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Norman C Waters

Publications and source records attributed to Norman C Waters.

17 recordsLinked to original sources
PubMed2006

Synthesis and biological evaluation of thiazolidine-2-one 1,1-dioxide as inhibitors of Escherichia coli beta-ketoacyl-ACP-synthase III (FabH).

A series of cyclic sulfones has been synthesized and their activity against beta-ketoacyl-ACP-synthase III (FabH) has been investigated. The compounds are selectively active against Escherichia coli FabH (ecFabH), but not Mycobacterium tuberculosis FabH (mtFabH) or Plasmodium falciparum KASIII (PfKASIII). The activity against ecFabH ranges from 0.9 to >100microM and follows a consistent general SAR trend. Many of the compounds were shown to have antimalarial activity against chloroquine (CQ)-sensitive (D6) P. falciparum (IC(50)=5.3microM for the most potent inhibitor) and some were active against E. coli (MIC=6.6microg/ml for the most potent inhibitor).

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2006

Identification of an effector protein and gain-of-function mutants that activate Pfmrk, a malarial cyclin-dependent protein kinase.

Cyclin-dependent protein kinases (CDKs) are key regulators of cell cycle control. In humans, CDK7 performs dual roles as the CDK activating kinase (CAK) responsible for regulating numerous CDKs and as the RNA polymerase II carboxyl-terminal domain (CTD) kinase involved in the regulation of transcription. Binding of an effector protein, human MAT1, stimulates CDK7 kinase activity and influences substrate specificity. In Plasmodium falciparum, CDKs and their roles in regulating growth and development are poorly understood. In this study, we characterized the regulatory mechanisms of Pfmrk, a putative homolog of human CDK7. We identified an effector, PfMAT1, which stimulates Pfmrk kinase activity in a cyclin-dependent manner. The addition of PfMAT1 stimulated RNA polymerase II CTD phosphorylation and had no effect on the inability of Pfmrk to phosphorylate PfPK5, a putative CDK1 homolog, which suggests that Pfmrk may be a CTD kinase rather than a CAK. In an attempt to abrogate the requirement for PfMAT1 stimulation, we mutated amino acids within the active site of Pfmrk. We found that two independent mutants, S138K and F143L, yielded a 4-10-fold increase in Pfmrk activity. Significant kinase activity of these mutants was observed in the absence of either cyclin or PfMAT1. Finally, we observed autophosphorylation of Pfmrk that is unaffected by the addition of either cyclin or PfMAT1.

Amino Acid Sequence↗
PubMed2006

Antiplasmodial flavonoids from Erythrina sacleuxii.

The acetone extracts of the root bark and stem bark of Erythrina sacleuxii showed antiplasmodial activities against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Chromatographic separation of the acetone extract of the root bark afforded a new isoflavone, 7-hydroxy-4'-methoxy-3'-prenylisoflavone (trivial name 5-deoxy-3'-prenylbiochanin A) along with known isoflavonoids as the antiplasmodial principles. Flavonoids and isoflavonoids isolated from the stem bark of E. sacleuxii were also tested and showed antiplasmodial activities. The structures were determined on the basis of spectroscopic evidence.

Animals↗
PubMed2005

Targeting malaria with specific CDK inhibitors.

Cyclin-dependent protein kinases (CDKs) are attractive targets for drug discovery and efforts have led to the identification of novel CDK selective inhibitors in the development of treatments for cancers, neurological disorders, and infectious diseases. More recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. CDKs are valid targets as they function as essential regulators of cell growth and differentiation. To date, several CDKs have been characterized from the genome of the malaria-causing protozoan Plasmodium falciparum. Our approach employs experimental and virtual screening methodologies to identify and refine chemical inhibitors of the parasite CDK Pfmrk, a sequence homologue of human CDK7. Chemotypes of Pfmrk inhibitors include the purines, quinolinones, oxindoles, and chalcones, which have sub-micromolar IC50 values against the parasite enzyme, but not the human CDKs. Additionally, we have developed and validated a pharmacophore, based on Pfmrk inhibitors, which contains two hydrogen bond acceptor functions and two hydrophobic sites, including one aromatic ring hydrophobic site. This pharmacophore has been exploited to identify additional compounds that demonstrate significant inhibitory activity against Pfmrk. A molecular model of Pfmrk designed using the crystal structure of human CDK7 highlights key amino acid substitutions in the ATP binding pocket. Molecular modeling and docking of the active site pocket with selective inhibitors has identified possible receptor-ligand interactions that may be responsible for inhibitor specificity. Overall, the unique biochemical characteristics associated with this protein, to include distinctive active site amino acid residues and variable inhibitor profiles, distinguishes the Pfmrk drug screen as a paradigm for CDK inhibitor analysis in the parasite.

