In the second part of the project, the immunology of PfMSP-133 was studied. During the invasion of merozoites, PfMSP--142 is processed into two fragments with molecular weight of 33kDa and 19kDa. The 19kDa fragment (PfMSP-119) originating from the carboxyl--terminal of PfMSP--142 is relatively more immuno-dominant in different malarial species such as P. falciparum, P. vivax and P. yoelii. In the past, only limited researches about PfMSP-1 33 were performed. Apart from its difficulty in expression, PfMSP-1 33 was also believed to be incapable of inducing protection. / Nevertheless, following the breakthrough of expressing recombinant PfMSP-1 33 in our laboratory, we have demonstrated in this study that recombinant MSP-133 can elicit antibodies with a titer up to a million. Also, we observed that MSP-133 can help MSP-119 to induce protective immunity and such effect is independent from the covalent linkage between these two proteins. Most importantly, our results show that recombinant PfMSP-133 can elicit the production of antibodies that can potentiate the inhibitory effect of anti-MSP-142 serum at high serum dilution. Results of this study give new insights in malarial vaccine development in terms of optimizing the use of adjuvant and immunization regimens. / The 42kDa carboxyl terminal fragment of Plasmodium falciparum Merozoite Surface Protein-1 (PfMSP--142) is one of the most promising candidate antigens in the development of malarial vaccine. In vivo experiments in the 1990's showed that Aotus monkeys immunized with PfMSP--142 were protected from malarial challenge. Later on, other experiments also demonstrated the possibility of using recombinant PfMSP-142 as candidate antigen for malarial vaccine. Previously, recombinant PfMSP-142 (Bvp42) was expressed with the baculovirus expression system and characterized in our laboratory. / The aim of the first part of this project is to improve the production of Bvp42. Experimental results have shown that the expression level of Bvp42 was increased under a BMN compatible baculovirus expression vector---pVL1393. Besides, a codon optimized MSP-142 nucleotide is constructed for the construction of a baculovirus carrying codon optimized MSP-142 gene and aimed for higher expression level. Unfortunately, no Bvp42 expression is observed in the transfection samples and the reason of this observation is unclear. Meanwhile, the purification of Bvp42 was also improved. Pretreatment of the hemolymph with Q--sepharose before affinity chromatography could enhance the purity of the final product. / Yuen, Sai-hang Don. / "July 2007." / Adviser: Walter K. K. Ho. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0220. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 183-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

No description available.

A emergência e a disseminação de cepas resistentes aos fármacos antimaláricos disponíveis na quimioterapia têm conduzido à busca por novos agentes potencialmente ativos. Neste sentido, derivados 4-hidroxiquinolínicos e 4-cloroquinolínicos foram sintetizados e submetidos à avaliação biológica frente à cepa AJ de Plasmodium chabaudi e à avaliação toxicológica frente a macrófagos peritoneais de camundongos BALB/c. O planejamento sintético consistiu na preparação de β-cetoésteres-α-alquenilados através de reação de acetoacetato de etila e brometo de alila ou brometo de cinamila. Posteriormente, β-enaminoésteres-α-alquenilados foram obtidos através de reações de -β-cetoésteres-α-alquenilados com amina aromática (anilina). Os derivados 2-metil-3-alil- ou 2-metil-3-cinamil-4-hidroxiquinolínicos foram obtidos através de termociclização de Conrad-Limpach, utilizando-se difeniléter como solvente reacional. Por fim, a cloração dos agentes hidroxilados com oxicloreto de fósforo rendeu 2-metil-3-alil- ou 2-metil-3-cinamil-4-cloroquinolinas. Dos quatro derivados quinolínicos avaliados, 2-metil-3-[(2E)-3-fenilprop-2-enil]quinolin-4-ol (11) mostrou-se 1,13 vezes mais efetivo que sulfato de cloroquina contra as formas intraeritrocíticas do parasita, 1,69 vezes menos tóxico para os macrófagos peritoneais em relação ao fármaco padrão e valor de índice de seletividade igual a 280, enquanto sulfato de cloro quina apresentou valor de 146,84. / The emergence and spread of resistance to antimalarial drugs have highlighted the need for the discovery and development of novel antimalarial molecules. To achieve this goal, 4-hydroxyquinoline and 4-chloroquinoline derivatives were prepared. Their biological activity was tested against the AJ Plasmodium chabaudi strain and their toxicity was evaluated toward BALB/c mouse peritoneal macrophages. The synthetic design was started by reacting ethyl-acetoacetate with allyl bromide or cinamyl bromide to obtain -α-alkenyl-β-ketoesters. The -α-alkenyl-β-enaminoesters were prepared by condensation of -α-alkenyl-β-ketoesters with aromatic amine (aniline). The derivatives 2-methyl-3-allyl- or 2-methyl-3-cinamyl-4-hydroxyquinolines were obtained by Conrad-Limpach ciclization in reacional solvent diphenyl ether. The 2-methyl-3-allyl- or 2-methyl-3-cinamyl-4-chloroquinoline derivatives had been prepared by chloration of hydroxyl group with phosphorous oxycloride. Among the quinoline compounds evaluated, 2-methyl-3-[(2E)-3-phenylpro-2-enyl]quinolin-4-ol (11) has shown more active than chloroquine sulphate (1, 13-fold) against the parasite intraerytrocytic stage. The compound 11 has presented less toxic than this drug (l,69-fold) to peritoneal macrophages. The selectivity index value has been 280, while the value to chloroquine sulphate has been 146,84.

