Identification of SARS-CoV-2 entry inhibitors among already approved drugs.

To discover effective drugs for COVID-19 treatment amongst already clinically approved drugs, we developed a high throughput screening assay for SARS-CoV-2 virus entry inhibitors using SARS2-S pseudotyped virus. An approved drug library of 1800 small molecular drugs was screened for SARS2 entry inhibitors and 15 active drugs were identified as specific SARS2-S pseudovirus entry inhibitors. Antiviral tests using native SARS-CoV-2 virus in Vero E6 cells confirmed that 7 of these drugs (clemastine, amiodarone, trimeprazine, bosutinib, toremifene, flupenthixol, and azelastine) significantly inhibited SARS2 replication, reducing supernatant viral RNA load with a promising level of activity. Three of the drugs were classified as histamine receptor antagonists with clemastine showing the strongest anti-SARS2 activity (EC50 = 0.95 ± 0.83 µM). Our work suggests that these 7 drugs could enter into further in vivo studies and clinical investigations for COVID-19 treatment.

PDCD4 as a marker of mTOR pathway activation and therapeutic target in mycobacterial infections.

Programmed cell death protein 4 (PDCD4) is instrumental in regulating a range of cellular processes such as translation, apoptosis, signal transduction, and inflammatory responses. There is a notable inverse correlation between PDCD4 and the mammalian target of rapamycin (mTOR) pathway, which is integral to cellular growth control. Activation of mTOR is associated with the degradation of PDCD4. Although the role of PDCD4 is well established in oncogenesis and immune response regulation, its function in mycobacterial infections and its interplay with the mTOR pathway necessitate further elucidation. This study investigates the modulation of PDCD4 expression in the context of mycobacterial infections, revealing a consistent pattern of downregulation across diverse mycobacterial species. This observation underscores the potential utility of PDCD4 as a biomarker for assessing mTOR pathway activation during such infections. Building on this finding, we employed a novel approach using PDCD4-based mTOR (Tor)-signal-indicator (TOSI) reporter cells for the high-throughput screening of FDA-approved drugs, focusing on mTOR inhibitors. This methodology facilitated the identification of several agents, inclusive of known mTOR inhibitors, which upregulated PDCD4 expression and concurrently exhibited efficacy in impeding mycobacterial proliferation within macrophages. These results not only reinforce the significance of PDCD4 as a pivotal marker in the understanding of infectious diseases, particularly mycobacterial infections, but also illuminate its potential in the identification of mTOR inhibitors, thereby contributing to the advancement of therapeutic strategies. IMPORTANCE: This study emphasizes the critical role of the mammalian target of rapamycin (mTOR) pathway in macrophage responses to mycobacterial infections, elucidating how mycobacteria activate mTOR, resulting in PDCD4 degradation. The utilization of the (Tor)-signal-indicator (TOSI) vector for real-time monitoring of mTOR activity represents a significant advancement in understanding mTOR regulation during mycobacterial infection. These findings deepen our comprehension of mycobacteria's innate immune mechanisms and introduce PDCD4 as a novel marker for mTOR activity in infectious diseases. Importantly, this research laid the groundwork for high-throughput screening of mTOR inhibitors using FDA-approved drugs, offering the potential for repurposing treatments against mycobacterial infections. The identification of drugs that inhibit mTOR activation opens new avenues for host-directed therapies, marking a significant step forward in combating tuberculosis and other mycobacterial diseases.

Novel metallo-ß-lactamases inhibitors restore the susceptibility of carbapenems to New Delhi metallo-lactamase-1 (NDM-1)-harbouring bacteria.

