Synthesis and evaluation of 2-pyridyl pyrimidines with in vitro antiplasmodial and antileishmanial activity

A series of 2-pyridyl pyrimidines, reported inhibitors of Plasmodium falciparum methionine aminopeptidase 1b were synthesized and evaluated for their antiplasmodial activities. An analysis of physicochemical properties demonstrated a link between lipophilicity and antiparasitic activity. Cross screening of the library against cultured Leishmania donovani parasites revealed this class of compounds as potent inhibitors of parasite development in vitro.     

Inhibitors of casein kinase 1 block the growth of Leishmania major promastigotes in vitro

Casein kinase 1 (CK1) is a family of multifunctional Ser/Thr protein kinases that are ubiquitous in eukaryotic cells. Recent studies have demonstrated the existence of, and role for, CK1 in protozoan parasites such as Leishmania, Plasmodium and Trypanosoma. The value of protein kinases as potential drug targets in protozoa is evidenced by the successful exploitation of cyclic guanosine monophosphate-dependent protein kinase (PKG) with selective tri-substituted pyrrole and imidazopyridine inhibitors. These compounds exhibit in vivo efficacy against Eimeria tenella in chickens and Toxoplasma gondii in mice. We now report that both of these protein kinase inhibitor classes inhibit the growth of Leishmania major promastigotes and Trypanosoma brucei bloodstream forms in vitro. Genome informatics predicts that neither of these trypanosomatids codes for a PKG orthologue. Biochemical studies have led to the unexpected discovery that an isoform of CK1 represents the primary target of the pyrrole and imidazopyridine kinase inhibitors in these organisms. CK1 from extracts of L. major promastigotes co-fractionated with [(3)H]imidazopyridine binding activity. Further purification of CK1 activity from L. major and characterization via liquid chromatography coupled tandem mass spectrometry identified CK1 isoform 2 as the specific parasite protein inhibited by imidazopyridines. L. major CK1 isoform 2 expressed as a recombinant protein in Escherichia coli displayed biochemical and inhibition characteristics similar to those of the purified native enzyme. The results described here warrant further evaluation of the activity of these kinase inhibitors against mammalian stage Leishmania parasites in vitro and in animal models of infection, as well as studies to genetically validate CK1 as a therapeutic target in trypanosomatid parasites.     

In vitro efficacy of 7-benzylamino-1-isoquinolinamines against Plasmodium falciparum related to the efficacy of chalcones

A series of 1,7-diaminoisoquinolinamines, that are expected to mediate antimalarial activity by the same mechanism employed by the chalcones, were produced. Six 7-benzylamino-1-isoquinolinamines were found to be submicromolar inhibitors in vitro of drug-resistant Plasmodium falciparum, with the best possessing activity comparable to chloroquine. Despite being developed from a lead that is a DHFR inhibitor, these compounds do not mediate their antimalarial effects by inhibition of DHFR.     

Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1)

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Structural basis for the regulation of cysteine-protease activity by a new class of protease inhibitors in Plasmodium

Plasmodium cysteine proteases are essential for host-cell invasion and egress, hemoglobin degradation, and intracellular development of the parasite. The temporal, site-specific regulation of cysteine-protease activity is a prerequisite for survival and propagation of Plasmodium. Recently, a new family of inhibitors of cysteine proteases (ICPs) with homologs in at least eight Plasmodium species has been identified. Here, we report the 2.6?? X-ray crystal structure of the C-terminal, inhibitory domain of ICP from P. berghei (PbICP-C) in a 1:1 complex with falcipain-2, an important hemoglobinase of Plasmodium. The structure establishes Plasmodium ICP as a member of the I42 class of chagasin-like protease inhibitors but with large insertions and differences in the binding mode relative to other family members. Furthermore, the PbICP-C structure explains why host-cell cathepsin B-like proteases and, most likely, also the protease-like domain of Plasmodium SERA5 (serine-repeat antigen 5) are no targets for ICP.     