A emergência e a disseminação de cepas resistentes aos fármacos antimaláricos disponíveis na quimioterapia têm conduzido à busca por novos agentes potencialmente ativos. Neste sentido, derivados 4-hidroxiquinolínicos e 4-cloroquinolínicos foram sintetizados e submetidos à avaliação biológica frente à cepa AJ de Plasmodium chabaudi e à avaliação toxicológica frente a macrófagos peritoneais de camundongos BALB/c. O planejamento sintético consistiu na preparação de β-cetoésteres-α-alquenilados através de reação de acetoacetato de etila e brometo de alila ou brometo de cinamila. Posteriormente, β-enaminoésteres-α-alquenilados foram obtidos através de reações de -β-cetoésteres-α-alquenilados com amina aromática (anilina). Os derivados 2-metil-3-alil- ou 2-metil-3-cinamil-4-hidroxiquinolínicos foram obtidos através de termociclização de Conrad-Limpach, utilizando-se difeniléter como solvente reacional. Por fim, a cloração dos agentes hidroxilados com oxicloreto de fósforo rendeu 2-metil-3-alil- ou 2-metil-3-cinamil-4-cloroquinolinas. Dos quatro derivados quinolínicos avaliados, 2-metil-3-[(2E)-3-fenilprop-2-enil]quinolin-4-ol (11) mostrou-se 1,13 vezes mais efetivo que sulfato de cloroquina contra as formas intraeritrocíticas do parasita, 1,69 vezes menos tóxico para os macrófagos peritoneais em relação ao fármaco padrão e valor de índice de seletividade igual a 280, enquanto sulfato de cloro quina apresentou valor de 146,84. / The emergence and spread of resistance to antimalarial drugs have highlighted the need for the discovery and development of novel antimalarial molecules. To achieve this goal, 4-hydroxyquinoline and 4-chloroquinoline derivatives were prepared. Their biological activity was tested against the AJ Plasmodium chabaudi strain and their toxicity was evaluated toward BALB/c mouse peritoneal macrophages. The synthetic design was started by reacting ethyl-acetoacetate with allyl bromide or cinamyl bromide to obtain -α-alkenyl-β-ketoesters. The -α-alkenyl-β-enaminoesters were prepared by condensation of -α-alkenyl-β-ketoesters with aromatic amine (aniline). The derivatives 2-methyl-3-allyl- or 2-methyl-3-cinamyl-4-hydroxyquinolines were obtained by Conrad-Limpach ciclization in reacional solvent diphenyl ether. The 2-methyl-3-allyl- or 2-methyl-3-cinamyl-4-chloroquinoline derivatives had been prepared by chloration of hydroxyl group with phosphorous oxycloride. Among the quinoline compounds evaluated, 2-methyl-3-[(2E)-3-phenylpro-2-enyl]quinolin-4-ol (11) has shown more active than chloroquine sulphate (1, 13-fold) against the parasite intraerytrocytic stage. The compound 11 has presented less toxic than this drug (l,69-fold) to peritoneal macrophages. The selectivity index value has been 280, while the value to chloroquine sulphate has been 146,84.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The work in this dissertation provides structural and kinetic insight into the mechanism of action of quinoline antimalarial drugs which may aid rational drug design. Quinoline antimalarial drug-ferrihaem (Fe(III)PPIX) complexes were investigated. Single crystal Xray diffraction (SCD) structures of the complexes formed between Fe(III)PPIX and the quinoline methanol antimalarials quinine, quinidine and mefloquine have been determined, and are the first observed structures of complexes formed between free Fe(III)PPIX and quinoline antimalarial compounds. Quinine, quinidine and mefloquine are shown to have a three-point binding mode to Fe(III)PPIX, which comprises direct coordination of the drug to the Fe(III) centre through its benzylic alcohol functionality, π-stacking between the drug and porphyrin aromatic systems, and intramolecular hydrogen bond formation between the porphyrin propionate group and the protonated quinuclidine nitrogen atom of the drug in the case of quinine and quinidine, and formation of an intramolecular hydrogen bonding network in the case of mefloquine. Extended X-ray absorption fine structure spectroscopy (EXAFS) was use to elucidate structural information of Fe(III)PPIX-drug complexes in solution, and indicates