Doctor of Philosophy, The Ohio State University, 2019, Pharmaceutical Sciences

The use of data-driven methods and machine learning has become increasingly pervasive in many industries, including drug discovery and design, as computing power and large amounts of data become increasingly available. In an effort to efficiently leverage this data, cheminformatics has emerged as a data-driven, interdisciplinary field that focuses on storing, accessing, and applying chemical information. Cheminformatics methods and tools facilitate the management and analysis of large annotated chemical datasets that would be difficult or impossible to do manually. A famous application of leveraging large amounts of chemical data was performed by Christopher A. Lipinski in 1997. Lipinski analyzed a large set of bioavailable synthetic drug molecules and identified trends in their molecular properties, which has since been referred to as the “Lipinski's Rule of 5”. While these rules are far from absolute, Lipinski's analysis demonstrates the utility of leveraging large amounts of chemical data to gain important insights. This thesis describes the application of cheminformatics methods to tackle two very different research problems: 1) the analysis and binding of a class of protein degraders called proteolysis targeting chimeras (PROTACs) and 2) the development of a target fishing application for the prediction of mechanism of action of natural products. PROTACs are a novel class of small molecule therapeutics that are garnering significant interest. Unlike traditional small molecule therapeutics, PROTACs simultaneously bind to both their protein target and an E3 ligase to induce degradation. The requirement to simultaneously bind two proteins necessitates a high molecular weight as PROTACs must contain two unique binding moieties that are connected by a linker. As a result, PROTAC molecules are expected to lie outside of the traditional drug-like chemical space described by Lipinski. To gain a better understanding of the physicochemical properties of PROTACs curre (open full item for complete abstract)

Committee: James Fuchs (Advisor); Xioalin Cheng (Advisor); Karl Werbovetz (Committee Member); Lara Sucheston-Campbell (Committee Member) Subjects: Chemistry; Computer Science; Molecular Chemistry; Molecules; Pharmacy Sciences

MS, University of Cincinnati, 2008, Engineering : Computer Science

Noroviruses have been recognized as the most important cause of non-bacterial epidemic acute gastroenteritis, affecting individuals of all ages. With the identification of trisaccharides' binding site(s) for norovirus (both VA387 and Norwalk virus strains), the door has turned open to find biologically active chemicals with better or equal binding affinity compared to trisaccharides. In this thesis as a first objective, trisaccharides binding site(s) on noroviruses were identified via computational docking and validated with experiments results. In addition to experimentally identified and computationally predicted binding site, a second stable binding site for Norwalk virus was also computationally predicted. Completion of the first goal paved the way for the second and most important aim of the research, which was to computationally identify lead candidates from a library of two million drug-like compounds that bind to the viral receptor pocket identified earlier, thus inhibiting the binding of host histo-blood group antigens (HBGA). Delivering on the second objective, a selection of 255 potential drug-like compounds were obtained successfully. Finally, an approach to score and cluster chemicals based on binding energy and binding similarity (specificity) to trisaccharides was developed and successfully implemented.

Committee: Yizong Cheng PhD (Committee Chair); Jarek Meller PhD (Committee Chair); Ali Minai PhD (Committee Chair) Subjects: Bioinformatics; Biomedical Research; Computer Science; Molecules; Virology

Doctor of Philosophy, The Ohio State University, 2012, Pharmacy

Discovery of novel drug candidates for a particular disease condition has traditionally been carried out by experimentally screening thousands of compounds for desired activity in anticipation of a few being successful. However, this approach is expensive because a large number of compounds need to be synthesized or purchased and tested for biological activity. The success rates of such blind screens are very poor. Recently, rational drug design methods have gained popularity due to progress in structural genomics, chemical biology, and computational chemistry. In particular, in silico methods have improved the screening efficiency in the past five years due to advancement in multi-core, multi-threaded computational hardware and software development. By utilizing rational approaches, we have developed and applied protocols capable of rapidly and inexpensively screen hundreds of thousands of compounds virtually to disease targets. A novel kinase, maternal embroyonic leucine zipper kinase (MELK), has been reported to be involved in tumor progression. We developed an ensemble virtual screening method utilizing multiple induced fit models of MELK to screen 500,000 compounds. We tested 23 compounds and identified one potent (15; Kd =0.37 µM), one moderate (9; Kd=3.2 µM), and several weak MELK inhibitors. Human protein arginine methyl transferase 5 (PRMT5) has been shown to methylate arginine residues of histone protein, subsequently silencing tumor suppressor genes. To target this aberrant epigenetic pathway, a comparative model of the PRMT5 was constructed. We computationally screened 10,000 compounds and eight small molecules were selected for biological assay. Enzyme inhibition assays show that two compounds were capable of selectively inhibiting PRMT5 activity. The compounds were also proven to inhibit cellular proliferation in several cancer cell lines. Leishmaniasis is a parasitic disease which affects millions of people all over the world. The Leishmania UDP-gluco (open full item for complete abstract)

