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A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit.

De Muylder G, Ang KK, Chen S, Arkin MR, Engel JC, McKerrow JH - PLoS Negl Trop Dis (2011)

Bottom Line: In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage.Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite.This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sandler Center for Drug Discovery, University of California San Francisco, San Francisco, California, United States of America. Geraldine.DeMuylder@ucsf.edu

ABSTRACT
The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

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Related in: MedlinePlus

Structure and activity of naloxonazine and naloxone.Lower panels: Dose response curve for naloxonazine (left) and naloxone (right) against intracellular amastigotes (black diamonds), promastigotes (black squares), axenic amastigotes (white diamonds) and THP-1 (white triangles) plotting the percentage of parasite growth inhibition.
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pntd-0001253-g004: Structure and activity of naloxonazine and naloxone.Lower panels: Dose response curve for naloxonazine (left) and naloxone (right) against intracellular amastigotes (black diamonds), promastigotes (black squares), axenic amastigotes (white diamonds) and THP-1 (white triangles) plotting the percentage of parasite growth inhibition.

Mentions: The Iconix collection contained two opioid receptor antagonists, naloxone and naloxonazine. The first was not selected as a hit in any of the screens described above while the latter showed specific activity against the intracellular amastigote stage. To confirm these primary observations, the activity of both compounds was tested against promastigotes, intracellular and axenic amastigotes. Naloxonazine exhibited specific activity against intracellular amastigotes (GI50 intracellular amastigote: 3.45 µM; GI50 THP-1: 33.8 µM; GI50 promastigote: >50 µM; GI50 axenic amastigote: >50 µM), while naloxone was inactive against all parasite forms and not toxic to the host macrophage (Figure 4). At a curative concentration, the selectivity window of naloxonazine was reduced (GI90 intracellular amastigote: 12.5 µM; GI90 THP-1: 50 µM), limiting the possibility of using naloxonazine for treatment.


A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit.

De Muylder G, Ang KK, Chen S, Arkin MR, Engel JC, McKerrow JH - PLoS Negl Trop Dis (2011)

Structure and activity of naloxonazine and naloxone.Lower panels: Dose response curve for naloxonazine (left) and naloxone (right) against intracellular amastigotes (black diamonds), promastigotes (black squares), axenic amastigotes (white diamonds) and THP-1 (white triangles) plotting the percentage of parasite growth inhibition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139667&req=5

pntd-0001253-g004: Structure and activity of naloxonazine and naloxone.Lower panels: Dose response curve for naloxonazine (left) and naloxone (right) against intracellular amastigotes (black diamonds), promastigotes (black squares), axenic amastigotes (white diamonds) and THP-1 (white triangles) plotting the percentage of parasite growth inhibition.
Mentions: The Iconix collection contained two opioid receptor antagonists, naloxone and naloxonazine. The first was not selected as a hit in any of the screens described above while the latter showed specific activity against the intracellular amastigote stage. To confirm these primary observations, the activity of both compounds was tested against promastigotes, intracellular and axenic amastigotes. Naloxonazine exhibited specific activity against intracellular amastigotes (GI50 intracellular amastigote: 3.45 µM; GI50 THP-1: 33.8 µM; GI50 promastigote: >50 µM; GI50 axenic amastigote: >50 µM), while naloxone was inactive against all parasite forms and not toxic to the host macrophage (Figure 4). At a curative concentration, the selectivity window of naloxonazine was reduced (GI90 intracellular amastigote: 12.5 µM; GI90 THP-1: 50 µM), limiting the possibility of using naloxonazine for treatment.

Bottom Line: In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage.Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite.This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sandler Center for Drug Discovery, University of California San Francisco, San Francisco, California, United States of America. Geraldine.DeMuylder@ucsf.edu

ABSTRACT
The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

Show MeSH
Related in: MedlinePlus