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#1 WCG Drug Search for Leishmaniasis

Post by Alez »

Drug Search for Leishmaniasis

Project Status and Findings:
Information about this project is provided on the web pages below and by the project scientists on the Drug Search for Leishmaniasis website. If you have comments or questions about this project, please visit the Drug Search for Leishmaniasis forum.

Mission
The mission of Drug Search for Leishmaniasis is to identify potential molecule candidates that could possibly be developed into treatments for Leishmaniasis. The extensive computing power of World Community Grid will be used to perform computer simulations of the interactions between millions of chemical compounds and certain target proteins. This will help find the most promising compounds that may lead to effective treatments for the disease.

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Significance
Leishmaniasis is one of the most neglected tropical diseases in the world. Each year this disease infects more than two million people in 97 countries. To date, there are no available vaccines to prevent the disease, in spite of multiple research efforts. Leishmaniasis is caused by a protozoan parasite (genus Leishmania) transmitted between human and animal hosts by female sand flies. One form of the disease, the "visceral" form caused by Leishmania infantum in America, mainly affects children, who can die if adequate treatment is not provided promptly. Existing control measures rely upon drug therapy, insect control and education in the affected communities. However, the number of human cases continues to increase in tropical countries such as Bangladesh, India, Sudan, Ethiopia, Brazil, Colombia, Peru and many others.

The classical treatments for all forms of Leishmaniasis can cause severe side effects, including death. Furthermore, drug resistant parasites are causing major problems in many endemic countries. For these reasons, there is an urgent need for new, safe and inexpensive anti-Leishmania drug compounds.

Approach
A software program called VINA from The Scripps Research Institute in La Jolla, California, will be used to perform the virtual chemistry experiments. These virtual experiments will search to find which of millions of drug compounds might be able to disable particular proteins, essential for the parasite's survival. Screening for the best potential drug compounds is an early step in the process of developing effective treatments for the disease. With enough computing power, this screening can be done much more quickly than using conventional laboratory experiments. However, existing computer facilities available to the researchers would require approximately 120 years to perform the screening. The power of World Community Grid can reduce the time required to less than one year. Information about the best candidate compounds will be published by the scientists, and this information will be available in the public domain for other scientists to build upon with their research. Further laboratory work using the best candidates identified by this project could lead to the development of better drugs to fight Leishmaniasis.
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#2 Re: WCG Drug Search for Leishmaniasis

Post by Alez »

About the Project

Leishmaniasis is a tropical disease caused by a parasite transmitted by specific insects. Infections of this disease have been increasing - with over two million people affected last year. Existing treatments can have severe side effects, including death. Currently, pharmaceutical companies have not been investing in extensive research to combat this disease. Therefore, the researchers at the University of Antioquia in Medellín, Colombia, are running a project on World Community Grid to search for chemical compounds which may lead to new drugs for treating this disease.

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Leishmaniasis is caused by a single celled protozoan parasite. The genus of this protozoan is Leishmania. It is transmitted between human and animal hosts via the female sand fly. In the Americas, the genus of the sand fly is Lutzomyia and elsewhere it is Phlebotomus. The insect injects humans or other animals with promastigotes, the infective stage of the parasite. Once injected into the skin, the promastigote infects immune system cells such as macrophages and other mononuclear phagocytic cells. Within these cells, the promastigote transforms into the tissue stage of the parasite, known as amastigote, which multiplies inside the cell by simple division, moving on to infect other phagocytic mononuclear cells. Various factors of the parasite and host determine which form of the diseases appears in the host. The insects become infected by sucking infected cells of the host during a blood meal. In the insect´s gut, the cells rupture releasing amastigotes, which are transformed back into promastigotes. They multiply and develop in the insect's gut. After several days, depending on the species, the parasites migrate to the mouthparts of the insect, where they are ready again to be transmitted to a host, during the next blood meal.

The disease has three clinical forms:

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  • cutaneous - affecting the surface skin consisting mostly of ulcerated lesions, warty lesions or spots.
  • mucocutaneous - affecting mucous membranes, particularly from the nose, laryngeal and pharynx.
  • visceral - affecting bone marrow or internal organs, such as the liver, spleen and lymph nodes. Symptoms can include anemia, clotting problems, weight loss, enlarging of the spleen, liver and lymph nodes.

