Quantitative Assay of Cyclin-Dependent Kinase Activity As a Sensitive Marker of Cell Proliferation in Marine Teleost Larvae

An evolutionarily conserved family of cyclin-dependent kinases (Cdks) regulates cell cycle progression in eukaryotes. Mitosis-promoting factor (MPF) is a transiently activated Cdk required for mitosis. We propose use of partially purified MPF kinase activity as a sensitive marker of cell proliferation in marine teleost larvae and describe a quantitative spin-filter assay of suc1-precipitated MPF activity. MPF extracted from embryos and larvae of red drum, Sciaenops ocellatus, phosphorylated histone H1 protein and a Cdk-specific peptide substrate. Kinetic analyses demonstrated that apparent K _m values of red drum MPF for adenosine triphosphate and the peptide substrate were 98.3 and 18.2 μM, respectively. A simple growth experiment showed that MPF activity was significantly greater in rapidly growing red drum larvae than in intermittently fed cohorts growing more slowly. Received July 23, 1998; accepted January 14, 1999     

A Distinct Peripheral Blood Monocyte Phenotype Is Associated with Parasite Inhibitory Activity in Acute Uncomplicated Plasmodium falciparum Malaria

Monocyte (MO) subpopulations display distinct phenotypes and functions which can drastically change during inflammatory states. We hypothesized that discrete MO subpopulations are induced during malaria infection and associated with anti-parasitic activity. We characterized the phenotype of blood MO from healthy malaria-exposed individuals and that of patients with acute uncomplicated malaria by flow cytometry. In addition, MO defense function was evaluated by an in vitro antibody dependent cellular inhibition (ADCI) assay. At the time of admission, the percentages and absolute numbers of CD16⁺ MO, and CCR2⁺CX3CR1⁺ MO, were high in a majority of patients. Remarkably, expression of CCR2 and CX3CR1 on the CD14^{high (hi)} MO subset defined two subgroups of patients that also differed significantly in their functional ability to limit the parasite growth, through the ADCI mechanism. In the group of patients with the highest percentages and absolute numbers of CD14^hiCCR2⁺CX3CR1⁺ MO and the highest mean levels of ADCI activity, blood parasitemias were lower (0.14±0.34%) than in the second group (1.30±3.34%; p = 0.0053). Data showed that, during a malaria attack, some patients'' MO can exert a strong ADCI activity. These results bring new insight into the complex relationships between the phenotype and the functional activity of blood MO from patients and healthy malaria-exposed individuals and suggest discrete MO subpopulations are induced during malaria infection and are associated with anti-parasitic activity.     

A Purine Analog-Sensitive Protein Kinase Activity Associates with Trk Nerve Growth Factor Receptors

Abstract: Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and ∼50–80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.     

A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella-like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro, PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process.     

An Exported Heat Shock Protein 40 Associates with Pathogenesis-Related Knobs in Plasmodium falciparum Infected Erythrocytes

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.     

Efficacy of Combined Histone Deacetylase and Checkpoint Kinase Inhibition in a Preclinical Model of Human Burkitt Lymphoma

Checkpoint kinase inhibition has been studied as a way of enhancing the effectiveness of DNA-damaging agents. More recently, histone deacetylase inhibitors have shown efficacy in several cancers, including non-Hodgkin lymphoma. To evaluate the effectiveness of this combination for the treatment of lymphoma, we examined the combination of AR42, a histone deacetylase inhibitor, and checkpoint kinase 2 (CHEK2) inhibitor II in vitro and in vivo. The combination resulted in up to 10-fold increase in potency in five Burkitt lymphoma cell lines when compared with either drug alone. Both drugs inhibited tumor progression in xenograft models, but the combination was more effective than either agent alone, resulting in regression of established tumors. No toxicity was observed. These results suggest that the combination of histone deacetylase inhibition and checkpoint kinase inhibition represent an effective and nontoxic treatment option that should be further explored in preclinical and clinical studies.     

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.     

Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum

Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.     

