A PDK-1 Allosteric Agonist Neutralizes Insulin Signaling Derangements and Beta-amyloid Toxicity in Neuronal Cells and In Vitro

The Alzheimer’s brain is affected by multiple pathophysiological processes, which include a unique, organ-specific form of insulin resistance that begins early in its course. An additional complexity arises from the four-fold risk of Alzheimer’s Disease (AD) in type 2 diabetics, however there is no definitive proof of causation. Several strategies to improve brain insulin signaling have been proposed and some have been clinically tested. We report findings on a small allosteric molecule that reverses several indices of insulin insensitivity in both cell culture and in vitro models of AD that emphasize the intracellular accumulation of β-amyloid (Aβi). PS48, a chlorophenyl pentenoic acid, is an allosteric activator of PDK-1, which is an Akt-kinase in the insulin/PI3K pathway. PS48 was active at 10 nM to 1 μM in restoring normal insulin-dependent Akt activation and in mitigating Aβi peptide toxicity. Synaptic plasticity (LTP) in prefrontal cortical slices from normal rat exposed to Aβ oligomers also benefited from PS48. During these experiments, neither overstimulation of PI3K/Akt signaling nor toxic effects on cells was observed. Another neurotoxicity model producing insulin insensitivity, utilizing palmitic acid, also responded to PS48 treatment, thus validating the target and indicating that its therapeutic potential may extend outside of β-amyloid reliance. The described in vitro and cell based-in vitro coupled enzymatic assay systems proved suitable platforms to screen a preliminary library of new analogs

Antibacterial properties of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile

The emergence of multidrug-resistant bacterial strains has heightened the need for new antimicrobial agents based on novel chemical scaffolds that are able to circumvent current modes of resistance. We recently developed a whole-animal drug-screening methodology in pursuit of this goal and now report the discovery of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC) as a novel antibacterial effective against resistant nosocomial pathogens. The minimum inhibitory concentrations (MIC) of PSPC against Staphylococcus aureus and Enterococcus faecium were 4 μg/mL and 8 μg/mL, respectively, whereas the MICs were higher against the Gram-negative bacteria Klebsiella pneumoniae (64 μg/mL), Acinetobacter baumannii (32 μg/mL), Pseudomonas aeruginosa (\u3e64 μg/mL), and Enterobacter spp. (\u3e64 μg/mL). However, co-treatment of PSPC with the efflux pump inhibitor phenylalanine arginyl β-naphthylamide (PAβN) or with sub-inhibitory concentrations of the lipopeptide antibiotic polymyxin B reduced the MICs of PSPC against the Gram-negative strains by \u3e4-fold. A sulfide analog of PSPC (PSPC-1S) showed no antibacterial activity, whereas the sulfoxide analog (PSPC-6S) showed identical activity as PSPC across all strains, confirming structure-dependent activity for PSPC and suggesting a target-based mechanism of action. PSPC displayed dose dependent toxicity to both Caenorhabditis elegans and HEK-293 mammalian cells, culminating with a survival rate of 16% (100 μg/mL) and 8.5% (64 μg/mL), respectively, at the maximum tested concentration. However, PSPC did not result in hemolysis of erythrocytes, even at a concentration of 64 μg/mL. Together these results support PSPC as a new chemotype suitable for further development of new antibiotics against Gram-positive and Gram-negative bacteria

Propyl-5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC): a new bacteriostatic agent against methicillin—resistant Staphylococcus aureus

The emergence of Staphylococcus aureus strains resistant to ‘last resort’ antibiotics compels the development of new antimicrobials against this important human pathogen. We found that propyl 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) shows bacteriostatic activity against S. aureus (MIC = 4 μg/ml) and rescues Caenorhabditis elegans from S. aureus infection. Whole-genome sequencing of S. aureus mutants resistant to the compound, along with screening of a S. aureus promoter-lux reporter array, were used to explore possible mechanisms of action. All mutants resistant to HMPC acquired missense mutations at distinct codon positions in the global transcriptional regulator mgrA, followed by secondary mutations in the phosphatidylglycerol lysyltransferase fmtC/mprF. The S. aureus promoter-lux array treated with HMPC displayed a luminescence profile that was unique but showed similarity to DNA-damaging agents and/or DNA replication inhibitors. Overall, HMPC is a new anti-staphylococcal compound that appears to act via an unknown mechanism linked to the global transcriptional regulator MgrA

Salicylanilide anthelmintic drugs prolong survival of <i>C</i>. <i>elegans</i> infected with MRSA.