Committee: Chenglong Li PhD (Advisor); Robert Baiocchi MD, PhD (Committee Member); Karl Werbovetz PhD (Committee Member) Subjects: Pharmaceuticals; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2024, Pharmaceutical Sciences

Cancers and leishmaniasis are distinct diseases, but the effects of each on people and communities are similarly devastating. Cancers cause over 10 million deaths worldwide each year, and are so widespread that nearly every person has lost a loved one to them, myself included. Leishmaniasis primarily affects tropical countries and in many places where access to medical care is limited, and the visceral form of the disease requires medical treatment to increase chances of survival above 5%. Both cancers and visceral leishmaniasis are diseases that the human immune system alone often cannot overcome, so the continued research into treatments is crucial to develop new and better tools to fight against these diseases. This dissertation details drug discovery efforts for two different projects, one against each disease; chapter 1 introduces readers to each disease state, chapter 2 describes the synthesis and biological evaluation of anticancer compounds, and chapter 3 describes the synthesis and biological evaluation of antileishmanial compounds. Following the serendipitous discovery of an antileukemia hit compound with an arylimidamide-azole scaffold, a series of analogs was synthesized to evaluate modifications to the scaffold. A robust structure-activity relationship (SAR) was developed through the synthesis of these compounds, and analysis of this relationship pointed to specific chemical modifications to the scaffold which improved their anticancer potency. Combining these favorable modifications led to compounds with >4-fold improved potency compared to the parent compound. Among the most potent compounds in this iv series was 2.9k, which displayed an IC50 value of 100 nM against the acute myeloid leukemia (AML) cell line OCI-AML3. Promising compounds in this series were then further evaluated for broad anticancer activity, pharmacokinetic properties, and mechanism of action as described in chapter 2. The antileishmanial compounds described in this dissertation (open full item for complete abstract)

Committee: Karl Werbovetz (Advisor); Xiaolin Cheng (Committee Member); James Fuchs (Committee Member) Subjects: Biology; Chemistry; Organic Chemistry; Pharmaceuticals; Pharmacology; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2024, Chemistry

Molecular docking and molecular dynamics simulations are two commonly used computational techniques for the in silico evaluation of receptor-ligand binding and characterization of biomacromolecules. This work presents two independent projects to elucidate the molecular interactions in different prospective protein targets and provide physical insights into structure-based drug design. The focus of the first part is the “interacts-with-Spt6” protein (IWS1), a eukaryotic transcription elongation factor present in the RNAP II polymerase (RNAP II) complexes. IWS1 is gaining increasing attention as recent works revealed its collaborative actions with many formerly identified transcription factors of known regulatory effects and its direct interaction with the catalytic alpha subunit of RNAP II (RPB1). However, having about 70% of residues in human IWS1 being intrinsically disordered, its structure and function are not fully understood. Motivated by the recently discovered connection between IWS1 and liposarcoma (LPS), a cancer that can grow and spread aggressively, we initiated the effort of finding drug molecules targeting IWS1 to suppress transcriptional activities in cancer cells. To begin with, we analyzed the supermolecular arrangement, the protein interactions within the structured core, and the potential interactions with chromatin in the intrinsically disordered region of hIWS1 based on various recent studies of the structures and biological functions of IWS1. We then identified a potential binding pocket on IWS1's interface with Spt6 for a drug molecule to act as a competitive inhibitor that downregulates the transcriptional activity of RNAP II by diminishing the association of IWS1/Spt6. Next, we designed and performed a series of molecular docking calculations. Among the top-ranked compounds from the virtual screening of FDA-approved drugs, Ketotifen and Desloratadine were selected for experimental validation and were shown to reduce the association of IWS (open full item for complete abstract)

Committee: Christopher Hadad (Advisor); Rafael Bruschweiler (Committee Member); Alexander Sokolov (Committee Member) Subjects: Chemistry

