Affinage · Plasmodium

PKG

PF3D7_1436600 · cGMP-dependent protein kinase

Annotated
2026-09-08
78 papers in source corpus 31 papers cited in narrative 31 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

PKG (PF3D7_1436600) is the sole cGMP-activated protein kinase and master signaling hub of Plasmodium, orchestrating the transitions of the parasite life cycle through cGMP-triggered phosphorylation of downstream effectors (PMID:12068803, PMID:26149123). Recombinant PfPKG is a cGMP-selective phosphotransferase whose activity is stimulated at least fivefold by cGMP but not by cAMP, and whose regulatory module contains four cyclic-nucleotide-binding sites, of which one dominant site (later assigned to the C-terminal CNB-D) governs activation while the degenerate fourth site contributes to full activity without binding cGMP (PMID:12068803, PMID:12817987, PMID:25646845). Structurally, full-length PfPKG adopts a pentagonal arrangement of the four CNBs around the kinase domain in which an N-terminal autoinhibitory segment docks into the substrate site; cooperative cGMP binding, initiated at the CNB nearest the catalytic domain, propagates conformational change through a structural relay to release autoinhibition and activate the kinase (PMID:31239348, PMID:29251493). In vivo, full function additionally requires cGMP occupancy of CNB-B and activation-loop phosphorylation at Thr695, both of which are dispensable for in vitro catalysis but essential in the parasite (PMID:38935780, PMID:32179592). Upstream, GCα is the sole cGMP synthase in asexual blood stages and PP1 phosphatase sets cGMP homeostasis by tuning GCα, positioning both as controllers of PKG activation (PMID:33500341, PMID:39808636). Once active, PKG drives phosphoinositide hydrolysis and intracellular Ca2+ mobilization—transduced in part through its tightly associated multipass membrane partner ICM1—to trigger PfSUB1 discharge, microneme release, parasitophorous vacuole membrane breakdown, host-cell egress, and erythrocyte invasion, and it phosphorylates a broad substrate set including PfCDPK1, actomyosin components, and the proteasome subunit RPT1 (PMID:23675297, PMID:24594931, PMID:33762339, PMID:26149123, PMID:28292906, PMID:30323024). This PKG-centered pathway operates across the life cycle, being required for asexual schizogony and egress, gametogenesis, ookinete and sporozoite motility, and liver-stage merosome release (PMID:19915077, PMID:18532880, PMID:19779564, PMID:19940133, PMID:27425827). PKG is a validated antimalarial drug target: potent selective inhibitors act across multiple stages, and resistance selection maps to gatekeeper mutations rather than loss of PKG itself (PMID:28874661, PMID:41277867, PMID:32359426).

Mechanistic history

Synthesis pass · year-by-year structured walk · 22 steps
  1. 2002 High

    Establishing that PF3D7_1436600 encodes a genuine cGMP-selective kinase was the founding question, defining it as a distinct second-messenger effector expressed in a stage-specific manner.

    Evidence Recombinant PfPKG expressed in E. coli assayed for cGMP-stimulated phosphotransferase activity, with stage expression by Western blot

    PMID:12068803

    Open questions at the time
    • Physiological substrates unknown
    • Cellular function untested
    • Structural basis of cGMP selectivity undefined
  2. 2003 High

    Which of the four nucleotide-binding sites drives activation was resolved, showing a hierarchical contribution and that the degenerate fourth site aids activation without binding cGMP.

    Evidence Systematic site-directed mutagenesis of each cGMP-binding site with in vitro Ka and Vmax measurements

    PMID:12817987

    Open questions at the time
    • No structural view of the activation mechanism
    • Relationship between sites in the intact protein unresolved
  3. 2005 Medium

    Native PfPKG activity was purified and shown to match recombinant enzyme in cooperativity, substrate preference, and inhibitor sensitivity, validating the recombinant tools and Compound 1 as a probe.

    Evidence cGMP-affinity and ion-exchange purification of native activity, plus recombinant expression in T. gondii with kinase assays

    PMID:16325279

    Open questions at the time
    • Endogenous substrates still unidentified
    • Cellular role not yet tested
  4. 2009 High

    Chemical-genetic experiments established PKG as essential for asexual schizont rupture and gametogenesis, moving it from a biochemical entity to a functional gatekeeper of key life-cycle transitions.

    Evidence Compound 1 inhibition rescued by inhibitor-insensitive mutant PKG in P. falciparum blood stages and gametocytes, with Ca2+ epistasis

    PMID:18532880 PMID:19915077

    Open questions at the time
    • Direct substrates driving egress/gametogenesis unknown
    • Mechanistic link to Ca2+ undefined
  5. 2009 High

    Work in P. berghei extended PKG's role to ookinete gliding and late liver-stage merosome release, and placed GCbeta/PDEdelta as the cGMP source/sink in ookinetes.

    Evidence Genetic epistasis with single/double cyclase and phosphodiesterase knockouts, PKG inhibition, motility assays, and conditional sporozoite disruption

    PMID:19779564 PMID:19940133

    Open questions at the time
    • Downstream motility substrates unidentified
    • How merosome release is controlled unresolved
  6. 2013 High

    PKG was placed at the apex of the protease-mediated egress pathway by showing it controls PfSUB1 discharge and microneme release, and that its hyperactivation triggers premature egress.

