Gustavo Benaim

Leishmaniasis is a parasitic human infection that constitutes a major global health challenge due to limited therapeutic options, toxicity, resistance, and high costs of the drugs approved as therapy. In this study, a group of 7‐chloroquinoline derivatives ( 5 – 12 ) was synthesized via an optimized route and evaluated against the human parasite Leishmania mexicana . Compounds were fully characterized and showed favorable in silico ADME/Tox profiles, including high predicted gastrointestinal absorption (>93%), although limited solubility and potential hepatotoxicity were observed for several derivatives. Biological assays demonstrated significant antileishmanial activity. Interestingly, intracellular amastigote assays revealed nanomolar potency and very high selectivity index (SI), with compound 12 exhibiting IC ₅₀ = 2.5 nM (SI = 21.030) and compound 9 IC ₅₀ = 5.2 nM (SI = 7.384). Mechanistic studies demonstrated concentration‐dependent mitochondrial membrane depolarization and acidocalcisome alkalinization. Since these two organelles are both involved in bioenergetics and in parasite intracellular Ca ²⁺ homeostasis, the results support the notion that intracellular Ca ²⁺ disruption is an essential target for different drug actions. Overall, these findings support a multitarget mechanism and position these quinoline derivatives as promising chemical scaffolds for the development of improved antileishmanial strategies.

Los parásitos Trypanosoma cruzi y Leishmania spp. generan enfermedades conocidas como enfermedad de Chagas y Leishmaniasis, respectivamente, que representan un grave problema de salud pública a nivel mundial. Los tratamientos para ambas enfermedades presentan problemas de toxicidad e ineficacia importantes, por lo que es necesario desarrollar tratamientos más eficaces. En nuestro laboratorio hemos venido trabajando en los últimos años en la búsqueda de nuevas drogas con potencial terapéutico contra la enfermedad de Chagas y la Leishmaniasis, concentrándonos en la reutilización de drogas aprobadas y utilizadas en combinación con otras. Entre las drogas con las que hemos trabajado se encuentran la miltefosina, la amiodarona y sus derivados, la pentamidina, el posaconazol y otros azoles, y el SQ 109. Este último, en Fase III para el tratamiento de la tuberculosis resistente, ha tomado mucha importancia recientemente, ya que es capaz de tener un efecto letal en varios otros organismos parasitarios, incluyendo al Trypanosoma cruzi y Leishmania spp. Un denominador común que hemos encontrado en todas estas drogas es que actúan perturbando la homeostasis intracelular del Ca2+ en estos parásitos. Este hecho es relevante, ya que hemos demostrado que la regulación intracelular de este catión en tripanosomatidios parásitos difiere de manera importante respecto de la de los humanos, lo cual tiene potencial importancia desde el punto de vista terapéutico. En este trabajo revisaremos el efecto de las diferentes drogas mencionadas en cuanto a su acción tripanocida y/o leishmanicida, así como su mecanismo de acción, señalando su efecto sobre la homeostasis intracelular del Ca2+.

Chalcones constitute a distinct family of compounds with intrinsic fluorescence properties that make them valuable tools for a variety of applications. In this work, a new chalcone core derivative, ( E )-1-(4-aminophenyl)-3-(1-benzyl-pyrrol-2-yl) prop-2-en-1-one, was synthesized via a Claisen–Schmidt condensation reaction and fully characterized by IR, NMR spectroscopy and GC-MS analysis. Also was subsequently subjected to comprehensive photophysical and computational studies. For the photophysical characterization, the molar absorptivity coefficients and fluorescence quantum yields in methanol, acetonitrile, and chloroform were determined, along with an analysis of solvatochromic and solvatofluorochromic effects in different solvents and the assessment of the Stokes shift. The compound exhibited positive solvatochromism and solvatofluorochromism, providing valuable information on its polarity-dependent behavior. Computational analysis based on density functional theory elucidated the energy gaps between the boundary molecular orbitals and the electrostatic potential map, which corresponded to the structure of the compound and the trends observed in the photophysical study.

We investigated the effects of the tuberculosis drug candidate SQ109 (8a) and of its analog MeSQ109 (8b) against Leishmania mexicana in promastigote and amastigote forms and against host cell macrophages finding potent activity (1.7 nM) for MeSQ109 against the intracellular forms, as well as low toxicity (∼61 μM) to host cells, resulting in a selectivity index of ∼36,000. We then investigated the mechanism of action of MeSQ109, finding that it targeted parasite mitochondria, collapsing the proton motive force, as well as targeting acidocalcisomes, rapidly increasing the intracellular Ca²⁺ concentration. Using an E. coli inverted membrane vesicle assay, we investigated the pH gradient collapse for SQ109 and 17 analogs, finding that there was a significant correlation (on average, R = 0.67, p = 0.008) between pH gradient collapse and cell growth inhibition in Trypanosoma brucei, T. cruzi, L. donovani, and Plasmodium falciparum. We also investigated pH gradient collapse with other antileishmanial agents: azoles, antimonials, benzofurans, amphotericin B, and miltefosine. The enhanced activity against intracellular trypanosomatids is seen with Leishmania spp. grown in macrophages but not with Trypanosoma cruzi in epithelial cells and is proposed to be due in part to host-based killing, based on the recent observation that SQ109 is known to convert macrophages to a pro-inflammatory (M1) phenotype.

Ca²⁺ plays a crucial role in signaling pathways in all eukaryotic cells, including trypanosomatids. These represent a large family of parasites including the causative agents of several human infectious diseases, such as Chagas' disease and leishmaniasis. Accordingly, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) is subject to rigorous regulation. In these parasites, the cytosolic concentration is maintained at approximately 100 nM by various intracellular organelles, including the single mitochondrion, the endoplasmic reticulum, and acidocalcisomes, which as compartments, are limited to capacity confines. It is therefore the responsibility of plasma membrane mechanisms to ensure the long-term regulation of [Ca²⁺]_i, whereas a plasma membrane Ca²⁺ channel is responsible for Ca²⁺ entry and a Ca²⁺-ATPase regulates extrusion. However, the identification of this channel has remained a challenge until the ligand that induces its opening was identified: the sphingolipid sphingosine. Miltefosine, the only oral medication currently approved for the treatment of leishmaniasis, has been shown to mimic sphingosine. This review outlines the history of the trypanosomatid Ca²⁺ channel, beginning with its initial discovery and concluding with its incorporation into giant liposomes. This enabled the channel to be characterized by electrophysiological studies using "patch clamp" techniques. These studies revealed similarities and significant differences when compared with the human orthologue, which could be exploited for therapeutic purposes. Given that previous research has indicated the potential existence of an L-type VGCC in various trypanosomatids, we conducted a comparative analysis of putative genomic sequences, which demonstrated that, despite the low level of primary identity, this Ca²⁺ channel exhibits functional and structural homology with the mammalian counterpart.

We investigated the effects of the tuberculosis drug candidate SQ109 ( 8a ) and of its analog MeSQ109 ( 8b ) against Leishmania mexicana in promastigote and amastigote forms, as well as against host cell macrophages finding potent activity (1.7 nM) for MeSQ109 against the intracellular forms, as well as low toxicity (∼61 µM) to host cells, resulting in a selectivity index of ∼36,000. We then investigated the mechanism of action of MeSQ109 finding that it targeted parasite mitochondria, collapsing the proton motive force, as well as targeting acidocalcisomes, rapidly increasing the intracellular Ca ²⁺ concentration. Using an E. coli inverted membrane vesicle assay, we investigated the pH gradient collapse for SQ109 and 17 analogs finding that there was a significant correlation (on average R=0.67, p∼0.008) between pH gradient collapse and cell growth inhibition in Trypanosoma brucei, T. cruzi, L. donovani and Plasmodium falciparum . We also investigated pH gradient collapse with other anti-leishmanial agents: azoles, antimonials, benzofurans, amphotericin B and miltefosine. The enhanced activity against intracellular trypanosomatids is seen with Leishmania spp. grown in macrophages but not with Trypanosoma cruzi in epithelial cells and is proposed to be due in part to host-based killing, based on the recent observation that SQ109 is known to convert macrophages to a pro-inflammatory (M1) phenotype.

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca²⁺ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca²⁺ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca²⁺ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine’s therapeutic effect extends beyond its impact on the parasite to also positively affect the host’s immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas Disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca2+ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca2+ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca2+ channel that responds to sphingosine, that is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine's therapeutic effect extends beyond its impact on the parasite to also affect positively the host's immune system. These findings enhance our understanding on its multi targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

A series of heterocyclic chloroquine hybrids, containing a chain of two carbon atoms at position four of the quinolinic chain and acting as a link between quinoline and several benzoyl groups, is synthesized and screened in vitro as an inhibitor of β-hematin formation and in vivo for its antimalarial activity against chloroquine-sensitive strains of Plasmodium berghei ANKA in this study. The compounds significantly reduced haeme crystallization, with IC₅₀ values < 10 µM. The values were comparable to chloroquine’s, with an IC₅₀ of 1.50 ± 0.01 µM. The compounds 4c and 4e prolonged the average survival time of the infected mice to 16.7 ± 2.16 and 14.4 ± 1.20 days, respectively. We also studied the effect of the compounds 4b, 4c, and 4e on another important human parasite, Leishmania mexicana, which is responsible for cutaneous leishmaniasis, demonstrating a potential leishmanicidal effect against promasigotes, with an IC₅₀ < 10 µM. Concerning the possible mechanism of action of these compounds on Lesihmania mexicana, we performed experiments demonstrating that these three compounds could induce the collapse of the parasite mitochondrial electrochemical membrane potential (Δφ). The in vitro cytotoxicity assays against mammalian cancerous and noncancerous human cell lines showed that the studied compounds exhibit low cytotoxic effects. The ADME/Tox analysis predicted moderate lipophilicity values, low unbound fraction values, and a poor distribution for these compounds. Therefore, moderate bioavailability was expected. We calculated other molecular descriptors, such as the topological polar surface area, according to Veber’s rules, and except for 2 and 4i, the rest of the compounds violated this descriptor, demonstrating the low antimalarial activity of our compounds in vivo.