Bachelor of Science (BS), Ohio University, 2022, Neuroscience

Lithium, a therapeutic first introduced in 1952, is still considered the gold standard for treatment of bipolar disorder. Although many new medications have been discovered over the years, lithium is the only therapeutic used exclusively for bipolar disorder-- indicating a separate mechanism of action from antipsychotic and antidepressive treatments (Kaidanovich-Beilin et al., 2011). Genetic and pharmacological studies have shown that lithium's therapeutic efficacy is at least partially due to inhibition of glycogen synthase kinase-3 beta, or GSK3b (Kaidanovich-Beilin et al., 2011). Development of small molecule therapeutics to target GSK3b with better drug like qualities such as higher affinity and selectivity can lead to advancements in our understanding and treatment of bipolar disorder. This study investigates the structure activity relationship of small molecule GSK3b inhibitors through ab initio calculations, protein kinase sequence alignment, and computational docking studies. We found that although the ATP binding pocket is highly conserved, a proline residue unique to GSK3 may contribute to selectivity of small molecule inhibitors. In addition, small molecule inhibitors may show increased selectivity for GSK3b through preferential SH-π interactions with cysteine residue 199. Finally, docking studies suggest that GSK3b may be too dynamic or ligand-specific for predictive docking. Overall, this work has improved understanding of how small molecule inhibitors may utilize the GSK3b ATP binding pocket and their potential to treat bipolar disorder.

Committee: Jennifer Hines (Advisor) Subjects: Neurosciences

Master of Science (MS), Ohio University, 2022, Chemistry and Biochemistry (Arts and Sciences)

The T-Box Riboswitch is an antibiotic target affecting Gram positive bacteria. The riboswitch senses and regulates many pathways regarding protein synthesis. Wanting to build peptides to inhibit the T-box riboswitch, we first created a library of 260,002 peptides. The peptides were docked to a conserved sequence in the T-box riboswitch and the top 11 peptides were chosen. Chosen peptides were chemically synthesized using the Fmoc/ T-Butyl synthesis and were characterized by MALDI TOF/TOF and RP-HPLC. Initial in-vitro studies show that the peptides will bind to the RNA, though they have not shown to be selective.

Committee: Jennifer Hines (Advisor); Justin Holub (Committee Member); Benjamin Bythell (Committee Member) Subjects: Biochemistry

Master of Science in Pharmaceutical Science (MSP), University of Toledo, 2021, Pharmaceutical Sciences (Industrial Pharmacy)

Patient-specific medicine is a growing area of treatment in the healthcare sector and additive manufacturing, or 3D printing technology is a recent pharmaceutical approach to confront the challenge of this individualized drug delivery system. The focus of this study was to investigate the feasibility of formulating a 3D printed personalized dosage form using fused deposition modelling (FDM) in combination with hot-melt extrusion (HME) process. Acetaminophen was selected as a model drug and a commercial polyvinyl alcohol (PVA) filament was used to fabricate 3D printed tablets with two different drug loading percentages. After screening several polyvinyl alcohols (PVA), the commercial PVA filament was selected to enhance the extrusion process. 5% and 15% acetaminophen loaded filaments were successfully extruded through a filament extruder and tablets were printed using an FDM 3D printer. Thermal analysis using DSC and TGA confirmed the thermal stability of 3D printed tablets. No endothermic events corresponding to acetaminophen were observed in the DSC thermograms of drug-loaded filaments and tablets indicating that the drug was amorphously dispersed in PVA. With TGA, the drug-loaded filaments and tablets did not show any appreciable weight loss at the printing temperature of 240 ˚C suggesting that the polymer was stabilizing the drug. Molecular interactions of acetaminophen and PVA on drug-loaded tablets were verified through FTIR analysis. SEM micrographs of cross-sectioned drug-loaded filaments appeared to have a rough surface in compare to the commercial PVA filament due to the inclusion of acetaminophen, which was consistent with the drug-loaded tablets as well. Physical and mechanical characterization was performed according to mandated standards. The 3D printed tablets passed the weight variation, friability, thickness, dimensions, and breaking force tests with minimal outliers. Drug content loss was analyzed using a validated HPLC method. HPLC data demonstrat (open full item for complete abstract)

Committee: Jerry Nesamony (Committee Chair); Joseph Lawrence (Committee Member); Gabriella Baki (Committee Member) Subjects: Pharmaceuticals; Pharmacy Sciences

Master of Science in Pharmaceutical Science (MSP), University of Toledo, 2021, Pharmaceutical Sciences (Industrial Pharmacy)