The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony

A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.Cyclic GMP (cGMP)-dependent protein kinases (PKGs) are the major intracellular mediators of cGMP signal transduction in eukaryotic cells. Mammalian PKGs regulate a number of physiological processes including smooth muscle relaxation, platelet aggregation, intestinal secretion and hippocampal and cerebellar learning (reviewed in reference 27). Unlike cAMP-dependent protein kinase (PKA), PKG comprises both a catalytic domain and a regulatory domain in a single polypeptide. In the inactivated state, part of the regulatory domain (a substrate-like sequence known as the autoinhibitory domain) is thought to be bound to the catalytic domain. This binding is released and the enzyme activated upon binding of cGMP to the regulatory domain and opening of the substrate-binding region within the catalytic domain (1, 14). The PKG of the malaria parasite Plasmodium falciparum (PfPKG) and orthologues from the related coccidian parasites Toxoplasma and Eimeria are encoded by a single-copy gene and have been shown to differ from the mammalian enzymes in several respects. These include a larger number of cGMP binding sites (three functional plus one degenerate) in the regulatory domain of the apicomplexan enzymes (6, 7, 17), highly cooperative stimulation by cGMP (8, 17), and relative insensitivity to 8-substituted cGMP analogues (6, 7, 26). The parasite isoforms also lack an N-terminal leucine zipper motif that mediates homodimerization in mammalian isoforms, and evidence suggests that they are monomeric (17). Interestingly, it has been shown that the coccidian PKGs exist as both cytosolic and membrane-associated (mediated by N-terminal myristoylation and palmitoylation) isoforms (12), but the amino acid motifs required for these modifications are absent in the PfPKG sequence, and it seems to lack acylated forms (8).The coccidian PKGs have been shown to be the target of a potent anticoccidial agent, 4-[2-(4-fluorphenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl]pyridine (compound 1), which is a competitive inhibitor of ATP binding (17). The selectivity of the compound for apicomplexan enzymes is attributed to the relative accessibility of a hydrophobic pocket that overlaps with the ATP binding site conferred by a critical threonine residue in the key “gatekeeper” position (T761 in Toxoplasma gondii; T770 in Eimeria tenella) (11). Access is thought to be prevented by the relatively bulky side chain of the residue that occupies the corresponding position in mammalian PKGs. Expression of compound 1-insensitive coccidian PKGs (harboring T/Q or T/M gatekeeper residue substitutions) in Toxoplasma abolished the effects of the inhibitor on the parasite, thus establishing that PKG is the primary target of compound 1 in T. gondii. These compound 1-insensitive mutants were used to determine a role for coccidian PKG in secretion of micronemal adhesive proteins, attachment to and invasion of host cells and gliding motility of E. tenella sporozoites and T. gondii tachyzoites. Compound 1 has also been shown to be a potent inhibitor of the native P. falciparum PKG enzyme (50% inhibitory concentration [IC₅₀] of 8.53 nM on cGMP-dependent kinase activity in purified fractions using a fixed ATP concentration of 5 μM in the assay) but has limited in vivo activity in the rodent malaria model P. berghei (8).P. falciparum causes the most serious form of malaria in humans and is responsible for the deaths of approximately one million people each year. The parasite life cycle is complex consisting of distinct phases in the mosquito vector and the human host. Pathogenesis is caused by asexual parasites which proliferate within red blood cells. The invasive form (merozoite) enters a red blood cell and forms a ring-stage parasite. This develops into a trophozoite which feeds on hemoglobin, and the resulting schizont releases up to 32 daughter merozoites in a 48-h cycle. A small proportion of the schizonts release merozoites that differentiate into male or female gamete precursors (gametocytes). These sexual cells are essential for malaria transmission and must be taken up by an Anopheles mosquito to continue the life cycle. Upon entering the insect midgut, environmental cues trigger gametogenesis. Recently, we reported that PfPKG plays an essential role in initiating this process. The initial morphological change after activation of P. falciparum gametocytes, from crescent-shaped to spherical (known as rounding up), is inhibited by compound 1. Genetically manipulated parasites expressing a mutant (T618Q gatekeeper substitution) compound 1-insensitive but fully active PKG, introduced by allelic replacement, were resistant to the effects of compound 1 on gametogenesis, demonstrating a direct role for the enzyme in the initiation of this process (23).There is also evidence suggesting a role for PKG in P. falciparum erythrocytic stages. It has been reported that PfPKG is expressed at both the mRNA and the protein level in asexual blood stages, as well as the sexual phase of the life cycle (6, 8). Although the PfPKG gene could be targeted for allelic replacement, we were unable to disrupt it, suggesting an essential role for PKG in P. falciparum asexual blood stage development since it is this life cycle stage on which transfection and drug selection of genetically modified parasites are performed. Furthermore, we and others (8, 23) have observed that compound 1 inhibits in vitro-cultured P. falciparum (IC₅₀s in the sub-low-μM range depending on the isolate). Although the role of PKG in the initiation of gametogenesis has been elucidated, its role in erythrocytic stages was unknown prior to the present study.We demonstrate here that PfPKG has a central role in the late stages of erythrocytic schizogony. Compound 1-treated P. falciparum parasites develop normally through the trophozoite stage but arrest late in schizont development, forming large dysmorphic schizonts. Genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG are resistant to the effects of both compound 1 and (a second imidazopyridine PKG inhibitor) compound 2 on schizogony but not to the broad-specificity serine/threonine kinase inhibitor staurosporine. This demonstrates that PfPKG is the primary target of the inhibitors and provides direct evidence that this enzyme is essential for progression of the asexual erythrocytic stage of the malaria parasite life cycle.     