Pharmacophoric studies of in vitro inhibition of Plasmodium falciparum growth

Malaria continues to be a scourge in India and the situation has been compounded by the emergence of resistant strains of Plasmodium falciparum which is the primary cause of fatality in this disease. Therefore, there is an urgent need to develop newer drugs. Molecular modeling and pharmacophoric determination have become predominant methods today in the design and synthesis of newer and more effective drugs. Many Plasmodium specific enzymes and proteins involved in crucial biochemical pathways have been identified and their structures have been determined by X-ray crystallography. These enzymes and proteins are excellent targets for newer antimalarial agents. Bisphosphonates have shown potent inhibitory activity against Plasmodium farneysl diphosphate synthase (FPPS) enzyme, which is vital to the protein prenylation pathway of the organism. In this study, a set of 26 bisphosphonate inhibitors, synthesized by Oldfield et al [J Med Chem (2008) 51, 7827-7833] were subjected to rigorous 3D-QSAR studies using the PHASE computational package. In vitro Plasmodium growth inhibition rather than direct enzyme inhibition was considered in the study for a more realistic approach. Good statistical correlations were obtained for the pharmacophoric model as revealed by the regression values, indicating good stability of the model. Three hydrogen bond acceptors and a hydrogen bond donor defined the pharmacophore from the present study. This pharmacophore, AAAD (A = Hydrogen bond acceptor and D = Hydrogen bond donor) was put through a search-run for matching structures from the SPECS database yielding four matching structures, which could function as starting points for more novel and potent antimalarials.     

Quinolylhydrazones as novel inhibitors of Plasmodium falciparum serine protease PfSUB1

Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action and consequently able to overcome the current problems of resistance to established chemotherapies. Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone 2 as a hit compound. Here we report a preliminary investigation of the structure-activity relationships for a class of PfSUB1 inhibitors related to our identified hit.     

Rational design of the first difluorostatone-based PfSUB1 inhibitors

The etiological agent of the most dangerous form of malaria, Plasmodium falciparum, has developed resistance or reduced sensitivity to the majority of the drugs available to treat this deadly disease. Innovative antimalarial therapies are therefore urgently required. P. falciparum serine protease subtilisin-like protease 1 (PfSUB1) has been identified as a key enzyme for merozoite egress from red blood cells and invasion. We present herein the rational design, synthesis, and biological evaluation of novel and potent difluorostatone-based inhibitors. Our bioinformatic-driven studies resulted in the identification of compounds 1a, b as potent and selective PfSUB1 inhibitors. The enzyme/inhibitor interaction pattern herein proposed will pave the way to the future optimization of this class of promising enzyme inhibitors.     

Tight-binding inhibitors efficiently inactivate both reaction centers of monomeric Plasmodium falciparum glyoxalase 1

Glucose consumption and therefore methylglyoxal production of human erythrocytes increase significantly upon infection with malaria parasites. The glyoxalase systems of the host-parasite unit cope with this metabolic challenge by catalyzing the removal of harmful methylglyoxal. Thus, glyoxalase 1 from the malaria parasite Plasmodium falciparum (PfGlo1) could be a promising drug target. However, the enzyme has two different active sites and their simultaneous inactivation is considered challenging. Here, we describe the inactivation of PfGlo1 by two glyoxalase-specific tight-binding inhibitors with nanomolar K(i)(app) values and noncompetitive inhibition patterns. The inhibitors do not discriminate between the high-affinity and the high-activity conformations of PfGlo1, but seem to stabilize or trigger a conformational change in analogy with the substrate. In summary, we have characterized the most potent inhibitors of PfGlo1 known to date.     

Influence of human p16(INK4) and p21(CIP1) on the in vitro activity of recombinant Plasmodium falciparum cyclin-dependent protein kinases.

The regulatory mechanisms of most cyclin dependent protein kinases (CDKs) are well understood and are highly conserved in eukaryotes. CDKs from the malaria parasite, Plasmodium falciparum, appear to be regulated in a similar manner with regard to cyclin binding and phosphorylation. In order to further understand their regulatory mechanisms, we examined two classes of cyclin dependent kinase inhibitors (CDIs) to inhibit a panel of plasmodial CDKs. We find that Pfmrk and PfPK5 are inhibited by heterologous p21(CIP1) with varying degrees of inhibition. In contrast, PfPK6, a kinase with sequence features characteristic of both a CDK and MAP kinase, is unaffected by this CDI. Furthermore, the CDK4/6 specific CDI, p16(INK4), fails to inhibit these plasmodial CDKs. Taken together, these results suggest that plasmodial CDKs may be regulated by the binding of inhibitory proteins in vivo.     

