Biological effects of trans, trans-farnesol in Leishmania amazonensis

IntroductionFarnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. Candida albicans predominantly secretes the trans, trans-farnesol (t, t-FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including Candida albicans, and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added trans, trans-farnesol on the proliferative ability of Leishmania amazonensis and to identify its presence in the lipid secretome of the parasite.MethodsThe study involved the addition of exogenous trans, trans-farnesol to evaluate its interference with the proliferation of L. amazonensis promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of trans, trans-farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of trans, trans-farnesol and related products derived from the precursor, farnesyl pyrophosphate. In silico analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the Leishmania genome.ResultsExogenously added trans, trans-farnesol was found to interfere with the proliferation of L. amazonensis promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of trans, trans-farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the Leishmania genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production.DiscussionThe findings collectively offer the first insights into the mechanism of action of farnesol on L. amazonensis. While trans, trans-farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous trans, trans-farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in Leishmania. Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against Leishmania infections

Avaliação da utilização do colesterol exógeno por Leishmania amazonensis como possível alvo farmacológico

Made available in DSpace on 2016-03-28T12:39:39Z (GMT). No. of bitstreams: 2 vater_neto_ioc_mest_2009.pdf: 2942126 bytes, checksum: 50b2b2273e9c6f75bd2fa9a09c6548a3 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilTodos os fármacos atualmente em uso para o tratamento da leishmaniose apresentam restrições, como toxicidade, graves efeitos colaterais, custo elevado, administração parenteral ou teratogenicidade. Desta forma, o desenvolvimento de fármacos mais eficazes e seletivos é de suma importância e a identificação de vias metabólicas exclusivas do parasito que possam ser usadas como alvo pode ser um ponto de partida interessante. Acredita-se que a leishmania não dependa da utilização do colesterol exógeno para sua sobrevivência, uma vez que sintetiza seus próprios esteróis. Porém, apesar disso, um percentual expressivo de colesterol é encontrado em suas membranas sendo, em alguns casos, o esterol majoritário indicando um papel biológico para essa molécula. O presente trabalho visa estudar a importância para a L. amazonensis da utilização do colesterol proveniente do soro, em diversas situações, avaliando o potencial desse sistema como um possível alvo farmacológico. A atividade dos inibidores da biossíntese do ergosterol (cetoconazol, miconazol, terbinafina e sinvastatina) foi avaliada, na presença de soro normal ou deslipidado. Foi observada que a privação das lipoproteínas do soro potencializa o efeito dos inibidores do ergosterol. Os promastigotas tratados com esses inibidores mostraram diferenças na sua composição lipídica, com acúmulo de colesterol nas células tratadas, principalmente, com cetoconazol e miconazol, indicando um possível mecanismo de compensação da leishmania, para suprir a inibição do ergosterol. Experimentos com