Summary



The Drug Search for Leishmaniasis researchers recently conducted lab testing on 10 compounds. The testing showed that none of the compounds were good potential treatments, and the researchers will turn their attention to additional compounds.



Sandflies, such as the P. papatasi shown above, are responsible for the spread of leishmaniasis.

Short description of the team’s latest findings

Leishmaniasis is one of the most neglected tropical diseases in the world, infecting more than two million people in 98 countries. The current treatments for all forms of leishmaniasis can cause severe side effects, including death. Furthermore, drug resistant parasites are causing major problems in many countries. For these reasons, there is an urgent need for new, safe, and inexpensive drug compounds.

The Drug Search for Leishmaniasis team has continued their lab testing since their last update. The most recent round of testing involved 10 compounds that had been identified as having potential to be safer, more effective treatments.

The compounds were tested first for toxicity, then for effectiveness against two common parasites that can cause leishmaniasis. Based on the testing, none of the compounds tested would be effective treatments for the disease.

The researchers will make these results public, as they have done with their data to-date. This will alert other scientists to the strong possibility that these particular compounds are not effective against leishmaniasis, and help them make decisions about testing other compounds. Once the team has obtained additional funding, they will test additional compounds that may be useful in treating leishmaniasis.

Anyone interested in a full scientific description of this latest round of testing can read below. Thanks to everyone who supported this project.

In vitro evaluation of the anti-leishmanial activity of predicted molecules by docking

In order to determine if in silico predicted molecules with potential leishmanicidal activity could have the possibility of passing to in vivo assays, the molecules must first pass cytotoxicity testing against human cells in vitro. Then, those molecules that show low or no cytotoxicity are evaluated for parasite growth inhibition in human macrophages and the effective concentration 50 (EC 50 ). The EC 50 is the concentration of a molecule that kills 50% of the parasites in vitro.

Evaluation of Cytotoxicity

The cytotoxic activity of the compounds was evaluated on the human cell line U937 (CRL-1593-2 ™ of ATCC). For the evaluations, the cells were used in logarithmic phase of growth and were cultured in 96-well culture plates, at a concentration of 100,000 cells/mL for U937 in RPMI-1640 medium supplemented with 10% fetal bovine serum (SFB) and 1% antibiotics (penicillin-streptomycin) (Sigma). Six serial dilutions prepared from each of the following concentrations: 200 - 100 - 50 - 25.0 and 1 μg / mL were made according to the compound to be evaluated. The cells were incubated at 37°C with 5% CO 2 for 72 hours in the presence of the compounds and, subsequently, the effect was determined using the MTT enzyme method. This method uses a dye which live cells metabolize reducing the coloring, which is measured as "Optical Density" (OD). The plates were incubated at room temperature for another 30 minutes and the formazan production (change of color) was measured at 570 nm in a spectrophotometer. As a control of viability, cells cultured under the same incubation conditions were used in the absence of the compounds. Doxorubicin was used as cytotoxicity control.

The cytotoxicity was determined according to the percentage of decrease in viability and therefore, of the decrease in the number of cells obtained for each compound and doxorubicin, according to the OD values obtained in each experimental condition. The decrease in cell viability, was calculated using the OD values for each condition, i.e., compound or control at the evaluated concentration, using the following equation: % Viability = [OD cells exposed to the compound or control cell / OD cells not exposed] × 100). The values of OD obtained for the cells in the absence of compounds correspond to 100% viability or live cells. Then, with the viability percentages, the mortality percentage was calculated, which corresponds to 100% viability. With the mortality percentages, lethal concentration 50 (LC 50 ) was calculated by the Probit3 program. The cytotoxicity of each compound was classified according to the LC 50 values using a proprietary scale: high cytotoxicity LC 50 <50 μg/mL; moderate cytotoxicity: 50 < LC 50 < 200 μg/mL and low cytotoxicity: LC 50 > 200 μg/mL.

Table 1 shows the results of cytotoxicity, where it is observed that one compound showed low cytotoxicity while three had moderate cytotoxicity for the U937 human cell line. As expected, doxorubicin, included as a toxicity control, showed high cytotoxicity.

Table 1. Evaluation of the in vitro cytotoxicity

Name of Product CL 50 (µg/ml) X + SD Cellular Line U-937 Toxicity Level ZINC12005520 16.5 ± 0.8 High ZINC16626805 135.7 ± 2.9 Moderate ZINC17135526 12.8 ± 1.8 High ZINC08598759 3.4 ± 0.8 High ZINC32951205 3.7 ± 1.3 High ZINC32951223 27.1 ± 6.1 High ZINC08587552 0.5 ± 0.6 High ZINC08971918 65.6 ± 7.4 Moderate ZINC04777075 >200 Low ZINC18222288 53.8 ± 3.0 Moderate Doxorubicin (Control) 0.5 ± 0.1 High

Evaluation of Anti-Leishmanial Activity

Prior to the determination of the effective concentration 50 (EC 50 ), all the compounds were pre-selected, by evaluating the effect on the percentage of infection in intracellular amastigotes in the U-937 cell line compared with amastigotes controls, in the absence of the compound.

In this test of leishmanicidal activity in vitro, the fluorescent strains of Leishmania panamensis (UA140-pIR (-) - eGFP) and Leishmania braziliensis (UA301-pIR (-) - eGFP) were used.

The activity of the compounds was evaluated on intracellular parasites (amastigote stage) obtained after in vitro infection of macrophages. The U-937 cells were infected with fluorescent promastigotes in stationary growth phase in a 30:1 parasite:cell ratio for the Leishmania panamensis UA140 strain and 20:1 for Leishmania braziliensis UA301 strain. The infected cells were exposed different concentration of the compounds for 72 hours (see the concentrations used for each compound, in a note below the Table 2). As infection control, infected cells were used in the absence of the compounds, and amphotericin B was used as a positive control. After 72 hours of incubation, the cells were carefully removed from the bottom of the dish and analyzed in a flow cytometer, reading at 488 nm excitation and 525 nm emission with an Argon4 laser.

The anti-Leishmania activity was determined based on the parasite load, which is the number of parasites in the infected cells exposed to the concentration selected for each compound or amphotericin B. The decrease in parasite load, called inhibition of infection was calculated using the fluorescence mean intensity values (MFI) and using the following formula: % Infection = [MFI cells infected and exposed to the compound or amphotericin B / MFI infected of unexposed cells] × 100). The MFI values obtained for the infected cells in the absence of drug or compound corresponds to 100% of the infection. In turn, the percentage of inhibition of the infection corresponds to 100% of the infection -% infection in the presence of the compound.

Table 2. Evaluation of the percentage of inhibition obtained with the tested compounds in intracellular parasites.

Name of Product % Inhibition X + SD L. panamensis UA140 L. braziliensis UA301 ZINC12005520 19.0 ± 3.0 2.6 ± 0.5 ZINC16626805 0 17.3 ± 6.5 ZINC08598759 12.3 ± 1.9 38.6 ± 1.2 ZINC32951205 8.0 ± 0.9 29.4 ± 6.6 ZINC32951223 1.2 ± 0.4 0 ZINC08587552 6.6 ± 3.3 0.3 ± 0.5 ZINC08971918 0 3.0 ± 5.9 ZINC04777075 0 33.4 ± 4.2 ZINC18222288 0 14.3 ± 5.0 Amphotericin B-Control 75.9 ± 5.5 71.6 ± 5.1

The EC 50 was not determined for any of the molecules, because none of the compounds showed an inhibition percentage greater than 50% in the two Leishmania species used (See Table 2).

Conclusion