    Evidence PKG inhibition and phosphodiesterase-mediated hyperactivation with SUB1 discharge, microneme, and egress readouts in P. falciparum

    PMID:23675297

    Open questions at the time
    • Direct PKG substrates in the discharge pathway unknown
    • Signal transduction to Ca2+ not yet mechanistic
  7. 2014 High

    The intermediate signaling logic was clarified: PKG controls phosphoinositide metabolism and PIP2 hydrolysis upstream of the Ca2+ signal required for gliding, gametocyte activation, and egress.

    Evidence Conditional PKG inhibition with quantitative phosphoproteomics, lipidomics, and Ca2+ imaging across P. berghei and P. falciparum

    PMID:24594931

    Open questions at the time
    • Enzyme(s) linking PKG to phosphoinositide hydrolysis unresolved
    • Identity of the Ca2+ channel unknown at this stage
  8. 2015 High

    The substrate landscape was mapped and PKG was shown to be required for invasion as well as egress, establishing it as a shared signaling hub.

    Evidence SILAC phosphoproteomics under chemical and genetic PKG inhibition identifying 69 dependent proteins including PfCDPK1 and actomyosin components, plus invasion assays in P. falciparum

    PMID:26149123

    Open questions at the time
    • Direct versus indirect substrates not fully distinguished
    • Functional consequence of most phosphosites untested
  9. 2015 High

    The structural gate for activation was defined: CNB-D is the high-affinity gatekeeper whose cGMP-induced capping triad is required for both in vitro activity and in vivo egress.

    Evidence Crystal structures of CNB-D with and without cGMP, mutagenesis of the capping triad, in vitro kinase assays, and parasite egress phenotyping in P. falciparum

    PMID:25646845

    Open questions at the time
    • Full-length activation mechanism not yet visualized
    • How CNB-D change reaches the catalytic domain unresolved
  10. 2017 Medium

    The mechanistic and cellular consequences of PKG activation were refined: CNB-D activates via an alphaC-helix relay distinct from human PKG, and PKG triggers a temporally defined PVM breakdown step during egress.

    Evidence Truncation constructs with kinase assays and structural modeling; and selective inhibitor dissection with multimodal imaging of egress steps in P. falciparum

    PMID:28292906 PMID:29251493

    Open questions at the time
    • Effectors executing PVM breakdown unknown
    • Structural relay inferred rather than seen in full-length enzyme
  11. 2016 High

    PKG's motility role in sporozoites was defined and separated from CDPK4, showing it acts as an independent second-messenger effector controlling TRAP secretion, invasion, and egress.

    Evidence PKG-specific inhibitors, inhibitor-insensitive mutant sporozoites, invasion/motility and TRAP secretion assays in P. berghei

    PMID:27425827

    Open questions at the time
    • Direct substrates driving TRAP secretion unknown
    • Molecular link between PKG and adhesin release unresolved
  12. 2018 High

    PKG's cooperative relationships with other kinases were mapped, placing it upstream of/parallel to PfCDPK5 in egress and revealing synthetic interaction with CDPK4 in invasion.

    Evidence PfCDPK5 knockdown with PKG hyperactivation epistasis; systematic kinase genetic interaction screen with hypomorphic PKG and CDPK4 deletion validated in both species

    PMID:29487234 PMID:30315162

    Open questions at the time
    • Shared versus distinct substrates of PKG and CDPKs unresolved
    • Nature of the parallel Ca2+ inputs undefined
  13. 2018 Medium

    A specific direct substrate/partner was identified, linking PKG to proteasome regulation through phosphorylation of the 19S subunit RPT1.

    Evidence Yeast two-hybrid, in vitro binding/competition, and in vitro peptide kinase assays with consensus-motif mining in P. falciparum

    PMID:30323024

    Open questions at the time
    • Functional consequence of RPT1 phosphorylation in vivo untested
    • Single-lab interaction without reciprocal in-parasite validation
  14. 2019 High

    The full-length inactive structure revealed the pentagonal architecture and autoinhibitory segment, defining the cooperative structural relay by which cGMP activates PKG.

    Evidence X-ray structures of full-length cGMP-free PfPKG and PvPKG with analytical ultracentrifugation

    PMID:31239348

    Open questions at the time
    • Active-state full-length structure not solved
    • Dynamics of the relay not directly observed
  15. 2020 High

    Regulatory subtlety was added by showing an allosteric antagonist stabilizes a distinct mixed intermediate rather than a simple off-state, refining the activation model.

    Evidence NMR chemical shift analysis, molecular dynamics, SPR, and CNB-D mutagenesis with 8-NBD-cGMP on P. falciparum PKG

    PMID:32317283

    Open questions at the time
    • Relevance of the intermediate state to cellular regulation unknown
  16. 2020 High

    Genetic dissection established that PKG acts purely catalytically at egress with no scaffolding role, and that phosphorylation of PKG itself is required for its in vivo function.

    Evidence DiCre conditional disruption with simultaneous allelic replacement, phosphorylation-deficient mutant, and egress assays; plus multi-omic target validation of MMV030084 in P. falciparum

    PMID:32179592 PMID:32359426

    Open questions at the time
    • Kinase(s) phosphorylating PKG not identified
    • Role in stages other than egress not addressed by these knockouts
  17. 2021 High

    The long-missing link from PKG to Ca2+ was identified as the membrane protein ICM1, a PKG-associated, PKG-phosphorylated channel required for calcium mobilization.