Adenosine Triphosphate↗
PubMed2005

Rational inhibitor design and iterative screening in the identification of selective plasmodial cyclin dependent kinase inhibitors.

New chemical classes of compounds must be introduced into the malaria drug development pipeline in an effort to develop new chemotherapy options for the fight against malaria. In this review we describe an iterative approach designed to identify potent inhibitors of a kinase family that collectively functions as key regulators of the cell cycle. Cyclin-dependent protein kinases (CDKs) are attractive drug targets in numerous diseases and, most recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. Our approach uses experimental and virtual screening methodologies to identify and refine chemical inhibitors and increase the success rate of discovering potent and selective inhibitors. The active pockets of the plasmodial CDKs are unique in terms of size, shape and amino acid composition compared with those of the mammalian orthologues. These differences exemplified through the use of screening assays, molecular modeling, and crystallography can be exploited for inhibitor design. To date, several classes of compounds including quinolines and oxindoles have been identified as selective inhibitors of the plasmodial CDK7 homologue, Pfmrk. From these initial studies and through the iterative rational drug design process, more potent, selective, and most importantly, chemically unique compound classes have been identified as effective inhibitors of the plasmodial CDKs and the malarial parasite.

Amino Acid Sequence↗
PubMed2005

Fatty Acid synthesis as a target for antimalarial drug discovery.

In biological systems, fatty acids can be synthesized by two related, but distinct de novo fatty acid synthase (FAS) pathways. Human cells rely on a type I FAS whereas plants, bacteria and other microorganisms contain type II FAS pathways. This difference exposes the type II FAS enzymes as potential targets for anti-microbial drugs that have little to no side effects in the human host. A number of inhibitors of type II FAS enzymes have been discovered - many of which have anti-bacterial activity. Extensive biochemical and structural studies have shed light on how these compounds inhibit their target enzymes, laying the foundation for the design of inhibitors with increased potency. Recent work has shown that malaria parasites do not contain a type I FAS and rely solely on a type II FAS for the de novo biosynthesis of fatty acids. The malaria FAS enzymes are therefore an exciting source of new drug targets, and are being actively exploited by several drug discovery efforts. Rapid progress has been made, largely due to the vast body of mechanistic and structural information about type II FAS enzymes from bacteria and the availability of inhibitors. Ongoing antimalarial drug discovery projects will be described in this review as well as background information about the well-studied bacterial type II FAS enzymes.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed2004

A three-dimensional in silico pharmacophore model for inhibition of Plasmodium falciparum cyclin-dependent kinases and discovery of different classes of novel Pfmrk specific inhibitors.

The cell division cycle is regulated by a family of cyclin-dependent protein kinases (CDKs) that are functionally conserved among many eukaryotic species. The characterization of plasmodial CDKs has identified them as a leading antimalarial drug target in our laboratory. We have developed a three-dimensional QSAR pharmacophore model for inhibition of a Plasmodium falciparum CDK, known as Pfmrk, from a set of fifteen structurally diverse kinase inhibitors with a wide range of activity. The model was found to contain two hydrogen bond acceptor functions and two hydrophobic sites including one aromatic-ring hydrophobic site. Although the model was not developed from X-ray structural analysis of the known CDK2 structure, it is consistent with the structure-functional requirements for binding of the CDK inhibitors in the ATP binding pocket. Using the model as a template, a search of the in-house three-dimensional multiconformer database resulted in the discovery of sixteen potent Pfmrk inhibitors. The predicted inhibitory activities of some of these Pfmrk inhibitors from the molecular model agree exceptionally well with the experimental inhibitory values from the in vitro CDK assay.

Adenosine Triphosphate↗
PubMed2004

The yeast Pho80-Pho85 cyclin-CDK complex has multiple substrates.

