Falciparum cGMP-dependent Protein Kinase Research Articles

Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials.

Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.

Characterization of Competitive Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is an enticing antimalarial drug target. Novel chemotypes are needed because existing inhibitors have safety issues that may prevent further development. This work demonstrates isoxazole-based compounds are potent ATP competitive inhibitors of PfPKG and discloses a new analogue in this series. Isoxazoles 3 and 5 had Ki values that are comparable to a known standard, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine. They also exhibited excellent selectivity for PfPKG over the human orthologue and the gatekeeper mutant T618Q PfPKG, which mimics the less accessible binding site of the human orthologue. The human orthologue's larger binding site volume is predicted to explain the selectivity of the inhibitors for the P. falciparum enzyme.

Divergent allostery reveals critical differences between structurally homologous regulatory domains of Plasmodium falciparum and human protein kinase G

Malaria is a life-threatening infectious disease primarily caused by the Plasmodium falciparum parasite. The increasing resistance to current antimalarial drugs and their side effects has led to an urgent need for novel malaria drug targets, such as the P. falciparum cGMP-dependent protein kinase (pfPKG). However, PKG plays an essential regulatory role also in the human host. Human cGMP-dependent protein kinase (hPKG) and pfPKG are controlled by structurally homologous cGMP-binding domains (CBDs). Here, we show that despite the structural similarities between the essential CBDs in pfPKG and hPKG, their respective allosteric networks differ significantly. Through comparative analyses of chemical shift covariance analyses, molecular dynamics simulations, and backbone internal dynamics measurements, we found that conserved allosteric elements within the essential CBDs are wired differently in pfPKG and hPKG to implement cGMP-dependent kinase activation. Such pfPKG versus hPKG rewiring of allosteric networks was unexpected because of the structural similarity between the two essential CBDs. Yet, such finding provides crucial information on which elements to target for selective inhibition of pfPKG versus hPKG, which may potentially reduce undesired side effects in malaria treatments.

Discovery of Imidazole-Based Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

The discovery of new targets for the treatment of malaria, in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this paper presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization, and cell-based antiparasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity, and cell-based antiparasitic activity against multiple Plasmodium species that appears to be correlated with the in vitro potency.

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in PfPKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the PfPKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the PfPKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central β-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design PfPKG-selective allosteric inhibitors as a viable treatment for malaria.

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays the most crucial role in PfPKG regulation. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking.

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

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.

Simultaneous multiple allelic replacement in the malaria parasite enables dissection of PKG function.

Over recent years, a plethora of new genetic tools has transformed conditional engineering of the malaria parasite genome, allowing functional dissection of essential genes in the asexual and sexual blood stages that cause pathology or are required for disease transmission, respectively. Important challenges remain, including the desirability to complement conditional mutants with a correctly regulated second gene copy to confirm that observed phenotypes are due solely to loss of gene function and to analyse structure-function relationships. To meet this challenge, here we combine the dimerisable Cre (DiCre) system with the use of multiple lox sites to simultaneously generate multiple recombination events of the same gene. We focused on the Plasmodium falciparum cGMP-dependent protein kinase (PKG), creating in parallel conditional disruption of the gene plus up to two allelic replacements. We use the approach to demonstrate that PKG has no scaffolding or adaptor role in intraerythrocytic development, acting solely at merozoite egress. We also show that a phosphorylation-deficient PKG is functionally incompetent. Our method provides valuable new tools for analysis of gene function in the malaria parasite.

FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons

Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.

