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Cyclosporin (Cs) A has pronounced antimalarial activity in vitro and in vivo. In
other organisms, the drug is thought to exert its effects either by inhibiting
the peptidylprolyl cis/trans isomerase activity of cyclophilin (CyP) or by
forming a CyP-CsA complex that inhibits the phosphatase activity of calcineurin.
We have cloned and overexpressed in Escherichia coli a gene encoding a CyP from
Plasmodium falciparum (PfCyP19) that is located on chromosome 3. The sequence of
PfCyP19 shows remarkable sequence identity with human CyPA and, unlike the two
previously identified CyPs from P. falciparum, PfCyP19 has no signal peptide or
N-terminal sequence extension, suggesting a cytosolic localization. All the
residues implicated in the recognition of the synthetic substrate
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide are conserved, resulting in
characteristically high Michaelis-Menten and specificity constants
(Km>>120 microM, kcat/Km=1.2x10(7) M-1.s-1 respectively). As the first
line in the functional characterization of this enzyme, we have assessed its
binding affinity for CsA. In accordance with its tryptophan-containing
CsA-binding domain, PfCyP19 binds CsA with high affinity (Kd=13 nM, Ki=6.9 nM).
Twelve CsA analogues were also found to possess Ki values similar to CsA, with
the notable exceptions of Val2-Cs (Ki=218 nM) and Thr2-Cs (Ki=690 nM). The
immunosuppressants rapamycin and FK506 were inactive as inhibitors, consistent
with other members of the CyP family of rotamases. The CsA analogues were also
assessed as inhibitors of P. falciparum growth in vitro. Although their
antimalarial activity did not correlate with inhibition of enzyme activity,
residues 3 and 4 of CsA appeared to be important for inhibition of parasite
growth and residues 1 and 2 for PfCyP19 inhibition. We propose that a malarial
CyP-CsA complex presents residues 3 and 4 as part of an 'effector surface' for
recognition by a downstream target, similar to the proposed mechanism for T-cell
immunosuppression.
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