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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) EFETIVIDADE DA ELIPTICINA E ARTEMISININA-atual.pdf: 1595775 bytes, checksum: b76659782dbf4751e7f12e9f356c42fe (MD5) Previous issue date: 2018-02-27 / Parasite resistance to commercially available antimalarial drugs is a reality, which makes it possible to search for new drugs and to develop researches with plants that have pharmacological properties. Numerous discussions on rapidlyreleasing antimalarial drugs have been gaining strength and adherence, as it has advantages related to its efficacy, specificity and patient tolerance in therapy. In this way, nanotechnology offers important tools for the development of new drugs and nanoparticles can contribute to a more specific therapy of selective action in the distribution and absorption of drugs. According to the Amazonian biodiversity, the active principle, ellipticina involved in research as antimalarial was isolated from Aspidosperma vargasii, whose popular name is Amarelão or Carapanaúba branca. Ellipticin exhibits antiplasmodial activity that has been demonstrated in in vitro test because it inhibited the growth of heme crystal that occurs in the digestive vacuole of Plasmodium ssp. and is associated with parasite growth. Another active principle, which is extracted from Artemisia annua L. is artemisinin. It is considered essential in the treatment of malaria, by its potency and its rapid action. Interventions of artemisinin-based combination therapy have reduced morbidity and mortality associated with malaria in several parts of the world. The combinations of artemeter-lumifantrine, artesunate-mefloquine and artesunateamodiaquine, are related to artoisinin's gametocytocidal properties that inhibit the transmission of parasites to probably reduce the development of antimalarial resistance. Recent studies show that artesunate may be a good alternative for the treatment of some types of cancer by the antiangiogenic mechanism. Its low toxicity represents a promising chemotherapeutic. It is important to note that artesunate is not carried by P-glycoproteins, so it is not involved in multidrug resistance. In the fight against these pathologies, several inconveniences are pointed out for the patient as some adverse effects and dosing schedule. Thus, it considers that the evaluation of new drug release systems can improve the therapeutic efficacy of future nanoformulations for antimalarial and antitumor drugs. In this sense, this work aims to develop, characterize and evaluate nanoparticles in their physico-chemical and cytotoxic aspect by means of bioassays against Plasmodium falciparum and antitumor cells. The substances used in the synthesis of the nanospheres were sodium tetrachloropalladium and ellipticine hydrochloride. The substances involved in the synthesis of nanofibers were artemisinin hydrochloride, polyvinylpyrrolidone and polybutylene. / A resistência parasitária aos fármacos antimaláricos comercialmente disponível é uma realidade, o que possibilita a busca por novas drogas e o desenvolvimento de pesquisas com plantas que possuem propriedades farmacológicas. As inúmeras discussões sobre as drogas antimaláricas de liberação rápida, vem ganhando força e adesão, pois apresenta vantagens relacionadas a sua eficácia, especificidade e tolerância por parte do paciente na terapia. Desta forma, a nanotecnologia oferece ferramentas importantes para o desenvolvimento de novos fármacos e as nanopartículas podem contribuir para uma terapia mais específica de ação seletiva na distribuição e absorção das drogas. De acordo com a biodiversidade Amazônica, o príncipio ativo, elipticina envolvida em pesquisas como antimalárico foi isolado da Aspidosperma vargasii, cujo nome popular é Amarelão ou Carapanaúba branca. A elipticina apresenta atividade antiplasmodial que foi demonstrado em teste in vitro, pois inibiu o crescimento do cristal de heme que ocorre no vacúolo digestivo do Plasmodium ssp. e está associado ao crescimento do parasito. Outro princípio ativo, que é extraído da Artemisia annua L. é a artemisinina. É considerada fundamental no tratamento da malária, por sua potência e sua rápida ação. As intervenções da terapia combinada baseada em artemisinina, diminuíram a morbidade e a mortalidade associadas a malária em várias partes do mundo. As combinações de artemeter-lumifantrine, artesunato-mefloquina e artesunato-amodiaquina, está relacionado a propriedades gametocitócidas da artemisinina que inibe a transmissão de parasitas para provavelmente reduzir o desenvolvimento de resistência antipalúdica. Recentes estudos mostram que o artesunato pode ser uma boa alternativa para o tratamento de alguns tipos de câncer, pelo mecanismo antiangiogênico. A sua baixa toxicidade representa um quimioterápico promissor. É importante salientar que o artesunato não é transportado por P-glicoproteínas, portanto não está envolvido em resistência a multidrogas. Na luta contra essas patologias, são apontados vários inconvenientes para o paciente como alguns efeitos adversos e horário de dosagens. Assim, considera que a avaliação de novos sistemas de liberação de medicamentos podem melhorar a eficácia terapêutica de futuras nanoformulações para fármacos antimaláricos e antitumorais. Neste sentido, este trabalho tem por objetivo desenvolver, caracterizar e avaliar as nanopartículas no seu aspecto físico-químico e citotóxico por meio de bioensaios contra Plasmodium falciparum e células antitumorais. As substâncias utilizadas na síntese das nanoesferas foram sódio tetracloropaládio e cloridrato de elipticina. As substâncias envolvidas na síntese das nanofibras foram o cloridrato de artemisinina, polyvinilpirrolidona e polibutileno.

