Structure-based drug design studies of the interactions of ent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparum sarco/endoplasmic reticulum Ca²âº-ATPase PfATP6.

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosa were tested in silico against the Plasmodium falciparum Ca2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

Nanoemulsion composed of 10-(4,5-dihydrothiazol-2-yl)thio)decan-1-ol), a synthetic analog of 3-alkylpiridine marine alkaloid: development, characterization, and antimalarial activity.

Malaria treatment is based on a reduced number of antimalarial drugs, and drug resistance has emerged, leading to the search for new antimalarial drugs incorporated into pharmaceutical formulations. In this study, 10-(4,5-dihydrothiazol-2-yl)thio)decan-1-ol) (thiazoline), a synthetic analog of 3-alkylpiridine marine alkaloid, and a potent antimalarial substance, was incorporated into O/W nanoemulsion. This formulation was prepared by a 23 factorial design. It was characterized by globule diameter, polydispersity index, zeta potential, encapsulation efficiency, in vitro thiazoline release at pH 2 and 6.86, and accelerated stability. In vitro and in vivo antimalarial activity was determined against P. falciparum and P. berghei, respectively. Thiazoline nanoemulsion showed 248.8 nm of globule diameter, 0.236 of polydispersity index, -38.5 mV of zeta potential, 96.92% encapsulation efficiency, and it was stable for 6 months. Thiazoline release profiles differed in acidic and neutral media, but in both cases, the nanoemulsion controlled and prolonged the thiazoline delivery. Thiazoline nanoemulsion exerted in vitro antimalarial activity against the parasite (IC50 = 1.32 µM), and it significantly reduced the in vivo parasitemia for 8 days without increasing the survival time of animals. Therefore, the thiazoline nanoemulsion represents a strategy to treat malaria combining an antimalarial candidate and a new nanocarrier.

Improvement of antimalarial activity of a 3-alkylpiridine alkaloid analog by replacing the pyridine ring to a thiazole-containing heterocycle: Mode of action, mutagenicity profile, and Caco-2 cell-based permeability.

The development of new antimalarial drugs is urgent to overcome the spread of resistance to the current treatment. Herein we synthesized the compound 3, a hit-to­lead optimization of a thiazole based on the most promising 3-alkylpyridine marine alkaloid analog. Compound 3 was tested against Plasmodium falciparum and has shown to be more potent than its precursor (IC50 values of 1.55 and 14.7 µM, respectively), with higher selectivity index (74.7) for noncancerous human cell line. This compound was not mutagenic and showed genotoxicity only at concentrations four-fold higher than its IC50. Compound 3 was tested in vivo against Plasmodium berghei NK65 strain and inhibited the development of parasite at 50 mg/kg. In silico and UV-vis approaches determined that compound 3 acts impairing hemozoin crystallization and confocal microscopy experiments corroborate these findings as the compound was capable of diminishing food vacuole acidity. The assay of uptake using human intestinal Caco-2 cell line showed that compound 3 is absorbed similarly to chloroquine, a standard antimalarial agent. Therefore, we present here compound 3 as a potent new lead antimalarial compound.

Target-Guided Synthesis and Antiplasmodial Evaluation of a New Fluorinated 3-Alkylpyridine Marine Alkaloid Analog.

The need to develop new alternatives for antimalarial treatment is urgent. Herein, we report the synthesis and antimalarial evaluation of a small library of synthetic 3-alkylpyridine marine alkaloid (3-APA) analogs. First, the compounds were evaluated in vitro against Plasmodium falciparum. The most active compound 5c was selected for optimization of its antimalarial properties. An in silico approach was used based on pure ab initio electronic structure prediction, and the results indicated that a substitution of the hydroxyl group by a fluorine atom could favor a more stable complex with heme at a molecular ratio of 2:1 (heme/3-APA halogenated). A new fluorinated 3-APA analog was synthesized (compound 7), and its antimalarial activity was re-evaluated. Compound 7 exhibited optimized antimalarial properties (P. falciparum IC50 = 2.5 µM), low genotoxicity, capacity to form a more stable heme/3-APA complex at a molecular ratio of 2:1, and conformity to RO5. The new compound, therefore, has great potential as a new lead antimalarial agent.