LDL-I125, no qual tanto a leishmania tratada com cetoconazol, quanto a tratada com sinvastatina, que inibe o início da via de biossíntese de ergosterol, aumentaram a captação da LDL A suramina, um inibidor da captação de LDL, mostrou uma diminuição do conteúdo de colesterol da leishmania e, em associação com a sinvastatina, obteve um efeito sinérgico, mostrando que o conteúdo da LDL, principalmente colesterol, pode estar envolvido na manutenção da integridade da membrana da célula. Em adição, a resistência ao cetoconazol induzida in vitro foi estudada. Foi observada uma relação direta entre o aumento da expressão gênica da C14-desmetilase com o nível de resistência induzido em promastigotas de L. amazonensis, sugerindo que esse seja o principal mecanismo de resistência a essa classe de fármacos. De uma maneira geral, a análise dos resultados como um todo sugere que o colesterol desempenhe um importante papel na atividade dos inibidores da biossíntese do ergosterol e que o bloqueio da sua utilização pode ser um possível alvo farmacológicoAll drugs currently in use for the treatment of lei shmaniasis present restrictions, such as toxicity, serious side effects, high cost, paren teral administration or teratogenicity. Thus, the development of more effective and selecti ve drugs is a priority and the identification of exclusive metabolic pathways of t he parasite that can be used as a target could be an interesting starting point. It i s believed that leishmania is not dependent on the use of exogenous cholesterol for s urvival, since it synthesizes its own sterols. But, nevertheless, a significant perce ntage of cholesterol is found in its membranes and, in some cases, as the major sterol, indicating a biological role for this molecule. This work aims to study the importance fo r L. amazonensis of the use of cholesterol from the serum in various situations, a ssessing the potential of this system as a possible drug target. The activity of the ergo sterol biosynthesis inhibitors (ketoconazole, miconazole, terbinafine and simvasta tin) was evaluated in the presence of normal or delipidated serum. It was observed tha t the deprivation of the serum lipoprotein potentiates the effect of inhibitors of ergosterol. The promastigotes treated with these inhibitors showed differences in their lipid composition, with accumulation of cholesterol in treated cells, mainl y with ketoconazole and miconazole, suggesting a compensation mechanism in leishmania, which may overcome ergosterol inhibition. Experiments with LDL-I 125 , indicated an increased uptake of LDL in leishmania treated with either ketoconazole or simv astatin, the latter inhibits an early step in the ergosterol biosynthectic pathway. Suram ine, an inhibitor of the uptake of LDL, showed a decrease in cholesterol content of th e leishmania and in combination with simvastatin, had a synergistic effect, showing that content of LDL, especially cholesterol, could be involved in maintaining the c ell membrane integrity. In addition, resistance to ketoconazole induced in vitro was studied. There was a direct relationship between increased gene expression of C 14-desmetilase and the level of resistance induced in promastigotes of L. amazonensis , suggesting that this is the main mechanism of resistance to this class of drugs. Tak en together, these results suggest that cholesterol plays an important role in the act ivity of ergosterol inhibitors biosynthesis and that the blocking of its use by le ishmania may be a possible drug target