Self-micro-emulsifying drug delivery systems (SMEDDS) have been proven to have improved drug stability, lower toxicity, and increase bioavailability of insoluble drugs. It is a drug delivery design that can prevent physical and chemical drug degradation. The goal of this study was to develop a solid formulation incorporating a self-micro-emulsifying drug delivery system (SMEDDS) for the oral delivery of Monodora myristica essential oil (MMEO). MMEO was extracted from the blended seeds of Monodora myristica using the hydro-distillation method. MMEO was characterized by evaluating the physicochemical properties to ascertain the quality and purity of the essential oil by comparing with MMEO data in the literature. The design of the experiment was done by using Fusion Pro by S-Matrix (Fusion Pro Software Version 9.9.0 Build690, S-Matrix Corporation (www.smatrix.com)) to compare a combination of MMEO/Tween 80/Transcutol HP and MMEO/ Kolliphor/ Labrasol 12 formulations. MMEO (10.92%) / Tween 80 (48%) /Transcutol HP (41.8%) was predicted to be the best formulation with desirable characteristics such as a mean particle size of 112.7 nm, the zeta potential of +5.10 mv, and a transparent emulsion. The emulsion formed was stable over 90 days without any form of emulsion instability or oil precipitation. The liquid-SMEDDS was adsorbed unto Neusilin US2 to form solid-SMEDDS. The solid-SMEDDS was added to cellulose, lactose, starch, talc, magnesium stearate to directly compress type 1 and type 2 tablets while the solid-SMEDDS was directly compressed to formulate type 3 tablets. Type 3 tablets had the highest drug loading capacity unlike type 1 and type 2 tablets. Also, type 3 had the highest breaking force and longest disintegration time. Using one-way ANOVA, the P-value obtained was below 0.05 for tablet thickness, tablet breaking force, and disintegration tests. Therefore, there was a statistically significant difference between type 1, type 2, and type 3 tablets properties su (open full item for complete abstract)

Committee: Jerry Nesamony Dr (Committee Chair); Gabriella Baki Dr (Committee Member); Liyanaaratchige Tillekeratne Dr (Committee Member) Subjects: Health Sciences; Intellectual Property

Doctor of Philosophy, University of Akron, 2019, Chemical Engineering

In the study of this Ph.D. dissertation, two research topics related to zwitterionic materials have been investigated. Even though the applications have different objectives, the unique properties that existed in zwitterionic materials have, including charge property and antifouling property, have been used in both of research. The first topic is the development of zwitterionic-peptides gene delivery system. The gene delivery system, with high efficacy, low toxicity, long blood circulation time and targeting the specific cancer cell, is investigated. The second topic is the functionalization of protein therapeutics with zwitterionic polymers. The protein therapeutics with better solubility, stability, and activity is developed. The non-viral gene delivery system is under research due to their low toxicity, low immunogenic and large DNA loading size in gene therapy. Peptides gene delivery system is reported with the cationic charge and buffering effect which overcomes the barrier and delivery DNA into the nucleus. In our group, the economic dextran-peptide hybrid gene delivery system was developed with high transfection efficiency and low toxicity. The first topic of my research was expanded as a continuous work under the same research interest. The effect of the design of peptides length, zwitterionic group and targeting group was studied for the optimization objects on achieving low toxicity, transfection efficiency and blood circulation time, which was summarized into three research projects under this topic. The system was adjusted by the peptides length for toxicity and economic purpose. The system was functionalized with the zwitterionic group for improved stability, enhanced endosomal escape and longer blood circulation time. The system was also conjugating with targeting ligand for targeting gene delivery. It was found that the shorter length of peptides will not provide enough charge to form stable micelle with report DNA. The zwitterionic functionalized (open full item for complete abstract)

Committee: Gang Cheng (Advisor); Jie Zheng (Advisor); George Chase (Committee Member); Lingyun Liu (Committee Member); Ge Zhang (Committee Member); Coleen Pugh (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering; Polymers