The classical treatments for all forms of Leishmaniasis are certain compounds of pentavalent antimony (e.g. sodium stibogluconate and meglumine antimoniate). These compounds can have severe side effects, including death. Furthermore, drug resistant parasites are causing major problems in many endemic countries. Several additional drugs such as Pentamidine and Amphotericin B have been used with variable success, but these drugs also have serious side effects and are expensive and difficult to administer, limiting their use as drugs of choice. More recently, Miltefosine (an oral drug) has been used with variable success in Central and South America against cutaneous Leishmaniasis and for visceral Leishmaniasis in India. A phase IV trial of this drug in India has shown an increase in the relapse rate, indicating that drug resistance may develop quickly. The visceral form mainly affects children, who can die if adequate treatment is not provided promptly.

The complete genomes of several Leishmania species have been decoded and are providing information about proteins and processes essential for the survival of the parasite. Certain Leishmania proteins have been identified as targets using information about the genomes and through prior laboratory experiments and computational work. If drugs can be developed to disable these proteins, they may prove to be an effective treatment for the disease. The first step in drug development is to find chemical compounds which attach to the target protein in a manner that disables the protein's function, thus preventing the progression of the disease. To accelerate the search for potential drugs against Leishmaniasis, the computing power of World Community Grid will be used to screen millions of potential chemical compounds as possible drug treatment candidates. Instead of performing expensive and time-consuming laboratory experiments, simulations of these millions of experiments will be performed using the software running on World Community Grid's member computers.

A software program called VINA from The Scripps Research Institute in La Jolla, California, will be used to perform the virtual chemistry experiments, more precisely known as molecular dockings. Molecular docking is the process of determining how well two chemical compounds (molecules) bind together. One of the molecules is designated as the target, in this case one of several proteins, essential for the parasite's survival. The other compound is from a collection of millions of compounds from various drug data bases, cataloging known compounds and their exact atomic structure. The docking experiments position the two compounds in all possible orientations and then compute the binding energy, which tells how well they stick together. If a compound binds to the target protein, it may be useful in disabling the function of that protein and thus reducing parasite multiplication and the progress of the disease.

The VINA calculations will be used to identify the most promising chemical compounds that may inhibit these proteins. The computer computations involved are very intensive and would take about 120 years to test the 12 million compounds against 70 Leishmania proteins, if using machines normally available to the researchers. World Community Grid will be able to reduce the time required to less than one year. Information about the best candidate compounds will be published by the scientists, and this information will be available in the public domain for other scientists to build upon with their research. Further laboratory work using the best candidates identified by the VINA computations could lead to the development of better drugs to fight Leishmaniasis.
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#3 Re: WCG Drug Search for Leishmaniasis

Post by Alez »

Researchers Publish Findings on Potential New Treatments for Leishmaniasis
By: Dr. Carlos Muskus López
Coordinator, Molecular Biology and Computational Unit, PECET University of Antioquia
8 Sep 2016

Summary
The Drug Search for Leishmaniasis team recently published their findings in the Journal of Computer-Aided Molecular Design. Using World Community Grid's computing power, they have identified several drug compounds which may lead to improved treatments for this neglected and sometimes deadly disease.

One of the best ligands bound to the dihydrorootate dehydrogenase (DHODH) from Leishmania major.
Image (a) Predicted binding within the crystal structure
(b) Superimposition of the ligand conformations predicted to bind to each of the DHODH protein conformations extracted from the MD simulation

Background

Leishmaniasis, one of the most neglected tropical diseases in the world, infects more than two million people every year. The disease is caused by a parasite (genus Leishmania) which is transmitted between humans and animals by female sand flies. The number of cases continues to increase in tropical countries such as Bangladesh, India, Sudan, Ethiopia, Brazil, Colombia, Peru and others.

The existing treatments for leishmaniasis can cause severe side effects. Additionally, drug-resistant parasites are causing major problems in many countries. For these reasons, there is an urgent need for new, safe and inexpensive anti-leishmaniasis drug compounds.

