Automated assay optimization with integrated statistics and smart robotics.

The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.

High-throughput screening: new technology for the 21st century.

New technologies in high-throughput screening have significantly increased throughput and reduced assay volumes. Key advances over the past few years include new fluorescence methods, detection platforms and liquid-handling technologies. Screening 100,000 samples per day in miniaturized assay volumes will soon become routine. Furthermore, new technologies are now being applied to information-rich cell-based assays, and this is beginning to remove one of the key bottlenecks downstream from primary screening.

A multiple-purpose design approach to the evaluation of risks from mixtures of disinfection by-products.

Drinking water disinfection has effectively eliminated much of the morbidity and mortality associated with waterborne infectious diseases in the United States. Various disinfection processes, however, produce certain types and amounts of disinfection by-products (DBPs), including trihalomethanes (THM), haloacetic acids, haloacetonitriles, and bromate, among others. Human health risks from the ubiquitous exposure to complex mixtures of DBPs are of concern because existing epidemiologic and toxicologic studies suggest the existence of systemic or carcinogenic effects. Researchers from several organizations have developed a multiple-purpose design approach to this problem that combines efficient laboratory experimental designs with statistical models to provide data on critical research issues (e.g., estimation of human health risk from low-level DBP exposures, evaluation of additivity assumptions as useful for risk characterization, estimation of health risks from different drinking water treatment options). A series of THM experiments have been designed to study embryonic development, mortality and cancer in Japanese medaka (Oryzias latipes) and liver and kidney endpoints in female CD-1 mice. The studies are to provide dose-response data for specific mixtures of the 4 THMs, for the single chemicals, and for binary combinations. The dose-levels and mixing ratios for these experiments were selected to be useful for development and refinement of three different statistical methods: testing for departures from dose-additivity; development of an interactions-based hazard index; and use of proportional-response addition as a risk characterization method. Preliminary results suggest that dose-additivity is a reasonable risk assessment assumption for DBPs. The future of mixtures research will depend on such collaborative efforts that maximize the use of resources and focus on issues of high relevance to the risk assessment of human health.

Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits interleukin-8-induced neutrophil migration.

Interleukin-8 (IL-8) and closely related Glu-Leu-Arg (ELR) containing CXC chemokines, including growth-related oncogene (GRO)alpha, GRObeta, GROgamma, and epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), are potent neutrophil chemotactic and activating peptides, which are proposed to be major mediators of inflammation. IL-8 activates neutrophils by binding to two distinct seven-transmembrane (7-TMR) G-protein coupled receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB), while GROalpha, GRObeta, GROgamma, and ENA-78 bind to and activate only CXCR2. A chemical lead, which selectively inhibited CXCR2 was discovered by high throughput screening and chemically optimized. SB 225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea) is the first reported potent and selective non-peptide inhibitor of a chemokine receptor. It is an antagonist of 125I-IL-8 binding to CXCR2 with an IC50 = 22 nM. SB 225002 showed >150-fold selectivity over CXCR1 and four other 7-TMRs tested. In vitro, SB 225002 potently inhibited human and rabbit neutrophil chemotaxis induced by both IL-8 and GROalpha. In vivo, SB 225002 selectively blocked IL-8-induced neutrophil margination in rabbits. The present findings suggest that CXCR2 is responsible for neutrophil chemotaxis and margination induced by IL-8. This selective antagonist will be a useful tool compound to define the role of CXCR2 in inflammatory diseases where neutrophils play a major role.

Menorrhagia in adolescents requiring hospitalization.

STUDY OBJECTIVE: This study was undertaken to assess the causes and treatments of menorrhagia in adolescents hospitalized for this menstrual disorder. DESIGN: A retrospective chart review was performed of all adolescents < or =20 years of age with menorrhagia admitted at the University of Michigan from 1979 to 1995. Information regarding medical history, hematologic parameters, treatment, and diagnosis was extracted from each chart. Pregnant and premenarchal patients were excluded. RESULTS: Thirty-seven adolescents with 46 admissions for menorrhagia were identified. The average age of menarche was 12.9 years and the average age at admission was 15.9 years. Nineteen adolescents had significant medical diseases. For the 46 admissions, causes of menorrhagia were anovulation (21), hematologic disease (15), chemotherapy-related (5), and infections (5). Transfusions of blood products were performed in 28 of the admissions. Treatments included oral contraceptive pills or progestins (30), intravenous conjugated estrogens (8), antibiotics (4), immune gammaglobulin (3), DDAVP (3), and prednisone (1). Twelve surgical procedures were performed, including eight dilatation and curettages (D&Cs), three laparoscopies, and one hysterectomy. CONCLUSIONS: Sixty-one percent of admissions for adolescent menorrhagia were in adolescents with significant medical problems. The patients with menorrhagia who required admission had severe anemia and were transfused in 63% of cases. The predominant causes for these admissions included anovulation in 46%, hematologic disease in 33%, chemotherapy in 11%, and infection in 11%. Hormonal regulation or suppression of menses should be considered in adolescents with significant medical disease.