Doctor of Philosophy, University of Toledo, 2018, Chemistry

Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of ß-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step for the biosynthesis of several essential amino acids in fungi and in bacteria. The absence of this enzyme in humans and other mammals will allow the selective targeting of any ASADH inhibitors against pathogenic microorganisms. We have accumulated significant structural and mechanistic information about the bacterial ASADHs, but have only limited knowledge of their fungal counterparts. To bridge this gap we have determined the high-resolution structures of three pathogenic fungal forms of ASADH, from C. neoformans, A. fumigatus, and from B. dermatitidis in both apo- and ligand-bound forms. While the overall structures of the fungal ASADHs are similar to the bacterial orthologs, some critical differences both in biological assembly and in secondary structural features can potentially be exploited for the development of species-selective drugs with selective toxicity against infectious fungal organisms. As an initial study ~1,000 compounds from our customized fragment libraries were screened against ASADH, followed by determination of the inhibition constant (Ki) values of the most potent hits. Encouragingly, these results showed that most of the hits obtained already have Ki values in the lower micromolar range. Several of these most potent and structurally diverse initial hits were selected for structural-activity relationship (SAR) evaluation, leading to optimized inhibitors against fungal ASADH with inhibition constants in the low micromolar/high nanomolar range, while still retaining high ligand efficiency values. The efficacy of these hits was further validated using (open full item for complete abstract)

Committee: Ronald Viola PhD (Committee Chair); Donald Ronning PhD (Committee Member); Jianglong Zhu PhD (Committee Member); Viranga Tillekeratne PhD (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry

Doctor of Philosophy, University of Toledo, 2018, Chemistry

Trehalose is a non-reducing disaccharide used in Mycobacterium tuberculosis (Mtb) as an intracellular carbon storage and stress protectant. It is produced through three different biosynthetic pathways, namely the OtsAB pathway, the TreXYZ pathway and the GlgE pathway. While the first enzyme of the OtsAB pathway, the trehalose-6-phosphate synthase (TPS/OtsA) revealed to be non-essential for growth, the knock out of its second enzyme, trehalose-6-phosphate phosphatase (TPP/OtsB2), was lethal to the cell due to accumulation of its substrate trehalose-6-phosphate (T6P). Also involved in trehalose biosynthesis, but mostly for its consumption, the GlgE pathway was identified in 2010. Part of the four-enzyme pathway, the maltosyltransferase GlgE is an interesting drug target in that it has no homologues in humans and its inhibition results in the death of the microorganism in 14 days. The mechanism of cell death is similar to that of the TPP knockout. The inhibitory activity of compounds targeting each of these enzymes was characterized and structure/function relationship studies were employed to inform the next generation of inhibitor design. For TPP, three inhibitors were designed mimicking T6P and transition-state analogs: trehalose phosphonamide, trehalose methyl phosphonate and trehalose ethyl phosphonate. While the first inhibitor had an IC50 similar to the Km of T6P, 646.90 ± 57.45 µM, the other two inhibitors exhibited IC50 values that were three-fold worse. To gain more information on the TPP active site, an apo X-ray structure of the M. lentiflavum TPP homolog, sharing 79 % identity, was solved at a resolution of 1.85 A. Analysis of TPP structure revealed the presence of an unusual N-terminal domain hypothesized to be a regulatory domain. This domain being absent from non-mycobacterial TPP, it was hypothesized to form protein-protein interactions with an unidentified protein or membrane-protein interactions, allowing for easier motion of the cap domain. These hyp (open full item for complete abstract)

Committee: Donald Ronning (Committee Chair); John Bellizzi (Committee Member); Steve Sucheck (Committee Member); Mary Jackson (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

Proteins play a major role in virtually every biological process. Thus, proteins are an ideal platform for the next generation of therapeutics. Over the last few decades, technological and scientific advances in protein production and engineering have led to a new wave of protein-based biologics used in clinical settings. In this body of work, we have engineered both a protein-based cancer diagnostic and an immunotherapeutic. Antibody-based biologics are becoming one of the most widely approved drug platforms and owe their success to their versatility in binding targets, high stability, and low toxicity. The anti-TAG-72 cancer-targeting antibody, 3E8, is one such molecule that shows great potential as a diagnostic. We have designed and biophysically characterized a library of 3E8 single chain antibody fragments (scFV) with varying linker composition and length as well as domain orientations. In this library, we have found substantial variation in protein stability, binding affinity, and oligomeric states. Surprisingly, a drastic difference in the oligomeric state of these constructs was seen between conventional IMAC purification and Protein L purification. Therefore, the literature rules for scFV linker design must be updated to include the dependencies on purification method. A single antibody construct with optimal biophysical properties (3E8.G4S) was further characterized and subjected to in vivo pharmacokinetic studies. Due to its multimeric composition, 3E8.G4S showed a longer and more favorable clearance time compared to that of a fast clearing scFV. Xenograft mouse imaging and biodistribution studies revealed successful targeting of a colorectal tumor by 3E8.G4S with little accumulation in normal tissues. To determine the versatility of 3E8-based diagnostics and therapeutic agents, an expansive immunohistochemical analysis of TAG-72 expression was performed in over 1,500 tumors spanning 18 different cancer types. The results of this study showed enh (open full item for complete abstract)

