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.

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.

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.

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.

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.

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.

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.

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.

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.

Targeting Chikungunya Virus Replication by Benzoannulene Inhibitors.

A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 µM and viral titer reduction (VTR) of 2.5 log at 10 µM with no observed cytotoxicity (CC50 = 169 µM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 µM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design.

The macrodomain of nsP3 (nsP3MD) is highly conserved among the alphaviruses and ADP-ribosylhydrolase activity of Chikungunya Virus (CHIKV) nsP3MD is critical for CHIKV viral replication and virulence. No small molecule drugs targeting CHIKV nsP3 have been identified to date. Here we report small fragments that bind to nsP3MD which were discovered by virtually screening a fragment library and X-ray crystallography. These identified fragments share a similar scaffold, 2-pyrimidone-4-carboxylic acid, and are specifically bound to the ADP-ribose binding site of nsP3MD. Among the fragments, 2-oxo-5,6-benzopyrimidine-4-carboxylic acid showed anti-CHIKV activity with an IC50 of 23 µM. Our fragment-based drug discovery approach provides valuable information to further develop a specific and potent nsP3 inhibitor of CHIKV viral replication based on the 2-pyrimidone-4-carboxylic acid scaffold. In silico studies suggest this pyrimidone scaffold could also bind to the macrodomains of other alphaviruses and coronaviruses and thus, have potential pan-antiviral activity.

The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules.

Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 µM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

Regular physical activity reduces the percentage of spinally projecting neurons that express mu-opioid receptors from the rostral ventromedial medulla in mice.

INTRODUCTION: Regular physical activity/exercise is an effective nonpharmacological treatment for individuals with chronic pain. Central inhibitory mechanisms, involving serotonin and opioids, are critical to analgesia produced by regular physical activity. The rostral ventromedial medulla (RVM) sends projections to the spinal cord to inhibit or facilitate nociceptive neurons and plays a key role in exercise-induced analgesia. OBJECTIVE: The goal of these studies was to examine if regular physical activity modifies RVM-spinal cord circuitry. METHODS: Male and female mice received Fluoro-Gold placed on the spinal cord to identify spinally projecting neurons from the RVM and the nucleus raphe obscurus/nucleus raphe pallidus, dermorphin-488 into caudal medulla to identify mu-opioid receptors, and were immunohistochemically stained for either phosphorylated-N-methyl-d-aspartate subunit NR1 (p-NR1) to identify excitatory neurons or tryptophan hydroxylase (TPH) to identify serotonin neurons. The percentage of dermorphin-488-positive cells that stained for p-NR1 (or TPH), and the percentage of dermorphin-488-positive cells that stained for p-NR1 (or TPH) and Fluoro-Gold was calculated. Physically active animals were provided running wheels in their cages for 8 weeks and compared to sedentary animals without running wheels. Animals with chronic muscle pain, induced by 2 intramuscular injections of pH 4.0, were compared to sham controls (pH 7.2). RESULTS: Physically active animals had less mu-opioid-expressing neurons projecting to the spinal cord when compared to sedentary animals in the RVM, but not the nucleus raphe obscurus/nucleus raphe pallidus. No changes were observed for TPH. CONCLUSIONS: These data suggest that regular exercise alters central facilitation so that there is less descending facilitation to result in a net increase in inhibition.

Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2.

Drug repurposing is a rapid approach to identifying therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drug and 49 investigational drugs. Among these confirmed compounds, the anti-SARS-CoV-2 activities of 230 compounds, including 38 approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set of drug repurposing screen for SARS-CoV-2 is useful for drug repurposing efforts including design of new drug combinations for clinical trials.

Testosterone protects against the development of widespread muscle pain in mice.

Chronic widespread pain conditions are more prevalent in women than men, suggesting a role for gonadal hormones in the observed differences. Previously, we showed that female mice, compared to male, develop widespread, more severe, and longer-duration hyperalgesia in a model of activity-induced muscle pain. We hypothesized testosterone protects males from developing the female pain phenotype. We tested whether orchiectomy of males before induction of an activity-induced pain model produced a female phenotype and whether testosterone administration produced a male phenotype in females. Orchiectomy produced longer-lasting, more widespread hyperalgesia, similar to females. Administration of testosterone to females or orchiectomized males produced unilateral, shorter-lasting hyperalgesia. Prior studies show that the serotonin transporter (SERT) is increased in the nucleus raphe magnus (NRM) in models of chronic pain, and that blockade of SERT in the NRM reduces hyperalgesia. We examined potential sex differences in the distribution of SERT across brain sites involved in nociceptive processing using immunohistochemistry. A sex difference in SERT was found in the NRM in the activity-induced pain model; females had greater SERT immunoreactivity than males. This suggests that testosterone protects against development of widespread, long-lasting muscle pain and that alterations in SERT may underlie the sex differences.

The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators.

SARS-CoV-02 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus' pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 µM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.

P2X4 Receptors on Muscle Macrophages Are Required for Development of Hyperalgesia in an Animal Model of Activity-Induced Muscle Pain.

Activity-induced pain is common in those with chronic musculoskeletal pain and limits participation in daily activities and exercise. Our laboratory developed a model of activity-induced pain and shows that depletion of muscle macrophages prevents development of hyperalgesia. Adenosine triphosphate (ATP) is released from fatiguing muscle and activates purinergic receptors (P2X), and P2X4 receptors are expressed on macrophages. We hypothesized that exercise releases ATP to activate P2X4 receptors on muscle macrophages, which subsequently release interleukin-1ß (IL-1ß) to produce hyperalgesia. In an animal model of activity-induced pain, using male and female C57BL6/J mice, we show increased expression of P2X4 on muscle macrophages, and blockade of P2X4 receptors in muscle prevented development of hyperalgesia. Using a lentivirus expressing an artificial micro-RNA to P2X4 under the control of a CD68 promoter, we decreased expression of P2X4 mRNA in cultured macrophages, decreased expression of P2X4 protein in muscle macrophages in vivo, and prevented development of activity-induced hyperalgesia. We further show that macrophages primed with LPS differentially released IL-1ß when treated with ATP in neutral or acidic pH. Lastly, blockade of IL-1ß in muscle prevented development of hyperalgesia in this model. Thus, our data suggest that P2X4 receptors could be a valid pharmacological target to control activity-induced muscle pain experienced by patients with chronic musculoskeletal pain.

Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2.

Drug repurposing is a rapid approach to identify therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drugs and 49 investigational drugs. The anti-SARS-CoV-2 activities of 230 of these confirmed compounds, of which 38 are approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA-approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set is a useful resource for drug repurposing efforts, including design of new drug combinations for clinical trials for SARS-CoV-2.

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.

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.