, WHO. World Health Report, 2002.
, Science, vol.298, p.124, 2002.
, J. Med. Chem, vol.46, p.542, 2003.
, Nature, p.498, 2002.
, Curr Med. Chem, vol.7, p.835, 2000.
, For reviews see: (a) Rosenthal, P. J. Curr. Opin. Hematol, vol.9, p.59, 2000.
, Swissprot accession number of PfA-M1: O96935
, Parasitology, vol.125, p.1, 2002.
, Biochem. Parasitol, vol.117, p.37, 1998.
Abstracts of Papers, 223rd ACS National Meeting, p.176, 2002. ,
, J. Biol. Chem, vol.257, p.1825, 1982.
, Bioorg. Med. Chem. Lett, p.2595, 2002.
, J. Med. Chem, p.339, 1998.
The assays were set up in 96-well plates. The compounds were tested at a final concentration of 10 mM. 33 mL of purified PfA-M1 were pre-incubated 10 min at rt with 33 mL of the inhibitor (30 mM in Tris-HCl buffer, 0.3% DMSO). 33 mL of the substrate Leu-pNA (K m =0.1 mM) 0.3 mM in Tris-HCl buffer were then added. The reaction kinetics was followed on a UV-microplate reader Multi-skanRC (Labsystems, Finland) at 405 nm. The control activity was determined by incubating the enzyme in the same conditions without inhibitor. Bestatin was used as the reference inhibitor. The statistical Z 0 factor for the test, Native PfA-M1 was purified according to the procedure described in ref 9, and diluted 10 times in Tris-HCl buffer (25 mM; pH 7.4) before use ,
, In vitro antiplasmodial activity was determined using a modification of the semi-automated micro-dilution technique of Desjardins et al. (Antimicrob. Agents Chemother. 1979, 16, 710). P. falciparum CQ-resistant FcB1 (Colombia) strain was used. Stock solutions of CQ-diphosphate and test compounds were prepared in sterile distilled water and DMSO, respectively. Drug solutions were serially diluted with culture medium and introduced to asynchronous parasite cultures (0.5% parasitemia and 1% final hematocrite) on 96-well plates for 24 h at 37 C prior to the addition of 0, P. falciparum strains were maintained continuously in culture on human erythrocytes as described by Trager and Jensen, 1976.
France) per well, for 24 h. The growth inhibition of each drug concentration was determined by comparison of the radioactivity incorporated into the treated culture with that in the control culture (without drug) maintained on the same plate. The IC 50 , as obtained from the drug concentration-response curve were expressed as meanAE standard deviations, determined from three independent experiments. The DMSO concentration never exceeded 0 ,
, The assays were set up in 96-well plates and proceeded like for PfA-M1 with the same substrate Leu-pNA (K m =0.13 mM). The Z 0 factor of the test was 0.80 allowing activities to be determined with a single point with a 95% confidence. The reference inhibitor was bestatin
, 95 (s; 1H); 8.50-8.56 (m; 2H), 1H, vol.7, p.30
M+H + ). 34. (2TFA): NMR (CD 3 OD) 1 H, vol.534 ,
, 6 Hz; 2H), vol.3, p.62
, 33 (m; 2H), pp.25-33
, M+H + ). 35. (2TFA): NMR (DMSO-d 6 ) 1 H, vol.519
, 26 (m; 5H), vol.1, pp.10-11
, Hz; 1H), vol.8, p.34
, , vol.10, p.75
, In-vitro inhibition of haem polymerization induced by lipids, unpublished data (see ref 3 for protocol)