Structure-based drug design studies of the interactions of ent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparum sarco/endoplasmic reticulum Ca2+-ATPase PfATP6

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosa were tested in silico against the Plasmodium falciparum Ca2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

Otimização da atividade antimalárica de análogos sintéticos de alcalóides 3-alquilpiridínicos utilizando uma abordagem teórica e experimental

Mais de três bilhões de pessoas vivem em áreas de risco de transmissão de malária. O desenvolvimento de novos compostos com ação antimalárica é uma necessidade urgente para combater a doença e diminuir a emergência de cepas resistentes de Plasmodium falciparum ao tratamento atual. Sem uma vacina eficaz, o controle da malaria é feito pela eliminação do mosquito vetor, o diagnóstico rápido e o tratamento dos pacientes acometidos. Nesse contexto, novas classes de compostos vêm sendo estudadas para o desenvolvimento de novos antimaláricos. Alcaloides 3-alquilpiridínicos (3-APA) são compostos sintéticos, análogos à Teoneladina C (natural, isolada de esponjas marinhas). Esses compostos possuem um anel piridínico e uma cadeia alquila na posição 3. As novas moleculas foram sintetizadas para a avaliação antimalárica devido às atividades biológicas já descritas para o alcaloide natural. Dos sete alcaloides inicialmente testados, o mais seletivo para P. falciparum (composto 5c, IC50=14,7µM IS=6,7) foi utilizado para posteriores otimizações e uma nova série de compostos foi sintetizadas, baseadas em estudos de modelagem molecular, que avaliaram a interação dos mesmos com a molécula de hematina. De maneira interessante, o composto 7 (com um átomo de flúor no final da cadeia alquila) apresentou IC50 (2,5µM) aproximadamente seis vezes menor que seu precursor com uma hidroxila na mesma posição (14,7µM). Esse composto foi mutagênico apenas em concentrações maiores que o dobro do IC50. Já o composto 3 (com a troca do anel tiazol no lugar do piridínico) apresentou atividade antiplasmodial quase 10 vezes maior (IC50 1,55µM) e a seletividade passou de 6,7 para 74,7 contra uma linhagem celular não tumoral. Dessa forma, o desenvolvimento e a otimização foram focados neste composto com anel tiazolínico. Esse novo composto não foi considerado mutagênico e o teste de genotoxicidade mostrou que apenas em concentrações mais de quatro vezes o IC50 houveram quebras no DNA. Composto 3 foi avaliado in vivo contra Plasmodium berghei cepa NK65. Houve redução de cerca de 65% da parasitemia no quinto dia avaliado na concentração de 50mg/Kg. Abordagens in silico apontaram que esse composto promove interação com a hematina, de maneira análoga à cloroquina. Dessa forma, foi avaliado por microscopia confocal que o mesmo possui a capacidade de alcalinizar o vacúolo digestivo, sugerindo que a atividade antimalárica possa ser análoga à da cloroquina, impedindo a formação do cristal inerte hemozoína. Foi avaliada também a penetração (uptake) do composto 3 em células intestinais Caco-2. Interessantemente, não houve diferença estatística quando comparado com o uptake para o antimalárico padrão cloroquina. Outras modificações foram feitas, tanto no anel quanto na cadeia alquila, e, de maneira interessante, alguns compostos possuem seletividade para linhagens tumorais, sendo considerados promissores candidatos ao desenvolvimento de compostos para o tratamento de câncer. Com essas otimizações, podemos concluir que o composto 3 é um potente candidato a antimalárico, com características de atividade antimalárica (in vitro e in vivo) e seletividade dentro dos parâmetros recomendados na literatura e, além disso, com modo de ação elucidado.