Triamterene Functions as an Effective Nonsense Suppression Agent for MPS I-H (Hurler Syndrome).

Mucopolysaccharidosis I-Hurler (MPS I-H) is caused by the loss of α-L-iduronidase, a lysosomal enzyme that degrades glycosaminoglycans. Current therapies cannot treat many MPS I-H manifestations. In this study, triamterene, an FDA-approved, antihypertensive diuretic, was found to suppress translation termination at a nonsense mutation associated with MPS I-H. Triamterene rescued enough α-L-iduronidase function to normalize glycosaminoglycan storage in cell and animal models. This new function of triamterene operates through premature termination codon (PTC) dependent mechanisms that are unaffected by epithelial sodium channel activity, the target of triamterene's diuretic function. Triamterene represents a potential non-invasive treatment for MPS I-H patients carrying a PTC.

Identification of Quinolinones as Antivirals against Venezuelan Equine Encephalitis Virus.

Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies.

Over the past two decades, ribosome biogenesis has emerged as an attractive target for cancer treatment. In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibitors of rRNA synthesis. These two compounds, Ribosome Biogenesis Inhibitors 1 and 2 (RBI1 and RBI2), induce a substantial decrease in the viability of A375 cells, comparable to the previously published ribosome biogenesis inhibitor CX-5461. Anchorage-independent cell growth assays further confirmed that RBI2 inhibits cell growth and proliferation. Thus, the RBI compounds have promising properties for further development as potential cancer chemotherapeutics.

Revisiting the ß-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach.

The suboptimal effectiveness of ß-lactam antibiotics against Mycobacterium tuberculosis has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a ß-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R. Sergysels, et al., Int J Tuberc Lung Dis 16:558-560, 2012, https://doi.org/10.5588/ijtld.11.0414). We hypothesized that the innate resistance of M. tuberculosis to ß-lactams is mediated in part by noncanonical accessory proteins that are not considered the classic targets of ß-lactams and that small-molecule inhibitors of those accessory targets might sensitize M. tuberculosis to ß-lactams. In this study, we screened an NIH small-molecule library for the ability to sensitize M. tuberculosis to meropenem. We identified six hit compounds, belonging to either the N-arylindole or benzothiophene chemotype. Verification studies confirmed the synthetic lethality phenotype for three of the N-arylindoles and one benzothiophene derivative. The latter was demonstrated to be partially bioavailable via oral administration in mice. Structure-activity relationship studies of both structural classes identified analogs with potent antitubercular activity, alone or in combination with meropenem. Transcriptional profiling revealed that oxidoreductases, MmpL family proteins, and a 27-kDa benzoquinone methyltransferase could be the targets of the N-arylindole potentiator. In conclusion, our compound-compound synthetic lethality screening revealed novel small molecules that were capable of potentiating the action of meropenem, presumably via inhibition of the innate resistance conferred by ß-lactam accessory proteins. ß-Lactam compound-compound synthetic lethality may be an alternative approach for drug-resistant tuberculosis.

Identification of Small Molecule Inhibitors of Human Cytochrome c Oxidase That Target Chemoresistant Glioma Cells.

The enzyme cytochrome c oxidase (CcO) or complex IV (EC 1.9.3.1) is a large transmembrane protein complex that serves as the last enzyme in the respiratory electron transport chain of eukaryotic mitochondria. CcO promotes the switch from glycolytic to oxidative phosphorylation (OXPHOS) metabolism and has been associated with increased self-renewal characteristics in gliomas. Increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure, and patients with primary glioblastoma multiforme and high tumor CcO activity have worse clinical outcomes than those with low tumor CcO activity. Therefore, CcO is an attractive target for cancer therapy. We report here the characterization of a CcO inhibitor (ADDA 5) that was identified using a high throughput screening paradigm. ADDA 5 demonstrated specificity for CcO, with no inhibition of other mitochondrial complexes or other relevant enzymes, and biochemical characterization showed that this compound is a non-competitive inhibitor of cytochrome c When tested in cellular assays, ADDA 5 dose-dependently inhibited the proliferation of chemosensitive and chemoresistant glioma cells but did not display toxicity against non-cancer cells. Furthermore, treatment with ADDA 5 led to significant inhibition of tumor growth in flank xenograft mouse models. Importantly, ADDA 5 inhibited CcO activity and blocked cell proliferation and neurosphere formation in cultures of glioma stem cells, the cells implicated in tumor recurrence and resistance to therapy in patients with glioblastoma. In summary, we have identified ADDA 5 as a lead CcO inhibitor for further optimization as a novel approach for the treatment of glioblastoma and related cancers.

