Cocaine is a widely abused and addictive drug, resulting in serious medical and social problems in modern society. Currently, there is no FDA-approved medication specific for cocaine abuse treatment. The disastrous medical and social consequences of cocaine abuse have made the development of an anti-cocaine medication a high priority. However, despite decades of efforts, traditional pharmacodynamic approach has failed to yield a truly useful small-molecule drug due to the difficulties inherent in blocking a blocker like cocaine without affecting the normal functions of the transporters or receptors. An alternative approach, i.e. pharmacokinetic approach, is to interfere with the delivery of cocaine to its receptors/transporters and/or accelerate its metabolism in the body. It would be an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites. Two natural enzymes may catalyze hydrolysis of cocaine: human butyrylcholinesterase (BChE) and bacterial cocaine esterase (CocE). However, the wild-type enzymes are not suitable as anti-cocaine therapeutics, due to the low catalytic activity, thermoinstability, or short biological half-life. In this investigation, we performed integrated computational-experimental studies to rationally design and discover mutants of these enzymes in order to improve the catalytic activity, thermostability, and/or biological half-life. To rationally design desirable mutants of the enzymes, we have successfully developed computational models, including those for BChE gating, glycosylated BChE structure, BChE-substrate complex structures, BChE dimer/tetramer structures, CocE monomer/dimer structures, and CocE-substrate complex structures. Development of the computational models enabled us to rationally design new amino-acid mutations that may improve the catalytic activity, thermostability, and/or prolonged biological half-life. The computational design was followed by wet experimental tests, including both in vitro and in vivo experiments, leading to discovery of new enzyme forms with not only a high catalytic efficiency against cocaine, but also an improved thermostability and/or prolonged biological half-life. The identified new mutants of BChE and CocE are expected to be valuable candidates for development of a more efficient enzyme therapy for cocaine abuse. The encouraging outcomes of the present study also suggest that the structure-and-mechanism-based design and integrated computational-experimental approach is promising for rational drug design and discovery.

No description available.

This thesis explores the effectiveness of the Food and Drug Administration’s implementation of Breakthrough Therapy Designation (BTD), focusing on the low number of approval rates and repercussions of BTD for the development of new drugs for patients suffering serious life threatening illnesses. BTD, as an expedited review process, shows potential for improvement in its guidelines for necessary qualifications for BTD. Cutting costs, through a shortening in development time, and raising profits, through first mover status of new to market drugs, BTD is regarded by pharmaceutical executives as a tool to insure not only return on investment but also the rewards that accompanies a profitable blockbuster drug. Lessons learned from activism from 1980’s HIV/AIDS crisis show how advocates and “activist-experts” can rebalance and refocus more attention on the necessary beneficence for patients. A policy stipulation that insures all members: corporate, regulatory, and patient advocate, sit together at the decision making table will insure a more balanced discussion in regards to drug development.

Microneedle (MN) enhanced drug delivery is a safe, effective and efficient enhancement method for delivery of drug molecules across the skin. The “poke (press) and patch” approach employs solid stainless steel MN to permeablize the skin prior to application of a regular drug patch over the treated area. It has been previously shown that MN can be used to deliver naltrexone (NTX) at a rate that provides plasma concentrations in the lower end of the therapeutic range in humans. The drug delivery potential of this technique is, however, limited by the re-sealing of the micropores in a 48-72h timeframe. The goal of the current research was to optimize the formulation for a 7 day MN enhanced delivery system for NTX either by adding a second active pharmacological moiety or by optimizing formulation characteristics alone. Three different formulation strategies were explored: formulation pH optimization with NTX; a codrug approach with NTX and a nonspecific cyclooxygenase inhibitor, diclofenac (DIC); and a topical/transdermal approach with NTX and an enzyme inhibitor of the cholesterol synthesis pathway, fluvastatin (FLU). The results indicated that formulation pH cannot be used to extend micropore lifetime, although formulation optimization leads to enhanced transport and thus drug delivery across MN treated skin. The codrug approach was successful in extending the micropore lifetime and further screening of codrug structures and formulation optimization helped in selection of a codrug candidate suitable for evaluation in animal pharmacokinetic studies. Local treatment with FLU helped to keep the micropores open and enabled delivery of NTX for an extended period. The pores re-sealed on removal of treatment within a 30-45 minute timeframe, indicating that infection/irritation should not be a major issue, as in the case of other topical chemical enhancers. Thus, overall it can be concluded that different formulation strategies can be utilized to extend micropore lifetime and enhance delivery of drug molecules across the skin.