    Evidence Co-immunoprecipitation, PKG-dependent phosphoproteomics, and conditional ICM1 depletion with Ca2+ imaging and egress/invasion assays in P. berghei and P. falciparum

    PMID:33762339

    Open questions at the time
    • Direct proof that ICM1 conducts Ca2+ not established
    • How ICM1 phosphorylation gates channel activity unknown
  18. 2021 High

    The upstream cGMP source and additional substrates were pinned down: GCα is the sole cGMP synthase feeding PKG in blood stages, and SEA1 is a PKG substrate acting in nuclear segregation upstream of egress.

    Evidence Conditional GCα KO with cGMP/Ca2+/egress readouts and cGMP chemical complementation; PKG-dependent phosphoproteomics with conditional SEA1 disruption in P. falciparum

    PMID:33500341 PMID:33688001

    Open questions at the time
    • How GCα activity is triggered at egress unresolved
    • Direct versus indirect SEA1 phosphorylation not distinguished
  19. 2024 High

    Dual in vivo regulatory requirements were defined: cGMP binding to CNB-B and activation-loop phosphorylation at Thr695 are each dispensable in vitro but essential in the parasite and shape substrate specificity.

    Evidence Genetic CNB-B and T695A mutants with in vitro kinase assays, quantitative phosphoproteomics, and egress assays in P. falciparum

    PMID:38935780

    Open questions at the time
    • Mechanism by which CNB-B and T695 tune specificity unknown
    • In vitro/in vivo discrepancy in requirement unexplained
  20. 2025 Medium

    The upstream homeostatic control of cGMP was extended by showing PP1 phosphatase modulates GCα basal activity and PV rounding, positioning PP1 above PKG in both asexual and sexual stages.

    Evidence Conditional PfPP1 knockout with cGMP measurement, PV morphology imaging, chemical epistasis, and transmission assays in P. falciparum blood and gametocyte stages

    PMID:39808636 PMID:40823845

    Open questions at the time
    • Whether PP1 acts directly on GCα or intermediaries unresolved
    • PP1 targets controlling cGMP homeostasis unidentified
  21. 2017 High

    PKG was advanced as an antimalarial target with defined inhibitor pharmacology, culminating in co-crystal structures and picomolar-potency compounds active across life-cycle stages.

    Evidence Co-crystal structures of PvPKG–ML10 and PfPKG–pyrrole inhibitors with in vitro kinase, multi-stage parasite, and in vivo mouse assays

    PMID:28874661 PMID:38372781

    Open questions at the time
    • Long-term resistance liability incompletely defined at this stage
  22. 2022 High

    Resistance genetics confirmed PKG as a bona fide drug target, with mutations mapping to gatekeeper residues rather than loss of PKG function.

    Evidence In vitro evolution, whole-genome sequencing, CRISPR editing of gatekeeper mutations, and biochemical assays in P. falciparum

    PMID:32359426 PMID:41277867

    Open questions at the time
    • Full spectrum of clinically relevant resistance mechanisms unknown

Open questions

Synthesis pass · forward-looking unresolved questions
  • How PKG achieves precise substrate selection and stage-specific outputs from a single cGMP input—including the direct effectors executing PVM breakdown and the biophysical proof of ICM1 as the Ca2+ conduit—remains open.
  • Direct substrates driving each cellular event not fully resolved
  • ICM1 channel activity not directly demonstrated
  • Active-state full-length structure and in-parasite dynamics undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
CDPK4 (SYNTHETIC GENETIC INTERACTION IN INVASION) [#17]GCΑ (GUANYLYL CYCLASE; SOLE BLOOD-STAGE CGMP SOURCE UPSTREAM OF PKG) [#24]ICM1 (MULTIPASS MEMBRANE CHANNEL, PKG-ASSOCIATED AND PKG-PHOSPHORYLATED) [#23]PFCDPK1 (PKG-DEPENDENT PHOSPHORYLATION) [#10]PFCDPK5 (COOPERATES IN EGRESS PATHWAY) [#16]PP1 PHOSPHATASE (UPSTREAM REGULATOR OF CGMP HOMEOSTASIS VIA GCΑ) [#28, #29]RPT1 (19S PROTEASOME REGULATORY SUBUNIT; DIRECT BINDING AND SUBSTRATE) [#18]SEA1 (PKG-DEPENDENT PHOSPHORYLATION, NUCLEAR SEGREGATION) [#25]
Complex memberships
PKG (PF3D7_1436600)–ICM1 membrane-associated complex [#23]