A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole-Induced Golgi Dispersal

The protein kinase D (PKD) family comprises multifunctional serine/threonine-specific protein kinases with three mammalian isoforms: PKD1, PKD2 and PKD3. A prominent PKD function is the regulation of basolateral-targeted transport carrier fission from the trans -Golgi network (TGN). To visualize site-specific PKD activation at this organelle, we designed a molecular reporter consisting of a PKD-specific substrate sequence fused to enhanced green fluorescent protein (EGFP), specifically targeted to the TGN via the p230 GRIP domain. Quantitative analyses using a phosphospecific antibody and ratiometric fluorescence imaging revealed that Golgi-specific phosphorylation of the reporter was strictly dependent on stimulation of endogenous PKD or transient expression of active PKD constructs. Conversely, PKD-specific pharmacological inhibitors and siRNA-mediated PKD knockdown suppressed reporter phosphorylation. Using this reporter we investigated a potential role for PKD in the regulation of Golgi complex morphology. Interestingly, nocodazole-induced Golgi complex break-up and dispersal was associated with local PKD activation as measured by reporter phosphorylation and this was efficiently blocked by expression of a dominant-negative PKD mutant or PKD depletion. Our data thus identify a novel link between PKD activity and the microtubule cytoskeleton, whereby Golgi complex integrity is regulated.     

Restoring Histone Deacetylase Activity by Waste Product Release. A View from Molecular Mechanics Simulations with Mammalian HDAC8

HDAC8 is a Zn^II‐based, single‐peptide mammalian histone deacetylase that is localized mainly in the cytoskeleton of smooth muscle cells, thus regulating muscle contractility. HDACs are also widely involved in cellular processes, ranging from cell differentiation to proliferation, senescence, and apoptosis; in particular, protecting a telomerase activator from ubiquitin‐mediated degradation. How HDACs can eliminate the hydrolytic reaction products, in order that the process of deacetylation of the acetyllysine moiety of histones can take place again, has long been debated in the scientific literature, without reaching any firm conclusion, however. This question is the subject of the present work, carried out along a theoretical line that is capable of describing the whole pathway followed by the acetate product (ACT). A model was built here on the crystal data for the Y306F‐mutated HDAC8 complex with a diacetylated peptide of the p53‐tumor‐suppressor class. That was followed by manually hydrolyzing the acetylated moiety bound to Zn^II and discharging the monoacetylated peptide product (MAP). The latter was replaced by a H₂O molecule bound to Zn^II, while ACT was left free in the reaction cage. This Zn^II cluster was DFT‐parameterized for the ff99SB force field without any further bias. As the result of random‐acceleration molecular dynamics (RAMD) simulations, egress of ACT from the reaction cage toward the aqueous environment can follow three pathways. Two of them utilize the channel for peptide (or histone) uptake and are preferred, if ACT leaves the reaction center before MAP (or the deacetylated histone). The third pathway, developing along the internal channel, is available to ACT even if MAP is still in place.     

Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu³⁵⁶ to Thr³⁷⁰) of the JD in the presence of calcium (Ca²⁺). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.     

Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED₅₀ values in the 4-day murine P. berghei efficacy model of 13–21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.