Licensing genomic amplification methods
Jay Epstein (FDA, Rockville, MD, USA) indicated that the position of regulatory agencies on the genomic detection of infectious agents is no different from their position on other types of tests. In addition to the routine requirements for specificity and sensitivity, a strong scientific basis for defining the intended use of the test is also required. The extreme sensitivity of genomic amplification forces manufacturers to redefine epidemiology and standards. The new
standards-called extended standards - include not only positives, accord- ing to reference methods, but also cases that have been confirmed by the consistent amplification of multiple conserved sequences of the same agent with the same assay for- mat, or amplification of the same sequence using different methods. The multiplicity of reagents and the risk of cross-contamination are ad- ditional difficulties.
Nice Lelie (Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) presented the
Accessing new resources for drug discovery
The increasing demand for new lead compounds within the pharmaceuti- cal and agrochemical industries has forced a dramatic change in the discovery process. The primary approaches that are now used for the identification of novel chemical leads are High Throughput Screening (HTS) -the automated simultaneous screening of very large numbers of samples - and general sources of molecular diversity (i.e. natural sources, including plants, soils and tissues; large compound collections gathered in the past 50 years by large pharmaceutical companies; and, most recently, combinatorial libraries). Automation and parallel and/or simultaneous multiple syn- thetic chemistry procedures have greatly accelerated the generation and screening of an increasing nun- ber of new compounds. However, innovative approaches to synthesis and screening are being developed that approach the limits and scope of molecular diversity. The advances in the identification of new sources of diversity, as well as the resulting technical issues, were discussed by over 40 speakers from academic and industrial laboratories at a recent meeting*.
l The meeting The 2nd European Confer-
ence on High Throughput Screening and Explomng Molecular Diversity was held m Budapest, Hungary, 17-20 May 1995.
0 1995, Elsevier Science Ltd 0167 - 7799/95/$9.50
Sources for HTS Large numbers of compounds that
are truly diverse in skeletal and func- tional array are essential for carrying out efficient HTS in the search for novel drugs. For many years, natural sources have been the primary supply of such diverse compounds; for example, more than 100 000 species of marine organisms are avail- able as natural sources of molecular diversity, and over 5000 different structures have been isolated for screening in the past 10 years (Matthew A. Sills; Ciba-Geigy, Summit, NJ, USA). However, the isolation and identification of samples are often cumbersome processes, and can delay the HTS process. Archival resources (i.e. collections of com- pounds that have been gathered by large pharmaceutical companies, either through internal synthesis pro- grams or from external acquisitions) represent a second source of com- pounds for HTS.
Innovations in multiple parallel and/or simultaneous organic syntheses have enabled the synthesis of large numbers of related analogues that can be processed using HTS. Future innovations in the acceleration of the simultaneous multiple synthesis of organic compounds are expected to be driven primarily by new develop- ments in organic chemistry using solid-phase approaches. Michael R. Pavia (Sphinx Pharmaceuticals, Cambridge, MA, USA) presented
results of a multicentre evaluation of panels for the detection of HBV- DNA and HCV-RNA using in- house and commercial methods. It revealed the poor performance of current assays, and provided a measure of what still needs to be done to achieve robust, user-friendly genomic detection methods.
the example of the simultaneous multiple synthesis, in a 96-well-plate format, of small quantities of indi- Meeting vidual benzamide- and urea-based compounds; the synthesis utilizes a report photocleavable linker that allows v~.~~~ii.i--ircxerrin~~~~~~~~~~ biphenyl scaffolds to be cleaved.
Solid-phase methods are now being used for the synthesis of com- binatorial libraries of organic com- pounds. Synthetic combinatorial libraries (SCLs) represent the most recently developed source of large numbers of diverse compounds that are being processed using HTS. While the efficiency of HTS relies on the screening rate, SCLs provide an excellent alternative to the simple screening of many individual samples. Using this approach, pools of com- pounds are combined in a selective and precise manner, and are screened as mixtures, instead ofbeing screened individually. The use of such mix- tures decreases the number of sam- ples to be screened, without decreas- ing the number of compounds being screened. This is beneficial, primarily for those assays that are complex, costly and difficult to automate. A number of combinatorial libraries of organic compounds have been prepared, including acylpiperidine and benzopyran libraries. These libraries were prepared on poly- styrene beads, and the active com- pounds identified through the decoding of an attached associated tag (Daniel Chelsky; Pharmacopeia, Princeton, NJ, USA). SCLs have been prepared for other organic compounds, including polyamines, acylated polyamines and peptido- mimetics (Sylvie E. Blondelle; Torrey Pines Institute for Molecular Studies, San Diego, CA, USA). These were developed using
TIBTECH OCTOBER 1995 WOL 13)
solid-phase methods, and were assayed following cleavage from their solid support. These com- pounds are pooled in such a manner that individual hits can be identified from the compound pools through a direct deconvolution process that requires no additional encoding tag or analytical steps, such as microsequencing. Future develop- ments in the simultaneous multiple synthesis of pools of organic com- pounds are expected to extend greatly the diversity of compounds that can be screened using HTS procedures.
Improving bioassay efficiency The use of HTS in all biological
areas represents a challenge for indus- try, and the key to its expedient use is the efficiency of bioassays. The potential of 96-well plates and, more recently, 384-well microplates, and the developments in instrumentation and robotics are powerful advances. The ability to automate assays in a 96-well format decreases the need to pre-select only the best compounds for screening, and enables every compound ofinterest to be screened. Furthermore, the standardization of bioassays in the 96-well format allows mother plates to be prepared and dispersed into daughter micro- plates for use in large numbers of screening assays. This flexibility enables the ready dissemination of HTS programs among different laboratories, which, in turn, leads to substantial increases in the efficiency of the drug discovery process (James S. Marks; Parke-Davis, Ann Arbor, MI, USA).