<p>Leonotis leonorus and Mentha longifolia are two herbs commonly used in South Africa, mostly in oral liquid dosage forms. Several disadvantages are associated with these traditional dosage forms which can perhaps be remedied by using an appropriate oral solid dosage form, provided the actual plant material in the latter still resemble, as closely as possible, the traditionally used material and provide products of suitable pharmaceutical quality. The objectives of this study were to prepare and evaluate the pharmaceutical suitability of the freeze-dried aqueous extracts of Leonotis Leonorus and Mentha Longifolia as plant raw material for the capsule dosage of these two therapies and to formulate and manufacture capsules of Leonotis Leonorus and Mentha Longifolia aqueous extract that would contain amounts of the plant materials equivalent to that found in their traditional liquid dosage forms, and have immediate release characteristics and suitability stability.</p>

Numerous studies have demonstrated the impact of organic cation (OCTs; SLC22 family) and anion transporters (OATs; SLC22 family) on the efficacy and safety of clinically important therapeutics. To be specific, OCTs and OATs have been identified as determinants for uptake into and secretion from enterocytes, hepatocytes and renal proximal tubular cells, and are frequent sites of drug-drug interaction (DDI). In addition, OCTs expressed in brain are components of the low-affinity, high capacity clearance pathway (uptake-2) for biogenic monoamine neurotransmitters. As a result, OCTs may represent novel targets for mood disorders. The inhibitory effects of several therapeutic agents, designed drugs and novel compounds were assessed on the function of OCTs/Octs and OATs/Oats. Among these compounds, the anthraquinone rhein showed significant inhibition on hOATs. While the antituberculosis drug ethambutol, the herbal products matrine and oxymatrine, synthetic cathinones, and all quinazoline and guanidine compounds produced significant inhibition on hOCT activity with most IC50 values in the micro- and even nanomolar ranges. Considering the clinically relevant unbound concentrations in biofluids, significant DDI potentials were found for rhein, ethambutol, matrine, oxymatrine and several synthetic cathinones affecting enterocytes, hepatocytes and/or proximal tubules. As hOCT2 and hOCT3 may participate in modulating neurotransmitter homeostasis in the CNS, these findings also suggested that the CNS pharmacological effects of synthetic cathinones, quinazoline and guanidine compounds might be due to their inhibitory effects on OCTs; although their impact may be limited solely to clearance of these compounds. Based upon their in vitro OCT/Oct inhibition profiles, three lead quinazoline and guanidine compounds were chosen for in vivo studies. Potent antidepressant-like effects of one lead hOCT-interacting compound (KEO-099) were re-confirmed in the tail suspension test. While in vivo results of the two newly identified hOCT-interacting lead compounds were somewhat less clear. Finally, homology modeling and docking studies for hOCT3 identified key amino acid residues that might be involved in interaction between hOCT3 and small molecules. Subsequent experiments confirmed a competitive mode of interaction between MPP+ and lead compounds on hOCT3. Thus, preliminary analysis indicates our hOCT3 homology model can be used to support rational drug design and high-throughput screening of novel hOCT substrates/inhibitors.