Evidence

Reading pass · 31 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2002 PKG (PF3D7_1436600) from P. falciparum is a 97.5 kDa protein containing three predicted cGMP-binding sites and a degenerate fourth site. Recombinant PfPKG (Met115-Phe853) expressed in E. coli displays phosphotransferase activity (substrate phosphorylation and auto-phosphorylation) stimulated at least fivefold by 1.0 µM cGMP, but not by cAMP or 8-pCPT-cGMP. Western blotting and kinase activity assays with native P. falciparum proteins show maximal expression predominantly in the ring stage of the asexual life cycle. Recombinant protein expression, in vitro kinase assay, Western blotting with stage-specific parasite lysates Molecular microbiology High 12068803
2003 Among the four cGMP-binding sites in PKG (PF3D7_1436600), site 3 plays the dominant role in activation: mutation of site 3 alone causes a 10–49-fold increase in Ka(cGMP) and a 45–55% decrease in maximal activity in vitro. Mutations at sites 1 and 2 have lesser effects. Mutation of the degenerate site 4 reduces maximal activity by ~40% without altering Ka(cGMP), indicating site 4 does not bind cGMP but contributes to full activation. Site-directed mutagenesis of each cGMP-binding site (individually and in combination), in vitro kinase activity assay The Biochemical journal High 12817987
2005 Native PKG (PF3D7_1436600) activity partially purified from P. falciparum by cGMP-affinity and ion-exchange chromatography is associated with a ~98 kDa protein detectable by anti-PKG antisera, and shows cGMP-stimulated kinase activity inhibitable by Compound 1. Recombinant PfPKG expressed in T. gondii (Pf(Tg)PKG) has kinase activity stimulated by cGMP with significant cooperativity (Hill coefficient of 2), and substrate preference and inhibitor sensitivity similar to native PfPKG and other apicomplexan PKGs. cGMP-affinity chromatography, ion-exchange chromatography, in vitro kinase assay, recombinant protein expression in T. gondii, Western blot Molecular and biochemical parasitology Medium 16325279
2008 PKG (PF3D7_1436600) is essential for xanthurenic acid (XA)- and zaprinast-induced gametogenesis in P. falciparum. Using PKG inhibitors together with transgenic parasites expressing an inhibitor-insensitive mutant PKG, it was shown that PKG activity is required for gametocyte-to-gamete differentiation. Intracellular Ca2+ signaling acts downstream of or in parallel with PKG activation during gametogenesis. PKG inhibitor treatment, inhibitor-insensitive mutant PKG transgenic parasites (genetic rescue), intracellular Ca2+ measurements PLoS biology High 18532880
2009 PKG (PF3D7_1436600) is required for blood-stage schizogony in P. falciparum. Compound 1 (trisubstituted pyrrole PKG inhibitor) blocked schizont rupture and merozoite liberation; transgenic parasites expressing a Compound 1-insensitive PfPKG completed schizogony in its presence, confirming PKG as the primary target. Treated schizonts became large and dysmorphic and were unable to rupture. PKG inhibitor treatment, inhibitor-insensitive mutant PKG transgenic parasites (genetic rescue), erythrocyte invasion assays, microscopy Eukaryotic cell High 19915077
2009 In P. berghei ookinetes, PKG (PF3D7_1436600 ortholog) is required for gliding motility. Inhibition of PKG blocks ookinete gliding, while enhanced cGMP signaling (via PDEdelta deletion) causes ookinete rounding. The cGMP for PKG activation in ookinetes is produced primarily by guanylyl cyclase beta (GCbeta) and degraded by PDEdelta, as demonstrated by genetic epistasis (PDEdelta/GCbeta double mutant suppresses PDEdelta-null rounding phenotype). Elevated cGMP/PKG signaling can partially rescue gliding defects in a CDPK3-null mutant. Genetic epistasis (single and double knockout), PKG inhibitor treatment, ookinete motility assay PLoS pathogens High 19779564
2009 Conditional disruption of P. berghei PKG (PF3D7_1436600 ortholog) in sporozoites blocks late liver stage development: mutant sporozoites invade hepatocytes and undergo intracellular development normally but fail to release merosomes into the medium. Conditional gene disruption in sporozoites, liver stage culture, merosome release assay The Journal of biological chemistry High 19940133
2012 In P. falciparum asexual blood stages, PKG (PF3D7_1436600) is expressed maximally at the late schizont stage. Subcellular fractionation and immunofluorescence of epitope-tagged PfPKG show a predominantly cytosolic location with partial overlap with ER markers and some association with the peripheral membrane fraction. PKG inhibition substantially changes the global protein phosphorylation pattern of schizonts, indicating a complex and broad role in schizogony. Transgenic epitope-tagged PKG expression, Western blot stage analysis, immunofluorescence assay (IFA), subcellular fractionation, global phosphoproteomics after PKG inhibition PloS one Medium 23139764
2013 PKG (PF3D7_1436600) activity is required for PfSUB1 discharge into the parasitophorous vacuole and for microneme release during merozoite egress. Pharmacological stimulation of PKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of immature, non-invasive parasites. This identifies PKG as the upstream regulator of the protease-mediated egress signaling pathway. PKG inhibitor treatment, phosphodiesterase inhibitor stimulation, immunofluorescence, Western blot for PfSUB1 discharge, egress assays PLoS pathogens High 23675297
2014 PKG (PF3D7_1436600) controls phosphoinositide biosynthesis and maintains elevated cytosolic Ca2+ required for gliding motility in P. berghei ookinetes. The same PKG-dependent pathway operates upstream of Ca2+ signals mediating P. berghei gametocyte activation and P. falciparum merozoite egress. Perturbation of PKG signaling in gliding ookinetes causes phosphoproteomic changes enriched in vesicular trafficking and phosphoinositide metabolism pathways. In P. falciparum, PKG inhibition blocks hydrolysis of phosphatidylinositol (4,5)-bisphosphate, linking PKG to phosphoinositide metabolism upstream of Ca2+ mobilization. Conditional PKG inhibition, quantitative phosphoproteomics, global phospholipid analysis, Ca2+ imaging, egress assays PLoS biology High 24594931
2015 Quantitative global phosphoproteomics in P. falciparum identified 69 proteins whose phosphorylation is directly or indirectly dependent on PKG (PF3D7_1436600) activity. These include PfCDPK1 (phosphorylated by PKG) and components of the actomyosin complex. PKG activity is also required for erythrocyte invasion (not only egress), establishing PKG as a signaling hub for both processes. Chemical and genetic PKG inhibition combined with quantitative global phosphoproteomics (SILAC-based), invasion assays Nature communications High 26149123