Overview

In our paper, we describe the detailed steps we took to identify protein drug targets, which included the screening of 600,000 molecules to determine which might lead to development of new treatments for the disease. Specifically, we searched for drugs which target the proteins which are essential for the survival of the parasite that causes leishmaniasis. By finding molecules that bind to these proteins, they can potentially be disabled, thus stopping the infection and curing the disease.

The paper describes how some of the protein targets can bend and change shape, which presents some challenges in finding good candidate molecules. We discuss our approach to dealing with these problems.

Using the results computed on World Community Grid, we selected the ten best drug candidates for test-tube experiments, which yielded very promising results. In particular, one of the compounds appears to be able to kill the leishmaniasis parasite without affecting human cells in vitro. Three additional compounds also showed promising results.

We now plan to work to modify the three promising identified drug compounds to improve their potency, solubility and to minimize toxicity. We will also evaluate other top candidates depending on the funds we can raise. Furthermore, we are developing an open data platform with World Community Grid´s results so that other researchers can mine our data to detect additional drug hits or leads.

You can read an abstract of the paper here. We are grateful to all the World Community Grid volunteers who made this research possible by donating their computing power to this project.
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#4 Re: WCG Drug Search for Leishmaniasis

Post by Alez »

Drug Search for Leishmaniasis Researchers Make Data Publicly Available
By: Dr. Carlos Muskus López
Coordinator, Molecular Biology and Computational Unit, PECET University of Antioquia
14 Nov 2016

Summary
The Drug Search for Leishmaniasis research team has created an online database of the highest-scoring results from their research on World Community Grid. This database will help accelerate the work of other scientists who are conducting similar research.

Image
Above is an illustration of how ligands (drugs) are predicted to bind to each of the DHODH protein conformations extracted from experiments simulated on World Community Grid. DHODH is one of the essential proteins associated with the survival of the parasite that causes leishmaniasis.

Background

The Drug Search for Leishmaniasis project was created to identify potential new treatments for leishmaniasis, a neglected tropical disease that infects more than two million people in 98 countries every year. The existing drug treatments for leishmaniasis can cause severe side effects, leading to an urgent need for new, safe, and inexpensive anti-leishmaniasis drug compounds.

With the help of massive computing power donated by more than 120,000 World Community Grid volunteers, they identified several drug compounds that may become improved treatments for this disease. The team has also created an open database of the project’s highest-scoring results, which is available to scientists and to the public.

New Database Links Proteins with Compounds

The project’s research team at PECET, University of Antioquia in Colombia used AutoDock VINA software to search the ZINC database of commercially available compounds for those which appeared to best bind to 50 selected target proteins involved in leishmaniasis.

You can access the database created by the Drug Search for Leishmaniasis team at:

http://ubmc-pecet.udea.edu.co/index.php/dsfl/

The database allows the selection of any protein studied in the virtual screening, based on its PDB (Protein Data Bank) ID or protein name. After selecting a protein, basic information about the protein appears together with a PDB link and a visualizer to explore the protein's structure without leaving the database. In the section beneath the protein information, the top 20 binding compounds for that protein are listed, with a link to each compound's details and the corresponding VINA docking score. At the bottom of the list, there is an option to download the data in CSV format.

Selecting one of the binding compounds from the list for a given protein target shows information about the molecule ( its physicochemical properties), a picture of its 2D structure, and its link to the ZINC database. A list of other proteins with significant docking scores with this compound is provided. This can also be downloaded in CSV format.

The researchers hope this information will be valuable to other scientists investigating treatments for this neglected tropical disease. Please contact us (rodrigo.ochoa@udea.edu.co or carlos.muskus@udea.edu.co) for information about lower-scoring compounds that are not listed in the database.

If you use information from this repository please cite: Ochoa, R, et al. "Drug search for leishmaniasis: a virtual screening approach by grid computing." Journal of Computer-Aided Molecular Design, 2016, 30(7):541-552.