Committee: Thomas Magliery (Advisor); Christopher Jaroniec (Committee Member); Edward Martin Jr. (Committee Member); Richard Swenson (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics; Immunology; Medicine; Molecular Biology

Doctor of Philosophy, University of Toledo, 2017, Chemistry

Canavan disease (CD), a fatal neurological disorder observed in newborns, is caused by an inherited genetic defect. CD is a leukodystrophy, causing disruption in the growth and maintenance of myelin sheath which is responsible for efficient transmission of nerve impulses. Therefore, infants affected with CD do not develop motor skills and speech, and they usually also develop conditions such as hypotonia and macrocephaly. This neurological disorder is caused by multiple mutations in the aspA gene that codes for aspartoacylase, an enzyme responsible for catalyzing the conversion of N-acetyl-L-aspartate (NAA) to L-aspartic acid and acetate (an important chemical in the biosynthesis of myelin sheaths). For several decades researchers primarily focused on the acetate deficiency as the major cause for the brain disorders that are associated with Canavan patients. Recently, a study has shown that a knockout of the Nat8l gene that codes for ANAT, the enzyme responsible for NAA synthesis, seems to overcome these adverse effects through normal myelination. In addition, overexpression of ANAT and the subsequent rise in NAA levels have been linked with different life-threatening cancers. High NAA levels were detected in a large percentage of adenocarcinoma and squamous cell carcinoma cells isolated from patients with late-stage lung cancer, and these elevated levels of NAA and high expression of ANAT was also identified in high-grade ovarian cancer tissue samples. In each case these elevated levels have been correlated with worse overall patient survival in these and in several other forms of cancers. This work is primarily focused on the synthesis of potent inhibitors against ANAT. In addition to the screening amino acids, metabolites, and constrained analog libraries, a library of 105 dioic acid and phthalate analogs were synthesized, out of which a 50 % hit rate of modest enzyme inhibitors was identified. An expansion of this synthetic inhibitor library is in progress b (open full item for complete abstract)

Committee: Viola Ronald (Committee Chair); Andreana Peter (Committee Member); Zhu Jianglong (Committee Member); Viranga Tillekeratne L.M (Committee Member) Subjects: Biochemistry; Chemistry; Organic Chemistry

Doctor of Philosophy, The Ohio State University, 2017, Pharmaceutical Sciences

IL-6 is a pleiotropic cytokine that participates in various cellular processes such as acute-phase response, immune response, and hematopoiesis. It is also involved in cell proliferation, survival, and apoptosis. Excessive IL-6 signaling leads to chronic inflammation and promotes malignancies. Blockade of IL-6 is therefore a potential strategy for developing cancer therapeutics. Most current successful anti-IL-6 signaling drugs are antibodies with very few small molecule drugs reported. More importantly, a structure-based rationale for IL-6/gp130 protein-protein interaction (PPI) inhibitor design was absent. Small molecule inhibitor design targeting PPI interfaces is very challenging. Lack of success against shallow PPI interfaces, as present in IL-6/gp130, suggests that more effective approaches are needed to tackle the problem. Computational modeling techniques are especially valuable in PPI inhibitor design. In this dissertation, varied computational methods were used to facilitate the rational design of small molecule inhibitors against the shallow IL-6/gp130 interface. We started from a natural product, Madindoline A (MDL-A), which was reported as a highly selective IL-6 inhibitor by binding to gp130 extracellular domains albeit with relatively weak binding affinity (288 µM) and limited inhibitory efficacy in cancer cellular assays. Through dynamics simulations and extensive free energy analyses, we identified two hot spots at the IL-6 site III/gp130 D1 domain interface and characterized the MDL-A binding mode on the D1 domain of gp130 (gp130-D1). Based on these findings, we optimized the MDL-A scaffold and designed several generations of inhibitors with three different strategies through structure-based approaches and ensemble molecular dockings. As compared to MDL-A, the latest generation of inhibitors (LLM-4x series) have 15-fold improved affinities, which were determined by surface plasmon resonance (SPR). Furthermore, we correlated our computed energy m (open full item for complete abstract)

Committee: Chenglong Li (Advisor); Werner Tjarks (Committee Chair); Karl Werbovetz (Committee Member) Subjects: Pharmacy Sciences