Molecular cloning and characterization of the human anaphylatoxin C3a receptor.

In a human neutrophil cDNA library, an orphan G-protein-coupled receptor, HNFAG09, with 37% nucleotide identity to the C5a receptor (C5a-R, CD88) was identified. A novel feature of this gene, unlike C5a-R and other G-protein-coupled receptors, is the presence of an extraordinarily large predicted extracellular loop comprised of in excess of 160 amino acid residues between transmembrane domains 4 and 5. Northern blot analysis revealed that expression of mRNA for this receptor in human tissues, while similar, was distinct from C5a-R expression. Although there were differences in expression, transcripts for both receptors were detected in tissues throughout the body and the central nervous system. Mammalian cells stably expressing HNFAG09 specifically bound 125I-C3a and responded to a C3a carboxyl-terminal analogue synthetic peptide and to human C3a but not to rC5a with a robust calcium mobilization response. HNFAG09 encodes the human anaphylatoxin C3a receptor.

Current and future risk assessment guidelines, policy, and methods development for chemical mixtures.

Humans are typically exposed to low doses of combinations of chemicals rather than to one or two chemicals at a time, yet most of the available toxicity data provide information on single chemicals or binary pairs, rather than on whole mixtures. The use of existing interactions study data for the quantitative risk assessment of chemical mixtures is problematic. These studies generally lack the necessary statistical characterizations to be useful in quantitative risk assessment procedures. The U.S. EPA developed guidelines for risk assessment for chemical mixtures in 1986 and is currently in the process of making revisions. Significant advances have been made in both the theoretical development and application of procedures such as dose addition, response addition, toxicity equivalence factors, comparative potency and interactions data characterizations. Details on the current revisions to the guidelines are given, along with information on the research efforts that have influenced these revisions or that represent future directions in chemical mixtures risk assessment.

Upper bound risk estimates for mixtures of carcinogens.

The excess cancer risk that might result from exposure to a mixture of chemical carcinogens usually is estimated with data from experiments conducted on individual chemicals. An upper bound on the total excess risk is estimated commonly by summing individual upper bound risk estimates. The degree to which this approach might overstate the true risk associated with the mixture has not been evaluated previously. This paper reports the results of a Monte Carlo simulation study on the degree of reduction in conservation that might be achieved using alternative methods for calculating mixture upper bounds. An unexpected finding is that for chemicals that exhibit strongly linear dose-response relationships, the summing of multistage-model-based upper bounds on excess risk can be anti-conservative, that is, it can provide less than the nominal 100(1-alpha)% coverage.

Chemical mixtures from a public health perspective: the importance of research for informed decision making.

When considered from a public health perspective, the central question regarding chemical mixtures is deceptively simple: Are current approaches to risk assessment for chemical mixtures affording effective (adequate) and efficient (cost-effective) protection for members of our society? Answering this question realistically depends on an understanding of the hierarchical goals of public health (i.e. prevention, intervention, treatment) and an accurate evaluation of the extent to which these goals are being achieved. To allow decision makers to make informed judgments about the health risks of chemical mixtures, adequate scientific knowledge and understanding must be available to support risk assessment activities, which are an integral part of the regulatory decision making process. Designing and implementing relevant research depends on the existence of a feedback loop between researchers and regulators, where the information needs of regulators influence the nature and direction of research and the information and understanding generated by researchers improves the scientific basis for public health decisions. A clear, consistent, commonly accepted taxonomy for describing important mixture-related phenomena is a key factor in creating and maintaining the necessary feedback loop. Ultimately, both researchers and regulators share a common goal with regard to chemical mixtures; improving the state-of-the-science so that we can make informed decisions about protecting public health. A survey of research issues and needs that are crucial to attaining this goal is presented.