Papel do colesterol exógeno nos mecanismos de adaptação de Leishmania spp a condições de estresse metabólico e farmacológico

Made available in DSpace on 2016-03-15T14:19:05Z (GMT). No. of bitstreams: 2 valter_neto_ioc_dout_2013.pdf: 5032159 bytes, checksum: 09d4b19d84fdc8d6bc1295312fe03ccd (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilOs tripanossomatídeos não sintetizam o colesterol e sim esteróis com o esqueleto ergostano, porém um percentual significativo de colesterol exógeno é encontrado em todas as espécies de Leishmania, sugerindo um papel biológico para esta molécula. Esta tese tem como objetivo estudar a importância do uso de colesterol para Leishmania spp. em várias situações, avaliando o potencial deste sistema como um possível alvo farmacológico. A atividade dos inibidores de biossíntese de ergosterol associado com inibidores de transporte de colesterol derivado de LDL, foi avaliada em promastigotas e amastigotas intracelulares. A associação entre LBqT01 e cetoconazol, miconazol ou terbinafina mostrou sinergia. A associação entre a imipramina ou progesterona e cetoconazol ou terbinafina indicaram um efeito aditivo. O cetoconazol e miconazol demonstraram uma diminuição de até duas vezes o valor de IC50 nas formas amastigotas, quando combinado com os inibidores de transporte de colesterol. Foi observado também alteração da biossíntese de ergosterol após tratamento dos parasitos com os inibidores de transporte de colesterol, demonstrado por CG/MS. A combinação de LBqT01 e cetoconazol mostrou ser mais ativa in vivo do que cada fármaco individualmente. Estudamos também o mecanismo de resistência desses inibidores, avaliando a modulação de enzimas da via de biossíntese de esteróis e a utilização de colesterol exógeno pelos parasitos. Promastigotas de Leishmania amazonensis, Leishmania braziliensis e Leishmania guyanensis foram cultivadas com concentrações crescentes de sinvastatina, terbinafina e miconazol Estes inibidores mostraram um índice de resistência de 2,5 - 8 vezes. A resistência cruzada também foi avaliada, com estes inibidores e fármacos de referência (miltefosina, anfotericina B e antimônio trivalente). A expressão de genes da biossíntese de esterol e utilização do colesterol exógeno entre as cepas selvagens e resistentes, foram avaliadas por PCR em tempo real e CG/MS, respectivamente. As enzimas HMGCoA e C -14 desmetilase foram as mais moduladas, independente do fármaco utilizado, variando sua expressão nas cepas resistentes. Promastigotas de L. braziliensis resistentes à terbinafina apresentaram alteração no perfil de esteróis, L. amazonensis resistente aos três inibidores e L. guyanensis resistente ao miconazol mostraram alteração da biossíntese de esteróis e aumento na absorção do colesterol exógeno. Os resultados mostram que alguns genes foram amplificados na cepa resistente em relação à cepa selvagem. Juntos estes resultados sugerem que o colesterol desempenha um papel importante na atividade e resistência aos inibidores da biossíntese de esteróis e que o bloqueio da sua utilização pela Leishmania spp. pode ser um alvo para a síntese de novos fármacos para o tratamento da leishmanioseThe trypanosomes do not synthesize cholesterol sterols but with ergostane skeleton, but a significant percentage of exogenous cholesterol is fou nd in all species of Leishmania , suggesting a biologica l rol e for this molecule. This work aims to study the importance of use cholesterol to Leishmania spp . in several cases , evaluating the potential of the s ystem as a possible drug target . The activity of the inhibitors of ergosterol biosynthesis inhibitors associated with transport of LDL cholesterol derivative was evaluated in intracellula r amastigotes and promastigotes . The association between LBqT01 and ketoconazole , miconazol e or terbinafine showed synergy . The association bet ween imipramine or progester one, and ketoconazole , or terbinafine indica ted an additive effect . The ketoconazole and miconazole showed a reduction of up to twice the IC50 value in amastigotes when combined with the inhi bitors of cholesterol transport . Change of ergosterol biosynthesi s of parasites after treatment with inhibitors of cholesterol transport as demonstrat ed by GC/MS was also observed . The combination of LBqT01 and ketoconazole was more active in vivo than either drug individually. We also studied the mechanism of resistanc e of these inhibitors by evaluating the modulation of enzymes of the sterol biosynthesis pathway and use of exo genous cholesterol by parasites . Promastigotes of Leishmania amazonensis , Leishmania braziliensis and Leishmania guyanensis were cultured with in creasing concentrations of simvastati n, terbinafine and miconazole . These inhibitors showed resis tance index from 2.5 to 8 times . Cross - resistance was evaluated with these inhibitors an d reference drugs (miltefosine , amphot ericin B and trivalent antimony) . The expression of sterol biosynthesis genes and use of exogenous cholesterol in the wild and resistant strains were analyz ed by real - time PCR and GC / MS , respectively. The HMGCoA C and - 14 demethyl ase enzymes were more modulated, regardless of the drug u sed , varying its expression in resistant strains . Promastigotes of L. braziliensis resistant to terbinafine pres ented changes in sterol profile, L. amazonensis resistant to all three inhibitors amazonensis and L. guyanensis miconazole resistant showed alte rations in the biosynthesis of sterols and increase in the absorption of exogenous cholesterol. The results show that some genes were amplified in the resistant strain c ompared to the wild type strain . Together these results suggest that cholesterol plays an important role in the activity and resistance to inhibitors of sterol biosynthesis and blocking their use Leishmania spp . may be a target for the synthesis of new drugs for the treatment of leishmaniasis

Oral effectiveness of PMIC4, a novel hydroxyethylpiperazine analogue, in Leishmania amazonensis

