Lynn Rasmussen

High Throughput Screening for Inhibitors of Mycobacterium tuberculosis H37Rv

There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high-throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein.

Antituberculosis Activity of the Molecular Libraries Screening Center Network Library

There is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against Mycobacterium tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein.

Characterization of virus-like particles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus

The entire gag gene of the bovine immunodeficiency-like virus (BIV) was inserted behind the strong polyhedron promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The resultant recombinant baculovirus (AcNPV-BIVgag) was used to infect insect cells in order to overexpress and characterize BIV gag gene products. The infection resulted in the high-level expression of a protein similar in size to the predicted BIV gag precursor (Pr53gag). BIV Pr53gag was detected in AcNPV-BIVgag-infected insect cells and in culture supernatants. Electron microscopy of these cells revealed an abundance of virus-like particles (VLPs) in the cytoplasm, budding from the cell membrane, and free in the culture medium. The size and morphology of the VLPs were similar to those of the immature forms of BIV observed in infected mammalian cells. The VLPs sedimented at a density of 1.16 g of sucrose per milliliter in linear gradients and were shown to contain the majority of the supernatant Pr53gag. Antigenic determinants on Pr53gag from VLPs were recognized by BIV and HIV-1 antiserum, and serum from rats immunized with VLPs reacted with recombinant and viral BIV Pr53gag and processed products. The protease (PR) activity in BIV virions was capable of processing recombinant Pr53gag; this activity was blocked by pepstatin A, a potent aspartyl PR inhibitor. Baculovirus-expressed BIV Pr53gag appears to be an excellent source of gag precursor; it may prove useful for structural studies and enable the development of assays to detect retroviral PR inhibitors. The data further suggest that unprocessed BIV Pr53gag plays a major role in the assembly of BIV particles. The expression of other BIV structural genes in insect cells may prove instructive in the study of molecular events involved in the assembly and processing of these BIV proteins.

Hantaan Virus Replication: Effects of Monensin, Tunicamycin and Endoglycosidases on the Structural Glycoproteins

The monovalent ionophore monensin, which interferes with cellular transport pathways, and the antibiotic tunicamycin, which prevents glycosylation of newly synthesized proteins, were used to examine Hantaan virus particle formation and polypeptide synthesis. Viral replication in the presence of either drug resulted in reduced antigen production as well as reduced yields of both intracellular and extracellular infectious virus. Analysis of viral polypeptides synthesized in the presence of the drugs suggested differential effects of monensin and tunicamycin on Hantaan virus. Although reduced levels of the three major structural proteins were detected with increasing concentrations of monensin, the electrophoretic migrations of the polypeptides synthesized were unaltered. In contrast, after tunicamycin treatment, G1 was no longer detectable and G2 displayed both a quantitative reduction and an apparent molecular weight reduction of approximately 3000. Both G1 and G2 were sensitive to endoglycosidases H and F with resultant electrophoretic mobility shifts corresponding to molecular weights of approximately 7000 for G1 and 3000 for G2. Oligosaccharides appeared to be mostly, but not entirely, of the high-mannose type.

A Novel Inhibitor of Mycobacterium tuberculosis Pantothenate Synthetase

Pantothenate synthetase (PS; EC 6.3.2.1), encoded by the panC gene, catalyzes the essential adenosine triphosphate (ATP)–dependent condensation of D-pantoate and β-alanine to form pantothenate in bacteria, yeast, and plants; pantothenate is a key precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). Because the enzyme is absent in mammals and both CoA and ACP are essential cofactors for bacterial growth, PS is an attractive chemotherapeutic target. An automated high-throughput screen was developed to identify drugs that inhibit Mycobacterium tuberculosis PS. The activity of PS was measured spectrophotometrically through an enzymatic cascade involving myokinase, pyruvate kinase, and lactate dehydrogenase. The rate of PS ATP utilization was quantitated by the reduction of absorbance due to the oxidation of NADH to NAD+ by lactate dehydrogenase, which allowed for an internal control to detect interference from compounds that absorb at 340 nm. This coupled enzymatic reaction was used to screen 4080 compounds in a 96-well format. This discussion describes a novel inhibitor of PS that exhibits potential as an antimicrobial agent.

Conservation of Antigenic Properties and Sequences Encoding the Envelope Proteins of Prototype Hantaan Virus and Two Virus Isolates from Korean Haemorrhagic Fever Patients

Viruses isolated from the blood of two Korean haemorrhagic fever patients were propagated in cell culture and compared to prototype Hantaan virus which was isolated from Apodemus mice. The antigenic properties of the human isolates were found to be closely related to Hantaan virus by plaque reduction neutralization, haemagglutination inhibition and fluorescent antibody staining with both polyclonal and monoclonal antibodies. The medium genome segment of each human isolate was sequenced and compared to that of Hantaan virus. Nucleotides comprising the Hantaan virus G1 and G2 envelope protein-coding regions differed from those of the other viruses by only 5.4% and 5.7%. The human isolates differed from one another by 1.6%. The nucleotide differences resulted in predicted amino acid variations of 1.3% to 2.3% among the three viruses, with the majority occurring as conservative substitutions in G1.

High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. (Journal of Biomolecular Screening 2008:879-887)

Assay development and high throughput antiviral drug screening against Bluetongue virus

Abstract Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5000cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z′-values ≥0.70, Coefficient of Variations ≥5.68 and signal-to-background ratio ≥7.10. This assay was further validated using a 9532 compound library. The fully validated assay was then used to screen the 194950 compound collection, which identified 693 compounds with >30% CPE inhibition. The 10-concentration dose response assay identified 185 structures with IC50 ≤100μM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease.

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.

ABSTRACT 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.

Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned

In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high-throughput screening (HTS) arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the HTS drug discovery platform, with special consideration being given to assay miniaturization, liquid-handling complications, and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell-plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry over, and incubator-induced artifacts.