A novel and potent antileishmanial agent: In silico discovery, biological evaluation and analysis of its structure -activity relationships

2007 2007

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Abstract (summary)

Leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, currently afflicts 12 million people worldwide with two million new cases occurring annually. Without effective treatment, visceral leishmaniasis is associated with a near 100% fatality rate while other forms can be severely disfiguring and debilitating. Novel therapies are urgently needed to treat this disease, as current treatments possess negative attributes such as toxicity, expense, inconvenience, and loss of effectiveness due to resistance.

Research efforts directed by Karl Werbovetz have previously demonstrated the promising antileishmanial activity of several dinitroaniline sulfonamide compounds. These compounds selectively inhibit parasite tubulin and arrest cells in mitosis. In this study, an evaluation of the quantitative structure-activity relationship (QSAR) of this class of compounds was performed using Catalyst software to generate a three-dimensional pharmacophore. The pharmacophore highlighted the specific functionalities in the compounds that confer antileishmanial activity, namely an aliphatic-hydrophobic group, an aromatic-hydrophobic group, an aromatic functionality and a hydrogen-bond acceptor in specific regions of space. The pharmacophore was then used to search the Maybridge database containing approximately 55,000 commercially available drug-like compounds. Over 200 compounds were identified that fit the pharmacophore, and nineteen of the most promising hits were tested for antileishmanial activity. Two compounds, BTB 06237 and BTB 06256, were highly active with IC50 values against L. donovani amastigotes of 0.5 ± 0.2 and 2.3 ± 0.8 μM, respectively. Another five compounds were moderately active with IC50 values between 21 and 39 μM. As BTB 06237 was the most potent of the series, its activity was further evaluated.

Unlike the parent dinitroaniline sulfonamides, BTB 06237 did not inhibit parasite tubulin polymerization or cause the accumulation of parasites in the G2/M phases of the cell cycle. However, transmission electron microscopy and fluorescence microscopy have shown that the single parasite mitochondrion becomes dilated and fragments into intensely staining, disjoined spheres when treated with a cationic, lipophilic, mitochondrion-specific dye (MitoTracker Red 580) following incubation with BTB 06237. Overall uptake of MitoTracker Green FM was reduced in BTB 06237-treated parasites. This was similar to the reduction observed in parasites pre-treated with the uncoupler FCCP, which disrupts the mitochondrial membrane potential. These data imply that the compound disrupts mitochondrial structure and function. The effects correlated more closely with necrosis than with passive cell death when BTB 06237-treated parasites were assayed for apoptosis (annexin V binding and TUNEL staining). BTB 06237 also reduced parasite burdens in L. mexicana -infected J774 macrophages at low micromolar concentrations. The presence of nitro groups on this compound implies that it may increase the level of reactive radical species (RRS; reactive oxygen derivatives and nitric oxide) in the parasites through redox cycling. RRS assays on BTB 06237-treated parasites showed increased levels of oxidative burst, intracellular reactive oxygen species and nitric oxide.

In addition to the mechanistic evaluation of this compound, a structure-activity relationship study was performed by synthesizing 13 key analogs of BTB 06237, purchasing 3 others, and evaluating the antileishmanial activity of all 16 analogs. It was observed that antileishmanial potency was preserved regardless of the substituents on the phenylsulfanyl ring, but loss of aromaticity lead to loss of activity. The dinitrobenzyl ring, on the other hand, was less flexible, requiring two nitro groups and an additional electron withdrawing group for activity against the parasites.

Taken together, these results indicate that BTB 06237 is an intriguing lead compound against Leishmania that likely participates in redox cycling. The redox cycling then induces RRS inside the parasites, interfering with mitochondrial function, and ultimately causing death by necrosis. This work also demonstrates the utility of in silico methods for identifying lead compounds against Leishmania parasites.

Indexing (details)

0419: Pharmacology
0491: Pharmacology
0572: Pharmaceuticals
Identifier / keyword
Health and environmental sciences; Pure sciences; Antileishmanial; Drug discovery; Leishmania; Pharmacophore; Structure-activity relationships
A novel and potent antileishmanial agent: In silico discovery, biological evaluation and analysis of its structure -activity relationships
Delfin, Dawn Athelsia
Number of pages
Publication year
Degree date
School code
DAI-B 68/04, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
Werbovetz, Karl A.; Li, Pui-Kai
The Ohio State University
University location
United States -- Ohio
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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