Atualmente, a malária é considerada a principal infecção parasitária existente e apresenta distribuição mundial. Dentre as alternativas terapêuticas utilizadas, destacam-se cloroquina (CQ), mefloquina (MQ) e, recentemente, arteméter (ART). Segundo a literatura, estudos farmacocinéticos destes fármacos têm sido dificultados pela ausência de métodos adequados de análise em fluidos biológicos e, no caso dos fármacos quirais (CQ e MQ), com capacidade de determinar os enantiômeros individualmente. Com isso, o objetivo deste trabalho foi avaliar o emprego da microextração em fase líquida (LPME) na preparação de amostras para a determinação destes três fármacos antimaláricos e respectivos metabólitos em plasma. O método para análise enantiosseletiva de CQ e metabólitos teve a LPME como técnica de preparação de amostras, a qual apresentou valores de recuperação no intervalo de 28-66%. Estes analitos foram separados na coluna Chirobiotic V em fase polar-orgânica, com posterior detecção por espectrometria de massas (MS), com interface de eletronebulização (ESI) no modo positivo. O método desenvolvido foi linear no intervalo de 5-500 ng mL-1 para todos os analitos avaliados. A disposição cinética de CQ e do principal metabólito monodesetilcloroquina (DCQ) em ratos sugere enantiosseletividade após administração do fármaco na forma racêmica, com maiores concentrações de (+)-(S)-CQ e (-)-(R)-DCQ. O método para análise dos enantiômeros de MQ e do metabólito aquiral carboximefloquina (CMQ) também foi desenvolvido empregando LPME na preparação das amostras. A extração destes analitos foi realizada em duas etapas para eficaz recuperação dos mesmos (valores entre 35-38%). Os analitos foram separados na coluna Chirobiotic T em fase polarorgânica, com detecção por absorção no ultravioleta em 285 nm. O método apresentou linearidade no intervalo de 50-1500 e 50-3000 ng mL-1 para os enantiômeros de MQ e CMQ, respectivamente. A disposição cinética de MQ em ratos indica enantiosseletividade com maiores concentrações de (+)-(RS)- MQ após administração do fármaco na forma racêmica. A separação cromatográfica em fase reversa de ART e metabólito diidroartemisinina (DHA) foi alcançada utilizando-se coluna contendo Si-Zr-PMTDS como fase estacionária e a detecção destes analitos foi realizada empregando-se MS no modo ESI positivo. O procedimento otimizado de LPME em duas fases para extração de ART e DHA em plasma resultou em valores de recuperação de 32 e 25%, respectivamente. O método desenvolvido foi linear no intervalo de 5- 1000 ng mL-1 para ambos os analitos. O estudo piloto de disposição cinética em ratos evidenciou maiores concentrações de DHA. Os resultados obtidos confirmam a viabilidade da LPME para extração destes antimaláricos e respectivos metabólitos em plasma. / Currently, malaria is the main parasitic infection and shows worldwide distribution. Among therapeutic options used, chloroquine (CQ), mefloquine (MQ) and, more recently, artemether (ART) have been standing out. According to the literature, pharmacokinetic studies of these drugs have been hampered by the lack of proper methods of analysis in biological fluids and, regarding the chiral drugs (CQ and MQ), with the ability to determine the individual enantiomers. Therefore, the aim of this work was to evaluate the utilization of liquid-phase microextraction as the sample preparation technique for the determination of these antimalarial drugs and their metabolites in plasma. The enantioselective analysis of CQ and its metabolites was carried out using LPME as technique of sample preparation, which yielded recovery rates within 28- 66%. These analytes were resolved on a Chirobiotic V column in the polarorganic mode and further detected using mass spectrometry (MS) with electrospray interface (ESI) in the positive mode. The developed method was linear in the range of 5-500 ng mL-1 for all analytes studied. The kinetic disposition of CQ and its main metabolite monodesethylchloroquine (DCQ) in rats suggests enantioselectivity following the administration of the racemic drug, with higher concentrations of (+)-(S)-CQ and (-)-(R)-DCQ. The method for the analysis of the