Doctor of Philosophy, Case Western Reserve University, 0, Chemistry

Ribonucleotide reductase (RR) is an essential enzyme found in all living organisms which catalyzes the rate-limiting step in dNTP synthesis. As expression levels of RR are high during cell replication, RR has long been considered an attractive drug target for a range of proliferative diseases, including cancer. Many drugs targeting RR, such as the standard treatment for pancreatic cancer gemcitabine, are nucleoside analogues which irreversibly inhibit RR and exhibit a wide range of off-target effects that lead to unwanted toxicity in healthy cells. Developing reversible, non-nucleoside inhibitors which target RR more specifically may reduce this unwanted toxicity. In order to identify non nucleoside inhibitors, a moderate throughput screening method was developed targeting a protein-protein interface on the inactive human RR hexamer. Through a combination of in silico screening and biochemical assays, ten unique non-nucleoside compounds were shown to inhibit RR with mid-micromolar enzymatic IC50s. One phthalimide-based compound was found to inhibit RR noncompetitively, in agreement with binding to the targeted protein-protein interface. Through this screening process, an acyl hydrazone inhibitor (NSAAH-E-3A) was identified to bind reversibly to the catalytic site of RR. While NSAAH-E-3A was shown to demonstrate nanomolar cytotoxicity in multiple cancer cell lines, additional studies in healthy bone marrow progenitor cells showed no cytoxicity until micromolar doses were used (data provided by Dr. John Pink of the Case Comprehensive Cancer Center). A library of 25 hydrazone analogues, designed using medicinal chemistry and structure-based drug design, were synthesized for the purpose of structure-activity relationship (SAR) studies. The SAR studies identified key chemical moieties in the hydrazone scaffold based on potency toward hRR in cell-free inhibition assays. These results were then used to design a second-generation library, (open full item for complete abstract)

Committee: Rajesh Viswanathan PhD (Advisor); Chris Dealwis PhD (Advisor) Subjects: Organic Chemistry

Doctor of Philosophy, Case Western Reserve University, 2017, Biochemistry

This thesis comprises the study of two pharmaceutically interesting lyase enzymes which catalyze breaking a bond between a saccharide moiety and another moiety, facilitated by the saccharide hydroxyl group, leading to the formation of an intra-saccharide cyclic product. The first lyase, soluble guanylyl cyclase (sGC), is a mammalian enzyme that can catalyze the conversion of GTP to the second messenger, cGMP. sGC is activated by NO leading to a ~200-fold enhanced cGMP production. The structure of the full length enzyme is unknown, limiting our understanding of the structural mechanisms that underlie activation of sGC. sGC consists of four domains, the HNOX domain, PAS domain, coiled coil domain and the catalytic domain. Previous research from our lab and others have shown that NO binds to the HNOX domain of the ß1 subunit and causes breakage of the heme-H105 bond, causing a downward shift of a-F helix in ß1 subunit. The structures of individual domains have either been solved from bacterial homologs of the domains, or from human sGC. Our initial goal was to obtain structural insights into the structure and mechanism of the full length sGC or multi-domain constructs. However, we were unsuccessful in expressing large amounts of sGC or obtaining diffraction quality crystals of purified sGC or its domains. Nevertheless, to obtain insights into the mechanism of sGC activation, we used the structure of the human sGC catalytic domain to find small molecules that may bind and prevent the reorientation of this domain. A pocket at the heterodimeric interface was targeted for in silico screening. Our hypothesis was that compound binding to this pocket can potentially affect the inter-subunit rotational activation mechanism. We found two inhibitors that inhibited NO-activated sGC at micromolar concentrations. Both compounds also inhibited activated and stimulated sGC activity. Additional biochemical analyses showed that one of the compounds also inhibited isolated heterodi (open full item for complete abstract)

Committee: Focco van den Akker (Advisor); Menachem Shoham (Committee Chair); Michael Harris (Committee Member); Vera Moiseenkova-Bell (Committee Member); Derek Taylor (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, University of Akron, 2015, Chemistry

