Organic cation transporters (OCTs) play a pivotal role in the absorption, tissue distribution, and excretion of a diverse array of substances, and currently the nature of the biochemical interactions between substrate and OCTs are unknown. Therefore, identifying which amino acid residues are critical for OCT-substrate interactions is of central importance to understanding and predicting interactions between drugs and OCTs. A three-dimensional (3-D) homology model of human OCT3 was generated using the crystal structure of a high affinity phosphate transporter from Piriformospora indica (PiPT) as template, and putative binding pocket for the prototypical hOCT3 ligand 1-methyl-4-phenylpyridinium (MPP+) was identified through docking studies. Five residues, Phe36, Val40, Trp358, Glu451 and Asp478, were identified as potentially mediating hOCT3-MPP+ interactions, and confirmed through in vitro studies. Additionally, 3-D homology modeling of the functional hOCT3 mutant Val40Leu, and all non-functional hOCT3 mutants, indicated changes in binding pocket architecture consistent with weakening of ligand-transporter interactions. Docking of structurally divergent hOCT3 substrates indicated binding interactions in the same general region as that identified for MPP+, albeit with mostly unique residues. Interspecies differences were explored by generating 3-D homology models for rat and murine Oct3. Results from docking studies using compounds exhibiting vastly different binding affinities (Km or IC50) towards the OCT3/Oct3 orthologs were consistent with varying strength in ligand-transporter binding pocket interactions. Finally, a series of novel compounds exhibiting anti-depressant-like activity was screened for OCT interaction in vitro, and demonstrated significant inhibitory effects on OCTs for many of the compounds.

The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome.

Prostate cancer is the most prevalent non-cutaneous cancer among men. Since the 19th century when Virchow first introduced the concept of inflammation in cancer, chemokines and their receptors have garnered a lot of interest. Chemokine receptor CCR5 has been especially implicated in many disease states and recently found to be over expressed in prostate cancer cell lines. Anibamine, a natural CCR5 antagonist discovered in 2004, has been found to have significant anti-prostate cancer activity at micromolar level. To optimize this compound and also discover a novel pharmacophore, exploration of the original structure was carried out. Significant modifications were made to the side chain in the original structure and ten different analogues were prepared by altering the original synthetic route. While cytotoxicity assay proved the compounds to be non toxic to normal cells, anti-proliferation assay displayed that having a bulky, hydrophobic group in the side chain of the parent compound is essential for the activity. Looking at this data, the third generation of analogues can be prepared that might generate a better lead compound for the treatment of prostate cancer.

As ferramentas computacionais de modelagem molecular e de QSAR estão integradas ao processo de planejamento de fármacos na busca inesgotável por novas moléculas bioativas de elevado interesse terapêutico. O trabalho em Química Medicinal realizado nesta dissertação de mestrado envolveu o estudo das relações entre a estrutura e atividade de inibidores da enzima purina nucleosídeo fosforilase (PNP) bovina e de Schistosoma mansoni. A potência de uma série de inibidores da PNP de S. mansoni foi determinada experimentalmente através da medida de valores de 1C50, empregando um ensaio cinético padronizado e validado. Conjuntos de dados padrões para inibidores da enzima PNP bovina e de S. mansoni foram organizados, contendo os dados de estrutura e atividade correspondentes. Estes conjuntos formaram a base científica para o desenvolvimento dos modelos preditivos de QSAR 2D, empregando o método holograma QSAR. Os modelos finais de HQSAR desenvolvidos possuem alta consistência interna e externa, apresentando bom poder preditivo. Estes modelos, em conjunto com as informações obtidas dos mapas de contribuição de HQSAR, são guias químico-medicinais importantes no planejamento de inibidores mais potentes e seletivos, candidatos a protótipos de novos fármacos na quimioterapia segura das doenças alvo deste trabalho / Computational tools for molecular modeling and QSAR are well-integrated into the drug design process in the search for new bioactive molecules of significant therapeutic interest. The Medicinal Chemistry work done in this dissertation involved structure-activity studies of inhibitors of bovine and Shistosoma mansoni purine nucleoside phosphorylase (PNP). The potency of a series of S. mansoni inhibitors was experimentally determined through measurements of IC50 values, employing a standard validated kinetic assay. Data sets for bovine and S. mansoni PNP were organized, encompassing the structural information and corresponding biological data. These data sets established the scientific basis for the development of the predictive QSAR models using the hologram QSAR method. The final HQSAR models generated possess both good internal and external consistency with good correlative and predictive power. These models and the information obtained from the HQSAR contribution maps should be useful in guiding future medicinal chemistry efforts designed to discover novel potent and selective inhibitors as drug candidates for the chemotherapy of the target diseases of this work

