10 research outputs found
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
SSAO-TbALDH3, contributed by both host and parasites, metabolizes the aminomethyl-benzoxaboroles.
<p>(<b>A</b>) HPLC-MS identification of AN3057 and derived metabolites from the FBS-TbALDH3 pathway. HPLC peaks are indicated with corresponding retention time (RT), m/z of ion precursors identified in MS, and proposed structural formula. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006850#ppat.1006850.s009" target="_blank">S9 Fig</a> for the detailed MS data. (<b>B</b>) The potency (EC<sub>50</sub>) of AN3057 and derived metabolites in <i>T</i>. <i>brucei</i>. The change in EC<sub>50</sub> upon TbALDH3 RNA<i>i</i> is presented for each molecule with the key moiety illustrated, i.e., methylamine, aldehyde, and carboxylic acid. (<b>C</b>) SSAO contributes to the AO activity in FBS. FBS-derived AO activity is represented by the luminescence intensity from Luciferin, the generation of which is coupled to the aldehyde-carboxylic acid conversion. The chemical inhibitors are listed as follows, Semicarbazide (Semi), 2-Bromopropylamine hydrobromide (Bromo), Tetraphenylphosphonium (TPP), N,N-Diethyldithiocarbamate (DDC), Mexiletine (Mexi) and Ammonium tetrathiomolybdate (ATTM). (<b>D</b>) The potency (EC<sub>50</sub>) of AN3057 in <i>T</i>. <i>brucei</i> upon SSAO inhibition. Prior to the assay, the complete media were incubated with indicated concentrations of inhibitor for 4 hours.</p
TbALDH3, encoded by Tb927.6.3050, is a member of FALDH subfamily.
<p>(<b>A</b>) The unrooted phylogenic tree was constructed based on the alignment of all ALDH orthologues in Trypanosomatida with the Opisthokont orthologues representing all the subfamilies of the ALDH superfamily categorized. Only representative orthologues are presented for simplicity. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006850#ppat.1006850.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006850#ppat.1006850.s002" target="_blank">S2</a> Figs for the complete data set. Each clade, in which the Trypanosomatida orthologues are identified with a Opisthokont subfamily, is assigned with the corresponding subfamily number, indicated in black; otherwise in grey. The family members in <i>T</i>. <i>brucei</i> are highlighted in a larger font with Tb927.6.3050 also in red. Strongly supported nodes (bootstrap proportion >70, Bayesian posterior probability >90) are indicated by a star. (<b>B</b>) The structure of TbALDH3 in cartoon representation. One subunit of the dimeric assembly is shown for clarity. Secondary structure elements are labeled and domains are indicated in color. Blue, catalytic domain; red, NAD-binding domain; green, oligomerisation domain. (<b>C</b>) The glycosomal localization of TbALDH3. The glycosomal localization is represented by the GAPDH signal, and the nucleus by DAPI stain. Scale bar = 1um.</p
Potencies of the phenoxy derivatives upon Tb927.6.3050 RNAi.
<p>The compounds are listed in pair according to structural similarity in the functional groups. The methylamine and carboxylic acid groups are highlighted with dot circles. Ref. stands for reference.</p
Tb927.6.3050 serves as a specific potency determinant for aminomethylphenoxy benzoxaboroles.
<p>(<b>A</b>) Tb927.6.3050 identification in RIT-seq from a genome-scale screening for potency determinants of benzoxaboroles. The gene locus is indicated in red, with flanking genes in black, in the relevant chromosomal context. Each peak represents an identification by sequencing and the relative height corresponds to the number of reads. A pair of short sequences were introduced in the original library [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006850#ppat.1006850.ref026" target="_blank">26</a>], flanking the individual RNA<i>i</i> targeting fragment in both directions, as a unique bar code to ensure sequencing specificity. The corresponding identifications are indicated in color, red for the forward sequence and blue for the reverse, distinguished from all other identification indicated in grey. The aminomethylphenoxy moieties in the respective compounds are indicated with the dotted rectangles. (<b>B</b>) The changes in potency (EC<sub>50</sub>) of chemically diverse benzoxaboroles upon Tb927.6.3050 RNA<i>i</i>.</p
Structural insight into interactions between the aldehyde metabolites and TbALDH3.