Discovery of Clinically Approved Agents That Promote Suppression of Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations.

RATIONALE: Premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Several agents are known to suppress PTCs but are poorly efficacious or toxic. OBJECTIVES: To determine whether there are clinically available agents that elicit translational readthrough and improve CFTR function sufficient to confer therapeutic benefit to patients with CF with PTCs. METHODS: Two independent screens, firefly luciferase and CFTR-mediated transepithelial chloride conductance assay, were performed on a library of 1,600 clinically approved compounds using fisher rat thyroid cells stably transfected with stop codons. Select agents were further evaluated using secondary screening assays including short circuit current analysis on primary cells from patients with CF. In addition, the effect of CFTR modulators (ivacaftor) was tested in combination with the most efficacious agents. MEASUREMENTS AND MAIN RESULTS: From the primary screen, 48 agents were selected as potentially active. Following confirmatory tests in the transepithelial chloride conductance assay and prioritizing agents based on favorable pharmacologic properties, eight agents were advanced for secondary screening. Ivacaftor significantly increased short circuit current following forskolin stimulation in cells treated with pyranoradine tetraphosphate, potassium p-aminobenzoate, and escin as compared with vehicle control. Escin, an herbal agent, consistently induced readthrough activity as demonstrated by enhanced CFTR expression and function in vitro. CONCLUSIONS: Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.

Identification of a novel HIV-1 inhibitor targeting Vif-dependent degradation of human APOBEC3G protein.

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(-) T cells and had an IC50 as low as 8.4 µM and a TC50 of >100 µM when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 µM). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Acid-sensing ion channel 3 deficiency increases inflammation but decreases pain behavior in murine arthritis.

OBJECTIVE: Through its location on nociceptors, acid-sensing ion channel 3 (ASIC-3) is activated by decreases in pH and plays a significant role in musculoskeletal pain. We recently showed that decreases in pH activate ASIC-3 located on fibroblast-like synoviocytes (FLS), which are key cells in the inflammatory process. The purpose of this study was to test whether ASIC-3-deficient mice with arthritis have altered inflammation and pain relative to controls. METHODS: Collagen antibody-induced arthritis (CAIA) was generated by injection of an anti-type II collagen antibody cocktail. Inflammation and pain parameters in ASIC-3(-/-) and ASIC-3(+/+) mice were assessed. Disease severity was assessed by determining clinical arthritis scores, measuring joint diameters, analyzing joint histology, and assessing synovial gene expression by quantitative polymerase chain reaction analysis. Cell death was assessed with a Live/Dead assay of FLS in response to decreases in pH. Pain behaviors in the mice were measured by examining withdrawal thresholds in the joints and paws and by measuring their physical activity levels. RESULTS: Surprisingly, ASIC-3(-/-) mice with CAIA demonstrated significantly increased joint inflammation, joint destruction, and expression of interleukin-6 (IL-6), matrix metalloproteinase 3 (MMP-3), and MMP-13 in joint tissue as compared to ASIC-3(+/+) mice. ASIC-3(+/+) FLS showed enhanced cell death when exposed to pH 6.0 in the presence of IL-1ß, which was abolished in ASIC-3(-/-) FLS. Despite enhanced disease severity, ASIC-3(-/-) mice did not develop mechanical hypersensitivity of the paw and showed greater levels of physical activity. CONCLUSION: Our findings are consistent with the hypothesis that ASIC-3 plays a protective role in the inflammatory arthritides by limiting inflammation through enhanced synoviocyte cell death, which reduces disease severity, and through the production of pain, which reduces joint use.

High-Throughput cell-based immunofluorescence assays against influenza.

