Drug Administration-approved Compounds Research Articles

In silico Screening of Food and Drug Administration-approved Compounds against Trehalose 2-sulfotransferase (Rv0295c) in Mycobacterium tuberculosis: Insights from Molecular Docking and Dynamics Simulations.

Tuberculosis (TB) remains a prominent global health challenge, distinguished by substantial occurrences of infection and death. The upsurge of drug-resistant TB strains underscores the urgency to identify novel therapeutic targets and repurpose existing compounds. Rv0295c is a potentially druggable enzyme involved in cell wall biosynthesis and virulence. We evaluated the inhibitory activity of Food and Drug Administration (FDA)-approved compounds against Rv0295c of Mycobacterium tuberculosis, employing molecular docking, ADME evaluation, and dynamics simulations. The study screened 1800 FDA-approved compounds and selected the top five compounds with the highest docking scores. Following this, we subjected the initially screened ligands to ADME analysis based on their dock scores. In addition, the compound exhibited the highest binding affinity chosen for molecular dynamics (MD) simulation to investigate the dynamic behavior of the ligand-receptor complex. Dihydroergotamine (CHEMBL1732) exhibited the highest binding affinity (-12.8 kcal/mol) for Rv0295c within this set of compounds. We evaluated the stability and binding modes of the complex over extended simulation trajectories. Our in silico analysis demonstrates that FDA-approved drugs can serve as potential Rv0295c inhibitors through repurposing. The combination of molecular docking and MD simulation offers a comprehensive understanding of the interactions between ligands and the protein target, providing valuable guidance for further experimental validation. Identifying Rv0295c inhibitors may contribute to new anti-TB drugs.

Novel chemical scaffold as potential drug against Leishmania donovani: Integrated computational and experimental approaches.

In this study, we have screened a large number of Food and Drug Administration-approved compounds for novel anti-leishmanial molecules targeting the citrate synthase enzyme of the parasite. Based on their docking and molecular dynamic simulation statistics, five compounds were selected. These compounds followed Lipinski's rule of five. Additionally, in vitro, antileishmanial and cytotoxicity studies were performed. The three compounds, Abemaciclib, Bazedoxifene, and Vorapaxar, had shown effective anti-leishmanial activities with IC50 values of 0.92 ± 0.02, 0.65 ± 0.09, and 6.1 ± 0.91 against Leishmania donovani promastigote and with EC50 values of 1.52 ± 0.37, 2.11 ± 0.38, 10.4 ± 1.27 against intramacrophagic amastigote without significantly harming macrophage cells. Among them, from in silico and antileishmanial activities studies, Abemaciclib had been selected based on their less binding energy, good antileishmanial activities, and also a significant difference in their binding energy with human citrate synthase for cell death mechanistic studies using flow cytometry and a DNA fragmentation assay. The action of this compound resulted in an increased reactive oxygen species production, depolarization of mitochondrial membrane potential, DNA damage, and an increase in the sub-G1 cell population. These properties are the hallmarks of apoptosis which were further confirmed by apoptotic assay. Based on the above result, this anticancer compound Abemaciclib could be employed as a potential treatment option for leishmaniasis after further confirmation.

Computational approach in searching for dual action multitarget inhibitors for osteosarcoma.

Osteosarcoma is a common primary malignant bone tumor that typically manifests in the second decade of life. This study aimed to identify osteogenic compounds that potentially serve as multitarget inhibitors for osteosarcoma. The study was a molecular docking study of nine Food and Drug Administration-approved compounds with osteogenic properties to the key membrane proteins of osteosarcoma. The ligands used were raloxifene, simvastatin, dexamethasone, risedronate, ibandronate, zoledronic acid, ascorbic acid, alendronate, and β-glycerophosphate, whereas the target proteins used were RET, fibroblast growth factor receptor 1, KIT, PDGFRA, VEGFR1, and VEGFR2. Chem3D version 15.0.0.106 was used for ligand preparation, and AutoDockTools version 1.5.6 was used for protein preparation, whereas molecular docking was conducted using AutoDock Vina. Raloxifene, simvastatin, and dexamethasone had the lowest binding activity to the target proteins. The binding affinity of raloxifene was from -8.4 to -10.0 kcal mol-1, that of simvastatin was -8.3 to -9.2 kcal mol-1, whereas dexamethasone ranged from -6.9 to -9.1 kcal mol-1. Most types of interactions were hydrophobically followed by hydrogen bonding. The current study suggests that raloxifene, simvastatin, and dexamethasone have the potential to act as multitarget inhibitors for osteosarcoma with the ability to induce bone remodeling.