Cancer constitutes a terrible burden on modern society. In the United States there are an estimated 1,658,370 new cancer diagnoses resulting in 589,430 deaths in 2015 alone.[1] An estimated 41,170 of these cases will be diagnosed right here in Virginia. With new cancer patients comes the expanding demand for new treatments. As we all know, many modern chemotherapeutics cause adverse reactions to patients. This is because the toxic nature of these therapies often affects normal tissue alongside the tumors that are infesting the body. Therefore, researching novel ways to make chemotherapeutics selective for cancer, while leaving healthy tissue unscathed, is of paramount importance. There are a few ways in which we have approached cancer-specific chemotherapeutics. Through the use of light controlled toxicity and drug release and the targeting of tumor phenotypes such as overexpressed proteins and the Warburg effect, we begin to tackle the problem of non-specificity of current chemotherapeutics. Combretastatin A-4 (CA4) is highly potent anticancer drug that acts as an inhibitor of tubulin polymerization.[2, 3] The core of the CA4 structure contains a cis-stilbene, and it is known that the trans isomer is significantly less potent. We prepared an azobenzene analog of CA4 (Azo-CA4) that shows 13-35 fold enhancement in potency upon external irradiation. GI50 values in the light were in the mid nM range. Due to its ability to thermally revert to the less toxic trans form, Azo-CA4 also has the ability to automatically turn its activity off with time. Therefore, this work establishes a novel strategy for switchable potency for cancer treatment. Doxorubicin (Adriamycin) is an anthracycline type of chemotherapeutic that intercalates double-stranded DNA.[4] Although this drug has played a huge role in the treatment of cancer, its usefulness declines in cases of cancer recurrence because of the impact this drug has on the cardiovascular system. Therefore, we prepared this drug as a cell impermeable conjugate that gains penetrability through the use of external radiation.[5] Folate receptor alpha (FRα) is overexpressed in a variety of cancer cells and accepts folic acid as a natural ligand.[6] Therefore, conjugation of drugs to folic acid introduces a promising way to bring these drugs to cancer cells with greater specificity. We took this concept one step further with the introduction of a photo-labile linker, connecting doxorubicin to folic acid, which offers dual-specificity through ligand targeting and light activation. Finally, many cancer cells produce adenosine triphosphate, the energy currency of a cell, through an abnormal upregulation of glycolysis.[7] This pathway results in a larger-than-normal production of lactic acid and lowers the pH of cancer cells through a phenomenon known as the Warburg Effect. We hypothesized that through the use of L-canavanine, an L-arginine analog, we could construct short peptides that would gain cell permeability in a low pH environment. Attaching a cargo to these peptides, such as doxorubicin will ultimately allow for targeting the low pH extracellular environment of cancer cells. Through the use of these strategies we have furthered the fight against cancer. Targeting cancer by taking advantage of its phenotypes or through the use of light is vital in reducing negative side-effects of current chemotherapeutics. The novel technologies offered above bring us a step closer to side-effect free treatment of cancer patients.

