Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase

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.

Allosteric inhibition explained through conformational ensembles sampling distinct “mixed” states

“…Recent studies on the mechanisms of partial agonists that target the CBDs of allosterically regulated enzymes show an emerging trend, where partial agonism is best accounted for in terms of sampling multi-state equilibria with mixed intermediate states [11] , [12] , [13] , [14] . A common feature shared by these mixed intermediate states is the differential allosteric response of the C-terminal helix and the PBC ( Fig.…”

“…This realization has prompted a plethora of studies for understanding allosteric communication and networks [3] , [4] , [5] , allosteric drug design [6] , [7] , [8] , [9] , as well as the mechanisms of allosteric inhibitors and partial agonists. The latter typically target the allosteric domains of enzymes such as kinases [10] , [11] , [12] , guanine nucleotide exchange factors [13] , [14] , tyrosine kinases [15] , [16] , and proteases [17] , [18] , or protein–protein interfaces such as molecular chaperone-client interactions [19] .…”

“…In the first part of the review, we will summarize and analyze recent findings on the mechanisms of partial agonists for representative allosteric signalling enzymes: the cAMP-dependent protein kinase (PKA), the cGMP-dependent protein kinase (PKG), and the exchange protein activated by cAMP (EPAC) [10] , [11] , [12] , [13] , [14] , [28] , [29] , [30] , [31] , [32] . The goal of this initial section is to reveal emerging trends, such as stabilization of novel “mixed” states that play essential roles in explaining the observed functional response.…”

“…This review does not claim to be exhaustive, and we will focus primarily on illustrative cases from our own work on cyclic nucleotide monophosphate (cNMP)-binding domains (CBDs) [10] , [11] , [12] , [13] , [14] . CBDs share a conserved fold architecture with a non-continuous α-subdomain and a continuous β-subdomain ( Fig.…”

Targeting the Malaria Parasite cGMP-Dependent Protein Kinase to Develop New Drugs

“…Apart from ATP-competitive inhibitors, cGMP analogs have also recently been explored as potential anti-malarials. It has been shown using biochemical and biophysical approaches that 8-NBD-cGMP, although having a similar affinity to cGMP, is a P. falciparum PKG antagonist with a 10-fold reduction of activation of PKG compared to cGMP (Byun et al, 2020). The effects on the parasite have so far not been reported.…”