that coordination persists in solution. The protocol for lipid-mediated formation of β-haematin, where monopalmitic glycerol was used as a model lipid, was successfully modified to incorporate antimalarial drugs into the aqueous layer in order to investigate drug activity under biologically-relevant conditions. Four compounds were chosen, namely chloroquine and amodiaquine, both 4- aminoquinolines and quinine and quinidine. IC50 values for the inhibition of β-haematin formation show good correlation with biological activities determined against a chloroquine-sensitive Plasmodium falciparum strain. The lipid-water interface system was further used to investigate the effects of quinine, quinidine chloroquine and amodiaquine on the kinetics of β-haematin formation. The results led to the development of a kinetic model based on the Avrami equation and the Langmuir isotherm. The data strongly support a mechanism of antimalarial drug action by adsorption to the growing face of haemozoin, with precipitation of Fe(III)PPIX at high drug concentrations accounting for decreased yields. Adsorptions constants (log Kads) determined for each drug show a strong correlation with biological activity. Finally, the first SCD structure of the μ-propionato dimer of Fe(III)PPIX, the structural unit of haemozoin, has been determined as its DMSO solvate. EXAFS suggests that this species is only formed upon nucleation, with the π-π dimer species being favoured in solution. / AFRIKAANSE OPSOMMING: Die werk in die dissertasie verleen struktuur en kinetiese insig in the meganisme van aktiwiteit vir kinolien antimalariamiddels wat kan bydra tot die ontwikkeling van nuwe medisyne. Kinolien antimalariamiddel-ferriheem (Fe(III)PPIX) komplekse was ondersoek. Navorsing is gedoen op die enkelkristal X-straaldiffraksie strukture van die komplekse gevorm tussen Fe(III)PPIX en die kinolien metanol antimalaria middels kinien, kinidien en mefloquine. Die strukture is die eerste komplekse wat waargeneem is tussen vrye Fe(III)PPIX en kinolien antimalariamiddels. Kinien, kinidien en mefloquine het ʼn driepunt bindingsvorm, direkte koördinasie met die Fe(III) deur die bensielalkohol groep, ʼn π- stapel tussen die middel en die porfirien aromatiese sisteem, ʼn intramolekulêre waterstofbinding tussen the porfirienpropionaat funksie en die geprotoneerde kinuklidien stikstofatoom (kinien en kinidien) en ʼn netwerk van intramolekulêre waterstof bindings (mefloquine) insluit. Uitgebreide X-straal absorpsie fyn struktuur spektroskopie (EXAFS) is gebruik om inligting oor Fe(III)PPIX-middel komplekse in oplossing te verkry en het aangedui dat die koördinasie in oplossing voorkom. Deur gebruik te maak van monopalmitiengliserol as die lipid in the lipid-water interfase sisteem, waar antimalariamiddels suksesvol in die buffer geïnkorporeer was, was die middel se aktiwiteit onder biologiese kondisies geondersoek. Vier middels was gekies naamlik, chloroquine en amodiaquine, albei 4-aminokinoliene en kinien en kinidien om die IC50-waarde vir inhibisie van β-hematien vorming te bepaal. Die IC50 waardes het ʼn goeie korrelasie met biologiese aktiwiteite teen die chloroquine-sensitiewe Plasmodium falciparum stam gewys. Die lipid-water interfase-sisteem was ook gebruik om die effek van kinien, kinidien, chloroquine en amodiaquine op die kineties effek op die vorming van β-hematien te ondersoek. Die resultate het gelei to die ontwikkeling van die kinetiese model gebaseer op die Avrami vergelyking en die Langmuir isoterm. Die data ondersteun ʼn meganisme van middel aksie waar die middel teen die groeiende vlak van hemosoïen kristal adsorbeer. Die neerslag van Fe(III)PPIX wat vorm by hoë konsentrasies, het gelei tot laer opbrengste. Die adsorpsiekonstante (log Kads) bepaal vir elke middel, het goeie korrelasie met biologiese aktiwiteit getoon. Enkelkristal X-straaldiffraksie strukture van μ- propionatodimeer van Fe(III)PPIX, die struktuur eenheid van hemosoïen, was bepaal as ʼn DMSO solvaat. EXAFS het aangedui dat die spesie slegs by kernvorming ontstaan en dat die π-π dimeerspesie in oplossing voorkom.