enantiomers of MQ and its achiral metabolite carboxymefloquine (CMQ) also had LPME as technique of sample preparation. The extraction of these analytes was carried out in two-steps to obtain efficient recovery rates (values within 35-38%). The analytes were resolved on a Chirobiotic T column in polar-organic mode and ultraviolet detection was performed at 285 nm. The method was linear in the range of 50-1500 and 50-3000 ng mL-1 for the enantiomers of MQ and CMQ, respectively. The kinetic disposition of MQ in rats indicates enantioselectivity with higher concentrations of (+)-(RS)-MQ following the administration of the racemic drug. The chromatographic resolution of ART and its metabolite dihydroartemisinin (DHA) in the reversed mode was achieved utilizing a column containing Si-Zr-PMTDS as stationary phase and their detection was conducted employing MS in the positive ESI mode. The optimized two-phase LPME procedure for the extraction of ART and DHA from plasma showed recovery values of 32 and 25%, respectively. The developed method was linear over the range of 5-1000 ng mL-1 for both analytes. The pilot study of kinetic disposition in rats showed higher concentrations of DHA. The obtained results confirm the feasibility of LPME for the extraction of these antimalarial drugs and their metabolites from plasma.

Orientador: Ronaldo Aloise Pilli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T09:48:15Z (GMT). No. of bitstreams: 1 Correia_ValquirioGraia_M.pdf: 5120551 bytes, checksum: 59d3f74f13394c176e7a8ee336417804 (MD5) Previous issue date: 2011 / Resumo: Neste trabalho sintetizamos alguns análogos à febrifugina (1), alcaloide inicialmente isolado a partir de extratos da erva Chang shan (Dichroa febrífuga) e detentor de potente atividade antimalárica. Foram sintetizados análogos na forma racêmica em que se variou o tamanho do anel heterocíclico saturado, a ausência de substituinte e presença de um átomo de bromo na posição C3¿, visando estudar a influência desses elementos na atividade contra P. falciparum e na citotoxicidade. A síntese dos análogos envolveu reações de a-amidoalquilação entre íons N-acilimínio e éteres enólicos de silício ou acetatos enólicos na etapa chave. Os análogos 11, 80 e 81 foram preparados em oito etapas, em bons rendimentos globais. Enquanto os análogos 82-84 foram preparados em seis etapas, utilizando-se metodologias diastereosseletivas, em rendimentos globais razoáveis / Abstract: In this work we synthesized some febrifugine analogs (1), alkaloid initially isolated from the extracts of the herb Chang Shan (dichroa febrifuga) wich displayed potent antimalarial activity. Analogs have been synthesized in racemic form, varying the size of the saturated heterocyclic ring, the absence of substituent and the presence of a bromine atom at C3¿ position aiming to study the influence of these elements in the activity against P. falciparum and the cell toxicity. To synthesize the analogues we have used a-amidoalkylation reaction between N-acyliminium ions and silylenolethers or enolacetates in the key step. Analogs 11, 80 and 81 were prepared in eight steps in good overall yields, while the analogs 82-84 were prepared in six steps, using a diastereoselective á-amidoalkylation reaction, in reasonable overall yields / Mestrado / Quimica Organica / Mestre em Química

Orientador: Fabio Trindade Maranhão Costa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T06:24:11Z (GMT). No. of bitstreams: 1 Lopes_StefanieCostaPinto_D.pdf: 63660430 bytes, checksum: 46ba12508eba9cd5aeed590ca748d9cd (MD5) Previous issue date: 2012 / Resumo: Este trabalho de tese está dividido em dois capítulos. Abaixo descreveremos brevemente os achados de cada um deles. O primeiro capítulo entitulado Desenvolvimento de Drogas Experimentais trata da atividade antimalárica de um composto natural, a violaceína e suas formulações geradas com o auxílio da nanotecnologia. Neste sentido, a violaceína foi eficiente em eliminar in vitro formas sanguíneas de P. falciparum 3D7 e P. falciparum S20, sendo este último isolado resistente à cloroquina. Quando testada in vitro em isolados frescos de P. vivax, a violaceína não foi