2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis:Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization

<i>Mycobacterium tuberculosis</i> (<i>MTb</i>) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose <i>MTb</i> whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the <i>ndh</i> encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in <i>MTb</i> was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by <i>ndhA</i> in <i>MTb</i>. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial <i>MTb</i> SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors

Finnlines Oyj:n liikuntaluotsitoiminnan kehittäminen

Tämän opinnäytetyön tavoitteena oli kehittää Finnlines Oyj:n liikuntavastaavien eli -luotsien toimintaa ja antaa heille konkreettisia työkaluja sekä neuvoja heidän toimintaansa laivaympä-ristössä. Projektin tarkoituksena oli, että luotsit pystyvät toiminnallaan jatkossa tavoittamaan laajemmin merihenkilöstöä sekä auttamaan henkilöstöä motivoitumaan liikkumiseen laivoilla. Tässä projektissa tutkittiin ensiksi kyselyllä merihenkilöstön motiiveja liikkumiseen laivatyö-jaksoilla sekä heidän kokemuksiaan liikuntaluotsien toiminnasta tähän asti. Kyselyyn vastasi yhteensä 83 henkilöä. Tulosten pohjalta suunniteltiin liikuntaluotseille kahden päivän koulutus, johon osallistui kahdeksan henkilöä Finnlines Oyj:n henkilöstöstä. Koulutuksen tavoitteena oli ohjatun oivaltamisen ja ongelmanratkaisujen avulla ohjata luotseja kehittämään yhdessä toimintatapoja merihenkilöstön liikuntamotivaation ja liikunta-aktiivisuuden edistämiseksi. Merihenkilöstöstä 54 % oli laivatyöjaksoilla liikunnallisesti passiivisia ja he motivoituivat liik-kumaan eniten sisäisistä motivaatiotekijöistä, kuten liikunnasta saadusta hyvän olon tunteesta. Vastaajista 43 % ei ollut kuullut liikuntaluotseista ja 83 % eivät olleet ollut luotsien kanssa tekemisissä. Koulutuksen tuloksena liikuntaluotsit suunnittelivat yhdessä laivoille toteutettaviksi konkreettiset liikuntakampanjat, jotka motivoivat laajalti merihenkilöstöä liikkumaan. Lisäksi he kehittivät tapoja tuoda omaa toimintaansa enemmän esille, kuten ilmoitustauluilla luotsien esittely, ja luotsien välisen yhteydenpitokanavan. Liikuntaluotsit ovat innokkaita ja liikunta-asioista kiinnostuneita vapaaehtoisia. He ovat oivaltaneet, että heidän aktiivisuudellaan on mahdollista saada paljon aikaan. Jatkossa luotsien ja heidän yhteyshenkilöidensä välinen yhteydenpito sekä suunniteltujen toimintojen toteuttaminen ovat merkittävässä asemassa luotsien toiminnan sekä motivaation ylläpitämiseksi

Modeling the structure of <i>B. malayi</i> NMT and structural comparison with <i>Leishmania major</i> NMT.

<p>(A) Ribbon diagram of the predicted crystal structure of NMT from <i>B. malayi</i>. (B) Comparison of <i>B. malayi</i> NMT (tan) with <i>L. major</i> NMT (blue: 2wsa). An overlay of the structure of these enzymes using UCSF Chimera <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003145#pntd.0003145-Pettersen1" target="_blank">[26]</a> reveals a nearly identical conformation of the binding sites for myristoyl-CoA (yellow) and inhibitor DDD85646 (magenta). The 2 small helixes (arrow) formed by an insertion of 21 amino acids in <i>L. major</i> NMT are replaced with a loop in <i>B. malayi</i> NMT.</p

Evidence for the presence of N-myristoyltransferase (NMT) in nematodes and conservation of NMT among prokaryotes and eukaryotes.

<p>Rooted phylogenetic tree analysis of NMT and various homologs. Deduced open reading frames of NMTs were used to generate the phylogram. Branch length was generated using ClustalW (<a href="http://www.genome.jp/tools/clustalw/" target="_blank">http://www.genome.jp/tools/clustalw/</a>). The percentage of amino acid identity and similarity between <i>B. malayi</i> NMT and orthologs are shown.</p

NMT is essential in <i>C. elegans</i>.