Thank you to all World Community Grid volunteers who donated computing time to this project.
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#5 Re: WCG Drug Search for Leishmaniasis

Post by Alez »

Potential New Treatments for Leishmaniasis Tested in Lab
By: Dr. Carlos Muskus López
Coordinator, Molecular Biology and Computational Unit, PECET University of Antioquia
1 Aug 2017

Summary
The Drug Search for Leishmaniasis team has begun lab testing potential treatments for this serious—yet neglected—tropical disease. In this update, Dr. Carlos Muskus discusses the results of topical (on the skin) testing of four compounds.

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

Background

Leishmaniasis is one of the most neglected tropical diseases in the world, infecting more than two million people in 98 countries. One form of the disease, caused by Leishmania infantum in America, mainly affects children, who can die if adequate treatment is not provided promptly. The classical treatments for all forms of Leishmaniasis can cause severe side effects, including death. Furthermore, drug resistant parasites are causing major problems in many endemic countries. For these reasons, there is an urgent need for new, safe, and inexpensive drug compounds.

Identifying Potential Compounds

After analyzing many compounds with the best docking score based on the data from the project on World Community Grid and our further testing, 10 compounds were finally selected to test in vitro. The in vitro evaluation involved cytotoxicity analysis against human-derived cell lines. (As part of the process of drug discovery, we do testing to ensure that the promising compounds do not affect human cells, thus decreasing the chance of side effects.) In addition, we evaluate the effectiveness of each compound against Leishmania, the parasites that cause leishmaniasis. The best compounds are those that kill the parasites at a low dose and do not affect the human cells even at higher doses.

Lab Testing of Potential Compounds

After in vitro testing, four compounds were selected to evaluate in vivo in hamsters. Hamsters and humans have similar reactions to leishmaniasis, so we can use hamsters to evaluate potential compounds.

Each compound was prepared as a topical formulation and applied daily for 10 days on the hamster's lesions. The hamsters were followed for two months. Groups of five hamsters per compound were used, and a summary of the compound, dose, and an outcome is presented in the table below. The different dosages used for each compound were selected based on the previous in vitro assays.

CompoundDose mg /dayResults (%)
11078180,14 / 10Improvement of the lesion between 11,1 - 15,7%
202874601,3 / 10Cure in 2 out 5 hamsters and improvement between 18,9-44,2%
203127192,8 / 10Improvement in 35,7%
203257670,6 / 10Improvement between 18,9-66%
Improvement: Percentage of reduction of the lesion size.
Cure: Complete re-epithelization of the lesion.

For the four compounds, different degrees of improvement were observed. The compound Amb 20287460 induced an almost complete curing of the lesions in two out of five hamsters. The compound 20312719 decreased the size of the lesions to various degrees. The remaining two compounds only induced small decreases in lesion size.

Next Steps

We still have a small remaining quantity of one of the compounds that produced some of the best result in the in vitro assay, but unfortunately the company that provided it did not synthesize it in sufficient quantity. We plan to contact another company to order more of this compound.

In addition, we are planning to re-test the compound that induced a complete cure in two of five hamsters. In future tests, we will probably change the method of administration to injection or oral, and closely monitor the outcomes.

Thank you to everyone who supported this project. We will keep you updated on our progress.
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#6 Re: WCG Drug Search for Leishmaniasis

Post by Alez »

Drug Search for Leishmaniasis Project Continues Quest for Better Treatments
By: Dr. Carlos Muskus López
Coordinator, Molecular Biology and Computational Unit, PECET University of Antioquia
20 Mar 2018

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.

Image
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 (EC50). The EC50 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% CO2 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 (LC50) was calculated by the Probit3 program. The cytotoxicity of each compound was classified according to the LC50 values €‹using a proprietary scale: high cytotoxicity LC50 <50 μg/mL; moderate cytotoxicity: 50 < LC50 < 200 μg/mL and low cytotoxicity: LC50> 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

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Evaluation of Anti-Leishmanial Activity
Prior to the determination of the effective concentration 50 (EC50), 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.

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The EC50 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
None of the 10 molecules evaluated showed promising anti-leishmanial results based on the in vitro cytotoxicity inhibition assays. And given this, the EC50 was not evaluated.
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