High-throughput screening of the Plasmodium falciparum cGMP-dependent protein kinase identified a thiazole scaffold which kills erythrocytic and sexual stage parasites

Antimalarial drug resistance compels the quest for new compounds that target alternative pathways to current drugs. The Plasmodium cyclic GMP-dependent protein kinase (PKG) has essential functions in all of the major life cycle developmental stages. An imidazopyridine PKG inhibitor scaffold was previously shown to clear P. falciparum infection in a rodent model in vivo and blocked transmission to mosquitoes providing proof of concept for this target. To find new classes of PKG inhibitors to serve as alternative chemical starting points, we performed a high-throughput screen of the GSK Full Diversity Collection using recombinant P. falciparum PKG. We developed a robust enzymatic assay in a 1536-well plate format. Promising compounds were then tested for activity against P. falciparum asexual blood stage growth, selectivity and cytotoxicity. By using a scoring system we selected the 66 most promising PKG inhibitors (comprising nine clusters and seven singletons). Among these, thiazoles were the most potent scaffold with mid-nanomolar activity on P. falciparum blood stage and gamete development. Using Kinobeads profiling we identified additional P. falciparum protein kinases targeted by the thiazoles that mediate a faster speed of the kill than PKG-selective compounds. This scaffold represents a promising starting point to develop a new antimalarial.

The role of a parasite-specific D-site in activation of Plasmodium falciparum cGMP-dependent protein kinase

Background Malaria is one of the most dangerous tropical diseases worldwide, resulting in approximately 1.5-2.7 million deaths per year [1]. Furthermore, malaria belongs to the four major infectious diseases also including HIV, tuberculosis and hepatitis. In humans, malaria is transmitted by four species of the genus Plasmodium. However, most malaria deaths are caused by Plasmodium falciparum [2]. The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is one of the key regulators of the malaria parasite life cycle in both sexual and asexual blood-stages. Inhibition of PfPKG stops differentiation and transmission of the parasites, indicating that this kinase is a promising drug target for malaria [3-5]. However, despite its physiological importance, the activation mechanism of PfPKG is not fully understood. Recently, our group discovered that disrupting cGMP binding at the C-terminal cyclic nucleotide-binding (CNB-D) domain almost completely abolishes the kinase activation [6]. Therefore, we investigate the functional role of the PfCNB-D in PfPKG activation.

Crystal structures of the carboxyl cGMP binding domain of the Plasmodium falciparum cGMP-dependent protein kinase reveal a novel capping triad crucial for merozoite egress.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG’s activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a “gatekeeper” for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation.

Crystal structures of the carboxyl cGMP binding domain of plasmodium falciparumcGMP-dependent protein kinase reveals a novel salt bridge crucial for activation

Background Plasmodium falciparum cGMP-dependent protein kinase (pfPKG) is a validated therapeutic target of malaria. As a key regulator of its life cycle, pfPKG plays a crucial role in both the sexual and asexual blood-stages that cause malaria pathology. Inhibiting pfPKG blocks proliferation and transmission of the parasite [1,2]. However the development of pfPKG-specific inhibitor has been greatly hampered by the lack of high-resolution structure information to guide drug design. Targeting the ATP binding site of pfPKG is an approach commonly associated with low specificity and toxicity [3]. Therefore, we aim to target a domain that is unique to this kinase, the cyclic nucleotide binding (CNB) domain. Since previous studies demonstrated the fourth-cyclic nucleotide binding (CNB-D) domain of pfPKG to be the most important for the kinase activation [4] we focused on this domain to understand its role in cGMP dependent activation.

Malaria Parasite cGMP-dependent Protein Kinase Regulates Blood Stage Merozoite Secretory Organelle Discharge and Egress

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

The role of the cGMP-dependent protein kinase in development of the malaria parasite

The life cycle of the malaria parasite Plasmodium is complex with distinct phases occurring in the human host and the mosquito vector. Proliferation of asexual parasites within blood cells leads to pathology whereas a distinct sexual stage is required to mediate transmission to the insect. There is a surprising lack of information about how progression of the life cycle is controlled. By analogy with other systems, it is likely that differentiation is regulated by intracellular signalling cascades involving specific phosphorylation/dephosphorylation events. Following an early report in the literature suggesting a role for the parasite cGMP signalling pathway in male gametogenesis, our recent work has investigated the role of the P. falciparum cGMP-dependent protein kinase (PfPKG) in the parasite life cycle. We have used specific inhibitors of the parasite PKG in conjunction with transgenic parasites expressing an inhibitor-insensitive PfPKG to provide direct evidence of a role for this kinase in gametogenesis [1]. Furthermore, we have used this approach recently to elucidate a central role for PfPKG in the late events of P. falciparum asexual blood stage schizogony. Using the P. berghei mouse malaria model we have also demonstrated a role for the kinase in gliding motility of the ookinete; the zygote form that borrows through the insect midgut wall. Discovery of essential functions for PfPKG in multiple developmental stages suggest that it may be a good target for new anti-malarial drugs.