Agonists of prostaglandin E2 receptors as potential first in class treatment for nephronophthisis and related ciliopathies

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.

Pinaverium Bromide Attenuates Lipopolysaccharide-Induced Excessive Systemic Inflammation via Inhibiting Neutrophil Priming

Abstract Dominant infiltration of neutrophils is a hallmark of many inflammatory diseases, especially in septic shock. IL-1β as one of the most early released proinflammatory cytokine in neutrophil, plays a pivotal role in the progress of sepsis. In this study, we built a high-throughput–compatible drug screen assay platform based on our newly constructed reporter C57BL/6 mice, pIL1-DsRed, expressing the DsRed gene under the control of the IL-1β promoter. After screening 1200 U.S. Food and Drug Administration–approved compounds, we found that pinaverium bromide (PVB) significantly suppressed the DsRed expression of primed neutrophil and improved the survival rate of mice given LPS in an endotoxin challenge analogous to sepsis, regardless of whether PVB was administered before or after LPS. PVB also protected the liver and lung from LPS-induced damage and reduced organ-specific inflammatory responses. PVB decreased the production of IL-1β, IL-6, and CXCL1 mRNA in the lungs of LPS-treated mice and decreased the serum levels of liver transaminases (alanine aminotransferase and aspartate aminotransferase) at multiple time points and doses tested. PVB can significantly suppress primed neutrophil-specific respiratory bursts and migration as well. Lastly, PVB affected neutrophils’ gene expression and phenotypic changes during neutrophil priming. PVB downregulated GM-CSF–induced expression of CD54 and dectin-2 (markers of fully primed neutrophils) at both mRNA and protein levels during late-phase neutrophil priming. In summary, we demonstrated that PVB can be used as a potential therapeutic agent for sepsis by inhibiting neutrophil priming.

Desloratadine inhibits heterotopic ossification by suppression of BMP2-Smad1/5/8 signaling.

Heterotopic ossification (HO) is a pathological condition in which ectopic bone forms within soft tissues such as skeletal muscle. Human platelet-derived growth factor receptor α positive (PDGFRα+) cells, which were proved to be the original cells of HO were incubated in osteogenic differentiation medium with Food and Drug Administration-approved compounds. Alkaline phosphatase activity was measured as a screening to inhibit osteogenic differentiation. For the compounds which inhibited osteogenic differentiation of PDGFRα+ cells, we examined dose dependency of its effect using alizarin red S staining and its cell toxicity using WST-8. In addition, regulation of bone morphogenetic proteins (BMP)-Smad signaling which is the major signal of osteogenic differentiation was investigated by Western blotting to elucidate the mechanism of osteogenesis inhibitory effect by the compound. In vivo experiment, complete transverse incision of Achilles tendons in mice was made and mice were fed the compound by mixing with drinking water after operation. Ten weeks after operation, we assessed and quantified HO by micro-computed tomography scan. Intriguingly, we discovered desloratadine inhibited osteogenic differentiation of PDGFRα+ cells using the drug repositioning method. Desloratadine inhibited osteogenic differentiation of the cells dose dependently without cell toxicity. Desloratadine suppressed phosphorylation of Smad1/5/8 induced by BMP2 in PDGFRα+ cells. In Achilles tenotomy mice model, desloratadine treatment significantly inhibited ectopic bone formation compared with control. In conclusion, we discovered desloratadine inhibited osteogenic differentiation using human PDGFRα+ cells and proved its efficacy using Achilles tenotomy ectopic bone formation model in vivo. Our study paved the way to inhibit HO in early clinical use because of its guaranteed safety.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 μM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

Future appeal of comparative studies on putative binding sites of HIV-1 virus-encoded proteolytic enzyme inhibitor of different Food and Drug Administration-approved compounds

