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. 2011 May 3;5(5):e1023.
doi: 10.1371/journal.pntd.0001023.

Mining a Cathepsin Inhibitor Library for New Antiparasitic Drug Leads

Free PMC article

Mining a Cathepsin Inhibitor Library for New Antiparasitic Drug Leads

Kenny K H Ang et al. PLoS Negl Trop Dis. .
Free PMC article


The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ∼ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Structural composition of the cathepsin inhibitor library.
Illustrated are significantly represented chemotypes at three positions: warhead/P1, P2, and P3. Indicated in parentheses are the approximate number of library members possessing each substructural chemotype.
Figure 2
Figure 2. Comparing percent inhibition of compounds between cysteine proteases.
Single-point inhibition data for rhodesain (Y-axis, % inhibition at 0.1 µM test compound) as compared to other parasite enzymes (X-axes), including: (A) cruzain (% inhibition at 0.1 µM), (B) falcipain-3 (% inhibition at 0.05 µM), (C) TbCatB (% inhibition at 1 µM), and (D) trypanathione reductase (% inhibition at 10 µM). Individual data points are colored according to warhead/P1 type as follows: vinylsulfones (red), nitriles unsubstituted or monosubstituted at P1 (blue), nitriles with geminal cyclic substitution at P1 (light blue), keto-heterocycles (green), hydroxy/alkoxy ketones (yellow) and others (grey). The shape of each data point corresponds to the P2 chemotype as follows: phenylalanine (triangle), benzyl cysteine or homphenylalanine (needle); naphthalene (circle), spirocyclic (open circle), dihalogenated phenylalanine (diamond), leucine/leucine-like (square) and others (open square).
Figure 3
Figure 3. Fifty percent inhibition concentrations.
IC50, (µM) against five parasite cysteine proteases as determined for library compounds that qualified as hits from the single point screens. Data points above the dashed line had no measurable IC50 (i.e., IC50>25 µM). Data points are colored and shaped by chemotype, as described in Figure 2.
Figure 4
Figure 4. Growth inhibition of parasites.
GI50 (µM) data for selected library members against cultured P. falciparum (W2 strain), T. brucei brucei, or T. cruzi parasites. Data points above the dotted line had no measurable GI50 (i.e., GI50>25 µM). Data points are colored and shaped by chemotype as described in Figure 2. The relative area of data points reflects cytotoxicity to mammalian cell lines (larger squares demoting increasing % growth inhibition relative to a 100% inhibition control). Cytotoxicity evaluations were performed in Jurkat cells for the T. brucei and P. falciparum actives (at 10 µM test compound) and in BESM cells for the T. cruzi actives (at 20 test µM compound).
Figure 5
Figure 5. Anti-plasmodium lead compounds.
Chemical structures of protease inhibitors discussed in the text. Associated enzyme inhibition and growth inhibition data is provided in tables 2– 4.
Figure 6
Figure 6. Anti-trypanosomal lead compounds.
Chemical structures of protease inhibitors discussed in the text. Associated enzyme inhibition and growth inhibition data is provided in tables 4–5.
Figure 7
Figure 7. T. cruzi growth inhibition of compounds 24.
Representative fluorescent images acquired with the IN Cell Analyzer 2000, taken with 20× objective magnification at Ex 350 nm/Em 460 nm, showing the host BESM nuclei (red outline) and surrounding smaller T. cruzi kinetoplastids (blue outline). (A) 1% DMSO as 0% inhibition control; (B) compound 24 at the highest assay concentration of 20 µM; (C) GI50 dose response plots of compound 24 against T. cruzi and cytotoxicity to host BESM cells.

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