As the profession of pharmacy has transitioned from a chemistry-centered profession to a patient-centered profession, the role of medicinal chemistry in the curriculum has evolved. There is decreased emphasis on memorization of chemical structures, and priority placed on relating these structures to ADME, physical properties, and pharmacodynamics. Simultaneously, the delivery of this content has shifted from traditional lecture format to other styles. Here we discuss some new approaches to teaching medicinal chemistry.

Familial hypercholesterolemias (FH) are inherited mutations that cause elevated total cholesterol and low-density lipoprotein cholesterol levels (LDL-C) which lead to premature coronary heart diseases. Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect the patient’s response to the drug. Single Nucleotide Morphism (SNP) mutations in the LDLR, apoB, LDRAP1, and PCSK9 genes are linked to familial hypercholesterolemia. The mutations in the LDLR gene are the most common while mutations in the apoB and PCSK9 genes are the least common in hypercholesterolemia patients. My research will study how pharmacogenetics can be used to diagnose and prescribe patients with FH who have apoB and PCSK9 double gene mutations. I will genotype and sequence the PCR amplified gene segments of the patients with FH to identify any of the 6 apoB SNPs and any of the 3 PCSK9 SNPs that are known. Then, I will provide 4 different treatments: placebo, antisense therapy (mipomersen), PCSK9 inhibitor (alirocumab), and a combination of mipomersen + alirocumab, and I will measure the LDL-C levels before and after a 12-week trial. I hypothesize that individuals with both apoB and PCSK9 gene mutations with the known SNPs that cause loss of function will be more responsive when given both treatments by observing a significant decrease in LDL-C levels.

Respiratory Syncytial Virus (RSV) is a potentially life-threatening respiratory pathogen that infects approximately 64 million children and immunocompromised adults globally per year. Currently, there is a need for prophylactic and therapeutic approaches effective against primary and secondary RSV infections. This project focuses on the development of a simple, smart, and scalable anti-RSV nanotherapeutic that combines novel cellular antiviral defense mechanisms targeting the inhibition of viral fusion and replication. An ICAM-1 targeted liposomal nanocarrier will be synthesized and coated with a layer of chitosan containing the anti-fusion HR2-D peptide as an extracellular defense mechanism. Additionally, chitosan complexed to dual expressing short hairpin RNA (shRNA) recombinant plasmids will be encapsulated within the nanocarrier, and provide an intracellular defense mechanism that will interfere with the expression of the NS1 and P proteins. In combination, both defense mechanisms are expected to induce a synergistic anti-RSV effect that will surpass those of conventional therapeutics. Through this research, the NS1 and P containing plasmid (pSH-NS1-P) was cloned, and the nanotherapeutic was successfully synthesized. Based on the acquired results, pSH-NS1-P was shown to express anti-RSV effects, and it was also concluded that both inserts were producing active shRNA. Additionally, the anti-RSV efficiency of HR2-D was confirmed. Overall, this research will lead to development of a dual-mechanistic anti-viral nanotherapeutic.

