Literature Search and Reviews

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<ul><li><p>ASSAY and Drug Development TechnologiesVolume 1, Number 2, 2003 Mary Ann Liebert, Inc.</p><p>Literature Search and Review</p><p>J. Fraser Glickman</p><p>In each issue of ASSAY and Drug Development Technologies, our Literature Editor, J. Fraser Glickman,Ph.D., selects several significant papers covering timely and pertinent topics that will keep our readers up-to-date on the ever-changing field of assay and drug development technologies. Dr. Glickman will provide rel-evant commentary on each of the cited abstracts.</p><p>High Throughput Mice</p><p>385</p><p>Peters LL, Cheever EM, Ellis HR, Magnani PA, Sven-son KL, Von Smith R, Bogue MA: Large-scale, high-throughput screening for coagulation and hemato-logic phenotypes in mice. Physiol Genomics 2002;11:185193.</p><p>Abstract: The Mouse Phenome Project is an internationaleffort to systematically gather phenotypic data for a de-fined set of inbred mouse strains. For such large-scale proj-ects the development of high-throughput screening proto-cols that allow multiple tests to be performed on a singlemouse is essential. Here we report hematologic and coag-ulation data for more than 30 inbred strains. Completeblood counts were performed using an Advia 120 analyzer.For coagulation testing, we successfully adapted the DadeBehring BCS automated coagulation analyzer for use inmice by lowering sample and reagent volume require-ments. Seven automated assay procedures were developed.Small sample volume requirements make it possible to per-form multiple tests on a single animal without euthanasia,while reductions in reagent volume requirements reducecosts. The data show that considerable variation in manybasic hematological and coagulation parameters exists</p><p>among the inbred strains. These data, freely available onthe World Wide Web, allow investigators to knowledge-ably select the most appropriate strain(s) to meet their in-dividual study designs and goals.</p><p>Commentary: Inbred mouse strains have become im-portant models for the understanding of the genetic ba-sis of complex diseases. For example, screening of ran-domly mutagenized inbred strains can reveal phenotypesthat match human disease states. However, in order tofully utilize sequence information from the mouse genomeproject, it is necessary to precisely define what is the nor-mal phenotype and rapidly identify what is an alteredphenotype among a large population of individuals. Pe-ters et al. present high-throughput methods to analyzeblood chemistry and biology in mice. Seven different as-say procedures are described that rapidly measure var-ious coagulation factors and complete blood cell counts,for example. The methods require low sample volume, sothat euthanasia is not necessary. The 42 most commoninbred strains were tested in these parameters. The sys-tem described would be useful in determining those mu-tations that have effects on hematological parameters.</p><p>Kinase Substrates</p><p>Brinkworth RI, Breinl RA, Kobe B: Structural basisand prediction of substrate specificity in protein ser-ine/threonine kinases. Proc Natl Acad Sci U S A2003;100:7479.</p><p>Abstract: The large number of protein kinases makesit impractical to determine their specificities and sub-strates experimentally. Using the available crystal struc-tures, molecular modeling, and sequence analyses of ki-</p><p>nases and substrates, we developed a set of rules gov-erning the binding of a heptapeptide substrate motif(surrounding the phosphorylation site) to the kinase andimplemented these rules in a web-interfaced programfor automated prediction of optimal substrate peptides,taking only the amino acid sequence of a protein kinaseas input. We show the utility of the method by analyz-ing yeast cell cycle control and DNA damage check-point pathways. Our method is the only available pre-</p></li><li><p>dictive method generally applicable for identifying pos-sible substrate proteins for protein serine/threonine ki-nases and helps in silico construction of signaling path-ways. The accuracy of prediction is comparable to theaccuracy of data from systematic large-scale experimen-tal approaches.</p><p>Commentary: The identification of increasing numbersof protein kinases that are important in human pathol-ogy has resulted in a demand for methods that rapidlyidentify their cognate substrates. This area of researchis also important to those seeking to map out signaltranduction pathways and to develop physiologicallyrelevant assays. Brinkworth et al. have identified a com-putational algorithm for predicting oligopeptides thatwill act as substrates for serine/threonine kinases. Con-ventional approaches to identifying kinase substrates</p><p>utilize phage display techniques, which can be very timeconsuming.