capaz de inibir o desenvolvimento parasitário. No entanto, acreditamos que novos ensaios são necessários para confirmar este dado, uma vez que o IC50 (concentração inibitória 50%) observado para cloroquina apresentou-se estranhamente elevado. Os experimentos in vivo utilizando camundongos infectados com Plasmodium chabaudi chabaudi AS revelaram uma potente atividade desta droga, inclusive quando o tratamento foi iniciado após o estabelecimento de infecção patente nos animais. Quando administrada em animais infectados com uma cepa murina letal (Plasmodium chabaudi chabaudi AJ), a violaceína protegeu 80% dos animais. Como a violaceína é um composto insolúvel em água, novas formulações com essa molécula foram feitas visando sua solubilidade e também potencializar sua atividade antimalárica. Neste sentido, tanto a violaceína encapsulada com co-polímero de ácido láctico e ácido glicólico quanto as formulações de violaceína com três diferentes nanotubos de carbono também foram capazes de inibir o crescimento parasitário in vitro contra P. falciparum, no entanto esta atividade não foi potencializada. Em síntese, este capítulo nos mostra que a violaceína apresenta capacidade de inibir o crescimento parasitário de Plasmodium sp., no entanto as suas formulações não foram capazes de potencializar a atividade antimalárica. O segundo capítulo entitulado Imunopatogênese na malária vivax trata da capacidade adesiva de eritrócitos infectados por P. vivax (Pv-EI) a diferentes receptores endoteliais e da capacidade deste parasita em formar rosetas. Uma vez que há uma baixa proporção de formas maduras principalmente esquizontes na circulação periférica de pacientes infectados por P. vivax, estabelecemos uma técnica de cultivo in vitro para obtenção destas formas maduras e investigamos a capacidade adesiva destas formas do parasita. Neste sentido, observamos que Pv- EI pós-amadurecimento apresentam maior capacidade adesiva em HLEC (células endoteliais de pulmão humano) que parasitas do mesmo isolado antes do amadurecimento. Quando avaliado os receptores envolvidos antes e após o amadurecimento, ensaios com células transfectadas ou uso de inibidores específicos, revelou a participação de ICAM-1 (molécula de adesão intercelular 1) e CD36 (cluster de diferenciação 36) na citoadesão deste parasita enquanto a participação de CSA (Condroitin Sulfato A) permanece a ser elucidada. Ao passo que ICAM-1 parece estar envolvida na citoadesão de todos os estágios do parasitas, o CD36 parece ser estritamente envolvido na citoadesão de formas maduras, essencialmente esquizontes. Além disso, a citoadesão de Pv-EI em HLEC foi potencializada na presença de soro do próprio paciente, sugerindo a existência no soro dos infectados de mediadores adesivos. Quando investigada a formação de rosetas por Pv-EI verificamos que a porcentagem de rosetas foi maior nas formas amadurecidas e também foi potencializada na presença de soro autólogo. Os dados aqui apresentados abrem perspectivas para novos estudos visando compreender o fenômeno patológico envolvido na malária vivax grave, incluindo a participação de mediadores plasmáticos e a identificação dos receptores celulares e dos ligantes parasitários envolvidos / Abstract: This thesis was divided into two chapters, and below we briefly describe the findings of each one. The first chapter entitled Experimental Drug Development investigates the antimalarial activity of a natural compound, violacein, and its formulations developed by nanotechnology. Violacein effectly killed in vitro blood stages of P. falciparum 3D7 and P. falciparum S20, a chloroquine resistant strain. When tested in vitro against P. vivax fresh isolates, violacein did not inhibit parasite development. However, further experiments are needed to confirm this finding, as chloroquine IC50 (inhibition concentratios 50%) was strangely high. The in vivo experiments using mice infected with Plasmodium chabaudi chabaudi AS revealed a potent activity of this drug, even when treatment was initiated after the establishment of patent infection in animals. When administered to lethal strain (Plasmodium chabaudi chabaudi AJ) infected mice, violacein