The interaction of a protein with endogenous ligands is at the heart of developing new chemotherapeutic agents; emulating how a ligand binds within a known target macromolecular active site is essential to drug discovery. In the following work, several aspects of drug design are enlightened, including structural-based drug design, structural determination and binding interface identification, and fragment-based drug discovery (FBDD). Primarily is the proposal of a new anti-viral drug candidate for the Influenza A virus, which is a structural-based drug design project employing a carbon-sulfur atom switch. This allows for a more suitable fit in the molecular space allotted in the binding site provided by the known target, the matrix 2 (AM2) proton channel. Understanding how the adamantanes interact with the binding site was essential in the design of this proposed drug candidate, 2,4,9-trithiaadamante-7-amine. Another aspect of drug design is the structural identification of a target, as seen in the second project; the work investigates epigenetic changes and how they occur via interactions between a known long non-coding RNA (lncRNA), the steroid receptor activator (SRA) RNA and the RNA recognition motif (RRM) 1 of the SMRT/HDAC1 Associated Repressor Protein SHARP with the distant objective of developing a small molecule chemotherapeutic agent. This involves the solution structure determination of SHARP1P82*, with the determination of the binding interface between the domain and the region of SRA RNA stem loop region 7 (STR7). Lastly, an FBDD project is explored using the glutaredoxin protein system as the known target. The ortholog glutaredoxins, human glutaredoxin 1 (hGRX), and bacterial Brucella melitensis (brmGRX) and Pseudomonas aergunisa (paGRX) are essential proteins with anti-oxidative roles. There are several health risks involving these proteins and the misbalance of the redox system, including Brucella ovis (Malta fever), and cystic fibrosis ( (open full item for complete abstract)

Committee: Thomas Leeper (Advisor) Subjects: Chemistry

MS, Kent State University, 2014, College of Arts and Sciences / School of Biomedical Sciences

Piperine has shown that it has wide range of effects, including monoamine oxidase B inhibitory effect which is one the targets in treating Parkinson disease. Piperine which is an alkaloid is the active ingredient of plant of black and white pepper grain, Piper nigrum. Several compounds related to piperine was screening using monoamine oxidase B and monoamine oxidase A assays. Z-factor statistical analysis showed that monoamine oxidase (A and B) assays was greater than (0.8). Most of test compounds were selective toward monoamine oxidase B enzyme, with the most potent compound with IC-50 of 498 micro molar . To obtain insight understanding of binding of test compounds to monoamine oxidase B enzyme, each test compound was docked to the crystal structure of monoamine oxidase B enzyme using Autodock 4.2 and Auto dock tools programs. Bovine serum albumin high throughput screening assay was performed to each test compound to understand their binding pattern to this carrier protein. To estimate blood brain barrier permeability, PAMPA method was used and it was showed that most test compounds be able to pass blood brain barrier. Taking together, the date obtained here may be useful in design selective monoamine oxidase B inhibitory compound without monoamine oxidase A activity that is able pass blood brain barrier and reach an active site.

Committee: Werner Geldenhuys (Advisor); Altaf Darvesh (Committee Member); Moses Oyewumi (Committee Member) Subjects: Biomedical Research; Pharmacology

PhD, University of Cincinnati, 2014, Arts and Sciences: Chemistry

DNA modifying agents are stalwarts of chemotherapeutic cancer treatments, but require vital design improvements to improve selectivity, lower side effects, and continue their widespread use. A key problem for DNA modifying agents is lack of specificity. To address this issue, our lab designs novel molecular scaffolds which are activated by a hallmark of some cancers: increased oxidative stress cause by reactive oxygen species (ROS). Oxidative stress, as measured by levels of ROS, oxidized biomolecules, and enzyme activity, is a hallmark of certain cancer cells. My work focuses on a potential path forward in the design of DNA modifying agents by exploiting the increased ROS into a pro-drug approach. Elevation of ROS has been linked to oncogenesis and has been found in several aggressive cancers, including renal cell carcinoma, melanoma, and leukemia. ROS occurs in four major endogenous forms within the cell: superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radical. ROS occur in cells via two discrete mechanisms; first, as a byproduct of metabolism. For example, mitochondria generate superoxide via complex I and III during oxidative phosphorylation. Secondly, ROS are known to derive from several enzymes. Especially important are NADPH oxidases that regulate the function of several tyrosine kinases involved in cell growth and survival. Amplified ROS results in increased DNA damage and mutation, tumor heterogeneity, the ability to self-replicate, and angiogenesis. In turn, these mutations cause enhanced activation of oncogenes. Consequently, it is no surprise that levels of ROS-induced DNA damage correlates with cancer prognosis. We utilize a design strategy wherein the pro-drug is stable, but upon ROS activation a reactive molecule is formed. This leads to more reactive forms of the molecule being present in cancer cells. Thus reactivity, and not uptake, is controlled to induce cytotoxicity more specifically in cancer cells and lower off-target reacti (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); David Smithrud Ph.D. (Committee Member); Pearl Tsang Ph.D. (Committee Member) Subjects: Biochemistry