BACKGROUND AND PURPOSE: The production of metallo-ß-lactamases is a major mechanisms adopted by bacterial pathogens to resist carbapenems. Repurposing approved drugs to restore the efficacy of carbapenems represents an efficient and cost-effective approach to fight infections caused by carbapenem resistant pathogens. EXPERIMENTAL APPROACH: The nitrocefin hydrolysis assay was employed to screen potential New Delhi metallo-lactamase-1 (NDM-1) inhibitors from a commercially available U.S. Food and Drug Administration (FDA) approved drug library. The mechanism of inhibition was clarified by metal restoration, inductively coupled plasma mass spectrometry (ICP-MS) and molecular dynamics simulation. The in vitro synergistic antibacterial effect of the identified inhibitors with meropenem was determined by the checkerboard minimum inhibitory concentration (MIC) assay, time-dependent killing assay and combined disc test. Three mouse infection models were used to further evaluate the in vivo therapeutic efficacy of combined therapy. KEY RESULTS: Twelve FDA-approved compounds were initially screened to inhibit the ability of NDM-1 to hydrolyse nitrocefin. Among these compounds, dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid were demonstrated to inhibit all tested metallo-ß-lactamases and showed an in vitro synergistic bactericidal effect with meropenem against metallo-ß-lactamases-producing bacteria. Dexrazoxane, embelin and candesartan cilexetil are metal ion chelating agents, while the inhibition of NDM-1 by nordihydroguaiaretic acid involves its direct binding to the active region of NDM-1. Furthermore, these four drugs dramatically rescued the treatment efficacy of meropenem in three infection models. CONCLUSIONS AND IMPLICATIONS: Our observations indicated that dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid are promising carbapenem adjuvants against metallo-ß-lactamases-positive carbapenem resistant bacterial pathogens.

High-Throughput Image-Based Aggresome Quantification.

Aggresomes are subcellular perinuclear structures where misfolded proteins accumulate by retrograde transport on microtubules. Different methods are available to monitor aggresome formation, but they are often laborious, time-consuming, and not quantitative. Proteostat is a red fluorescent molecular rotor dye, which becomes brightly fluorescent when it binds to protein aggregates. As this reagent was previously validated to detect aggresomes, we have miniaturized its use in 384-well plates and developed a method for high-throughput imaging and quantification of aggresomes. Two different image analysis methods, including one with machine learning, were evaluated. They lead to similar robust data to quantify cells having aggresome, with satisfactory Z' factor values and reproducible EC50 values for compounds known to induce aggresome formation, like proteasome inhibitors. We demonstrated the relevance of this phenotypic assay by screening a chemical library of 1280 compounds to find aggresome modulators. We obtained hits that present similarities in their structural and physicochemical properties. Interestingly, some of them were previously described to modulate autophagy, which could explain their effect on aggresome structures. In summary, we have optimized and validated the Proteostat detection reagent to easily measure aggresome formation in a miniaturized, automated, quantitative, and high-content assay. This assay can be used at low, middle, or high throughput to quantify changes in aggresome formation that could help in the understanding of chemical compound activity in pathologies such as protein misfolding disorders or cancer.

Identification of FDA-Approved Drugs with Activity against Stationary Phase Bartonella henselae.

Bartonella henselae can cause various infections in humans, ranging from benign and self-limiting diseases to severe and life-threatening diseases as well as persistent infections that are difficult to treat. To develop more effective treatments for persistent Bartonella infections, in this study, we performed a high-throughput screen of an FDA-approved drug library against stationary phase B. henselae using the SYBR Green I/propidium iodide (PI) viability assay. We identified 110 drug candidates that had better activity against stationary phase B. henselae than ciprofloxacin, and among the top 52 drug candidates tested, 41 drugs were confirmed by microscopy to have higher activity than the current frontline antibiotic erythromycin. The identified top drug candidates include pyrvinium pamoate, daptomycin, methylene blue, azole drugs (clotrimazole, miconazole, sulconazole, econazole, oxiconazole, butoconazole, bifonazole), aminoglycosides (gentamicin and streptomycin, amikacin, kanamycin), amifostine (Ethyol), antiviral Lopinavir/ritonavir, colistin, nitroxoline, nitrofurantoin, verteporfin, pentamidine, berberine, aprepitant, olsalazine, clinafloxacin, and clofoctol. Pyrvinium pamoate, daptomycin, methylene blue, clotrimazole, and gentamicin and streptomycin at their respective maximum drug concentration in serum (Cmax) had the capacity to completely eradicate stationary phase B. henselae after 3-day drug exposure in subculture studies. While the currently used drugs for treating bartonellosis, including rifampin, erythromycin, azithromycin, doxycycline, and ciprofloxacin, had very low minimal inhibitory concentration (MIC) against growing B. henselae, they had relatively poor activity against stationary phase B. henselae, except aminoglycosides. The identified FDA-approved agents with activity against stationary phase B. henselae should facilitate development of more effective treatments for persistent Bartonella infections.

Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells.

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs-Nitazoxanide, Closantel Sodium, and Closantel-with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs.

A Cytotoxic Three-Dimensional-Spheroid, High-Throughput Assay Using Patient-Derived Glioma Stem Cells.