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Abduljaleel Z, Athar M, Al-Allaf FA, Al-Dehlawi S, Melebari S, El-Huneidi W. Future appeal of comparative studies on putative binding sites of HIV-1 virus-encoded proteolytic enzyme inhibitor of different Food and Drug Administration-approved compounds. HIV & AIDS Review. International Journal of HIV-Related Problems. 2020;19(2):78-86. doi:10.5114/hivar.2020.96402. APA Abduljaleel, Z., Athar, M., Al-Allaf, F. A., Al-Dehlawi, S., Melebari, S., & El-Huneidi, W. (2020). Future appeal of comparative studies on putative binding sites of HIV-1 virus-encoded proteolytic enzyme inhibitor of different Food and Drug Administration-approved compounds. HIV & AIDS Review. International Journal of HIV-Related Problems, 19(2), 78-86. https://doi.org/10.5114/hivar.2020.96402 Chicago Abduljaleel, Zainularifeen, Mohammad Athar, Faisal A Al-Allaf, Saied Al-Dehlawi, Sami Melebari, and Waseem El-Huneidi. 2020. "Future appeal of comparative studies on putative binding sites of HIV-1 virus-encoded proteolytic enzyme inhibitor of different Food and Drug Administration-approved compounds". HIV & AIDS Review. International Journal of HIV-Related Problems 19 (2): 78-86. doi:10.5114/hivar.2020.96402. Harvard Abduljaleel, Z., Athar, M., Al-Allaf, F., Al-Dehlawi, S., Melebari, S., and El-Huneidi, W. (2020). Future appeal of comparative studies on putative binding sites of HIV-1 virus-encoded proteolytic enzyme inhibitor of different Food and Drug Administration-approved compounds. HIV & AIDS Review. International Journal of HIV-Related Problems, 19(2), pp.78-86. https://doi.org/10.5114/hivar.2020.96402 MLA Abduljaleel, Zainularifeen et al. "Future appeal of comparative studies on putative binding sites of HIV-1 virus-encoded proteolytic enzyme inhibitor of different Food and Drug Administration-approved compounds." HIV & AIDS Review. International Journal of HIV-Related Problems, vol. 19, no. 2, 2020, pp. 78-86. doi:10.5114/hivar.2020.96402. Vancouver Abduljaleel Z, Athar M, Al-Allaf F, Al-Dehlawi S, Melebari S, El-Huneidi W. Future appeal of comparative studies on putative binding sites of HIV-1 virus-encoded proteolytic enzyme inhibitor of different Food and Drug Administration-approved compounds. HIV & AIDS Review. International Journal of HIV-Related Problems. 2020;19(2):78-86. doi:10.5114/hivar.2020.96402.

Microfluidic Synthesis of Gd-Based Nanoparticles for Fast and Ultralong MRI Signals in the Solid Tumor.

Clinically used magnetic resonance imaging contrast agents (MRI CAs) for solid tumors suffer from short life spans and low accumulation at the tumor for their low molecular weights. A good solution is to incorporate these MRI CAs into nanoparticles. Food and Drug Administration-approved compounds, poly(lactic-co-glycolic acid) (PLGA) and lipids, are chosen to assemble these nanoparticles. PLGA/lipid hybrid nanoparticles are assembled in microfluidic channels with a suitable size distribution for imaging tumors. These nanoparticles achieve clearly enhanced MRI contrast at the tumor at 0.5 h postinjection. The enhanced MRI contrast is sustained for 16 h. They can margin the tumor with as good an enhanced MRI contrast as clinical MRI CAs (which visualize the whole tumor) of the solid tumor with much less Gd. They are particularly useful for monitoring the solid tumor after therapy within a day and without repeated administration as clinical MRI CAs.

Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Suppresses Human Adenovirus Gene Expression and Replication.