Made available in DSpace on 2015-05-04T16:34:36Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) claudia_levyetal_IOC_2014.pdf: 620953 bytes, checksum: e1bae1d82f29d4be01678c3fa2ec7a2c (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Síntese de Fármacos. Rio de Janeiro, RJ, Brasil.Universidade Federal de Pelotas. Centro de Ciências Químicas, Farmacêuticas e de Alimentos. Laboratório de Química Aplicada à Bioativos. Pelotas, RS, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Síntese de Fármacos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Síntese de Fármacos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Pentavalent antimonials have saved the lives of thousands of Leishmania-infected patients more than seventy years but, unfortunately, they are highly toxic and require parenteral delivery. Therefore, the search for safer and orally delivered alternative is a need. This paper describes the antileishmanial properties of PMIC4, a novel hydroxyethylpiperazine analogue. PMIC4 showed potent activity against intracellular amastigotes of Leishmania amazonensis, with IC50 of 1.8 lM and selectivity index higher than 100-fold, calculated in relation to the toxicity on the host cell. Following laboratory animal welfare policies, we analyzed the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties and calculated the Lipinski’s rule of five of PMIC4 before proceeding to in vivo tests. PMIC4 satisfied Lipinski’s rule of five and presented high probability of human intestinal absorption, suggesting a good chance of druglikeness and oral bioavailability. For in vivo studies, PMIC4 was administered via intralesional injection (3.4 mg/kg/day, three times a week) or orally (34.0 mg/kg/day, five times a week) to L. amazonensisinfected BALB/c mice throughout the 98 day experiments. At the end of the treatment period, serum markers of toxicity were measured. When administered orally, PMIC4 controlled the lesions in L. amazonensis- infected BALB/c mice without altering serological markers of toxicity. These results demonstrate that PMIC4 is a promising molecular scaffold, orally effective against experimental leishmaniasis

Effect of 3-Carene and the Micellar Formulation on <i>Leishmania (Leishmania) amazonensis</i>

Leishmaniases are neglected tropical diseases caused by obligate intracellular protozoa of the genus Leishmania. The drugs used in treatment have a high financial cost, a long treatment time, high toxicity, and variable efficacy. 3-Carene (3CR) is a hydrocarbon monoterpene that has shown in vitro activity against some Leishmania species; however, it has low water solubility and high volatility. This study aimed to develop Poloxamer 407 micelles capable of delivering 3CR (P407-3CR) to improve antileishmanial activity. The micelles formulated presented nanometric size, medium or low polydispersity, and Newtonian fluid rheological behavior. 3CR and P407-3CR inhibited the growth of L. (L.) amazonensis promastigote with IC50/48h of 488.1 ± 3.7 and 419.9 ±1.5 mM, respectively. Transmission electron microscopy analysis showed that 3CR induces multiple nuclei and kinetoplast phenotypes and the formation of numerous cytosolic invaginations. Additionally, the micelles were not cytotoxic to L929 cells or murine peritoneal macrophages, presenting activity on intracellular amastigotes. P407-3CR micelles (IC50/72 h = 0.7 ± 0.1 mM) increased the monoterpene activity by at least twice (3CR: IC50/72 h >1.5 mM). These results showed that P407 micelles are an effective nanosystem for delivering 3CR and potentiating antileishmanial activity. More studies are needed to evaluate this system as a potential therapeutic option for leishmaniases

Monocyclic Nitro-heteroaryl Nitrones with Dual Mechanism of Activation:Synthesis and Antileishmanial Activity

[Image: see text] 5-Nitro-furan nitrones (1) and 5-nitro-thiophene nitrones (2) were synthesized in one step. Compounds 1a–c had the most potent leishmanicidal activity against intracellular amastigote forms of Leishmania amazonensis and L. infantum (from 0.019 to 2.76 μM), with excellent selectivity (from 39 to 5673). The comparison of the leishmanicidal activity in promastigotes of wild type L. donovani with those overexpressing nitroreductases NRT1 or NRT2 shows that 1a,b are activated by both, which could slow the development of resistance. Their redox potential (E(redox)) obtained by cyclic voltammetry (−0.67 and −0.62 V) shows that the reduction of the nitro group is modulated by the nitrone group. Oral administration of 1b to mice infected by L. infantum reduced the parasite load on the spleen by 76.6 and 95.0% with doses of 50 and 100 mg/kg, respectively, administered twice a day, for 5 days. In the liver, the parasite load suppression was above 75% with either treatment

Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization, In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07 &plusmn; 0.20; 0.83 &plusmn; 0.12; 2.02 &plusmn; 0.27 and 5.83 &plusmn; 1.2 &micro;M against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 &micro;M). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of &gt;100 &micro;M for intracellular amastigotes, as well as against axenic amastigotes (94.12 &plusmn; 2.8 &micro;M for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets