Synthetic analogs of a molecule found in plants of the species Piper malacophyllum, a member of the pepper family occurring only in the Southeast and South of Brazil (common name pariparoba-murta), displayed up to 40 times more potent anti-parasite activity than the standard drugs used to combat the protozoans that cause Chagas disease and visceral leishmaniasis.
The results of the Brazilian researchers’ in vitro assays, performed at the Federal University of São Paulo (UNIFESP) with FAPESP’s support, were reported in early August during a workshop on “The momentum and perspectives of drug discovery and development in Brazil,” held at the University of São Paulo’s Biomedical Science Institute (ICB–USP) by the Center for Applied Mass Spectrometry (CEMSA).
The findings were also published in the journal Bioorganic & Medicinal Chemistry Letters.
“A compound called gibbilimbol, with anti-parasite action, has been found in this plant, and our group has now synthesized more than 15 analogs. We made minor changes to the molecule’s structure to enhance its efficacy,” said João Paulo dos Santos Fernandes, a professor at UNIFESP’s Environmental, Chemical & Pharmaceutical Science Institute (ICAQF) and a co-author of the article.
Two versions of the molecule originally isolated from P. malacophyllum, gibbilimbol A and B, were characterized as part of a projectcoordinated by João Henrique Ghilardi Lago, also a professor at ICAQF and a co-author of the article. The project aims to find compounds with anti-parasite action in plants native to the Atlantic Rainforest.
The synthetic analogs with boosted activity were developed under Fernandes’s supervision during Marina Themoteo Varela’s scientific initiation project and Master’s research, both supported by FAPESP. Varela is the first author of the article.
The in vitro assays to evaluate the efficacy of the compounds against the parasites were led by Andre Gustavo Tempone Cardoso at Adolfo Lutz Institute (IAL) under a research grant from FAPESP. Cardoso is also a co-author of the article.
The first round of in vitro assays, which involved the natural compounds, showed that gibbilimbol B was more effective against the parasites than gibbilimbol A.
“When we studied the chemical structures of the two molecules, we found that the only difference between them was the position of a double bond, which was closer to the aromatic ring in gibbilimbol B. We then synthesized analogs with other substitutions close to the aromatic ring and additional functional groups designed to achieve specific interactions with parasite cells so as to boost the analogs’ activity,” Fernandes said.
Many of the synthetic analogs produced to date have proven more effective against the parasites in question than the natural compounds, he added. The molecule found most promising in the laboratory has been named LINS03003 by the researchers.
Besides these comparisons with natural prototypes, the researchers also performed tests to compare the performance of the synthetic analogs with that of the standard drugs used to combat Trypanosoma cruzi, the parasite that causes Chagas disease, and Leishmania infantum, which causes visceral leishmaniasis. “We used a method that measures potency in terms of IC50 values, meaning the amount of the compound necessary to kill 50% of the parasites. Generally speaking, values below 10 micromolar [μM] are considered promising,” Fernandes explained.
Against the amastigote form of T. cruzi (the intracellular morphological stage, which has to be combated in order to control chronic Chagas disease), LINS03003 proved 40 times more effective than benznidazole, the drug currently used to treat chronic Chagas. A concentration of only 5.5 μM eliminated 50% of the parasites. Against the trypomastigote (the infective stage, in which the protozoan migrates between host cells and the bloodstream), it was 26 times more effective than benznidazole.
“Another significant advantage we observed was low relative toxicity,” Fernandes said. “Our compound was four times more toxic to T. cruzi amastigotes than to human cells, meaning the dose needed to kill the parasites would have to be multiplied fourfold to kill 50% of the human host’s cells. In the case of benznidazole, this selectivity is the same or lower.”
In the study of L. infantum, the compounds were compared with miltefosine, used mainly in India to treat visceral leishmaniasis. LINS03003 was 10 times more potent against the amastigote (intracellular) stage, with only 1.8 μM eliminating 50% of the parasites. Against the promastigote (found in the insects that transmit the parasite and in the human host’s bloodstream after inoculation), its efficacy was similar to that of miltefosine, with an IC50 of 28 μM.
With regard to safety, LINS03003 was 13 times more toxic to the amastigote stage of L. infantum than to cultured human cells. This makes it roughly equivalent to miltefosine, which is 14 times more toxic to these parasites than to human cells.
Varela is currently developing new synthetic variants of gibbilimbol, also as part of her Master’s research, by modifying other portions of the molecule in search of a more effective and less toxic compound. The new analogs will also be tested in vitro and, later, in vivo.
“The compounds appear to be highly promising. They’re small and easy to synthesize. Their chemical structure points to low toxicity,” Fernandes said. “However, only the first tests in animals will show with any degree of certainty how they behave in a living organism: how they’re metabolized and whether they’re able to reach the parasite, among other factors.”