<p>(<b>A</b>) TbALDH3 catalytic site. All key residues are in stick representation, while NAD and AN3057-aldehyde are shown with electron density (blue chicken wire), in which non-carbon atoms are marked in color, oxygen in red, nitrogen in blue, and phosphorus in orange. Potential hydrogen bonds are depicted as black dashed lines; selected water as blue spheres. (<b>B</b>) The comparison of substrate binding funnel between TbALDH3, HsALDH3A2 and RnALDH3A1. The cross-section structures are depicted from a top view with indicated features. NAD and AN3057-aldehyde are shown in solid stick representation with TbALDH3, while transparent superimposed to HsALDH3A2 and RnALDH3A1. (<b>C</b>) The structural superposition of TbALDH3 with mammalian homologues, RnALDH3A1 (Rat, blue) and HsALDH3A2 (Human, black). Two orientations are presented with indicated features. TbALDH3- RnALDH3A1 alignment: 1.4Å (rmsd); 445 (C<sub>α</sub>-atoms); 55 (Z-score); 47% (identity). TbALDH3-hALDH3A2: 1.8Å (rmsd); 454 (C<sub>α</sub>-atoms); 54 (Z-score); 47% (identity).</p
Model for aminomethylphenoxy benzoxaboroles metabolism <i>in vivo</i>.
<p>In the host, the aminomethylphenoxy benzoxaboroles are first oxidized by SSAO in the intravascular system. The resulting aldehyde metabolites are taken up by the parasites, and further oxidized into carboxylic acids that give rise to the final active trypanocides. The activation of the compounds requires both enzymatic activities in the host and parasites, with the loss of drug potency when either enzymatic activity is compromised. The key chemical moieties undergoing biochemical transitions through this process are highlighted with dotted boxes.</p
AN3057 is metabolized by MAO-TbALDH3.
<p>(<b>A</b>) Schematic view of the enzymatic reactions with MAOa-TbALDH3 in metabolizing AN3057. (<b>B</b>) HPLC-MS identification of AN3057-derived metabolites from the MAO-TbALDH3 pathway. HPLC peaks are indicated with corresponding retention time (RT) under acidic condition, m/z of ion precursors identified in MS, and proposed structural formula. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006850#ppat.1006850.s012" target="_blank">S2 Table</a> for the RTs with acidic eluent or basic eluent; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006850#ppat.1006850.s003" target="_blank">S3 Fig</a> for the detailed MS data. (<b>C</b>) The potency (EC<sub>50</sub>) of AN3057 and derived metabolites in <i>T</i>. <i>brucei</i>. The change in EC<sub>50</sub> upon TbALDH3 RNA<i>i</i> is presented for each molecule with the key moiety illustrated, i.e., methylamine, aldehyde, and carboxylic acid.</p
Lead Optimization of a Pyrazole Sulfonamide Series of Trypanosoma brucei <i>N</i>‑Myristoyltransferase Inhibitors: Identification and Evaluation of CNS Penetrant Compounds as Potential Treatments for Stage 2 Human African Trypanosomiasis
Trypanosoma brucei <i>N</i>-myristoyltransferase
(<i>Tb</i>NMT) is an attractive therapeutic
target for the treatment of human African trypanosomiasis (HAT). From
previous studies, we identified pyrazole sulfonamide, DDD85646 (<b>1</b>), a potent inhibitor of <i>Tb</i>NMT. Although
this compound represents an excellent lead, poor central nervous system
(CNS) exposure restricts its use to the hemolymphatic form (stage
1) of the disease. With a clear clinical need for new drug treatments
for HAT that address both the hemolymphatic and CNS stages of the
disease, a chemistry campaign was initiated to address the shortfalls
of this series. This paper describes modifications to the pyrazole
sulfonamides which markedly improved blood–brain barrier permeability,
achieved by reducing polar surface area and capping the sulfonamide.
Moreover, replacing the core aromatic with a flexible linker significantly
improved selectivity. This led to the discovery of DDD100097 (<b>40</b>) which demonstrated partial efficacy in a stage 2 (CNS)
mouse model of HAT
Neither mycorrhizal inoculation nor atmospheric CO<sub>2</sub> concentration has strong effects on pea root production and root loss
Chagas’
disease, caused by the protozoan parasite Trypanosoma
cruzi, is the most common cause of cardiac-related
deaths in endemic regions of Latin America. There is an urgent need
for new safer treatments because current standard therapeutic options,
benznidazole and nifurtimox, have significant side effects and are
only effective in the acute phase of the infection with limited efficacy
in the chronic phase. Phenotypic high content screening against the
intracellular parasite in infected VERO cells was used to identify
a novel hit series of 5-amino-1,2,3-triazole-4-carboxamides (ATC).
Optimization of the ATC series gave improvements in potency, aqueous
solubility, and metabolic stability, which combined to give significant
improvements in oral exposure. Mitigation of a potential Ames and hERG liability ultimately led to two promising compounds, one of which demonstrated significant suppression of parasite burden in a mouse model of Chagas’ disease