Human adenovirus (HAdV) causes minor illnesses in most patients but can lead to severe disease and death in pediatric, geriatric, and immunocompromised individuals. No approved antiviral therapy currently exists for the treatment of these severe HAdV-induced diseases. In this study, we show that the pan-histone deacetylase (HDAC) inhibitor SAHA reduces HAdV-5 gene expression and DNA replication in tissue culture, ultimately decreasing virus yield from infected cells. Importantly, SAHA also reduced gene expression from more virulent and clinically relevant serotypes, including HAdV-4 and HAdV-7. In addition to SAHA, several other HDAC inhibitors (e.g., trichostatin A, apicidin, and panobinostat) also affected HAdV gene expression. We determined that loss of class I HDAC activity, mainly HDAC2, impairs efficient expression of viral genes, and that E1A physically interacts with HDAC2. Our results suggest that HDAC activity is necessary for HAdV replication, which may represent a novel pharmacological target in HAdV-induced disease.IMPORTANCE Although human adenovirus (HAdV) can cause severe diseases that can be fatal in some populations, there are no effective treatments to combat HAdV infection. In this study, we determined that the pan-histone deacetylase (HDAC) inhibitor SAHA has inhibitory activity against several clinically relevant serotypes of HAdV. This U.S. Food and Drug Administration-approved compound affects various stages of the virus lifecycle and reduces virus yield even at low concentrations. We further report that class I HDAC activity, particularly HDAC2, is required for efficient expression of viral genes during lytic infection. Investigation of the mechanism underlying SAHA-mediated suppression of HAdV gene expression and replication will enhance current knowledge of virus-cell interaction and may aid in the development of more effective antivirals with lower toxicity for the treatment of HAdV infections.

The Experimental Oxime K027-A Promising Protector From Organophosphate Pesticide Poisoning. A Review Comparing K027, K048, Pralidoxime, and Obidoxime.

Poisoning with organophosphorus compounds (OPCs) is a major problem worldwide. Standard therapy with atropine and established oxime-type enzyme reactivators (pralidoxime, obidoxime) is unsatisfactory. In search of more efficacious broad-spectrum oximes, new bispyridinium (K-) oximes have been synthesized, with K027 being among the most promising. This review summarizes pharmacokinetic characteristics of K027, its toxicity and in vivo efficacy to protect from OPC toxicity and compares this oxime with another experimental bisquaternary asymmetric pyridinium aldoxime (K048) and two established oximes (pralidoxime, obidoxime). After intramuscular (i.m.) injection, K027 reaches maximum plasma concentration within ∼30 min; only ∼2% enter the brain. Its intrinsic cholinesterase inhibitory activity is low, making it relatively non-toxic. In vitro reactivation potency is high for ethyl-paraoxon-, methyl-paraoxon-, dichlorvos-, diisopropylfluorophosphate (DFP)- and tabun-inhibited cholinesterase. When administered in vivo after exposure to the same OPCs, K027 is comparable or more efficacious than pralidoxime and obidoxime. When given as a pretreatment before exposure to ethyl-paraoxon, methyl-paraoxon, DFP, or azinphos-methyl, it is superior to the Food and Drug Administration-approved compound pyridostigmine and comparable to physostigmine, which because of its entry into the brain may cause unwanted behavioral effects. Because of its low toxicity, K027 can be given in high dosages, making it a very efficacious oxime not only for postexposure treatment but also for prophylactic administration, especially when brain penetration is undesirable.

Phenotypic Screens Reveal Posaconazole as a Rapidly Acting Amebicidal Combination Partner for Treatment of Primary Amoebic Meningoencephalitis.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration-approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 μM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri-infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.

Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.

Apelin signaling plays an important role during embryo development and regulates angiogenesis, cardiovascular activity, and energy metabolism in adulthood. Overexpression and hyperactivity of this signaling pathway is observed in various pathologic states, such as cardiovascular diseases and cancer, which highlights the importance of inhibiting apelin receptor (APJ); therefore, we developed a cell-based screening assay that uses fluorescence microscopy to identify APJ antagonists. This approach led us to identify the U.S. Food and Drug Administration-approved compound protamine-already used clinically after cardiac surgery-as an agent to bind to heparin and thereby reverse its anticlotting activity. Protamine displays a 390-nM affinity for APJ and behaves as a full antagonist with regard to G protein and β-arrestin-dependent intracellular signaling. Ex vivo and in vivo, protamine abolishes well-known apelin effects, such as angiogenesis, glucose tolerance, and vasodilatation. Remarkably, protamine antagonist activity is fully reversed by heparin treatment both in vitro and in vivo Thus, our results demonstrate a new pharmacologic property of protamine-blockade of APJ-that could explain some adverse effects observed in protamine-treated patients. Moreover, our data reveal that the established antiangiogenic activity of protamine would rely on APJ antagonism.-Le Gonidec, S., Chaves-Almagro, C., Bai, Y., Kang, H. J., Smith, A., Wanecq, E., Huang, X.-P., Prats, H., Knibiehler, B., Roth, B. L., Barak, L. S., Caron, M. G., Valet, P., Audigier, Y., Masri, B. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.