<p>(A) Phenotypic analyses were performed using the mutant strain <i>nmt-1(tm796)/hT2[qla48]</i>. Predicted genomic organization of <i>CeNMT</i> and location of the deletion are shown. Boxes and lines denote exons and introns respectively. To verify the deletion genomic DNA was prepared from a single mutant animal “−/−”, and one worm from heterozygous “+/−” and wild-type “+/+” strains using standard methods. (B) Primers were designed to flank the deletion site and the deletion was confirmed on the basis of the change in size of a PCR product. Bands of the expected sizes were obtained. (C) RNAi knockdown of NMT in <i>C. elegans</i>. Three <i>C. elegans</i> strains were used for RNAi knockdown of NMT: <i>C. elegans</i> wild-type and two RNAi sensitive <i>C. elegans</i> strains, one containing a mutation in <i>rrf-3</i>, and a second strain carrying mutations in both <i>eri-1</i> and <i>lin-15B</i>. RNAi was performed by feeding worms <i>E. coli</i> expressing dsRNA corresponding to NMT, or pL4440 plasmid vector without <i>CeNMT</i>.</p

Biochemical analysis of recombinant <i>C. elegans</i> NMT and <i>B. malayi</i> NMT.

<p>Purified recombinant <i>B. malayi</i> NMT and <i>C. elegans</i> NMT enzymes myristoylate several synthetic peptide substrates (ARL-1 ADP ribosylation factor related protein; ABL-1 tyrosine kinase and SRC-1 tyrosine kinase). No activity was detected in the absence of peptide. Enzyme activity is expressed as radioactivity in disintegrations per minute (DPM).</p

Structures of the NMT inhibitors, DDD85646 and DDD100870.

<p>Structures of the NMT inhibitors, DDD85646 and DDD100870.</p

Inhibition of <i>C. elegans</i> (A) and <i>B. malayi</i> (B) NMTs using DDD85646 and DDD100870.

<p>Enzyme mixtures were preincubated with various concentrations of each compound (0.0025–1 µM). Reactions performed in the absence of inhibitor (positive control) or peptide (negative control), were included. Background radioactivity values generated from ‘non-peptide’ control samples were subtracted from the values obtained from experimental samples. Percent activity relative to the positive control ± propagation of error (<a href="http://laffers.net/blog/2010/11/15/error-propagation-calculator/" target="_blank">http://laffers.net/blog/2010/11/15/error-propagation-calculator/</a>) was determined. Assays were performed in triplicate.</p

Effect of NMT inhibitors on <i>C. elegans</i> growth.

<p>Wild-type <i>C. elegans</i> were grown on NGM plates seeded with OP50 <i>E. coli</i>. Compound screening (DDD85646 and DDD100870) commenced with L4-staged worms placed into a well of a sterile 96-well micro titer plate (Falcon 3072) containing a 100 µL suspension of previously frozen HB101 <i>E. coli</i> bacteria in S medium. Various concentrations (25, 50 or 100 µM) of compound or DMSO (control) were then added. Plates were maintained at 20°C in a humidity chamber for 7 days. Worm growth and development was scored daily by measuring a decrease in OD_{600 nm} resulting from consumption of <i>E. coli</i>, (A) and by microscopic examination of the number of F1 progeny produced and size of worms treated with DDD100870 (B). For each condition, 10 L4-stage worms were used, and the average ± standard deviation was plotted.</p

Listing of predicted <i>B. malayi</i> NMT substrates.

<p>Survey of potential targets for N-terminal glycine myristoylation in <i>B. malayi</i>. Predicted myristoylated proteins in the proteome of <i>C. elegans</i> (145 clusters) were retrieved from MYRbase and used to query the <i>B. malayi</i> genome. Homologs with a BLASTP E-Value −10 were analyzed using the MYR predictor <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003145#pntd.0003145-Bowyer2" target="_blank">[55]</a> to predict myristoylation sites. Protein sequences that were scored as “Reliable” with a positive score or “Twilight zone” with a negative score were retained and duplicates were discarded. <i>C. elegans</i> homologs that showed any abnormal phenotype in WormBase were marked with *. Unlikely candidates such as multi-pass integral membrane proteins were removed.</p><p>Listing of predicted <i>B. malayi</i> NMT substrates.</p