Solid-phase syntheses of two 14-membered ring peptidomimetics were done to determine whether or not a beta-turn structure can facilitate macrocyclization. NMR methods, together with CD and QMD calculations, do not fully support this assumption. However, cyclizations of more ordered structures like those of compounds 2 were more efficient than those for highly strained ring systems like 1. A small library of 18-membered ring peptidomimetics that accommodate an extra amino acid residue was synthesized on resin. Their syntheses were not complicated by head-to-tail dimer impurity, unlike those for previously synthesized 14-membered systems. These larger macrocycles exhibit beta-turn structures as verified by NMR, CD and QMD techniques. Moreover, two compounds in this series (3a and 3g) were shown to have agonistic properties for TrkC in cell survival assays. Dimerization of monovalent mimics was achieved first by modifying the organic template so that monovalent mimics with requisite functional groups can be synthesized. Second, the monovalent units were dimerized using sequential nucleophilic substitutions on fluorescently labeled dichlorotriazine. Our rationale to make bivalent compounds out of monovalent ones was justified when compound 4 was shown to bind TrkA with a 20 nM affinity. Reactions of amino acids with NH4SCN under acylating conditions produced 2-thiohydantoins in which the nitrogen of the amino acid (N1) was acylated. This was proven by 2-D NMR which showed no cross-peak between the NH signal observed and the Cα±-H of the amino acid. When the compound was deacylated, a new NH signal appeared and the corresponding cross-peak with the Cα±-H was observed. Solution-phase syntheses of non-peptidic mimics were achieved by doing a double substitution on a dihalogenated nitrobenzene scaffold. Sonogashira and SNAr reactions were done to install the required side-chains to give the desired compounds. These non-peptidic compounds can be easily adapted to our DTAF-Inp dimerization protocol since the nitro groups can be easily reduced. Attempts to make a spirotetracyclic peptidomimetic with three side chain mimics were done by synthesizing the spirocyclic diketopiperazine precursor. The synthesis of the DKP was achieved by making the cyclic quaternary amino acid that was coupled to another amino acid via the HOAt-EDC method. This protocol gave dipeptides in high yields. These dipeptides were deprotected and cyclized to the DKP under mildly acidic conditions in toluene.

Cancer is responsible for one fourth of the total deaths and is the second leading cause of death, behind heart disease, in the United States. However, there are as many approaches to curing cancer as there are types of cancer. One important issue in solid tumors is hypoxia, a lack of oxygen, which promotes angiogenesis and anaerobic metabolism, which can increase cancer progression and metastasis. The HIF transcription factor is responsible for the mediation of many processes involved during hypoxia and is linked to poor patient prognosis, increased cancer progression, and invasiveness of tumors. With this in mind, the HIF pathway has become an attractive target for small molecule inhibition. Herein, we describe the design and synthesis of small molecules that inhibit the HIF pathway. These compounds are based off an initial “hit” compound, KCN-1, from screening of a 10,000 compound library. KCN1 is both highly effective and has a low toxicity profile. Over 200 compounds have been synthesized by the Wang lab, with the best compound IVSR64b having an IC50 of 0.28 μM. Of special interest is that these compounds do not appear to have any inherent toxicity toward healthy tissues, but only affect cancer cells. Moreover, x-ray crystal structures for both KCN-1 and IVSR64b were obtained and used as the basis for computational modeling, which is still in progress.

Alzheimer’s disease (AD) is a highly complex and rapidly progressive neurodegenerative disorder characterized by the systemic collapse of cognitive function and formation of dense amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). AD pathology is derived from the cholinergic, amyloid and tau hypotheses, respectively. Current pharmacotherapy with known anti-cholinesterases, such as Aricept ® and Exelon ®, only offer symptomatic relief without any disease-modifying effects (DMEs). It is now clear that in order to prevent the rapid progression of AD, new therapeutic treatments should target multiple AD pathways as opposed to the traditional “one drug, one target” approach. This research project employed medicinal chemistry tools to develop multifunctional small organic molecules against three key targets of AD pathology – the cholinesterases (AChE and BuChE), AChE-induced and self-induced Aβ1-40 aggregation and generation (β-secretase). A chemical library composed of 112 derivatives was generated to gather structure-activity relationship (SAR) data. The derivatives were based on a novel, non-fused, 2,4-disubstituted pyrimidine ring (2,4-DPR) template with substituents at the C-2 and C-4 position varying in size, steric and electronic properties. Molecular modeling was utilized to investigate their binding modes within the target enzymes and along with the acquired SAR, the chemical library was screened to identify lead multifunctional candidates.