A malária é a doença tropical com maior taxa de mortalidade global. O surgimento de resistência às terapias de primeira linha reforça a necessidade do desenvolvimento de novos candidatos a fármacos. O objetivo deste trabalho foi a descoberta de inibidores e otimização da atividade destas moléculas como candidatos a compostos líderes para o desenvolvimento de novos agentes antimaláricos. Nesse sentido, realizamos a triagem da coleção Malaria Box e identificamos 11 moléculas de diferentes classes químicas como candidatos a inibidores da enzima enolase de Plasmodium falciparum (Pfeno). Em seguida, determinamos a potência inibitória contra a enzima alvo (IC50 entre 11 &ndash; >1.000 &mu;M), atividade antiplasmodial in vitro contra a forma eritrocítica do parasito (EC503D7 entre 5,6&ndash;1.600 nM) e citotoxicidade (MCL50 > 19 &mu;M) do subconjunto de 11 compostos do Malaria Box (1&ndash;11). Ensaios de combinação com o antimalárico artesunato e estágio de ação foram conduzidos para avaliar o potencial de associação e qual fase do ciclo eritrocítico seria mais suscetível as moléculas desse subconjunto. Os resultados obtidos indicam que alguns compostos apresentam caráter aditivo (2 e 9) ou antagônico (1, 3&ndash;8, 10 e 11) em relação ao artesunato e ação rápida (1&ndash;3, 5, 7, 9&ndash;11) ou lenta (4, 6 e 8) no desenvolvimento do parasita. Diante desses resultados, um conjunto de critérios estruturais e de atividade biológica foi estabelecido para a seleção de um candidato para estudos de relação estrutura-atividade (SAR). O derivado de hidrazinobenzimidazol (4) foi selecionado como hit inicial e 24 derivados (12&ndash;35) foram planejados, sintetizados e tiveram a atividade de inibição enzimática (IC50 entre 44 &ndash; >200 &mu;M), atividade antiplasmodial contra cepas sensível (EC503D7 entre 0,19&ndash;14 &mu;M) e resistente (EC50K1 entre 0,15&ndash;2,4 &mu;M) e citotoxicidade (MCL50 > 3,7 &mu;M) determinadas no primeiro ciclo de SAR. Os dados obtidos sugerem que a enzima Pfeno não é o alvo principal de ação dos derivados hidrazinobenzimidazol, contudo, a atividade antiplasmodial da série indicou razoável distribuição em termos de potência e variabilidade estrutural que permitiram a construção de um modelo HQSAR (q2= 0,64 e r2 = 0,93) adequado para o planejamento e predição da atividade inibitória de oito novos derivados hidrazinobenzimidazol (36&ndash;43). Os novos derivados foram sintetizados e tiveram sua atividade antiplasmodial (EC503D7 entre 0,10&ndash;2,3&mu;M), citotoxicidade (MCL50 > 3 &mu;M) e seletividade (SI > 5) determinadas. Os dados de validação prospectiva do modelo indicaram boa correlação entre a atividade real e predita para os novos derivados. Além disso, neste segundo ciclo de SAR foi descoberto o composto 41 como o mais potente (EC50 = 0,1 &mu;M) e seletivo (SI > 2.000) da série investigada neste trabalho. Nossos resultados indicam que os derivados hidrazinobenzimidazol são moléculas atrativas para a descoberta de compostos líderes para o desenvolvimento de candidatos a fármacos antimaláricos. / Malaria is the tropical disease with the highest overall mortality rate. The emergence of resistance to first-line therapies reinforces the need for the development of new drug candidates. The main goal of this work was the discovery and optimization of these molecules as lead candidates for the development of new antimalarial agents. In this sense, we screened Malaria Box collection and identified 11 molecules from different chemical classes as inhibitor candidates of Plasmodium falciparum enolase enzyme (Pfeno). Then, we determined the inhibitory potency against the target enzyme (IC50 between 11 &ndash; >1.000 &mu;M), in vitro antiplasmodial activity against the erythrocytic form of the parasite (EC503D7 between 5,6&ndash;1.600 nM) and cytotoxicity (MCL50 > 19 &mu;M) of the 11 compounds subset from Malaria Box (1&ndash;11). Combination with artesunate and stage of action assays were conducted to evaluate the potential of association and which erythrocytic stage would be more susceptible to the molecules of this subset. The results obtained indicate that some compounds showed additive (2 and 9) or antagonistic (1, 3&ndash;8, 10 and 11) effect with artesunate and fast (1&ndash;3, 5, 7, 9&ndash;11) or slow (4, 6 and 8) acting on parasite development. In view of these, a set of structural and biological activity criteria was established for the selection of a candidate for structure-activity relationship (SAR) studies. The hydrazinobenzimidazole derivative (4) was selected as hit and 24 derivatives (12&ndash;35) were designed, synthesized and had the enzyme inhibitory activity (IC50 between 44 &ndash; >200 &mu;M), antiplasmodial activity against sensitive strains (EC503D7 between 0,19&ndash;14 &mu;M) and resistant (EC50K1 between 0,15&ndash;2,4 &mu;M) and cytotoxicity (MCL50 > 3,7 &mu;M) determined in the first round of SAR. The collected data suggest that Pfeno is not the main target of the hydrazinobenzimidazole derivatives. However, the antiplasmodial activity of the series indicated a reasonable distribution in terms of potency and structural variability which allowed us the development of a HQSAR model (q2 = 0,64 and r2 = 0,93) suitable for the design and prediction of the inhibitory activity of eight new hydrazinobenzimidazole derivatives (36-43). The new derivatives were synthesized and had the antiplasmodial activity (EC503D7 between 0,10&ndash;2,3&mu;M), cytotoxicity (MCL50 > 3 &mu;M) and selectivity (SI > 5) evaluated. The prospective validation of the HQSAR model indicated good correlation between the actual and predicted activity for the new derivatives. Moreover, in the second round of SAR, compound 41 was discovered as the most potent (EC50 = 0,1 &mu;M) and selective (SI > 2,000) in these series. Our results indicate that the hydrazinobenzimidazole derivatives are attractive molecules for the discovery of new lead compounds for the development of antimalarial drug candidates.