Characterization of Plasmodium falciparum cGMP-dependent protein kinase ( PfPKG): Antiparasitic activity of a PKG inhibitor

Cyclic GMP-dependent protein kinase (PKG) has been biochemically and genetically validated in Toxoplasma gondii as a primary target responsible for the antiparasitic activity of the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1 H pyrrol-3-yl] pyridine (Compound 1) [Biftu T, Feng D, Ponpipom M, et al. Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents. Bioorg Med Chem Lett 2005;15:3296–301; Gurnett AM, Liberator PA, Dulski PM, et al. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J Biol Chem 2002;277:15913–22; Donald RGK, Allocco J, Singh SB, et al. Toxoplasma gondii cyclic GMP-dependent kinase: Chemotherapeutic targeting of an essential parasite protein kinase. Eukaryotic Cell 2002;1:317–28; Nare B, Allocco J, Liberator PA, Donald RGK. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine Toxoplasmosis: Gamma interferon is required for efficacy. Antimicrob Agents Chemother 2002;46:300–7]. Compound 1 inhibits the growth of several related protozoan parasites of the subphylum Apicomplexa. Native PKG activity has been partially purified by cGMP-affinity and MonoQ ion exchange chromatography from Plasmodium falciparum ( PfPKG). Biochemical fractions enriched for a 98 kDa protein detected using anti-PKG antisera, contain cGMP-induced protein kinase activity that is sensitive to inhibition by Compound 1. To enable a more thorough characterization of PfPKG we expressed a synthetic cDNA incorporating T. gondii codon preference ( Pf Tg PKG) in T. gondii parasites. The protein kinase activity of purified recombinant Pf Tg PKG is stimulated by cGMP, with significant cooperativity as demonstrated by a Hill coefficient of 2. Both substrate preference and inhibition of Pf Tg PKG kinase activity by Compound 1 are similar to that seen with native PfPKG, as well as PKG enzymes from Eimeria spp. and T. gondii. We conclude that PfPKG has biochemical and pharmacological properties that are similar to previously characterized apicomplexan PKG enzymes. Compound 1 is active against blood cell stages of P. falciparum cultured in vitro. In a Plasmodium berghei mouse model of infection, Compound 1 delays the onset of parasitemia but does not cure the parasite infection.

The role of two novel regulatory sites in the activation of the cGMP-dependent protein kinase from Plasmodium falciparum.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) uniquely contains three cGMP binding sites, but also has a 'degenerate' fourth site. The role of each cGMP-binding site in PfPKG activation remains unknown. We have analysed the effect of mutation of each cGMP-binding site (individually and in combination) on PfPKG activation in vitro. The most striking result was that mutation of cGMP site 3 resulted in a 10-49-fold increase in the K (a((cGMP))) value and a 45-55% decrease in maximal activity compared with wild-type. Mutations involving only cGMP-binding sites 1 and 2 had less effect on both the K (a((cGMP))) values and the maximal activities. These results suggest that, although all three cGMP-binding sites are involved in PfPKG activation, cGMP-binding site 3 has the greatest influence on activation. A mutation in the fourth, degenerate cGMP-binding site decreased PfPKG maximal activity by 40%, but did not change the K (a((cGMP))) value for the PfPKG mutant, suggesting that this site does not bind cGMP, but is required for full activation of PfPKG. The distinct activation properties of PfPKG from mammalian isoforms may be exploitable in the design of a parasite-specific inhibitor and development of a novel anti-malarial drug.