Glioblastoma (GBM) is the most aggressive primary brain cancer with an average survival time after diagnosis of only 12-14 months, with few (<5%) long-term survivors. A growing body of work suggests that GBMs contain a small population of glioma stem cells (GSCs) that are thought to be major contributors to treatment resistance and disease relapse. Identifying compounds that modulate GSC proliferation would provide highly valuable molecular probes of GSC-directed signaling. However, targeting GSCs pharmacologically has been challenging. Patient-derived GSCs can be cultured as neurospheres, and in vivo these cells functionally recapitulate the heterogeneity of the original tumor. Using patient-derived GSC-enriched cultures, we have developed a 1536-well spheroid-based proliferation assay and completed a pilot screen, testing ~3300 compounds comprising approved drugs. This cytotoxic and automation-friendly assay yielded a signal-to-background (S/B) ratio of 161.3 ± 7.5 and Z' of 0.77 ± 0.02, demonstrating its robustness. Importantly, compounds were identified with anti-GSC activity, demonstrating the applicability of this assay for large-scale high-throughput screening (HTS).

Screening of FDA-approved drugs identifies sutent as a modulator of UCP1 expression in brown adipose tissue.

BACKGROUND: The pharmacological activation of thermogenesis in brown adipose tissue has long been considered promising strategies to treat obesity. However, identification of safe and effective agents remains a challenge. In this study, we addressed this challenge by developing a cellular system with a fluorescence readout, and applied in a high-throughput manner to screen for FDA-approved drugs that may activate endogenous UCP1 expression in adipocytes. METHODS: We have generated a Ucp1-2A-GFP reporter mouse, in which GFP intensity serves as a surrogate of the endogenous expression level of UCP1 protein; and immortalized brown adipocytes were derived from this mouse model and applied in drug screening. Candidate drugs were further tested in mouse models either fed with normal chow or high fat diet to induce obesity. FINDINGS: By using the cellular screening platform, we identified a group of FDA-approved drugs that can upregulate UCP1 expression in brown adipocyte, including previously known UCP1 activators and new candidate drugs. Further studies focusing on a previously unreported drug-sutent, revealed that sutent treatment could increase the energy expenditure and inhibit lipid synthesis in mouse adipose and liver tissues, resulting in improved metabolism and resistance to obesity. INTERPRETATION: This study offered an easy-to-use cellular screening system for UCP1 activators, and provided a candidate list of FDA-approved drugs that can potentially treat obesity. Further study of these candidates may shed new light on the drug discovery towards obesity. FUND: National Key Research and Development Program and the Strategic Priority Research Program of the Chinese Academy of Sciences, etc. (250 words).

Identification of novel activity against Borrelia burgdorferi persisters using an FDA approved drug library.

Although antibiotic treatment for Lyme disease is effective in the majority of cases, especially during the early phase of the disease, a minority of patients suffer from post-treatment Lyme disease syndrome (PTLDS). It is unclear what mechanisms drive this problem, and although slow or ineffective killing of Borrelia burgdorferi has been suggested as an explanation, there is a lack of evidence that viable organisms are present in PTLDS. Although not a clinical surrogate, insight may be gained by examining stationary-phase in vitro Borrelia burgdorferi persisters that survive treatment with the antibiotics doxycycline and amoxicillin. To identify drug candidates that can eliminate B. burgdorferi persisters more effectively, we screened an Food and Drug Administration (FDA)-approved drug library consisting of 1524 compounds against stationary-phase B. burgdorferi by using a newly developed high throughput SYBR Green I/propidium iodide (PI) assay. We identified 165 agents approved for use in other disease conditions that had more activity than doxycycline and amoxicillin against B. burgdorferi persisters. The top 27 drug candidates from the 165 hits were confirmed to have higher anti-persister activity than the current frontline antibiotics. Among the top 27 confirmed drug candidates from the 165 hits, daptomycin, clofazimine, carbomycin, sulfa drugs (e.g., sulfamethoxazole), and certain cephalosporins (e.g. cefoperazone) had the highest anti-persister activity. In addition, some drug candidates, such as daptomycin and clofazimine (which had the highest activity against non-growing persisters), had relatively poor activity or a high minimal inhibitory concentration (MIC) against growing B. burgdorferi. Our findings may have implications for the development of a more effective treatment for Lyme disease and for the relief of long-term symptoms that afflict some Lyme disease patients.