Planejamento, síntese e avaliação biológica de inibidores de falcipaína 2 como candidatos a antimaláricos / Design, synthesis and biological evaluation of falcipain 2 inhibitors as candidates for antimalarials

A malária, doença causada pelo protozoário do gênero Plasmodium, está entre as doenças que mais causam mortes os países subdesenvolvidosn. O hospedeiro é infectado por meio da picada do mosquito do gênero Anopheles, que introduz o parasita durante a hematofagia. As formas mais graves são causadas pelo Plasmodium vivax e o Plasmodium falciparum. As regiões mais afetadas por estas formas são África Subsaariana, Ásia, América Central e Sul. Desde o começo do século XXI, a Organização Mundial de Saúde (OMS) busca erradicar a doença, porém o P.falciparum se mostrou resistente aos fármacos antimaláricos existentes, dificultando a eficácia do tratamento. Isto, entre outros fatores, como mortalidade e alto índice de infecção, tornam necessárias novas pesquisas para a descoberta de novos fármacos mais seguros e eficazes contra a malária. Estudos têm mostrado como um alvo promissor para a criação de novos antimaláricos, a cisteína protease falcipaína, a qual se apresenta em três isoformas no parasita, sendo elas, falcipaína 1, 2 e 3. A falcipaína 2 está ligada com a hidrólise da hemoglobina, e seus inibidores vem sendo estudados como alternativas na busca de agentes antimaláricos. Derivados de semicarbazona, tais como o nitrofural e o hidroximetilnitrofural demonstraram atividade inibitória de cisteíno proteases parasitárias. Utilizando estratégias modernas de planejamento de fármacos e por meio da integração entre técnicas computacionais e experimentais, realizou-se o planejamento, síntese e avaliação biológica de compostos derivados dos ditiocarbazatos e tiossemicarbazonas, bioisosteros de semicarbazona, como inibidores da cisteíno protease falcipaína 2, no intuito de obter novos antimaláricos. Aplicaram-se técnicas de modelagem molecular em três séries de compostos (A, B e C), sendo a A e B derivados dos ditiocarbazatos e a C das tiossemicarbazonas. Estes estudos sugerem, três compostos da série A, quatro na série B e três na C com maior potencial para inibição da falcipaína 2. Isso devido aos resultados teóricos indicarem condições favoráveis ao ataque nucleofílico da cisteína 42 catalítica da falcipaína 2 às tiocarbonilass presentes nos compostos planejados. Estes derivados foram sintetizados, analisados por espectroscopia de ressonância magnética de 1H e 13C, espectroscopia de IV, ponto de fusão e pureza caracterizando sua formação. Após a obtenção, os compostos foram enviados para ensaios biológicos frente ao parasita P. falciparum. Os compostos testados não apresentaram inibição, porém é sabido que muitos inibidores enzimáticos não são ativos contra o parasita mesmo tendo alta potência contra a enzima, isto devido às barreiras a serem ultrapassadas até chegar ao alvo bioquímico, deste modo faz-se necessário ensaios contra a enzima para validar nossa hipótese

