Abstract
Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog-bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive "mixed" intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.
Highlights
Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum
Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not by reverting a twostate active versus inactive equilibrium, but by sampling a distinct inactive “mixed” intermediate
The apo assignments were obtained by transferring the holo assignments to the apo spectrum through 2D Nz-exchange (Fig. 2, B and D) and 2D difference Nz-exchange experiments acquired in the presence of substoichiometric amounts of cGMP (Fig. 2, C and E)
Summary
Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum. Its life cycle is regulated by a cGMPdependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. We hypothesize that the stabilization of the holo-inactive state arise from a reversal of the two-state inactive/active conformational equilibrium (Fig. 1F) or from a multistate equilibrium sampling a distinct intermediate conformation that exhibits components of both the inactive and the active states of PfD in different regions (Fig. 1G) To test these hypotheses and understand the mechanism of action of allosteric partial agonists and antagonists that serve as leads for new PfPKG-selective inhibitors, we use comparative NMR to analyze PfD in its apo, cGMP-bound, and cGMP analog– bound forms. Our results reveal that 8-NBDcGMP and 8-pCPT-cGMP significantly reduce the kinase activity and induce major perturbations throughout PfD by stabilizing an additional intermediate conformer that is distinct from both the native apo-inactive and holo-active conformations This “mixed” intermediate state exhibits inhibitory potential comparable with that of the apo-inactive state, as the C-terminal lid is disengaged, but it preserves an affinity similar to that of cGMP, as the key cGMP-binding regions, including the PBC and the adjacent ␣B, are still engaged. The stabilization of an allosterically mixed intermediate emerges as a new strategy for selectively inhibiting PfPKG with high potency and efficacy
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