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

Staphyloccocus aureus is an opportunisitic pathogen that causes soft skin and tissue infections (SSTI) such as endocarditis, osteomyelitis and meningitis. In recent years, the re-emergence of antibiotic-resistant S. aureus such as MRSA presents a formidable challenge for infection management worldwide. Amidst this global epidemic of antimicrobial resistance, several research efforts have turned their focus towards exploiting the cell-wall biosynthesis pathway for novel anti-bacterial targets. Recently, the lipoteichoic acid (LTA) biosynthesis pathway has emerged as a potential anti-bacterial target. LTA is an anionic polymer found on the cell envelope of Gram-positive bacteria. It comprises of repeating units of glycerol-phosphate (GroP) and is important for bacterial cell physiology and virulence. For example, it is critically involved in regulating ion homeostasis, cell division, host colonization and immune system invasion. Several reports showed that bacteria lacking LTA are unable to grow. At the same time, they suffer from severe cell division defects and also exhibit aberrant cell morphologies. The key protein involved in the LTA biosynthesis pathway is the Lipoteichoic acid synthase (LtaS). LtaS is located on the cell membrane of Gram-positive bacteria and can be divided into two parts: a transmembrane domain and an extra-cellular domain responsible for its enzymatic activity (annotated eLtaS). Given that LtaS is important for bacterial survival and there are no known eLtaS homologues in eukaryotic cells, this protein is an attractive antibacterial target. In 2013, a small molecule eLtaS inhibitor (termed 1771) was discovered. Although 1771 was able to deplete LTA production, the binding mechanism of 1771 to eLtaS remains unknown. Additionally, 1771 could only prolong the survival of infected mice temporarily because of its in vivo instability. Therefore, the need for finding more potent and metabolically stable inhibitors of eLtaS still remains. Computational-aided drug design (CADD) is a cost-effective and useful approach that has been widely integrated into the drug discovery process. The protein eLtaS lends itself to be a good target for CADD since its crystal structure and a known inhibitor (with limited structure-activity data) is available. In this work, I have targeted eLtaS using CADD methodology followed by prospective validation using various biophysical, biochemical and microbiological assays. My project can broadly be sub-divided into three phases: (a) identification of small molecule binding “hot spots”, (b) optimization of existing inhibitor and (c) discovery of new hits. Through a systematic use of different computational approaches, I modelled a plausible 1771-bound eLtaS complex and used the structural insights to generate new inhibitors against eLtaS. To this end, I discovered EN-19, which is a more potent inhibitor of eLtaS. Additionally, by targeting transient cryptic pockets predicted by Molecular Dynamic simulations, I have discovered a new inhibitor chemotype that seems to exhibit a different mode of action against eLtaS. Taken together, my work presents a computational platform for future drug design against eLtaS and reinforces the notion that targeting eLtaS is a viable strategy to combat Gram-positive infections.