protected 80% of animals. As violacein is insoluble in water, new formulations were developed to enhance its solubility and also its antimalarial activity. However, violacein encapsulated with PLGA (poly(lactic-co-glycolic acid)) and violacein formulations with three different carbon nanotubes did not enhanced violacein activity in vitro against P. falciparum. In summary, this chapter shows that violacein has the capacity to inhibit Plasmodium sp. growth; still their formulations were not able to potentiate violacein antimalarial activity. The second chapter entitled Immunopathogenesis of vivax malaria investigates the adhesive capacity of P. vivax infected erythrocytes (Pv-IE) in different endothelial cell receptors and the ability of this parasite to form rosettes. Since there are a low proportion of mature forms, especially schizonts, in peripheral circulation of malaria vivax patients, we established an in vitro technique to obtain these mature forms and investigate their adhesive capacity in endothelial cells. Here we showed that post-maturation Pv-IE presented more adhesive capacity in HLEC (human lung endothelial cells) than the same isolate before maturation. The search for receptors involved in cytoadhesion before and after maturation, using transfected cells or specific inhibitors, showed that ICAM-1 (intracellular adhesion molecule 1) and CD36 (cluster of differentiation 36) were involved in Pv-IE cytoadhesion whereas CSA (chondroitin sulfate A) involvement remains to be elucidated. While ICAM-1 seems to be involved in all stages of Pv-IE cytoadhesion, CD36 seems to be strictly involved in mature forms cytoadhesion, essentially schizonts. Furthermore, Pv-IE cytoadhesion in HLEC was enhanced in patient's serum presence, suggesting the existence of adhesive mediators in Pv-IE infected serum. Rosette formation by Pv- IE was also higher in mature forms and rosette formation was enhanced in autologous serum presence. These data collectively open new perspectives to study the pathological phenomenon involved in severe vivax malaria, including the involvement of plasma mediators and identification of cell receptors and parasite ligands involved in Pv-IE cytoadhesion / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.

Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.

This project has focused on exploring the application of Baylis-Hillman (BH) {a.k.a. Morita-Baylis-Hillman (MBH)} scaffolds in the construction of various compounds with medicinal potential. A series of 2-nitrobenzaldehydes has been treated under BH conditions, with two different activated alkenes, viz., (MVK) and methyl acrylate, using (DABCO) or (3-HQ) as catalyst. While most of the BH reactions were carried out at room temperature, some reactions were conducted using microwave irradiation. The resulting BH adducts have been subjected to dehydration, conjugate addition and allylic substitution to obtain appropriate intermediates, which have been used in turn, to synthesize possible lead compounds, viz., cinnamate esters as HIV-1 integrase inhibitors, 3-(aminomethyl)quinolines and quinolones as anti-malarials and cinnamate ester-AZT conjugates as dual-action HIV-1 integrase-reverse transcriptase (IN-RT) inhibitors. Conjugate addition reactions of methyl acrylate-derived BH β-hydroxy esters with the amines, piperidine, propargylamine and 2-amino-5-(diethylamino)pentane, has afforded a range of products as diastereomeric mixtures in moderate to excellent yields. Catalytic hydrogenation of the aminomethy β-hydroxy esters derivatives, using a palladium-oncarbon (Pd-C) catalyst, has afforded the corresponding, novel 3-aminomethyl-2- quinolone derivatives in moderate yields. Effective allylic substitution reactions of the MVK-derived BH β-hydroxy ketones (via a conjugate addition-elimination pathway) using in situ-generated HCl has afforded the corresponding α-chloromethyl derivatives, which have been reacted with various amines, including piperidine, piperazine, propargylamine and 2-amino-5-(diethylamino)pentane, to yield α-aminomethyl derivatives. Catalytic hydrogenation of selected α-aminomethyl derivatives, using a Pd-C