The Pho85-Pho80 cyclin-CDK (cyclin-dependent protein kinase) complex of Saccharomyces cerevisiae functions as a key regulator of the phosphate-repressible acid phosphatase system. We have further characterized the Pho85-Pho80 kinase complex and identified the Pho80 cyclin subunit and the Pho81 CDK inhibitor as substrates of the Pho85 protein kinase. The phosphorylation sites within Pho80 have been identified at Ser234 and Ser267. Of the two sites, phosphorylation of Ser234 is required for Pho80 function, to form an active kinase complex and repress acid phosphatase expression. Evidence suggests that the activity of Pho81 is regulated by a post-translational modification and therefore that Pho85-mediated phosphorylation of Pho81 may alter its ability to function as a CDK inhibitor. Thus, the control of acid phosphatase expression involves the phosphorylation of several of the regulatory components of the system.

Cyclin-Dependent Kinases↗
PubMed2004

Anti-plasmodial flavonoids from the stem bark of Erythrina abyssinica.

The ethyl acetate extract of the stem bark of Erythrina abyssinica showed anti-plasmodial activity against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum with IC(50) values of 7.9+/-1.1 and 5.3+/-0.7 microg/ml, respectively. From this extract, a new chalcone, 2',3,4,4'-tetrahydroxy-5-prenylchalcone (trivial name 5-prenylbutein) and a new flavanone, 4',7-dihydroxy-3'-methoxy-5'-prenylflavanone (trivial name, 5-deoxyabyssinin II) along with known flavonoids have been isolated as the anti-plasmodial principles. The structures were determined on the basis of spectroscopic evidence.

Animals↗
PubMed2004

Yellow fever outbreak, Imatong, southern Sudan.

In May 2003, the World Health Organization received reports about a possible outbreak of a hemorrhagic disease of unknown cause in the Imatong Mountains of southern Sudan. Laboratory investigations were conducted on 28 serum samples collected from patients in the Imatong region. Serum samples from 13 patients were positive for immunoglobulin M antibody to flavivirus, and serum samples from 5 patients were positive by reverse transcription-polymerase chain reaction with both the genus Flavivirus-reactive primers and yellow fever virus-specific primers. Nucleotide sequencing of the amplicons obtained with the genus Flavivirus oligonucleotide primers confirmed yellow fever virus as the etiologic agent. Isolation attempts in newborn mice and Vero cells from the samples yielded virus isolates from five patients. Rapid and accurate laboratory diagnosis enabled an interagency emergency task force to initiate a targeted vaccination campaign to control the outbreak.

Adolescent↗
PubMed2003

Oxindole-based compounds are selective inhibitors of Plasmodium falciparum cyclin dependent protein kinases.

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.

Amino Acid Sequence↗
PubMed2003

The initiating steps of a type II fatty acid synthase in Plasmodium falciparum are catalyzed by pfACP, pfMCAT, and pfKASIII.

Malaria, a disease caused by protozoan parasites of the genus Plasmodium, is one of the most dangerous infectious diseases, claiming millions of lives and infecting hundreds of millions of people annually. The pressing need for new antimalarials has been answered by the discovery of new drug targets from the malaria genome project. One of the early findings was the discovery of two genes encoding Type II fatty acid biosynthesis proteins: ACP (acyl carrier protein) and KASIII (beta-ketoacyl-ACP synthase III). The initiating steps of a Type II system require a third protein: malonyl-coenzyme A:ACP transacylase (MCAT). Here we report the identification of a single gene from P. falciparum encoding pfMCAT and the functional characterization of this enzyme. Pure recombinant pfMCAT catalyzes malonyl transfer from malonyl-coenzyme A (malonyl-CoA) to pfACP. In contrast, pfACP(trans), a construct of pfACP containing an amino-terminal apicoplast transit peptide, was not a substrate for pfMCAT. The product of the pfMCAT reaction, malonyl-pfACP, is a substrate for pfKASIII, which catalyzes the decarboxylative condensation of malonyl-pfACP and various acyl-CoAs. Consistent with a role in de novo fatty acid biosynthesis, pfKASIII exhibited typical KAS (beta-ketoacyl ACP synthase) activity using acetyl-CoA as substrate (k(cat) 230 min(-1), K(M) 17.9 +/- 3.4 microM). The pfKASIII can also catalyze the condensation of malonyl-pfACP and butyryl-CoA (k(cat) 200 min(-1), K(M) 35.7 +/- 4.4 microM) with similar efficiency, whereas isobutyryl-CoA is a poor substrate and displayed 13-fold less activity than that observed for acetyl-CoA. The pfKASIII has little preference for malonyl-pfACP (k(cat)/K(M) 64.9 min(-1)microM(-1)) over E. coli malonyl-ACP (k(cat)/K(M) 44.8 min(-1)microM(-1)). The pfKASIII also catalyzes the acyl-CoA:ACP transacylase (ACAT) reaction typically exhibited by KASIII enzymes, but does so almost 700-fold slower than the KAS reaction. Thiolactomycin did not inhbit pfKASIII (IC(50) > 330 microM), but three structurally similar substituted 1,2-dithiole-3-one compounds did inhibit pfKASIII with IC(50) values between 0.53 microM and 10.4 microM. These compounds also inhibited the growth of P. falciparum in culture.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2003

