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CIMB, Vol. 44, Pages 2089-2106: Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization, In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor

Current Issues in Molecular Biology doi: 10.3390/cimb44050141

Authors:
Pollyanna Stephanie Gomes
Monique Pacheco Duarte Carneiro
Patrícia de Almeida Machado
Valter Viana de Andrade-Neto
Alessandra Marcia da Fonseca-Martins
Amy Goundry
João Vitor Marques Pereira da Silva
Daniel Claudio Oliveira Gomes
Ana Paula Cabral de Araujo Lima
Vítor Ennes-Vidal
Ana Carolina Rennó Sodero
Salvatore Giovanni De-Simone
Herbert L. de Matos Guedes

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 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 µM). 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 >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM 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.

Avocado toast has become the favored breakfast of the healthy and fit, and now new research suggests their choice may protect their hearts.

People who ate half an avocado twice a week had a 16% lower risk of cardiovascular disease and a 21% lower risk of heart disease, compared with people who never or rarely ate the fruit, researchers found.

“This study provides further evidence that the intake of plant-sourced unsaturated fats can improve diet quality and is an important component in cardiovascular disease prevention in the general population,” says lead researcher Lorena Pacheco, a postdoctoral research fellow in the Department of Nutrition at the Harvard T.H. Chan School of Public Health in Boston.

“Our results are timely, since the nationwide consumption of avocado has risen steeply in the U.S. in the last 20 years,” she notes.

This type of observational study cannot prove definitively that eating avocados lowered the risk for cardiovascular disease, only that there might be a connection, Pacheco cautions. Funding for the study came from the U.S. National Institutes of Health and the U.S. National Institute of Diabetes and Digestive and Kidney Diseases.

Why avocados are good for the heart

Avocados are rich in dietary fiber, unsaturated fats like monounsaturated fat (healthy fats) and other components that have been linked with good cardiovascular health.

Avocados can be a part of a heart-healthy diet, Pacheco shares, but “it is certainly not a magical bullet in itself.”

The findings may be also skewed because participants themselves reported the amount of avocado they ate, so some may have misremembered.

For the study, Pacheco and her colleagues collected data on more than 110,000 men and women who took part in the Nurses’ Health Study (nearly 69,000 women) and the Health Professionals Follow-up Study (nearly 42,000 men).

Over more than 30 years of follow-up, more than 9,100 participants developed heart disease and more than 5,200 suffered a stroke.

The study also found that replacing half a serving daily of margarine, butter, egg, yogurt, cheese or processed meats with