A number of new techniques that allow the implementation of bioassays with a 96-weµplate format have been developed. For example, FlashPlate (developed and patented by Packard) is a 96-well microplate that has been precoated with scintil- lant; this allows the detection of the B-emitting isotopes of radiolabeled ligands that bind to molecules initially coated on the plate. The advantage of this method is the elimination of the harvesting and wash steps, which accelerates the screening process. This technique can be applied to radioimmuno- assays, radioreceptor assays (prefer- ably using pure soluble receptors), enzymatic assays and live-cell assays (Beverly A. Brown; E.I. DU Pont De Nemours, Boston, MA, USA).
Standard radioisotopic and colori- metric techniques have been sup-
nlemented with innovative lumi- 1
nescent and fluorescent methods that can be readilv adanted to HTS. Therefore, long-lived fluorescent rare-earth cryptates (Gerard Mathis; CIS Bio International, Bagnols sur C&e, France) or lanthanide chelates (Stuart A. Webb; Wallac Oy, Turku, Finland), which allow multilabel assays to be designed, are becoming more common for screening in non- radiolabeled receptor-ligand assays, and in cell adhesion, cytotoxicity, signal transduction and DNA hybrid- ization assays. The use of fluor- escently labeled ligands offers the advantages of extending the decay time, and eliminating the hazard and disposal problems that are related to radiolabeling methods.
Following the identification of a drug candidate, there are many steps before the development process can be completed. In particular, although HTS leads to the rapid identification of hits, only a small number of these compounds will be considered as lead candidates. Often, one of the main determinants of a new drug candidate is its ability to cross the blood-brain barrier. or other mem- brane biological barriers, in order to reach its target. New high- throughput methods that are aimed at screening large numbers of com- nounds for their abilitv to cross these biological barriers have recently been developed (Alexandros Makriyannis; University of Connecticut, Storrs, CT, USA). Such approaches will accelerate this first phase of dntg development, and allow the high- throughput secondary selection of compounds to be developed further.
Measuring diversity Owing to the diversity of drug
candidates that can be screened, there is a requirement for design strategies that maximize diversity for broad screening, and that focus the chemi- cal diversity toward a given target for lead optimization. One strategy involves characterizing the lipo- philicity, topology, chemical func- tionality, recognition features and three-dimensional shape descriptors of each monomer. A simple graphical approach (called flower plot graph- ics) for representing these descriptors allows different potential compounds to be compared (Eric Martin; Chiron, Emeryville, CA, USA). Therefore, the efficient design of libraries for broad screening should reduce redundancy by using diverse com-
ponents from each diversity-descrip- tor database. Conversely, this algo- rithm can be used to prepare focused libraries for targeted screening by identifying similarity to known ligands. Such descriptors can also be used to learn about the possible toxicity and the potential stability of newly designed compounds (Richard D. Cramer; Tripos, St Louis, MO, USA). Similar approaches use other features to describe molecular similarity, includ- ing three-dimensional atomic con- figuration to mark interaction centers (Keith Davis; Chemical Design, Chipping Norton, UK). Dissimilar- ities between newly designed mol- ecules and existing databases can also drive the selection of compounds (Michael S. Lajiness; Upjohn, Kalamazoo, MI, USA). Descriptors can also be used to select specific properties of interest for the gener- ation of new structures having such properties. At present, however, it appears to be extremely difficult to reconstruct a structure from core topo- logical properties or indices. Research is being carried out to solve this reversibility problem. A further approach is the design of new libraries from computational studies that are based on databases of conventional chemical reactions for the synthesis of series of compounds (Liszl6 Kovics, ComGenex, Budapest, Hungary).
Towards automation The main challenge in drug dis-
covery programs is to identify rapidly new lead compounds (the main requirement for patenting) that are more effective and cheaper. It is poss- ible that HTS will be able to screen 10 million samples per year, which would be impossible without automation. The level ofautomation to be implemented will vary depend- ing on the size, need and expertise of the discovery company or academic department. The end point of such automation relies not only on the ability to speed the screening process by decreasing the number of manual steps, but also on facilitating the use of the derived information. While robotics have had a tremendous impact in speeding-up HTS, data management remains the bottleneck in most HTS programs. Therefore, the generation of growing numbers of data sets through HTS has brought about tremendous logistical prob- lems. Slight errors in large data sets can rapidly lead to a large number of
TIBTECH OCTOBER 1995 WOL 131
incorrect values, which must be located and corrected. Recursive partitioning analysis of the data sets has been proposed; this allows the trends in general activity to be deter- mined and visualized (S. Stanley Young; Glaxo, Research Triangle Park, NC, USA). Future develop-
ments in this area are needed in order these developments will be the key to optimize automated HTS processes. to a new era in drug discovery
In conclusion, HTS, combined programs. with the recent explosion in combi- natorial chemistry approaches, repre- Sylvie E. Blondelle sents an essential resource, both for Tomy Piw3 htitute,fir Moldar Studies, the pharmaceutical industry and for 3550 Cerwral Atnmm Courr, Sm Diego, biomedical research. In the future, CA 92121, USA.
Plant-product and crop biotechnology
Trends in Biotechnology Special Issue (vol. 13, no. 9)
Plant biotechnology R & D - the next 10 years, Commercializing the products of plant biotechnology, Gene-transfer and
plant-regeneration techniques, Transgene expression, Controlling plant development, Genetic engineering for pollination control,
Plant-microbe interactions, Disease and pestresistant transgenic plants,
Manipulating plant metabolism, Plants as bioreactors, Vaccine production in
plants, Genetic engineering in floriculture, Phytoremediation, Field trialling
transgenic crops, Plant biotechnology and developing countries
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