2015 The C-terminal (carboxyl) cGMP-binding domain (CNB-D) of PKG (PF3D7_1436600) has the highest cGMP affinity and selectivity and functions as a gatekeeper for PKG activation. Crystal structures of this domain with (1.9 Å) and without (2.0 Å) cGMP reveal a PfPKG-specific capping triad that forms upon cGMP binding. Disruption of the capping triad reduces kinase activity by 90% in vitro. Mutating these residues in the parasite prevents blood-stage merozoite egress, confirming functional essentiality in vivo. X-ray crystallography, site-directed mutagenesis, in vitro kinase assay, parasite genetic engineering with egress phenotype readout PLoS pathogens High 25646845
2016 In P. berghei, PKG (PF3D7_1436600 ortholog) is required for sporozoite invasion of hepatocytes and sporozoite motility. Chemical inhibition of PKG with specific inhibitors decreases TRAP secretion (an adhesin essential for motility) by sporozoites, and chemical inhibition also reduces parasite egress from hepatocytes. Sporozoites expressing a mutated, inhibitor-insensitive form of PbPKG are defective in hepatocyte invasion. PKG and CDPK4 act as independent second-messenger effectors for sporozoite invasion. PKG-specific inhibitors, inhibitor-insensitive mutant PKG sporozoites, hepatocyte invasion assays, TRAP secretion assay, merosome egress assay Molecular microbiology High 27425827
2017 PKG (PF3D7_1436600) triggers the PKG-dependent breakdown of the parasitophorous vacuole membrane (PVM) into multilayered vesicles during egress. PKG-dependent PVM breakdown is temporally distinct from an initial cysteine-protease-independent PVM permeabilization step (which precedes PKG-triggered breakdown by 10–30 min). An abrupt host erythrocyte cytoskeleton collapse occurs in a narrow window just before PVM breakdown, not gradually during schizont development. Selective PKG inhibitor and cysteine protease inhibitor treatment combined with video microscopy, electron tomography, electron energy loss spectroscopy, soft X-ray tomography Proceedings of the National Academy of Sciences of the United States of America High 28292906
2017 A co-crystal structure of Plasmodium vivax PKG (PvPKG, ortholog of PF3D7_1436600) bound to the imidazopyridine inhibitor ML10 reveals intimate molecular contacts explaining potency and selectivity of this inhibitor series. The compound inhibits PKG with an IC50 of 160 pM (P. falciparum PKG assay) and acts on merozoite egress, erythrocyte invasion, and gametocyte transmission. X-ray co-crystal structure of PvPKG–inhibitor complex, in vitro PfPKG kinase assay, P. falciparum blood-stage and transmission assays, in vivo P. falciparum SCID mouse model Nature communications High 28874661
2017 The cGMP-binding domain D (CNB-D) of PKG (PF3D7_1436600) controls kinase activation through a mechanism distinct from the pseudosubstrate autoinhibitory sequence used by human PKG. A construct containing only CNB-D and the catalytic domain is inactive without cGMP and efficiently activated by cGMP. cGMP binding to CNB-D induces a conformational change at the αC-helix; the inactive state involves an interaction between Asp597 (catalytic domain) and Arg528 (αC-helix of CNB-D), while the active state involves Arg528 stabilizing cGMP via Tyr480 of the phosphate binding cassette. Truncation constructs, in vitro kinase assays, structural analysis based on existing crystal data ACS infectious diseases Medium 29251493
2018 PKG (PF3D7_1436600) cooperates with PfCDPK5 to fully activate the protease cascade critical for parasite egress. Hyperactivation of PKG (by phosphodiesterase inhibitor treatment) overcomes the egress block caused by PfCDPK5 knockdown and restores microneme release, placing PKG upstream of or in parallel with PfCDPK5 in the egress signaling pathway. PfCDPK5 knockdown, PKG hyperactivation (phosphodiesterase inhibitor), microneme release assay, egress assay mBio Medium 29487234
2018 In P. berghei, a hypomorphic allele of PKG (PF3D7_1436600 ortholog) combined with CDPK4 deletion causes a synthetic negative interaction for erythrocyte invasion, conserved also in P. falciparum. When PKG-dependent Ca2+ signals are attenuated, CDPK4 becomes critical for phosphorylating proteins important for inner membrane complex stability, which anchors the acto-myosin motor required for motility and invasion. Genetic interaction screen (294 kinase combinations), hypomorphic PKG allele, CDPK4 deletion, P. falciparum genetic validation, invasion assays, phosphoproteomics Nature communications High 30315162
2018 PKG (PF3D7_1436600) directly interacts with and phosphorylates RPT1, a subunit of the 19S regulatory particle of the P. falciparum proteasome. PfPKG and RPT1 interact in vitro (yeast two-hybrid and in vitro binding confirmed). A peptide from the RPT1 interacting region contains a PfPKG consensus phosphorylation site and is efficiently phosphorylated by PfPKG, and competes with full-length RPT1 fragment for binding to PfPKG. Phosphoproteomic mining using the consensus motif identified additional likely direct PKG substrates. Yeast two-hybrid library screen, in vitro binding/competition assay, in vitro kinase assay with peptide substrates, consensus phosphorylation site analysis Infection and immunity Medium 30323024
2019 Crystal structures of full-length PKG (PF3D7_1436600) from P. falciparum and P. vivax in the cGMP-free (inactive) state reveal an overall pentagonal arrangement of the four CNBs and kinase domain, with an N-terminal autoinhibitory segment (AIS) docked in the substrate site of the kinase domain in a swapped-domain dimer. The AIS is necessary to maintain the inactive state. A helix serves the dual role of N-terminal helix of the kinase domain and capping helix of the neighboring CNB. Cooperative cGMP binding, beginning at the CNB closest to the kinase domain, transmits conformational changes around the pentagonal molecule via a structural relay mechanism to activate PKG. X-ray crystallography of full-length PfPKG and PvPKG (cGMP-free), structural analysis, analytical ultracentrifugation (monomer/dimer determination) Proceedings of the National Academy of Sciences of the United States of America High 31239348
