SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A

<div><p>In order to expand the repertoire of antifungal compounds a novel, high-throughput phenotypic drug screen targeting fungal phosphatidylserine (PS) synthase (Cho1p) was developed based on antagonism of the toxin papuamide A (Pap-A). Pap-A is a cyclic depsipeptide that binds to PS in the membrane of wild-type <i>Candida albicans</i>, and permeabilizes its plasma membrane, ultimately causing cell death. Organisms with a homozygous deletion of the <i>CHO1</i> gene (<i>cho1ΔΔ</i>) do not produce PS and are able to survive in the presence of Pap-A. Using this phenotype (i.e. resistance to Pap-A) as an indicator of Cho1p inhibition, we screened over 5,600 small molecules for Pap-A resistance and identified SB-224289 as a positive hit. SB-224289, previously reported as a selective human 5-HT_1B receptor antagonist, also confers resistance to the similar toxin theopapuamide (TPap-A), but not to other cytotoxic depsipeptides tested. Structurally similar molecules and truncated variants of SB-224289 do not confer resistance to Pap-A, suggesting that the toxin-blocking ability of SB-224289 is very specific. Further biochemical characterization revealed that SB-224289 does not inhibit Cho1p, indicating that Pap-A resistance is conferred by another undetermined mechanism. Although the mode of resistance is unclear, interaction between SB-224289 and Pap-A or TPap-A suggests this screening assay could be adapted for discovering other compounds which could antagonize the effects of other environmentally- or medically-relevant depsipeptide toxins.</p></div

Compounds that conferred Pap-A resistance to <i>C</i>. <i>albicans</i> wild-type yeast.

<p>A) Structures of hits identified from the screen. B) Comparison of the ability of the hits to confer Pap-A resistance to wild type <i>C</i>. <i>albicans;</i> SB-224289, MG-624, and DMSO as a control.</p

Screen of FDA-approved bioactive compounds for those that confer Pap-A resistance.

<p>5,760 compounds were screened for their effects on the growth of wild-type <i>C</i>. <i>albicans</i> (open black circles) in the presence of 4 μg/ml Pap-A. Cell growth was measured by transformation of the dye Alamar Blue over approximately 3 hours at 37°C. The <i>cho1ΔΔ</i> positive control cells growth in the presence of Pap-A with no compounds from the library are represented by green circles. Compounds that allowed wild-type cells to display >90% (above the blue line) of the growth of <i>cho1ΔΔ</i> control were designated with filled-in blue circles. Around 95% of the tested compounds showed growth levels closer to the negative control, wells which contained no cells or drugs (open red circles). The horizontal lines show the 99th quantile (purple) where 99% of the compounds exhibited growth and the 95th quantile (yellow) 95% of the compounds lie. The vertical lines divide the compounds by the 384-well plate in which they were screened which correlate to plate numbers along the bottom. A full description of the screening method is found in Materials and Methods.</p

Resistance to Pap-A correlates with decreases in PS.

<p>The <i>cho1ΔΔ</i> mutant is resistant to all concentrations of papuamide A (Pap-A) indicating a total lack of PS. The <i>cho1ΔΔ</i>::<i>CHO1</i> reintegrant strain is more resistant to Pap-A than the wild-type (WT), but less resistant than the <i>cho1ΔΔ</i> mutant.</p

The full structure of SB-224289 is required to provide Pap-A resistance.

<p><b>(A)</b> and <b>(B)</b> show the structures of purchased or synthesized analogs of SB-224289, respectively. In <b>(C)</b> and <b>(D)</b> 1x10⁴ cells/ml were treated with a serial dilution of SB-224289, test compounds or DMSO control, and then allowed to incubate for 6 hours at 37°C. Pap-A was added at either 4 μg/ml or 5 μg/ml after 6 hours the plate was incubated at 37°C overnight. Alamar Blue was added and fluorescence signal measured at 590 nm indicating survival of cells.</p

SB-224289 and MG-624 Do Not Inhibit the Activity of Cho1p.

<p>PS synthase activity shown as counts per minute per milligram of protein, quantifying ³H-l-serine incorporated into PS. Addition of varying concentrations of SB-224289 (A) and MG-624 (B) did not have an inhibitory effect on PS production.</p

Pentacyclic Nitrofurans with <i>In Vivo</i> Efficacy and Activity against Nonreplicating <i>Mycobacterium tuberculosis</i>

<div><p>The reductively activated nitroaromatic class of antimicrobials, which include nitroimidazole and the more metabolically labile nitrofuran antitubercular agents, have demonstrated some potential for development as therapeutics against dormant TB bacilli. In previous studies, the pharmacokinetic properties of nitrofuranyl isoxazolines were improved by incorporation of the outer ring elements of the antitubercular nitroimidazole OPC-67683. This successfully increased stability of the resulting pentacyclic nitrofuran lead compound Lee1106 (referred to herein as <b>9a</b>). In the current study, we report the synthesis and antimicrobial properties of <b>9a</b> and panel of <b>9a</b> analogs, which were developed to increase oral bioavailability. These hybrid nitrofurans remained potent inhibitors of <i>Mycobacterium tuberculosis</i> with favorable selectivity indices (>150) and a narrow spectrum of activity. <i>In vivo</i>, the pentacyclic nitrofuran compounds showed long half-lives and high volumes of distribution. Based on pharmacokinetic testing and lack of toxicity <i>in vivo,</i><b>9a</b> remained the series lead. <b>9a</b> exerted a lengthy post antibiotic effect and was highly active against nonreplicating <i>M. tuberculosis</i> grown under hypoxia. <b>9a</b> showed a low potential for cross resistance to current antitubercular agents, and a mechanism of activation distinct from pre-clinical tuberculosis candidates PA-824 and OPC-67683. Together these studies show that <b>9a</b> is a nanomolar inhibitor of actively growing as well as nonreplicating <i>M. tuberculosis</i>.</p></div

Murine model of acute tuberculosis infection.

<p>Log₁₀ reduction provided by compound <b>9a</b> in lungs (black bars) and spleen (grey bars) after 9 days of daily oral administration of 300 mg/kg was determined by calculating the difference between bacillary loads in organs from the untreated group and <b>9a</b> dissolved in (<b>1</b>) 0.5% methylcellulose in DI-H₂O (<b>2</b>) 30% captisol in DI-H₂O (<b>3</b>) 10% vitamin E TPGS in DI-H₂O (<b>4</b>) 0.5% Tween 80 in DI-H₂O (<b>5</b>) 20% cyclodextrin in DI-H₂O or (<b>6</b>) cold PEG (50∶35∶15 H₂O:PEG300:PG). Error bars indicate SEM within treatment groups of 5–7 mice per group.</p

Known nitroaromatic antimicrobial drugs and nitrofurans explored in this study.

<p>Known nitroaromatic antimicrobial drugs and nitrofurans explored in this study.</p

Cross Resistance Profiles for Compounds 9a Resistant Clones.

<p>Abbreviations: S, susceptible; <b>R</b>, resistant.</p