catalyst, has afforded the corresponding, novel 3- (aminomethyl)-2-methylquinoline derivatives in low to moderate yields. A bioassay, conducted on a 6-hydroxy-2-methyl-3-[(piperidin-1-yl)methyl]quinoline isolated early in the study indicated anti-malarial activity and prompted further efforts in the synthesis of analogous compounds. Reaction of the methyl acrylate-derived BH adducts with POCl3 has provided access to α-(chloromethyl)cinnamate ester derivatives, which have been aminated to afford α- (aminomethyl)cinnamate ester derivatives as potential HIV-1 integrase inhibitors. The α- (propargylaminomethyl)cinnamates were used, in turn, as substrates for the “click chemistry” reaction with 3'-azido-3'-deoxythymidine (AZT– an azide and an established reverse transcriptase HIV-1 inhibitor) to afford cinnamate ester-AZT conjugates as potential dual-action HIV-1 integrase-reverse transcriptase (IN-RT) inhibitors. Computer modelling and docking studies of a cinnamate ester-AZT conjugate into the HIV-1 integrase and reverse transcriptase active-sites revealed potential hydrogen-bonding interactions with amino acid residues within the receptor cavities. The isolated products have been appropriately characterized using IR, 1- and 2-D NMR and HRMS techniques, while elucidation of the stereochemistry of the double bond in the BH-derived halomethyl derivatives has been assigned on the basis of NOE, computer modelling and X-ray crystallographic data.

Malaria, caused by an intracellular Plasmodium parasite, remains a devastating disease, having claimed approximately 655 000 lives worldwide in 2010. The Medicines for Malaria Venture suggests a "single-dose radical cure" as the ideal malaria treatment since rapid clearance of blood-stage parasites and symptom relief improves patient compliance and limits drug resistance. Thus, novel antimalarials should be rapid-acting and assessing their rate of activity is critical to drug discovery. Traditional evaluation of this rate by morphological assessments is flawed by highly subjective, operator-specific interpretations, mainly due to heterogeneous parasite morphology under routine culture conditions. This study aimed to develop an alternative, quantitative assay. Energy is vital for the growth and maintenance of all living organisms. Commercially available kits allow rapid quantification of the cell's energy currency, ATP. Therefore, quantification of parasite ATP shows potential for diagnosing abnormal parasite metabolism and the kinetics of drug action. In this study, a rapid protocol for detecting ATP in Plasmodium falciparum parasites using a luminescence-based kit was developed and optimised. Furthermore, luciferase-expressing transgenic parasites, in which luciferase activity is detected using a similar kit, were acquired. The utility of both methods for evaluating the rate of drug-induced stress was explored using antimalarials with varying modes of action and, presumably, rates of activity. Results showed that parasite ATP remained unchanged, increased or decreased during drug exposure. Morphological examinations by light microscopy and a Recovery assay, aided interpretation of the drug-induced changes in parasite ATP. These investigations suggested that unchanged parasite ATP levels reflect poor drug action, increased ATP levels indicate a stress response and partially compromised viability, while significantly reduced ATP reflects severely compromised viability. Concerning the Luciferase assay, parasite luciferase activity decreased during drug exposure, even in the presence of proteasome inhibitors. Changes in parasite ATP and luciferase activity occurred at rates which suggested that chloroquine is slow-acting, mefloquine has a moderate rate of activity and artemisinin is rapid-acting. These findings are compatible with the expected rates of activity of these established antimalarials. Hence, measurement of parasite ATP and/or luciferase activity may support assessments of parasite health and the kinetics of antimalarial action during drug discovery

Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.

Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.