a peptide drug conjugate (PDC). Antibody mimics were found to specifically bind and internalized by EGFR overexpressing cell lines with three to four folds higher than control cells. Antibody mimics showed binding in nanomolar range with Pep11 with binding affinity (K D ) of 252nM as shown by SPR studies. EGFR phosphorylation studies also showed that antibody mimics were able to inhibit the binding of EGF to the EGFR in a similar fashion as Cetuximab. Specific binding, affinity and functional activity of the antibody mimics demonstrated that these peptides were able to mimic all the three important characteristics of antibodies. Peptide drug conjugate (PDC) was found to be around 10 fold more potent as compared to the drug itself towards EGFR overexpressed cancer cells. PDC also showed more than 100 fold low potency against control cells. These studies demonstrated that a rational design of molecules to mimic the antibody characteristics is feasible. The antibody mimics were also successfully applied and used as targeting moiety to design peptide drug conjugates for efficient targeted drug delivery system than antibody drug conjugates.

Guanine-rich telomeric DNA at the end of chromosomes can form a unique DNA tertiary structure - G-quadruplex, which is known to inhibit the binding of telomerase to telomeric regions in cancer cells and thus regulate unrestricted cancer cell growth. Hence, G-quadruplex DNA has recently become a promising target in oncology. The formation of G-quadruplex structures is greatly facilitated by G-quadruplex binding ligands such as Thiazole orange (TO). Compared with other G-quadruplex binding ligands, TO has an intriguing tunable fluorescence property. Upon binding to DNA, the fluorescence of TO can increase up to 1000-fold, making it an attractive probe for studying ligand-DNA interactions. However, the poor binding affinity and minimal binding selectivity towards different DNA conformations greatly limit its applications. My research focuses on developing G-quadruplex binding ligands using TO as a scaffold. In the first part of this work, we investigated the feasibility of increasing the TO binding affinity and selectivity toward G-quadruplex DNA by introducing side chains to the molecule. TO derivatives containing various side chains were successfully synthesized and characterized. Biophysical and biochemical studies with duplex and G-quadruplex DNA showed that tethering side chains to TO is an effective approach to tune its ability of binding to duplex or G-quadruplex DNA. Possible binding modes of the effective derivatives were studied using AutoDock. Their inhibition of telomerase activities was studied using the TRAP assay. The cytotoxicity of these derivatives toward three cancer cell lines was also investigated using the MTS assay. The second part of this work focuses on development of TO-based G-quadruplex DNA binding ligands that can bind to DNA via the dual recognition mode. TO was tethered with pyrene, naphthalene diimide, and anthraquinone respectively to yield three novel conjugates. Further investigation suggested that the conjugate of TO with naphthalene diimide (TO-NF) gave the best G-quadruplex binding affinity. It binds to G-quadruplex DNA via the end stack mode and strongly inhibits the telomerase activity. The cytotoxicity results will also be discussed in this presentation.

Dioxins such as TCDD are environment pollutants whose toxic effects are mediated via aryl hydrocarbon receptor (AhR) signaling pathway. AhR is a ligand sensitive transcription factor. The unbound AhR resides in cytoplasm as a complex containing p23, Hsp90 and XAP2. Upon ligand binding, AhR undergoes conformational change and translocates into the nucleus. Once the AhR dimerizes with AhR nuclear translocator (Arnt), the chaperone proteins in the complex get dissociated followed by the activation of transcription of various genes such as CYP1A1 and CYP1A2 by AhR-ARNT heterodimer. Various cancers have altered levels of AhR in the absence of ligand. Our current knowledge is only limited in the regulation of AhR protein levels in its ligand bound state. However, the mechanism involved in the regulation of AhR protein levels in the absence of ligand is still unknown. To make the study of AhR signaling pathway possible, our lab has been working on the expression of various AhR constructs in E.coli using recombinant DNA technology. As AhR forms inclusion bodies due to its poor solubility in the cytoplasm of the host bacteria, it is tagged as a “difficult to express” protein. Therefore, it is challenging to generate functional recombinant AhR protein. My thesis documents the expression of human AhR construct amino acid 108-400 using two different solubility enhancing tags (thioredoxin and maltose binding protein). Western blot data revealed that the soluble expression of the human AhR construct by thioredoxin solubility enhancing tag has outperformed the other.