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.     

Plasmodium DDI1 is a potential therapeutic target and important chromatin-associated protein

DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control.     

Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay

ABSTRACT: BACKGROUND: There is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT), an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors. METHODS: Production of recombinant Plasmodium falciparum SHMT (PfSHMT) and Plasmodium vivax SHMT (PvSHMT), using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-beta-D-galactoside (LB-IPTG) induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-ethylenetetrahydrofolate dehydrogenase (MTHFD). The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate. RESULTS: Auto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L) compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR); thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. Plasmodium SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone. CONCLUSIONS: Auto-induction media offers a cost-effective method for the production of Plasmodium SHMTs and should be applicable for other Plasmodium enzymes. The SHMT-MTHFD coupled assay is equivalent to the SHMT-MTHFR coupled assay, but is more convenient for inhibitor screening and other studies of the enzyme. In addition to inhibitors of malarial SHMT, the development of species-specific, anti-SHMT inhibitors is plausible due to the presence of differential active sites on the Plasmodium enzymes.     

Synthesis and in vitro antiprotozoal activities of 5-phenyliminobenzo[a]phenoxazine derivatives

A series of 5-phenyliminobenzo[a]phenoxazine derivatives were synthesized. The in vitro antiprotozoal activities were evaluated against Plasmodium falciparum K1, Trypanosoma cruzi, Leishmania donovani and Trypanosoma brucei rhodesiense. N,N-Diethyl-5-((4-methoxyphenyl)imino)-5H-benzo[a]phenoxazin-9-amine shows IC(50)=0.040 μmol L(-1) with a selective index of 1425 against Plasmodium falciparum K1.     

In Silico Screening on the Three-dimensional Model of the Plasmodium vivax SUB1 Protease Leads to the Validation of a Novel Anti-parasite Compound

Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. We characterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had K_i values of <50 μm for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1 (compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial.     

A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.     

Combined inhibition of Chk1 and Wee1: In vitro synergistic effect translates to tumor growth inhibition in vivo

Targeting Chk1 protein kinase can enhance the antitumor effects of radio- and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. To identify genes in synthetic lethality with Chk1, we did a high-throughput screening using a siRNA library directed against 719 human protein kinases in the human ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibitors. Wee1 tyrosine kinase was the most significant gene in synthetic lethality with Chk1. Treatment with non-toxic concentrations of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) confirmed the marked synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate), independently of the p53 status. Detailed molecular analysis showed that the combination caused cancer cells to undergo premature mitosis before the end of DNA replication, with damaged DNA leading to cell death partly by apoptosis. In vivo treatment of mice bearing OVCAR-5 xenografts with the combination of Chk1 and Wee1 inhibitors led to greater tumor growth inhibition than with the inhibitors used as single agents with no toxicity. These data provide a strong rationale for the clinical investigation of the combination of a Chk1 and a Wee1 inhibitor.     

Modeling of human M1 aminopeptidases for in silico screening of potential Plasmodium falciparum alanine aminopeptidase (PfA-M1) specific inhibitors

Plasmodium falciparum alanine M1-aminopeptidase (PfA-M1) is a validated target for anti-malarial drug development. Presence of significant similarity between PfA-M1 and human M1-aminopeptidases, particularly within regions of enzyme active site leads to problem of non-specificity and off-target binding for known aminopeptidase inhibitors. Molecular docking based in silico screening approach for off-target binding has high potential but requires 3D-structure of all human M1-aminopeptidaes. Therefore, in the present study 3D structural models of seven human M1-aminopeptidases were developed. The robustness of docking parameters and quality of predicted human M1-aminopeptidases structural models was evaluated by stereochemical analysis and docking of their respective known inhibitors. The docking scores were in agreement with the inhibitory concentrations elucidated in enzyme assays of respective inhibitor enzyme combinations (r2≈0.70). Further docking analysis of fifteen potential PfA-M1 inhibitors (virtual screening identified) showed that three compounds had less docking affinity for human M1-aminopeptidases as compared to PfA-M1. These three identified potential lead compounds can be validated with enzyme assays and used as a scaffold for designing of new compounds with increased specificity towards PfA-M1.