Large-scale Drug Screening Research Articles

Fluorescence correlations, single molecule detection and large number screening Applications in biotechnology

Fluorescence correlation spectroscopy (FCS), when carried out under conditions with low background as obtained in very small volume elements, is a powerful tool for examining molecular interactions as well as their time dependence. Interactions of biological importance which can be analyzed are hybridization between nucleic acid primers and DNA or RNA targets, between peptide ligands and isolated as well as cell-bound receptors, between antigen and antibodies. Since the interaction can be analyzed rapidly in small volumes without the need for separating unbound from bound ligand, an important application of FCS is envisaged in large-scale drug screening. The sensitivity has been advanced to the point that detection of single dye molecules is possible in the submillisecond range. This opens up the possibility for detecting rare events such as the appearance of pathogens in the early phase of infection or mutants exhibiting unusual properties when screening combinatorial libraries.

A Comparison of Meconium, Maternal Urine and Neonatal Urine for Detection of Maternal Drug Use During Pregnancy

A large scale drug screening study was done to determine the prevalence of drug use in a large metropolitan, obstetric population. Meconium and first voided urine, as well as maternal urine were collected from 423 consecutive deliveries. Urine samples and methanolic extracts of meconium were initially screened by Enzyme Multiplied Immunoassay Technique (EMIT) and then confirmed by Gas Chromatography/Mass Spectrometry (GC/MS). Analysis of cocaine metabolite as benzoylecogonine, cannabinoid as carboxy-THC, codeine, morphine and methadone were included in the study. The positive rate for benzoylecgonine was virtually identical for meconium, maternal urine and neonatal urine (12%). Analysis of meconium was found to be more reliable than analysis of maternal or neonatal urine for the detection of benzoylecgonine. Meconium did not appear to offer an advantage over maternal or neonatal urine for detection of cannabinoid, codeine, morphine, or methadone.

A rapid microtiter plate method for the detection of lysozyme release from human neutrophils

An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng ml . Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC 50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC 50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t- Boc- l-methionyl- l-leucyl- l-phenylalanine ( N-t-Boc) , LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC 50 of 2 μM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC 50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC 50 of 2 μM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively. The adaptation of this assay, as demonstrated here, to a microplate format provides a simple, reproducible, and high throughput method that is ideally suited for largescale drug screening.

Validation of clinical predictive value of in vitro colorimetric chemosensitivity assay in head and neck cancer

For chemosensitivity testing, a rapid in vitro colorimetric method (MTT assay) was used. Eleven head and neck cancer cell lines were investigated to distinguish five known active agents from five compounds inactive in phase II studies. Evaluation of the reliability of the assay for assessing drug sensitivity in this tumor cell population was done by correlating the in vitro results with reported in vivo response data. Methotrexate and cisplatin (clinically active) and vindesine and doxorubicin (less active clinically) were recognized in vitro as active and correlated well with clinical experience. Bleomycin (clinically active) was ineffective against some cell lines. The in vitro findings for the clinically inactive drugs (deoxyazacytidine, lomustine, and carmustine) also corresponded. Amsacrine and etoposide, contrary to clinical experience, showed activity in vitro. Further comparison of MTT assay results with clinical data is warranted and essential before its use in large-scale drug screening studies.

Microtiter Assay Useful for Screening of Cell-Differentiation Agents1

Promyelocytic leukemia HL-60 cells induced to differentiate along the granulocytic and monocytic pathways respond to stimulation with phorbol myristate acetate by producing superoxide radicals. The amount of superoxide radical generation can be monitored by spectrophotometric measurement of cytochrome c reduction. We have developed a microtiter assay that assesses differentiation of HL-60 cells on the basis of cytochrome c reduction. HL-60 cells were incubated with known standards or unknown samples, including crude fermentation broths, for 6 days; then cytochrome c reduction was quantified as a function of increasing absorbance at 550 nm on a microtiter plate reader. HL-60 cells induced to differentiate showed up to a 10-fold increase in absorbance over that of control cells. Differentiation was confirmed by morphological assessment and by flow cytometric analysis of the DNA cell-cycle distribution and the cell-surface transferrin receptor. Analysis of 198 crude fermentation broth samples confirmed the feasibility of using this assay for large-scale drug screening.

Differential response of T-lymphoblastoid cell lines to infection with HIV and to antiviral drugs: Implications for large-scale anti-HIV drug screening : O. Weislow, R. Shoemaker, R. Kiser, D. Fine and M. Boyd. Program Resources, Inc. and Developmental Therapeutics Program, DCT, NCI-Frederick Cancer Research Facility, Frederick, MD 21701-1013

Differential response of T-lymphoblastoid cell lines to infection with HIV and to antiviral drugs: Implications for large-scale anti-HIV drug screening : O. Weislow, R. Shoemaker, R. Kiser, D. Fine and M. Boyd. Program Resources, Inc. and Developmental Therapeutics Program, DCT, NCI-Frederick Cancer Research Facility, Frederick, MD 21701-1013

Yellow Rows of Test Tubes: Due Process Constraints on Discharges of Public Employees Based on Drug Urinalysis Testing

As the problem of employee drug abuse gains increasing attention from the popular' and business2 news media, many public and private employers have instituted medical testing programs to detect drug use among employees.3 In the public sector, large-scale drug screening began in the United States military in 1981.' Currently, agencies at all levels of government have testing programs;5 moreover, on September 15, 1986, President Reagan signed Executive Order 12,564 mandating a drug-free federal workplace and requiring the head of each executive agency to establish a drug testing program within the parameters of the executive order.6 This order is estimated to affect more than one million federal employees.7 Clearly, drug testing is an issue many public employees now or will soon face. Drug testing is usually carried out by urinalysis, the easiest and least expensive method.8 However, drug urinalysis testing has generated much opposition, primarily on the ground that it violates the privacy rights of the persons tested.9 This Comment focuses on testing of

A computerized multichannel platelet aggregometer system

Commercially available instrumentation for conducting platelet aggregation studies in clinical and research laboratories consists of one-, two-, or four-channel aggregometers used in conjunction with strip chart recorders. These instruments have limited utility in large-scale drug screening and evaluation of the mode of action of drugs or in the clinical diagnosis of platelet disorders. A new instrument, a computerized multichannel aggregometer system (CMPAS) has been developed to collect, display, and analyze platelet aggregation data. The system is comprised of a 24-channel Born-type aggregometer, interfaced to a Rockwell AIM-65 micro-computer through an analogue-to-digital converter and an Epson dot-matrix printer. Each channel is individually calibrated, and aggregation data can be collected on up to 24 different platelet-rich plasma samples simultaneously. Conversational programs written in BASIC prompt the user for the addition of agonists and inhibitors. The tracings for each channel are displayed simultaneously, and a program automatically analyzes the data to generate the following parameters: baseline optical density, maximum aggregation response, positive and negative slopes, time to peak aggregation, and percentage response. Computerized multichannel aggregometer system data outputs are comparable to data generated by a standard Chronolog aggregometer unit. The advantages of the system include multichannel capability, simultaneous display of all channels allowing relative comparisons between control and experimental groups, and time savings and improved efficiency in conducting and analyzing aggregation experiments.