2020 8-NBD-cGMP is an effective allosteric antagonist of PKG (PF3D7_1436600) that inhibits PfPKG not by a simple two-state active/inactive switch, but by stabilizing a distinct inactive 'mixed' intermediate state sharing features of both apo and cGMP-bound forms. This was demonstrated by NMR chemical shift analysis, molecular dynamics simulations, and site-directed mutagenesis of the key regulatory CNB-D domain. NMR chemical shift perturbation analysis, molecular dynamics simulations, site-directed mutagenesis, surface plasmon resonance The Journal of biological chemistry High 32317283
2020 PKG (PF3D7_1436600) activity is required for P. falciparum asexual blood stage egress, hepatocyte invasion by sporozoites, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies combined with conditional knockdown parasites, molecular docking, and recombinant kinase assays identified PKG as the primary cellular target of MMV030084. Resistance selection and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under selection pressure. Metabolomics, phosphoproteomics, chemoproteomics, conditional knockdown, molecular docking, recombinant kinase assay, resistance selection, gene editing Cell chemical biology High 32359426
2020 Using the DiCre system with multiple lox sites, simultaneous conditional disruption and allelic replacement of PKG (PF3D7_1436600) demonstrated that PfPKG has no scaffolding or adaptor role in intraerythrocytic development, acting solely at merozoite egress. A phosphorylation-deficient PfPKG (with key phosphorylation sites mutated) is functionally incompetent, showing that phosphorylation of PKG itself is required for its in vivo function. DiCre conditional disruption, multiple simultaneous allelic replacement, egress assays, phosphorylation-deficient mutant Life science alliance High 32179592
2021 A multipass membrane protein ICM1 is tightly associated with PKG (PF3D7_1436600) in both asexual blood stages and transmission stages of Plasmodium. Multiple ICM1 phosphorylation events are dependent on PKG activity (shown by phosphoproteomics). Conditional depletion of P. berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization; conditional loss of P. falciparum ICM1 results in severely reduced calcium mobilization, defective egress, and lack of invasion. ICM1 is proposed as the missing link transducing PKG-dependent signals to intracellular Ca2+ release. Co-immunoprecipitation (PKG–ICM1 interaction), phosphoproteomics (PKG-dependent ICM1 phosphorylation), conditional stage-specific depletion (P. berghei and P. falciparum), Ca2+ imaging, egress and invasion assays Science advances High 33762339
2021 GCα is the sole enzyme responsible for cGMP production in P. falciparum asexual blood stages and is required for PKG (PF3D7_1436600)-driven Ca2+ mobilization and merozoite egress. GCα-null parasites cannot synthesize cGMP or mobilize calcium; chemical complementation with a cGMP analogue rescues egress. Point mutagenesis of a conserved residue in the P4-ATPase domain of GCα abolishes cGMP synthesis, showing P4-ATPase activity is upstream of and linked to cGMP synthesis and hence PKG activation. Conditional KO of GCα, cGMP measurement, Ca2+ mobilization assay, egress assay, chemical complementation with cGMP analogue, site-directed mutagenesis of P4-ATPase domain mBio High 33500341
2021 SEA1 (PF3D7_1021800) undergoes phosphorylation dependent on PKG (PF3D7_1436600) activity, placing SEA1 as a PKG substrate during schizont development. However, SEA1 does not play a direct mechanistic role in egress per se; instead it functions upstream of egress as a regulator of correct nuclear segregation into daughter merozoites. PKG-dependent phosphoproteomics, conditional SEA1 disruption, egress and nuclear segregation phenotyping mBio Medium 33688001
2024 PKG (PF3D7_1436600) is regulated by two distinct mechanisms in P. falciparum asexual blood stages: (1) cGMP occupancy of cyclic nucleotide binding domain B (CNB-B) — reduced cGMP binding to CNB-B does not affect in vitro kinase activity but abolishes parasite egress in vivo; (2) phosphorylation of Thr695 in the activation loop — T695 phosphorylation is dispensable for in vitro kinase activity but essential for in vivo PKG function, with loss of T695 phosphorylation causing aberrant proteome-wide phosphorylation events and altered substrate specificity. Genetic engineering of CNB-B cGMP-binding mutants and T695A activation-loop mutants, in vitro kinase assay, quantitative phosphoproteomics, egress assay PLoS pathogens High 38935780
2024 The crystal structure of PfPKG (PF3D7_1436600) bound to a pyrrole-series inhibitor was solved, revealing the structural basis for inhibitor binding at the ATP-binding site and enabling structure-activity relationship analysis for the pyrrole series. X-ray crystallography of PfPKG–inhibitor co-crystal, structure-based modeling, in vitro recombinant PfPKG kinase assay, gatekeeper mutant transgenic parasite selectivity assay Journal of medicinal chemistry Medium 38372781
2025 PP1 phosphatase controls the homeostasis of cGMP in P. falciparum by modulating the basal activity of guanylyl cyclase alpha (GCα), thereby acting upstream of PKG (PF3D7_1436600) and PKG-dependent downstream Ca2+ signaling. PP1 also regulates the rounding-up of the parasitophorous vacuole, the first step in the egress cascade, a process reliant on Ca2+ and occurring upstream of PKG-driven membrane breakdown. Conditional PfPP1 knockout (iKO), fluorescent reporters of PV morphology, live-microscopy, cGMP measurement, egress kinetics with PKG pathway inducers/inhibitors, Ca2+ ionophore rescue PLoS pathogens Medium 39808636
2025 In P. falciparum gametocytes, PfPP1 controls crucial steps of gametocyte activation through stimulation of the cGMP/PKG (PF3D7_1436600) pathway, placing PfPP1 upstream of PKG activation during sexual stage transmission. Conditional PfPP1 depletion, cGMP measurement, gametocyte activation assays, mosquito transmission experiments mBio Medium 40823845
2022 PKG (PF3D7_1436600) resistance mutations in P. falciparum mapped to the gatekeeper residue T618I upon in vitro selection with PKG-targeting compounds TCMDC-141334 and TCDMC-140674, consistent with direct interaction of these compounds with the PKG active site. CRISPR editing of T618I in naive parasites confirmed the resistance phenotype and thus validated PKG as the target. In vitro evolution of resistance, whole-genome sequencing, CRISPR editing, in vitro activity assays with recombinant proteins, molecular docking mBio High 41277867