A rapid drug discovery response to influenza outbreaks with the potential to reach pandemic status could help minimize the virus's impact by reducing the time to identify anti-influenza drugs. Although several anti-influenza strategies have been considered in the search for new drugs, only a few therapeutic agents are approved for clinical use. The cytopathic effect induced by the influenza virus in Madin Darby canine kidney (MDCK) cells has been widely used for high-throughput anti-influenza drug screening, but the fact that the MDCK cells are not human cells constitutes a disadvantage when searching for new therapeutic agents for human use. We have developed a highly sensitive cell-based imaging assay for the identification of inhibitors of influenza A and B virus that is high-throughput compatible using the A549 human cell line. The assay has also been optimized for the assessment of the neutralizing effect of anti-influenza antibodies in the absence of trypsin, which allows testing of purified antibodies and serum samples. This assay platform can be applied to full high-throughput screening campaigns or later stages requiring quantitative potency determinations for structure-activity relationships.

A high-throughput response to the SARS-CoV-2 pandemic.

Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.

A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus.

BACKGROUND: Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial. RESULTS: We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 µM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9. CONCLUSIONS: A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.

P2X7-NLRP3-Caspase-1 signaling mediates activity-induced muscle pain in male but not female mice.

ABSTRACT: We developed an animal model of activity-induced muscle pain that is dependent on local macrophage activation and release of interleukin-1ß (IL-1ß). Activation of purinergic type 2X (P2X) 7 receptors recruits the NOD-like receptor protein (NLRP) 3 and activates Caspase-1 to release IL-1ß. We hypothesized that pharmacological blockade of P2X7, NLRP3, and Caspase-1 would prevent development of activity-induced muscle pain in vivo and release of IL-1ß from macrophages in vitro. The decrease in muscle withdrawal thresholds in male, but not female, mice was prevented by the administration of P2X7, NLRP3, and Caspase-1 inhibitors before induction of the model, whereas blockade of IL-1ß before induction prevented muscle hyperalgesia in both male and female mice. Blockade of P2X7, NLRP3, Capsase-1, or IL-1ß 24 hours, but not 1 week, after induction of the model alleviated muscle hyperalgesia in male, but not female, mice. mRNA expression of P2X7, NLRP3, Caspase-1, and IL-1ß from muscle was increased 24 hours after induction of the model in both male and female mice. Using multiplex, increases in IL-1ß induced by combining adenosine triphosphate with pH 6.5 in lipopolysaccharide-primed male and female macrophages were significantly lower with the presence of inhibitors of P2X7 (A740003), NLRP3 (MCC950), and Caspase-1 (Z-WEHD-FMK) when compared with the vehicle. The current data suggest the P2X7/NLRP3/Caspase-1 pathway contributed to activity-induced muscle pain initiation and early maintenance phases in male but not female, and not in late maintenance phases in male mice.

Selective androgen receptor modulator microparticle formulation reverses muscle hyperalgesia in a mouse model of widespread muscle pain.

ABSTRACT: Chronic pain is a significant health problem associated with disability and reduced quality of life. Current management of chronic pain is inadequate with only modest effects of pharmacological interventions. Thus, there is a need for the generation of analgesics for treating chronic pain. Although preclinical and clinical studies demonstrate the analgesic effects of testosterone, clinical use of testosterone is limited by adverse androgenic effects. Selective androgen receptor modulators (SARMs) activate androgen receptors and overcome treatment limitations by minimizing androgenic side effects. Thus, we tested whether daily soluble SARMs or a SARM-loaded microparticle formulation alleviated muscle hyperalgesia in a mouse-model of widespread pain (male and female C57BL/6J mice). We tested whether the analgesic effects of the SARM-loaded microparticle formulation was mediated through androgen receptors by blocking androgen receptors with flutamide pellets. In vitro and in vivo release kinetics were determined for SARM-loaded microparticles. Safety and toxicity of SARM treatment was determined using serum cardiac and liver toxicity panels, heart histology, and conditioned place preference testing. Subcutaneous daily SARM administration, and 2 injections, 1 week apart, of SARM-loaded microparticles alleviated muscle hyperalgesia in both sexes and was prevented with flutamide treatment. Sustained release of SARM, from the microparticle formulation, was observed both in vitro and in vivo for 4 weeks. Selective androgen receptor modulator treatment produced no cardiac or liver toxicity and did not produce rewarding behaviors. These studies demonstrate that SARM-loaded microparticles, which release drug for a sustained period, alleviate muscle pain, are safe, and may serve as a potential therapeutic for chronic muscle pain.

