lated. Based on 7 independent e periments carried out on the 3D7 strain with 3 different batches selleck EPZ-5676 of synthetic peptides, IC50s were 23. 76 uM for KTISW and 9. 72 uM for KVVRW containing peptides respectively. When the effect of P1 and P5 peptides was tested on the HB3 strain, the IC50s were 14. 99 uM and 8. 79 uM respectively. Finally, the to icity of these peptides was evaluated by their capacity to block the proliferation of stimulated mouse spleen cells in vitro. The calculated IC50 was 45. 3 uM for the KTISW containing peptide and 59. 32 uM for the KVVRW containing peptide, showing a selectivity inde of 2 to 6 fold for P. falciparum according to the peptide tested. To further e plore the uptake of active P1 and P5 pep tides by blood parasite stages, FITC labeled peptides were used.
As shown in Figure 8F, FITC P1 was only accumu lated within free merozoites, while FITC P5 penetrated infected red blood cells and concentrated within intra cellular parasites as well as free merozoites. Uninfected red blood cells did not accumulate any FITC peptide. Discussion The Pf Inhibitor2 gene encodes Inhibitors,Modulators,Libraries a protein of 144 amino acids related to the I2 proteins of different organ isms, which are known to inhibit PP1c activity in vitro. Of the three central regions identified in the I2 protein as binding motifs to PP1, the KGILK, RV F, and HYNE mo tifs, PfI2 contained only a consensus RV F and the 102HYNE105 sequences. The lack of KGILK in PfI2 was supported by bioinformatics analysis indicating the absence of this sequence in all potential open reading frames upstream of the PfI2 gene and was further con firmed by a 5 cDNA walking approach.
The KGILK motif present in vertebrate I2 was found to be involved in the interaction Inhibitors,Modulators,Libraries with PP1 through the region of amino acids 50 59 in PP1c. In addition, deletion of the N terminal side of I2 containing this site and mutation of the first Lys or the Ile dramatically reduced the inhibition cap acity of I2. These Inhibitors,Modulators,Libraries observa tions emphasize the importance of this site in the binding and activity of vertebrate I2, which represents a major dif ference compared with PfI2, which lacks this motif. Concerning the RV F site, vertebrate I2 does not contain the canonical motif falling within the consensus sequence 0 1 0 1. However, studies on the crys tal structure of PP1c I2 revealed that the Inhibitors,Modulators,Libraries sequence KSQKW, where the consensus Val Ile residue is replaced by a Gln is docked in the PP1 groove, which usually binds the RV F motif.
Structure activity studies on the Batimastat im plication of KSQKW site showed that the mutation of Trp in mammalian I2 drastically reduced the inhibitory inhibitor Gefitinib activity of I2. It is worth noting that almost all I2 proteins contain Gln at the position of Val Ile. However, in P. falciparum, the I2 protein does contain an Ile in the RV F motif, a second important dissimilarity between PfI2 and other I2 proteins. The comparison of PfI2 with putative I2 of To oplasma gondii or Neospora canium, revealed the presence of the con sensus RV