Anti-plasmodial activities and X-ray crystal structures of rotenoids from Millettia usaramensis subspecies usaramensis.

The dichloromethane extract of the stem bark of Millettia usaramensis subspecies usaramensis showed anti-plasmodial activity against the chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum. Chromatographic separation of the extract led to the identification of a new rotenoid, (6aR,12aS)-2,3-methylenedioxy-9-methoxy-8-(3,3-dimethylallyl)-12a-hydroxyrotenoid (trivial name, usararotenoid C) along with known flavonoids (usararotenoid A, 12a-epimillettosin, 6a,12a-dehydromillettone, barbigerone and 4'-O-geranylisoliquiritigenin) as the anti-plasmodial principles. The structures were determined by spectroscopic analyses. CD and X-ray analyses established absolute configurations.

Animals↗
PubMed2003

Flavonoids and isoflavonoids with antiplasmodial activities from the root bark of Erythrina abyssinica.

From the root bark of Erythrina abyssinica a new pterocarpene [3-hydroxy-9-methoxy-10-(3,3-dimethylallyl)pterocarpene] and a new isoflav-3-ene [7,4'-dihydroxy-2',5'-dimethoxyisoflav-3-ene] were isolated. In addition, the known compounds erycristagallin, licoagrochalcone A, octacosyl ferulate and triacontyl 4-hydroxycinnamate were identified. The structures were determined on the basis of spectroscopic evidence. The crude extract and the flavonoids and isoflavonoids obtained from the roots of this plant showed antiplasmodial activities.

Animals↗
PubMed2003

Cyclin-dependent protein kinases as therapeutic drug targets for antimalarial drug development.

Cyclin-dependent protein kinases (CDKs) have been attractive drug targets for the development of anticancer therapies due to their direct and crucial role in the regulation of cellular proliferation. Following this trend, CDKs have been pursued as potential drug targets for several other diseases. Structure-based drug design programmes have focused on the plasmodial CDKs to develop new candidate antimalarial compounds. This review discusses the most recent advances relating to three Plasmodium falciparum CDKs (PfPK5, PfPK6 and Pfmrk) as they are developed as antimalarial drug targets. CDKs are highly conserved, and focus must be placed upon the amino acid differences between human and plasmodial CDKs in order to develop specific inhibitors. Comparisons of the active sites of human and parasite CDKs reveal sequence and potential structural variations. Using sequence analysis, molecular modelling and in vitro drug screening, it is possible to identify and develop inhibitors that specifically target the plasmodial CDKs. These efforts are aimed at identifying new classes of CDK inhibitors that may be exploited for antimalarial drug development.

Amino Acid Sequence↗
PubMed2002

Functional characterization of the acyl carrier protein (PfACP) and beta-ketoacyl ACP synthase III (PfKASIII) from Plasmodium falciparum.

The genome of the malaria parasite, Plasmodium falciparum, appears to contain the proteins necessary for a Type II dissociated fatty acid biosynthetic system. Here we report the functional characterization of two proteins from this system. Purified recombinant acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase III (KASIII) from P. falciparum are soluble and active in a truncated form. Malarial ACP is activated by the addition of a 4'-phosphopantetheine prosthetic group derived from coenzyme A, generating holo-PfACP. Holo-PfACP is an effective substrate for the transacylase activity of PfKASIII, but substitution of a key active site cysteine in PfKASIII to alanine or serine abolishes enzymatic activity. During the schizont stage of parasite development, there is a significant up-regulation of the mRNAs corresponding to these proteins, indicating an important metabolic requirement for fatty acids during this stage.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