Resistance training protects against muscle pain through activation of androgen receptors in male and female mice.

ABSTRACT: Resistance training-based exercise is commonly prescribed in the clinic for the treatment of chronic pain. Mechanisms of aerobic exercise for analgesia are frequently studied, while little is known regarding resistance training mechanisms. We developed a resistance training model in mice and hypothesized resistance training would protect against development of muscle pain, mediated through the activation of androgen receptors. Activity-induced muscle hyperalgesia was produced by 2 injections of pH 5.0 stimuli with fatiguing muscle contractions. Resistance training was performed by having mice climb a ladder with attached weights, 3 times per week. Resistance training acutely increased blood lactate and prolonged training increased strength measured via forepaw grip strength and 1 repetition maximum, validating the exercise program as a resistance training model. Eight weeks of resistance training prior to induction of the pain model blocked the development of muscle hyperalgesia in both sexes. Resistance training initiated after induction of the pain model reversed muscle hyperalgesia in male mice only. A single resistance training bout acutely increased testosterone in male but not female mice. Administration of the androgen receptor antagonist flutamide (200 mg pellets) throughout the 8-week training program blocked the exercise-induced protection against muscle pain in both sexes. However, single administration of flutamide (1, 3, 10 mg/kg) in resistance-trained animals had no effect on existing exercise-induced protection against muscle pain. Therefore, resistance training acutely increases lactate and testosterone and strength overtime. Eight weeks of resistance training prevents the development of hyperalgesia through the activation of androgen receptors in an animal model of muscle pain.

Novel Compound Inhibitors of HIV-1NL4-3 Vpu.

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.

A high-throughput screening assay for inhibitors of bacterial motility identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene expression in Vibrio cholerae.

Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot(-)) phenotype and is specific for the Na(+)-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na(+) bioenergetics, such as the function of the primary Na(+) pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na(+)-driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.

Identification of novel small molecule activators of nuclear factor-κB with neuroprotective action via high-throughput screening.

Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity.

Innovative university partnership meets neonatal nurse practitioner program need amid faculty shortage.

This nation is facing a significant nursing faculty shortage not only at the undergraduate but also at the graduate level. As nursing faculty, we must be innovative in considering ways of consolidating resources in this time of demand for advanced practice nurses. With the apparent need for qualified neonatal nurse practitioners in Iowa, the University of Iowa understood the importance of starting a neonatal nurse practitioners program. The University of Iowa College of Nursing and the University of Missouri-Kansas City formed a partnership to educate neonatal nurse practitioners. Although this partnership concentrates on a specific subspecialty population, it can serve as a pilot project that could be adapted by those who care for other diverse populations. The nurse faculty shortage requires innovative solutions, and a model such as this one may provide solutions for other institutions.

Fighting SARS-CoV-2 with green seaweed Ulva sp. extract: extraction protocol predetermines crude ulvan extract anti-SARS-CoV-2 inhibition properties in in vitro Vero-E6 cells assay.

Due to the global COVID-19 pandemic, there is a need to screen for novel compounds with antiviral activity against SARS-COV-2. Here we compared chemical composition and the in vitro anti- SARS-COV-2 activity of two different Ulva sp. crude ulvan extracts: one obtained by an HCl-based and another one by ammonium oxalate-based (AOx) extraction protocols. The composition of the crude extracts was analyzed and their antiviral activity was assessed in a cytopathic effect reduction assay using Vero E6 cells. We show that the extraction protocols have a significant impact on the chemical composition, anti- SARS-COV-2 activity, and cytotoxicity of these ulvan extracts. The ulvan extract based on the AOx protocol had a higher average molecular weight, higher charge, and 11.3-fold higher antiviral activity than HCl-based extract. Our results strongly suggest that further bioassay-guided investigation into bioactivity of compounds found in Ulva sp. ulvan extracts could lead to the discovery of novel anti-SARS-CoV-2 antivirals.