Malaria, a disease caused by the protozoan of the genus Plasmodium, is among the most deadly diseases in poor countries. The host is infected through the bite of the mosquito of the genus ,i>Anopheles, which introduces the parasite during hematophagy. The most severe forms are caused by Plasmodium vivax and Plasmodium falciparum. The regions most affected by these forms are Sub-Saharan Africa, Asia, Central and South America. Since the beginning of the 21st century, the World Health Organization (WHO) has sought to eradicate the disease, but P. falciparum has been resistant to antimalarial drugs treatment. Among other factors, such as mortality and high infection rates, new research is needed to find new, safer and more effective drugs against malaria. Studies have shown as a promising target for the creation of new antimalarial drugs, the cysteine protease falcipain, which is present in three isoforms in the parasite: falcipain 1, 2 and 3. Falcipain 2 is linked to the hydrolysis of hemoglobin, and its inhibitors have been studied as alternatives in the search for antimalarial agents. Derivatives of semicarbazone such as nitrofural and hydroxymethylnitrofural demonstrated inhibitory activity of parasitic cysteine proteases. Using modern strategies for drug design and the integration of computational and experimental techniques, the design, synthesis and biological evaluation of compounds derived from dithiocarbazates and thiossemicarbazones, semicarbazone biosynthesis as inhibitors of cysteine protease falcipain 2 were carried out in order to new antimalarials. Molecular modeling studies were performed in three series of compounds (A, B and C), with A and B being derived from dithiocarbazates and C from thiossemicarbazones. These studies suggest three compounds in the A series, four in the B series, and three in the C group with the greatest potential for inhibition of falcipain 2. This is due to the theoretical results indicating favorable conditions for the nucleophilic attack of the catalytic cysteine of falcipain 2 on thionyls present in the compounds planned. These derivatives were synthesized, analyzed by 1H and 13C magnetic resonance spectroscopy, IR spectroscopy and melting point, characterizing their formation. After being obtained, the compounds were sent for biological assays against the P. falciparum parasite. The compounds tested did not show inhibition, but it is known that many enzyme inhibitors are not active against the parasite even though they have high potency against the enzyme, this is due to the barriers to be overcome until reaching the biochemical target, thus enzyme to validate our hypothesis

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1270-290, 3D7610-630, G650-690, while 2 variants, K1150-170, and 3D7670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.

Evolution of Genetic Polymorphisms of Plasmodium falciparum Merozoite Surface Protein (PfMSP) in Thailand

Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.

Plasmodium falciparum: detection and strain identification of Indian isolates by polymerase chain reaction.

The polymerase chain reaction (PCR) was employed for detection and strain identification of P. falciparum in a comparative field study of Indian isolates. The primers were selected from highly conserved regions flanking the variable, tandemly repeated regions of highly polymorphic cell surface antigens, major merozoite surface antigen-1 (MSP-1), major surface antigen-2 (MSP-2), circumsporozoite surface antigen (CSP) and ring-infected erythrocyte surface antigen (RESA). Out of the 52 microscopically positive P. falciparum infected field samples, 47 samples were positive by PCR. Variation in the size of the amplified products was observed using MSP-1, MSP-2 specific primers respectively in different field isolates of P. falciparum, but CSP and RESA did not exhibit any variation in size of the amplified product. The multiplex PCR results demonstrated that amplified products from these surface antigens vary in size and there is a specific pattern for each strain and this could be utilized to identify a particular field isolate. One P. falciparum infected field sample detected by the above PCR method was found to be a mixed infection by two different strains. Five microscopically positive P. vivax infeced samples were also analyzed by PCR method using P. falciparum cell surface antigen (MSP-2) specific primers. PCR results showed one P. vivax infected sample was positive when P. falciparum specific primers were used, this could be due to inaccurate and reduced limit of detection of Plasmodial species by microscopic examination.

Plasmodium falciparum : taxonomie et genetique; apercu des questions actuelles

La structure genetique des populations naturelles de plasmodium falciparum est actuellement mal connue; et la nouvelle hypothese generale concernant l'importance de la reproduction clonale chez les protozoaires parasites qui est maintenant proposee pour ce plasmodium par certains protozoologistes; peut apporter une nouvelle vision des structures de populations de cet hematozoaire. Si elle se confirme; cette hypothese aura obligatoirement de grandes consequences taxonomiques et medicales

Multiple strains of Plasmodium falciparum are necessary for the growth inhibition assay.

Sixty-seven serum samples from individuals living in a malaria endemic area and sixty sera from healthy blood donors living in Bangkok (non-malarious area) were tested for growth inhibition activity against 3 strains (SO, SN and G-112) of Plasmodium falciparum. Forty-eight percent of the sera from the endemic area were positive when all 3 strains were tested. Among the positive sera, positive rates of 90.6% were observed for the SO and SN strain combination, 87.5% for the SO and G-112 combination, and 50% for the SN and G-112 combination. It is therefore recommended that multiple parasite strains should be tested in the growth inhibition assay. If facilities are limited, a minimum of two strains should be used, one of which is the SO strain or its equivalent.