Source papers

Stage 0 corpus · 78 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2013 Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS pathogens 217 23675297
2008 Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase. PLoS biology 193 18532880
2014 Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites. PLoS biology 178 24594931
2009 The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony. Eukaryotic cell 168 19915077
2009 A cyclic GMP signalling module that regulates gliding motility in a malaria parasite. PLoS pathogens 160 19779564
2015 Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion. Nature communications 141 26149123
2017 A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission. Nature communications 136 28874661
2009 Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development. The Journal of biological chemistry 84 19940133
2015 Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites. ACS chemical biology 82 25555060
2017 Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress. Proceedings of the National Academy of Sciences of the United States of America 81 28292906
2020 Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity. Cell chemical biology 75 32359426
2005 Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): antiparasitic activity of a PKG inhibitor. Molecular and biochemical parasitology 73 16325279
2018 Calcium-Dependent Protein Kinase 5 Is Required for Release of Egress-Specific Organelles in Plasmodium falciparum. mBio 64 29487234
2016 Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4. Molecular microbiology 62 27425827
2002 A novel cyclic GMP-dependent protein kinase is expressed in the ring stage of the Plasmodium falciparum life cycle. Molecular microbiology 60 12068803
2017 Cyclic nucleotide signalling in malaria parasites. Open biology 59 29263246
2018 ISP1-Anchored Polarization of GCβ/CDC50A Complex Initiates Malaria Ookinete Gliding Motility. Current biology : CB 57 30146157
2018 Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites. Nature communications 55 30315162
2016 Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling. The Journal of biological chemistry 55 26933036
2021 Ca2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG. Science advances 51 33762339
2018 Plasmodial Kinase Inhibitors: License to Cure? Journal of medicinal chemistry 49 29771541
2015 Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development. Antimicrobial agents and chemotherapy 47 26711771
2019 High-throughput screening of the Plasmodium falciparum cGMP-dependent protein kinase identified a thiazole scaffold which kills erythrocytic and sexual stage parasites. Scientific reports 46 31065005
2020 An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites. Nature communications 45 32273496
2003 The role of two novel regulatory sites in the activation of the cGMP-dependent protein kinase from Plasmodium falciparum. The Biochemical journal 44 12817987
2012 The role of cGMP signalling in regulating life cycle progression of Plasmodium. Microbes and infection 39 22613210
2020 Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization of Plasmodium falciparum and Plasmodium knowlesi asexual blood-stage parasites. PloS one 38 32673324
2019 FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons. Communications biology 37 31701023
2016 Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G. mBio 37 27923926
2019 Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation. Proceedings of the National Academy of Sciences of the United States of America 36 31239348
2022 The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages. Science translational medicine 35 36260689
2013 cGMP-Dependent Protein Kinase Inhibitors in Health and Disease. Pharmaceuticals (Basel, Switzerland) 33 24275951
2020 Simultaneous multiple allelic replacement in the malaria parasite enables dissection of PKG function. Life science alliance 30 32179592
2010 Activity of a trisubstituted pyrrole in inhibiting sporozoite invasion and blocking malaria infection. Antimicrobial agents and chemotherapy 28 20643897
2021 Plasmodium falciparum Guanylyl Cyclase-Alpha and the Activity of Its Appended P4-ATPase Domain Are Essential for cGMP Synthesis and Blood-Stage Egress. mBio 25 33500341
2019 Development of Chemical Entities Endowed with Potent Fast-Killing Properties against Plasmodium falciparum Malaria Parasites. Journal of medicinal chemistry 25 31566384
2015 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. PLoS pathogens 25 25646845
2021 Malaria Parasite Schizont Egress Antigen-1 Plays an Essential Role in Nuclear Segregation during Schizogony. mBio 24 33688001
2020 Targeting the Malaria Parasite cGMP-Dependent Protein Kinase to Develop New Drugs. Frontiers in microbiology 24 33391223
2018 Trisubstituted thiazoles as potent and selective inhibitors of Plasmodium falciparum protein kinase G (PfPKG). Bioorganic & medicinal chemistry letters 23 30174152
2012 Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase. PloS one 19 23139764
2021 Plasmodium falciparum cGMP-Dependent Protein Kinase - A Novel Chemotherapeutic Target. Frontiers in microbiology 17 33613463
