Screening methods for identifying Plasmodium proteases inhibitors

Patent metadata

Field	Value
Publication number	US-9404142-B2
Application number	US-201013989844-A
Country	US
Kind code	B2
Filing date	Nov 29, 2010
Priority date	Nov 29, 2010
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

The invention relates to the field of parasitology. Methods and peptidic substrate for screening and identifying inhibitors of Plasmodium are described. Also described are compounds identified by these screening methods, including more particularly inhibitors of Plasmodium subtilisin-like proteases. The invention also concerns anti-malaria compounds, anti-malaria compositions, and uses thereof for preventing, treating, improving, and/or alleviating a Plasmodium infection in a subject, and more Plasmodium vivax and/or by Plasmodium falciparum human infections.

First claim

The invention claimed is:

1. A peptidic substrate comprising a peptide having the amino acid sequence K-L-V-G-A-D-D-V-S-L-A (SEQ ID NO: 9), a fluorophore, and a quencher, wherein the fluorophore and the quencher are bound directly to the peptide on opposite sides of the aspartic acid residues, and wherein the peptidic substrate is cleavable by a protease comprising SEQ ID NO: 4.

2. The peptidic substrate of claim 1 , wherein the quencher is Dabsyl or DYQ60 and wherein the fluorophore is EDANS or DY630.

3. The peptidic substrate of claim 2 , wherein the substrate comprises one of the two following combinations of quencher and fluorophore: i) Dabsyl and EDANS; or ii) DYQ660 and DY630.

4. The peptidic substrate of claim 1 , wherein said peptidic substrate consists of Dabsyl-K-L-V-G-A-D-D-V-S-L-A-EDANS (Dabsyl-SEQ ID NO: 9-EDANS) or DYQ660-K-L-V-G-A-D-D-V-S-L-A-K-DY630 (DYQ660-SEQ ID NO: 10-DY630).

5. An in vitro screening method for identifying inhibitors of Plasmodium , comprising:
a) incubating a peptidic substrate according to claim 1 with malarial subtilisin-like protease 1 (SUB1) comprising SEQ ID NO: 4 in the presence of a candidate compound,
b) detecting cleavage of the peptidic substrate by the protease comprising SEQ ID NO: 4; and
c) comparing cleavage of the peptidic substrate by the protease in the presence of the candidate compound to cleavage of the peptidic substrate by the protease in the absence of the candidate compound.

6. The in vitro screening method of claim 5 , wherein an inhibition of said cleavage in the presence of the candidate compound compared to the cleavage in the absence of the candidate compound identifies said candidate compound as a potential inhibitor of Plasmodium life cycle.

7. The in vitro screening method of claim 6 , wherein said inhibition of cleavage identifies said candidate compound as a potential inhibitor of Plasmodium vivax, Plasmodium falciparum and/or Plasmodium berghei.

8. The in vitro screening method of claim 5 , wherein said screening method is a high-throughput method.

9. The in vitro screening method of claim 5 , wherein the cleavage in step b) is detected by fluorescence assay.

10. The in vitro screening method of claim 9 , wherein the fluorescence assay is Fluorescent Resonance Energy Transfer (FRET) assay.

11. The in vitro screening method of claim 5 , further comprising:
selecting a candidate compound capable of inhibiting cleavage of said peptidic substrate; and
testing ex vivo said selected candidate compound on a culture of one or more species of Plasmodium and/or testing in vivo said selected candidate compound in at least one Plasmodium -infected animal.

12. An isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 4.

13. A cell expressing the isolated polypeptide of claim 12 .

14. A kit for in vitro screening test comprising a peptidic substrate as defined in claim 1 , and a protease comprising SEQ ID NO: 4.

Assignees

• Barale Jean-Christophe
• Bouillon Anthony
• Giganti David
• Gorgette Olivier Louis Gabiel
• Stoven Veronique
• Nilges Michael
• Puijalon Odile
• Pasteur Institut

Inventors

• Barale Jean-Christophe
• Bouillon Anthony
• Giganti David
• Gorgette Olivier Louis Gabiel
• Stoven Veronique
• Nilges Michael
• Puijalon Odile

Classifications

• C12Q1/37 Primary

  involving peptidase or proteinase · CPC title

• C07K7/08

  having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• G01N33/56905

  Protozoa · CPC title