<p>A 384 well assay plate was co-inoculated with nematodes, bacteria and either DMSO (negative control), vancomycin (positive control) or the salicylanilide anthelmintic drugs. Serial dilutions of the drugs were tested to adjust the concentration between 0.78–50 μg/ml. <b>(A)</b> Sytox Orange stained and bright field images of assay wells containing a gradient of the tested drugs. Dead worms take up the vital dye Sytox Orange and fluoresce. <b>(B)</b> Percent survival of infected worms was calculated from intensity of fluorescence measured in each well of the assay plate.</p

Cytotoxicity of niclosamide and oxyclozanide to HepG2.

<p>The viability of HepG2 cells was measured after treatment with serially diluted concentrations (1–64 μg/ml) of niclosamide, oxyclozanide or the mitochondrial toxin rotenone (positive control). Cell viability was measured spectrophotometrically by detecting degradation of WST-1 dye into formazan by viable cells, which produces an intense color.</p

Repurposing Salicylanilide Anthelmintic Drugs to Combat Drug Resistant <i>Staphylococcus aureus</i>

<div><p><i>Staphylococcus aureus</i> is a Gram-positive bacterium that has become the leading cause of hospital acquired infections in the US. Repurposing Food and Drug Administration (FDA) approved drugs for antimicrobial therapy involves lower risks and costs compared to <i>de novo</i> development of novel antimicrobial agents. In this study, we examined the antimicrobial properties of two commercially available anthelmintic drugs. The FDA approved drug niclosamide and the veterinary drug oxyclozanide displayed strong <i>in vivo</i> and <i>in vitro</i> activity against methicillin resistant <i>S</i>. <i>aureus</i> (minimum inhibitory concentration (MIC): 0.125 and 0.5 μg/ml respectively; minimum effective concentration: ≤ 0.78 μg/ml for both drugs). The two drugs were also effective against another Gram-positive bacteria <i>Enterococcus faecium</i> (MIC 0.25 and 2 μg/ml respectively), but not against the Gram-negative species <i>Klebsiella pneumoniae</i>, <i>Acinetobacter baumannii</i>, <i>Pseudomonas aeruginosa</i> and <i>Enterobacter aerogenes</i>. The <i>in vitro</i> antimicrobial activity of niclosamide and oxyclozanide were determined against methicillin, vancomycin, linezolid or daptomycin resistant <i>S</i>. <i>aureus</i> clinical isolates, with MICs at 0.0625-0.5 and 0.125-2 μg/ml for niclosamide and oxyclozanide respectively. A time-kill study demonstrated that niclosamide is bacteriostatic, whereas oxyclozanide is bactericidal. Interestingly, oxyclozanide permeabilized the bacterial membrane but neither of the anthelmintic drugs exhibited demonstrable toxicity to sheep erythrocytes. Oxyclozanide was non-toxic to HepG2 human liver carcinoma cells within the range of its <i>in vitro</i> MICs but niclosamide displayed toxicity even at low concentrations. These data show that the salicylanilide anthelmintic drugs niclosamide and oxyclozanide are suitable candidates for mechanism of action studies and further clinical evaluation for treatment of staphylococcal infections.</p></div

Comparative time-kill kinetics of niclosamide and oxyclozanide against MRSA.

<p>The survival of MW2 in a broth culture treated with DMSO, niclosamide or oxyclozanide at a concentration of 4 times the MIC. (MIC values: niclosamide 0.125 μg/ml, oxyclozanide 0.5 μg/ml).</p