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1.
J Comput Aided Mol Des ; 34(11): 1117-1132, 2020 11.
Article in English | MEDLINE | ID: mdl-32833084

ABSTRACT

There is a pressing need to improve the efficiency of drug development, and nowhere is that need more clear than in the case of neglected diseases like malaria. The peculiarities of pyrimidine metabolism in Plasmodium species make inhibition of dihydroorotate dehydrogenase (DHODH) an attractive target for antimalarial drug design. By applying a pair of complementary quantitative structure-activity relationships derived for inhibition of a truncated, soluble form of the enzyme from Plasmodium falciparum (s-PfDHODH) to data from a large-scale phenotypic screen against cultured parasites, we were able to identify a class of antimalarial leads that inhibit the enzyme and abolish parasite growth in blood culture. Novel analogs extending that class were designed and synthesized with a goal of improving potency as well as the general pharmacokinetic and toxicological profiles. Their synthesis also represented an opportunity to prospectively validate our in silico property predictions. The seven analogs synthesized exhibited physicochemical properties in good agreement with prediction, and five of them were more active against P. falciparum growing in blood culture than any of the compounds in the published lead series. The particular analogs prepared did not inhibit s-PfDHODH in vitro, but advanced biological assays indicated that other examples from the class did inhibit intact PfDHODH bound to the mitochondrial membrane. The new analogs, however, killed the parasites by acting through some other, unidentified mechanism 24-48 h before PfDHODH inhibition would be expected to do so.


Subject(s)
Antimalarials/chemistry , Enzyme Inhibitors/chemistry , Malaria, Falciparum/drug therapy , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Plasmodium falciparum/drug effects , Quinolones/chemistry , Antimalarials/adverse effects , Antimalarials/pharmacokinetics , Dihydroorotate Dehydrogenase , Drug Design , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/pharmacokinetics , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Molecular Structure , Quantitative Structure-Activity Relationship , Quinolones/adverse effects , Quinolones/pharmacokinetics
2.
Nat Commun ; 8(1): 430, 2017 09 05.
Article in English | MEDLINE | ID: mdl-28874661

ABSTRACT

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.


Subject(s)
Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Imidazoles/therapeutic use , Malaria/enzymology , Malaria/transmission , Pyridines/therapeutic use , Animals , Cell Line , Crystallography, X-Ray , Culicidae , Cyclic GMP-Dependent Protein Kinases/chemistry , Cyclic GMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Female , Humans , Imidazoles/pharmacology , Life Cycle Stages/drug effects , Malaria/drug therapy , Mice, Inbred BALB C , Models, Molecular , Plasmodium chabaudi/drug effects , Plasmodium falciparum/drug effects , Plasmodium falciparum/growth & development , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/pharmacology , Treatment Outcome
3.
Anal Biochem ; 506: 13-21, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27133204

ABSTRACT

Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.


Subject(s)
Enzyme Inhibitors/analysis , Enzyme Inhibitors/chemistry , Fluorescence , High-Throughput Screening Assays , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Plasmodium/enzymology , Dihydroorotate Dehydrogenase , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Structure-Activity Relationship
4.
Proc Natl Acad Sci U S A ; 104(8): 2709-14, 2007 Feb 20.
Article in English | MEDLINE | ID: mdl-17296936

ABSTRACT

Mutations in the human methyl-CpG-binding protein gene MECP2 cause the neurological disorder Rett syndrome and some cases of X-linked mental retardation (XLMR). We report that MeCP2 interacts with ATRX, a SWI2/SNF2 DNA helicase/ATPase that is mutated in ATRX syndrome (alpha-thalassemia/mental retardation, X-linked). MeCP2 can recruit the helicase domain of ATRX to heterochromatic foci in living mouse cells in a DNA methylation-dependent manner. Also, ATRX localization is disrupted in neurons of Mecp2-null mice. Point mutations within the methylated DNA-binding domain of MeCP2 that cause Rett syndrome or X-linked mental retardation inhibit its interaction with ATRX in vitro and its localization in vivo without affecting methyl-CpG binding. We propose that disruption of the MeCP2-ATRX interaction leads to pathological changes that contribute to mental retardation.