Malaria is considered as one of the most prevalent and debilitating diseases affecting humans. Plasmodium falciparum is the most virulent form of the parasite which developed resistance to several antimalarial drugs. Chloroquine is one of the most successful antimalarials developed that is safe, effective, and cheap. However, its use has been limited due to the emergence of drug resistance. Click chemistry, particularly, the copper(I)-catalyzed reaction between azides and alkynes has shown to have a cutting-edge advantage in medicinal chemistry by its reliability, selectivity and biocompatibility. Triazole-based antimalarials were synthesized via copper(I)-catalyzed alkyne-azide cycloaddition reaction by modifying the aliphatic chains terminal of chloroquine. The compounds synthesized contain triazole ring directly connected to an aromatic ring or via a piperazine linker. When tested for their in vitro antimalarial activity against D6, Dd2 and 7G8 strains of P. falciparum, 12 out of 28 compounds showed better activity against chloroquine resistant strains. Particularly, PL403 and PL448 exhibited potent activity than chloroquine against CQ-resistant strains Dd2 and 7G8, with IC50 values of 12.8 & 14.5 nM, and 15.2 & 11 nM respectively. The efficiency of synthesizing several triazole-based antimalarials have proven click chemistry to be fast and efficient reaction. Generally, para-substitutions and di-substitutions with electron-withdrawing groups were found to be beneficial for having better antimalarial activity for these group of click compounds. Moreover, the incorporation of piperazine linker has brought an enhanced antimalarial activity.

Malaria is a mosquito borne infectious disease caused by a unicellular Apicomplexan parasite of the Plasmodia genus. The emergence and subsequent spread of drug resistance in the highly virulent Plasmodium falciparum parasite has been a major setback in eradicating malaria, which affects an estimated 216 million individuals and causes 445,000 deaths annually worldwide. Chloroquine (CQ) was once used as the first-line antimalarial drug treatment, until CQ-resistant parasites emerged in endemic regions including Africa, Southeast Asia, and South America. More recently, parasites have developed resistance to the current first line drug piperaquine (PPQ), used in combination with dihydroartemisinin (DHA) in Southeast Asia. Plasmodium falciparum chloroquine resistance transporter (PfCRT), a member of the drug/metabolite transporter (DMT) superfamily, is a 49-kDa integral transmembrane protein localized in the digestive vacuole (DV) of the pathogenic parasite. Mutations in PfCRT have been identified as the core determinants of Plasmodium falciparum resistance to CQ and PPQ by mediating the efflux of these antimalarial drugs. All CQ resistance-conferring PfCRT isoforms share the K76T mutation, which is widely used as a molecular marker for CQ resistance. Despite the significance in the impact of drug-resistant malaria, a detailed understanding of PfCRT physiological function and the molecular basis of PfCRT-mediated drug resistance have been hampered by a lack of high-resolution structural information. This dissertation describes the first structure of PfCRT and reveals the interaction of drugs with the purified and reconstituted protein. We determined the structure of the 49-kDa PfCRT 7G8, a clinically relevant CQ-resistant isoform found in South America, to 3.2 Å resolution by single-particle cryo-electron microscopy (cryo-EM), in complex with a specific antigen-binding fragment (Fab) to overcome current size limitations in cryo-EM. Our PfCRT structure displays an inward-open conformation, consists of 10 transmembrane (TM) helices with an inverted topology, and has unique elements including two juxtamembrane helices and a highly conserved cysteine-rich loop between TM helix 7 and 8. The architecture of PfCRT is similar to other members of the DMT superfamily. TM helices 1-4 and 6-9 in PfCRT form a central cavity which is a potential binding site for both CQ and PPQ. A striking feature is that virtually all the CQ resistance mutations, identified from decades of investigation into PfCRT variants that have evolved independently across the malaria-endemic world, map around this central, negatively-charged cavity. Distinct mutations that have been proposed to cause high-level PPQ resistance in parasites, which cause a loss of CQ resistance, form a planar ring that also lines this cavity. Functional experiments with various purified PfCRT isoforms or mutants provide evidence that drug resistance is possibly due to pH- and membrane potential-dependent drug transport. We also show that PfCRT CQ-resistant isoforms bind and transport arginine, suggesting that positively charged amino acids may be putative transport substrates for CQ-resistant PfCRT. This work provides a structural and functional framework to understand the mechanism of PfCRT-mediated drug resistance in the malaria parasite.