</p><p>The computational method relies upon input of the com-plete amino acid sequence of the kinase, and uses a modelbased on the known three-dimensional structures of thecatalytic domain of several classes of serine/threonine ki-nases bound to cognate substrates. The known kinaseshave certain specificity determinants that can be used astemplates for the unknown kinase. The system was able topredict with high probability a good peptide substrate forthe enzymes, and also to probe the human genome forprobable physiological protein substrates. The system wastested against a known phage-display methodology andwas found to agree well with this technique. Such meth-ods would be useful to those interested in developing ser-ine/threonine kinase assays, and needing a tool for rapidlydesigning peptide substrates for synthesis.</p><p>Literature Search and Review386</p><p>Zero-Mode Waveguides</p><p>Levene MJ, Korlach J, Turner SW, Foquet M, Craig-head HG, Webb WW: Zero-mode waveguides for sin-gle-molecule analysis at high concentrations. Science2003;299:682686.</p><p>Abstract: Optical approaches for observing the dynamicsof single molecules have required pico- to nanomolar con-centrations of fluorophore in order to isolate individual mol-ecules. However, many biologically relevant processes oc-cur at micromolar ligand concentrations, necessitating areduction in the conventional observation volume by threeorders of magnitude. We show that arrays of zero-modewaveguides consisting of subwavelength holes in a metalfilm provide a simple and highly parallel means for study-ing single-molecule dynamics at micromolar concentra-tions with microsecond temporal resolution. We present ob-servations of DNA polymerase activity as an example ofthe effectiveness of zero-mode waveguides for performingsingle-molecule experiments at high concentrations.</p><p>Commentary: Levene et al. have developed methods thatare alternatives to confocal fluorescence correlation spec-troscopic techniques for observing single-molecule bind-ing and enzymatic processes. The events are observedthrough zero-mode waveguides and are more suited tomeasuring the activity of enzymes and ligands with mil-limolar affinity, because the analysis is performed at vol-umes much lower than those of confocal detectors. Themethod is based on the assays being performed in small(subwavelenth) holes in a metal film deposited in a mi-croscope coverslip, upon which millions of such holes canbe made. The fluorescence correlation approach for mea-suring ligand binding depends upon the measurement ofaltered diffusion times in a very small volume of mea-surementthe difficulty in its high throughput being the</p><p>long times for data acquisition and the susceptibility to ar-tifacts. By using zero-mode waveguides, the observationvolumes are reduced to such a small volume that the timeit takes for diffusion out of the chamber is extremely re-duced, and thus the temporal resolution is improved, al-lowing for faster acquisition of fluorescence correlationdata. In proof-of-concept experiments, fluorescence cor-relation was carried out on DNA polymerase activity us-ing fluorescently coumarin-labeled dCTP as a probe. Thesystem seems to extend the ability to measure enzyme ac-tivities that require high concentrations of substrate. Thetechnology is still in its infancy and would require somedevelopment in order to bring it to the level of robustnessneeded for routine assay development.</p><p>Illumination</p><p>Dichroic filter</p><p>Collected fluorescence</p><p>Fluorescent ligand</p><p>Enzyme</p><p>Metal film</p><p>Fused silica</p><p>Reprinted from Levene MJ, Korlach J, Turner SW, Foquet M, Craig-head HG, Webb WW: Zero-mode waveguides for single-molecule anal-ysis at high concentrations. Science 299:682686. 2003.</p></li><li><p>Jaiswal JK, Mattoussi H, Mauro JM, Simon SM:Long-term multiple color imaging of live cells usingquantum dot bioconjugates. Nat Biotechnol 2003;21:4751.</p><p>Abstract: Luminescent quantum dots (QDs)semicon-ductor nanocrystalsare a promisingalternative to or-ganic dyes for fluorescence-based applications. We havedeveloped procedures for using QDs to label live cellsand have demonstrated their use for long-term multicolorimaging of live cells. The two approaches presented are(i) endocytic uptake of QDs and (ii) selective labeling ofcell surface proteins with QDs conjugated to antibodies.Live cells labeled usingthese approaches were used forlong-term multicolor imaging. The cells remained stablylabeled for over a week as they grew and developed.These approaches should permit the simultaneous studyof multiple cells over long periods of time as they pro-ceed through growth and development.</p><p>Wu X, Liu H, Liu J, Haley KN, Treadway JA, Lar-son JP, Ge N, Peale F, Bruchez MP: Immunofluores-cent labeling of cancer marker Her2 and other cellu-lar targets with semiconductor quantum dots. NatBiotechnol 2003;21:4146.