Subject(s)
DNA Helicases/metabolism , Intellectual Disability/genetics , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Mutation/genetics , Nuclear Proteins/metabolism , Animals , Brain/cytology , Brain/metabolism , Cells, Cultured , DNA/metabolism , DNA Helicases/chemistry , DNA Methylation , Humans , Methyl-CpG-Binding Protein 2/deficiency , Mice , Nuclear Proteins/chemistry , Protein Binding , Protein Transport , Two-Hybrid System Techniques , X-linked Nuclear Protein
5.
EMBO J ; 22(18): 4689-98, 2003 Sep 15.
Article in English | MEDLINE | ID: mdl-12970181

ABSTRACT

The genetic inactivation of the atypical protein kinase C (aPKC) inhibitor, Par-4, gives rise to increased NF-kappaB activation and decreased stimulation of JNK in embryo fibroblasts. Here we have characterized the immunological phenotype of the Par-4(-/-) mice and found that the loss of this gene leads to an increased proliferative response of peripheral T cells when challenged through the TCR. This is accompanied by a higher increase in cell cycle entry and inhibition of apoptosis, with enhanced IL-2 secretion but normal CD25 synthesis. Interestingly, the TCR-triggered activation of NF-kappaB was augmented and that of JNK was severely abrogated. Consistent with previous data from knock outs of different JNKs, NFATc1 activation and IL-4 secretion were augmented in the Par-4-deficient CD4+ T cells, suggesting that the loss of Par-4 drives T-cell differentiation towards a Th2 response. This is compelling evidence that Par-4 is a novel modulator of the immune response through its ability to impact aPKC activity, which translates into lower JNK signaling.


Subject(s)
Carrier Proteins/physiology , Intracellular Signaling Peptides and Proteins , Lymphocyte Activation/physiology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Animals , Apoptosis , Apoptosis Regulatory Proteins , Carrier Proteins/genetics , Cell Cycle , Cell Differentiation , Cell Division/genetics , Gene Deletion , JNK Mitogen-Activated Protein Kinases , Lymphocyte Activation/genetics , MAP Kinase Signaling System , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , NFATC Transcription Factors , Protein Kinase C/deficiency , Protein Kinase C/genetics , Protein Kinase C/physiology , Receptors, Antigen, T-Cell/physiology
6.
EMBO Rep ; 4(3): 307-12, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12634851

ABSTRACT

The Par4 gene was first identified in prostate cells undergoing apoptosis after androgen withdrawal. PAR4 was subsequently shown to interact with, and inhibit, atypical protein kinase C isoforms, functioning as a negative regulator of the NF-kappaB pathway. This may explain its pro-apoptotic function in overexpression experiments. To determine the physiological role of PAR4, we have derived primary embryonic fibroblasts (EFs) from Par4(-/-) mice. We show here that loss of PAR4 leads to a reduction in the ability of tumour necrosis factor-alpha (TNF-alpha) to induce apoptosis by increased activation of NF-kappaB. Consistent with recent reports demonstrating the antagonistic actions of NF-kappaB and c-Jun amino-terminal kinase (JNK) signalling, we have found that Par4(-/-) cells show a reduced activation of the sustained phase of JNK and p38 stimulation by TNF-alpha and interleukin 1. Higher levels of an anti-apoptotic JNK-inhibitor protein, X-chromosome-linked inhibitor of apoptosis, in Par4(-/-) EFs might explain the inhibition of JNK activation in these cells.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Receptors, Thrombin/deficiency , Receptors, Thrombin/physiology , Animals , Apoptosis/drug effects , Embryo, Mammalian , Fibroblasts/physiology , Gene Expression Regulation , Interleukin-1/pharmacology , JNK Mitogen-Activated Protein Kinases , MAP Kinase Signaling System/genetics , Mice , Mice, Knockout , Receptors, Thrombin/genetics , Restriction Mapping , Tumor Necrosis Factor-alpha/pharmacology , X Chromosome , p38 Mitogen-Activated Protein Kinases
8.
Microbiology (Reading) ; 148(Pt 7): 2111-2123, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12101299

ABSTRACT

The DUP240 gene family of Saccharomyces cerevisiae is composed of 10 members. They encode proteins of about 240 amino acids which contain two predicted transmembrane domains. Database searches identified only one homologue in the closely related species Saccharomyces bayanus, indicating that the DUP240 genes encode proteins specific to Saccharomyces sensu stricto. The short-flanking homology PCR gene-replacement strategy with a variety of selective markers for replacements, and classical genetic methods, were used to generate strains deleted for all 10 DUP240 genes. All of the knock-out strains were viable and had similar growth kinetics to the wild-type. Two-hybrid screens, hSos1p fusions and GFP fusions were carried out; the results indicated that the Dup240 proteins are membrane associated, and that some of them are concentrated around the plasma membrane.


Subject(s)
Membrane Proteins/metabolism , Multigene Family , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Cell Membrane/metabolism , Gene Deletion , Genes, Essential , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Proteins/genetics , Recombinant Fusion Proteins , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Analysis, DNA , Subcellular Fractions/metabolism , Tandem Repeat Sequences/genetics , Transformation, Genetic , Two-Hybrid System Techniques
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