The emergence of resistance in Plasmodium
falciparum to frontline artemisinin-based combination
therapies has raised
global concerns and emphasized the identification of new drug targets
for malaria. Cysteine protease falcipain-2 (FP2), involved in host
hemoglobin degradation and instrumental in parasite survival, has
long been proposed as a promising malarial drug target. However, designing
active-site-targeted small-molecule inhibitors of FP2 becomes challenging
due to their off-target specificity toward highly homologous human
cysteine cathepsins. The use of proteinaceous inhibitors, which have
nonconserved exosite interactions and larger interface area, can effectively
circumvent this problem. In this study, we report for the first time
that human stefin-A (STFA) efficiently inhibits FP2 with K
i
values in the nanomolar range. The
FP2–STFA complex crystal structure, determined in this study,
and sequence analyses identify a unique nonconserved exosite interaction,
compared to human cathepsins. Designing a mutation Lys68 > Arg
in
STFA amplifies its selectivity garnering a 3.3-fold lower K
i
value against FP2, and the
crystal structure of the FP2–STFAK68R complex shows stronger
electrostatic interaction between side-chains of Arg68 (STFAK68R)
and Asp109 (FP2). Comparative structural analyses and molecular dynamics
(MD) simulation studies of the complexes further confirm higher buried
surface areas, better interaction energies for FP2–STFAK68R,
and consistency of the newly developed electrostatic interaction (STFA–R68–FP2–D109)
in the MD trajectory. The STFA–K68R mutant also shows higher K
i
values against human cathepsin-L
and stefin, a step toward eliminating off-target specificity. Hence,
this work underlines the design of host-based proteinaceous inhibitors
against FP2, with further optimization to render them more potent
and selective.
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