The identification and testing of interaction patterns.

This paper presents a method for identifying and assessing the significance of interaction patterns among various chemicals and chemical classes of importance to regulatory toxicologists. To this end, efforts were made to assemble and evaluate experimental data on toxicologically significant interactions, to use this information to characterize the consistency of toxicological interactions, and to define classes of compounds that display similar toxicological interactions. The motivation for this effort is to be able to propose hypotheses, which can be validated by experimentation, on how 2 or more chemicals will interact.

Kinetic characterization of the interaction of biotinylated human interleukin 5 with an Fc chimera of its receptor alpha subunit and development of an ELISA screening assay using real-time interaction biosensor analysis.

The interaction of biotinylated human interleukin 5 ([BT]hIL5) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring an ELISA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5R alpha-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor alpha subunit to the constant domain (Fc) of immunoglobulin G), were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisfactory and led to use of the Fc chimera attached by oriented immobilization via protein A. Hence, shIL5R alpha-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BT]hIL5 to protein A/shIL5R alpha-Fc receptor complex. The binding was high affinity (Kdapp = 6 nM), reversible and saturable. The affinity of [BT]hIL5 was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BT]hIL5 with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing a microtiter plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BT]hIL5 to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors.

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.

A statistical test of compatibility of data sets to a common dose-response model.

Quantitative estimates of cancer risk generally involve low-dose extrapolation based on an exponential dose-response model for dichotomous response data. Frequently more than one data set is available. If a careful analysis of the biological issues indicates that more than one of the available data sets could be used in the quantitative estimate of cancer risk, it is reasonable to think of combining the data. Before combining data, however, it would be prudent to test whether the data sets are compatible with a common dose-response model. If they are not, it could be concluded that an underlying biological factor is responsible. If they are statistically compatible, the decision to combine data sets based on biological issues would be reinforced. A statistical test based on the generalized likelihood ratio method is proposed for evaluating the compatibility of different data sets with a common dose-response model. This method of constructing a statistical test and the associated asymptotic theory is consistent with the approach used by GLOBAL86 (R. B. Howe, K. S. Crump, and C. Van Landingham, GLOBAL86: A Computer Program to Extrapolate Quantal Animal Toxicity Data to Low Doses, K. S. Crump & Co., Ruston, LA, 1986) for estimating the confidence limits that are used as a basis for quantitative estimates.

Whole cell assays in screening for biologically active substances.

This past year has seen a number of new developments in recombinant cell based assays. Lead compounds are beginning to be identified using mechanism-based whole cell assays to discover inhibitors of cellular processes, such as transactivation and signaling. These assays offer advantages for many targets over subcellular assay systems with purified macromolecules.

Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content.

A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.

Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site.

Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.

Development of a stable camptothecin-resistant subline of P388 leukemia with reduced topoisomerase I content.

A camptothecin-resistant subline of P388 leukemia (P388/CPT) was developed by repeated transplantation of P388 cells in mice treated with therapeutic doses of camptothecin. In mice bearing the resistant tumor, a maximally tolerated dose of camptothecin produced no net reduction in tumor cell burden, in contrast to a 5-log cell kill in the parental P388 (P388/S). The IC50 of camptothecin, as determined by colony formation assays of cultured cells, was 8 times greater for the cloned P388/CPT cell line than for P388/S. P388/CPT cells were not cross-resistant to other antineoplastic agents, including topoisomerase II inhibitors. The type I topoisomerases purified from P388/CPT and P388/S cells were identical with respect to molecular weight, specific activity, in vitro camptothecin sensitivity, and DNA cleavage specificity. Camptothecin induced fewer protein-associated DNA single-strand breaks in the resistant cells than in the wild-type P388 cells. Topoisomerase I mRNA, immunoreactivity, and extractable enzymatic activity were 2-4 times lower for P388/CPT cells than for P388/S cells. As resistance to camptothecin developed, topoisomerase I extractable activity decreased, concomitant with an increase in topoisomerase II extractable activity. Furthermore, the appearance of camptothecin resistance was associated with specific rearrangements of the topoisomerase I gene. These results suggest that development of resistance to inhibitors of topoisomerase I can occur by down-regulation of the target enzyme, thus reducing the production of lethal enzyme-mediated DNA damage. The enhanced topoisomerase II activity in these cells suggests that resistance to camptothecin may be overcome by co-treatment with topoisomerase II inhibitors.