</p><p>Abstract: Semiconductor quantum dots (QDs) are amongthe most promising emerging fluorescent labels for cellu-lar imaging. However, it is unclear whether QDs, whichare nanoparticles rather than small molecules, can specifi-cally and effectively label molecular targets at a subcellu-lar level. Here we have used QDs linked to immunoglob-ulin G (IgG) and streptavidin to label the breast cancermarker Her2 on the surface of fixed and live cancer cells,to stain actin and microtubule fibers in the cytoplasm, andto detect nuclear antigens inside the nucleus. All labelingsignals are specific for the intended targets and are brighterand considerably more photostable than comparable or-ganic dyes. Using QDs with different emission spectra con-jugated to IgG and streptavidin, we simultaneously detectedtwo cellular targets with one excitation wavelength. The re-sults indicate that QD-based probes can be very effectivein cellular imaging and offer substantial advantages overorganic dyes in multiplex target detection.</p><p>Literature Search and Review 387</p><p>Commentary: Quantum dots are nanometer-sized crys-tals of inorganic semiconductors that have unusual flu-orescent properties. Unlike small organic fluorophoresand fluoresent proteins, these crystals can be excited atbroad wavelengths and have much narrowed emissionspectra. The dots can be successfully conjugated to pro-teins and nucleic acids, thus expanding the potential forfluorescent probes of cellular and biomolecular function.Because of the unusual fluorescent properties associatedwith these nanocrystals, novel optical setups (detectors)can be envisioned, and, at the very least, these moleculeswill add to the palette of fluorescent labeling tools usedin developing cellular and biochemical assays.</p><p>Quantum Dots</p><p>(A) Nuclear antigens in the nuclei of human epithelial cells were labeledwith ANA, anti-human IgG-biotin and QD 630-streptavidin. (B) Whennormal human IgGs were used in place of ANA, no detectable stain wasobserved. (C) The nuclei 3T3 cell was stained with ANA, anti-humanIgG-biotin, and QD 630-streptavidin (red). The microtubules were la-beled with mouse-anti-a-tubulin antibody, anti-mouse IgG-biotin, andQD 535-streptavidin (green). (D) Her2 on the surface of SK-BR-3 cellswas stained green with mouse anti-Her2 antibody and QD 535-IgG(green). Nuclear antigens were labeled with ANA, anti-human IgG-bi-otin and QD 630-streptavidin (red). Filter sets ex 420 nm/em 535-10 nmand ex 560. Reprinted from Wu X, et al: Immunofluorescent labeling ofcancer marker Her2 and other cellular targets with semiconductor quan-tum dots. Nature Biotechnology 21:4146. 2003.</p></li><li><p>Literature Search and Review388</p><p>Spark</p><p>Polacek N, Swaney S, Shinabarger D, Mankin AS:SPARKa novel method to monitor ribosomal pep-tidyl transferase activity. Biochemistry 2002;41:1160211610.</p><p>Abstract: The key enzymatic activity of the ribosome iscatalysis of peptide bond formation. This reaction is atarget for many clinically important antibiotics. However,the molecular mechanisms of the peptidyl transfer reac-tion, the catalytic contribution of the ribosome, and themechanisms of antibiotic action are still poorly under-stood. Here we describe a novel, simple, convenient, andsensitive method for monitoring peptidyl transferase ac-tivity (SPARK). In this method, the ribosomal peptidyltransferase forms a peptide bond between two ligands,one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachmentof the biotin moiety to a tritiated compound. The amountof the reaction product is then directly quantified usingthe scintillation proximity assay technology: binding ofthe tritiated radioligand to the commercially availablestreptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioac-tivity. The reaction is readily inhibited by known antibi-otics, inhibitors of peptide bond formation. The methodwe developed is amenable to simple automation, whichmakes it useful for screening for new antibiotics. The</p><p>method is useful for different types of ribosomal research.Using this method, we investigated the effect of muta-tions at a universally conserved nucleotide of the activesite of 23S rRNA, A2602 (Escherichia coli numbering),on the peptidyl transferase activity of the ribosome. Theactivities of the in vitro reconstituted mutant subunits,though somewhat reduced, were comparable with thoseof the subunits assembled with the wild-type 23S rRNA,indicating that A2602 mutations do not abolish the abil-ity of the ribosome to catalyze peptide bond formation.Similar results were obtained with double mutants car-rying mutations at A2602 and another universally con-served nucleotide in the peptidyl transferase center,A2451. The obtained results agree with our previous con-clusion that the ribosome accelerates peptide bond for-mation primarily through entropic rather than chemicalcatalysis.</p><p>Commentary: SPA technologies first made their impacton the world of HTS; however, one can envision this typeof technology being useful in the understanding of thebasic mechanisms of enzymes, which have been difficultto measure using older technologies. Polacek et al. pres-ent a novel method for easily measuring peptidyl trans-ferase activity using the scintillation proximity principle.The peptidyl transferase activity in the ribosome ofprokaryotic organisms is essential for protein synthesis</p><p>SPARK-PmnA</p><p>f-M e t</p><p>E</p><p>70S</p><p>P A</p><p>P m n</p><p>AU G</p><p>37 Cf-M e t Pm n</p><p>f-M e t</p><p>E</p><p>70S</p><p>50S</p><p>P A</p><p>AU G A AA</p><p>37...</p></li></ul>