2020 Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase. The Journal of biological chemistry 17 32317283
2020 Cyclic nucleotide selectivity of protein kinase G isozymes. Protein science : a publication of the Protein Society 17 33271627
2020 New Amidated 3,6-Diphenylated Imidazopyridazines with Potent Antiplasmodium Activity Are Dual Inhibitors of Plasmodium Phosphatidylinositol-4-kinase and cGMP-Dependent Protein Kinase. ACS infectious diseases 16 33319990
2022 A G-Protein-Coupled Receptor Modulates Gametogenesis via PKG-Mediated Signaling Cascade in Plasmodium berghei. Microbiology spectrum 15 35404079
2018 Potent inhibitors of malarial P. Falciparum protein kinase G: Improving the cell activity of a series of imidazopyridines. Bioorganic & medicinal chemistry letters 15 30553738
2016 Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II. The Journal of biological chemistry 14 26769964
2017 cGMP Binding Domain D Mediates a Unique Activation Mechanism in Plasmodium falciparum PKG. ACS infectious diseases 12 29251493
2024 Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials. Journal of medicinal chemistry 11 38372781
2023 Structure-based virtual screening against multiple Plasmodium falciparum kinases reveals antimalarial compounds. Molecular diversity 11 38127294
2019 Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency, selectivity and structural novelty. Bioorganic & medicinal chemistry letters 11 31471167
2018 Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome. Infection and immunity 10 30323024
2021 Discovery of Imidazole-Based Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase. ACS medicinal chemistry letters 8 34917261
2014 Targeting Plasmodium falciparum protein kinases with adenosine analogue-oligoarginine conjugates. Experimental parasitology 8 24534615
2025 The malaria parasite PP1 phosphatase controls the initiation of the egress pathway of asexual blood-stages by regulating the rounding-up of the vacuole. PLoS pathogens 7 39808636
2019 Discovery of isoxazolyl-based inhibitors of Plasmodium falciparum cGMP-dependent protein kinase. RSC medicinal chemistry 6 33479608
2014 Development of a transgenic Plasmodium berghei line (Pb pfpkg) expressing the P. falciparum cGMP-dependent protein kinase, a novel antimalarial drug target. PloS one 5 24805991
2022 Characterization of Competitive Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase. Chembiochem : a European journal of chemical biology 4 35044710
2022 Synchronisation of Plasmodium falciparum and P. knowlesi In Vitro Cultures Using a Highly Specific Protein Kinase Inhibitor. Methods in molecular biology (Clifton, N.J.) 4 35881342
2023 Exploring potential Plasmodium kinase inhibitors: a combined docking, MD and QSAR studies. Journal of biomolecular structure & dynamics 3 37599462
2014 Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress. Bio-protocol 3 29075653
2022 Divergent allostery reveals critical differences between structurally homologous regulatory domains of Plasmodium falciparum and human protein kinase G. The Journal of biological chemistry 2 35143840
2021 Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G. The journal of physical chemistry. B 2 34115498
2020 Plasmodium falciparum protein kinase as a potential therapeutic target for antimalarial drugs development. Tropical biomedicine 2 33612795
2026 Small Molecule Kinase Inhibitors with In Vitro β-Hematin Formation and Plasmodium falciparum Protein Kinase G Inhibitory Activity Identified Using Machine Learning. ChemMedChem 1 41693340
2025 Versatile Imidazole Scaffold with Potent Activity against Multiple Apicomplexan Parasites. ACS infectious diseases 1 40339062
2024 Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites. PLoS pathogens 1 38935780
2024 Another decade of antimalarial drug discovery: New targets, tools and molecules. Progress in medicinal chemistry 1 39370241
2026 Computational screening of natural inhibitors against Plasmodium falciparum kinases: Toward novel antimalarial therapies. PloS one 0 41529029
2026 TEpiNom: A computational framework integrating population data to prioritize Plasmodium falciparum T cell epitopes. Vaccine 0 41775037
2026 Accessing invasion-competent Plasmodium vivax merozoites reveals antibody-dependent NK cell activation. Research square 0 41960345
2026 An orally available PfPKG inhibitor blocks Plasmodium's infection of the liver. PLoS pathogens 0 42531257
2025 Medicinal Chemistry Progression of Sapanisertib, the Anticancer and Dual Plasmodium Phosphatidylinositol 4-Kinase Beta and cGMP-Dependent Protein Kinase Inhibitor, for Malaria. Journal of medicinal chemistry 0 40377111
2025 Loss of serine/threonine protein phosphatase 6 severely impairs sexual stage development in malaria parasite Plasmodium berghei. PLoS pathogens 0 40623036
2025 Plasmodium falciparum PP1 phosphatase is a key regulator of malaria parasite transmission. mBio 0 40823845
2025 Mechanistic insights into dual-active liver and blood-stage antiplasmodials. mBio 0 41277867
2020 Keep a lid on it: A troika in kinase allostery. The Journal of biological chemistry 0 32561554