Drug Repurposing Screen Identifies Foxo1-Dependent Angiopoietin-2 Regulation in Sepsis.

The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). Research laboratories of Hannover Medical School and Harvard Medical School. Septic patients/C57Bl/6 mice and human endothelial cells. Food and Drug Administration-approved library screening. In a cell-based screen of more than 650 Food and Drug Administration-approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2's dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.

Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment

Dravet syndrome (DS) is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterise zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviors. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviors and electrographic seizures. This approach represents a new direction in modeling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder.

Boosting innate immunity: Development and validation of a cell-based screening assay to identify LL-37 inducers

Innate immunity, the front line of our defence against pathogens, relies, to a great extent, on the production of antimicrobial peptides (AMPs). These peptides exhibit antimicrobial activity and immunomodulatory properties. In humans, AMPs include the defensins (α- and β-families) and the cathelicidin, LL-37. Bacterial resistance against antibiotics is a growing concern, and novel antimicrobial strategies are needed urgently. Hence, the concept of strengthening immune defences against infectious microbes by inducing AMP expression may represent novel or complementary pharmaceutical interventions in the treatment or prevention of infections. We have developed and validated a robust cell-based reporter assay for LL-37 expression, which serves as a marker for a healthy epithelial barrier. This reporter assay can be a powerful tool for high-throughput screenings. We first employed our assay to screen a panel of histone deacetylase inhibitors and derivatives, and then the Prestwick Chemical Library of Food and Drug Administration-approved compounds. After hit confirmation and independent validation in the parental cell line we identified five novel inducers of LL-37. This reporter assay will help to identify novel drug candidates for the treatment and prevention of infections. Importantly, the pattern of hits obtained may suggest cellular pathways and key mediators involved in the regulation of AMP expression.

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.

A High Content Drug Screen Identifies Ursolic Acid as an Inhibitor of Amyloid β Protein Interactions with Its Receptor CD36

A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid β (Aβ) in the brain. Aβ binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of Aβ binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of Aβ binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled Aβ to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, Aβ concentration, time required for Aβ binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of Aβ binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of Aβ to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 μm, with a maximal inhibition of 64%. Ursolic acid also blocked binding of Aβ to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of Aβ to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block Aβ-CD36 interactions.

A High-Throughput Fluorescence Polarization Assay for Inhibitors of Gyrase B

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-µL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

Sanguinarine Suppresses Prostate Tumor Growth and Inhibits Survivin Expression

Prostate cancer is a frequently occurring disease and is the second leading cause of cancer-related deaths of men in the United States. Current treatments have proved inadequate in curing or controlling prostate cancer, and a search for agents for the management of this disease is urgently needed. Survivin plays an important role in both progression of castration-resistant prostate cancer and resistance to chemotherapy. Altered expression of survivin in prostate cancer cells is associated with cancer progression, drug/radiation resistance, poor prognosis, and short patient survival. In the present study, the authors performed a cell-based rapid screen of the Prestwick Chemical Library consisting of 1120 Food and Drug Administration-approved compounds with known safety and bioavailability in humans to identify potential inhibitors of survivin and anticancer agents for prostate cancer. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified as a novel inhibitor of survivin that selectively kills prostate cancer cells over "normal" prostate epithelial cells. The authors found that sanguinarine inhibits survivin protein expression through protein degradation via the ubiquitin-proteasome system. Sanguinarine induces apoptosis and inhibits growth of human prostate cancer cells and in vivo tumor formation. Administration of sanguinarine, beginning 3 days after ectopic implantation of DU145 human prostate cancer cells, reduces both tumor weight and volume. In addition, sanguinarine sensitized paclitaxel-mediated growth inhibition and apoptosis, offering a potential therapeutic strategy for overcoming taxol resistance. These results suggest that sanguinarine may be developed as an agent either alone or in combination with taxol for treatment of prostate cancer overexpressing survivin.