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reviews 34 Tretmak, W., Leiner, M.J.P. and Wolfl3eis, O. S. (1988) Biosensors 4, 15-26 35 Trettnak, W. and Woltbeis, O. S. (1989) Anal. CIffm. Acta 221,195-203 36 Bauer, B. and Floyd, T. A. (1987) Anal. Chim. Acta 197, 295-301 37 Schultz,J. S. (1987) in Biasensors:FundamentalsandApplicatiom (Turner, A. P. F., Kambe, I. and Wilson, G. S., eds), Oxford Umversity Press 38 Schultz, J. S., Mansouri, S. and Goldstein, I. (1982) Diabetes Care 5, 245-253 39 Douglas, S. D., Kanfin, R. M. and Fudenberg, H. H. (1969) .J. lnlmunol. 103, 1185-1195 40 Zukin, R. S., Strange, P. G., Heavey, L. R. and Koshland, D. E. (1977) Biochemistry 16, 381-386

41 Weber, S. G. and Weber, A. (1993) Anal. Chem. 65, 223 230 42 Lakowicz, J. R., Szmacinski, H., Nowaczyk, K. and Johnson, M. L. (1992) Cell Calcium 13, 131-147 43 Szmacinski, H., Gwczynski, I. and Lakowicz,J. R. (1993) Photochem. Photobiol. 58, 341-345 44 Szmacinski, H. and Lakowicz, J. R.. (1994) in Probe Des(w and Chemiml Sensing, Topics in Fluorescence Spectroscopy Volume 4 (Lakowicz,J. R., ed.), pp. 295-334, Plenum Publishing Corporation 45 Lakowicz, J. R. and Szmacinski, H. (1993) Sensors and Actuators B 11, 133-143 46 Spencer, R. D. and Weber, G. (1969) Ann. N Y Acad. Sci. 158, 361 376

Identifying small-molecule lead compounds: the screening approach to drug discovery Paul Bevan, Hamish Ryder and lan Shaw A number of new technologies that enable high-throughput, cost-effective screening of potential drug candidates have been developed in recent years. Such compounds may be derived from the large proprietary collections held by pharmaceutical companies, from new synthetic approaches such as combinatorial chemistry, or from natural sources. The latter remain a major source of new chemicals: many are already used in human treatment and many others are currently undergoing evaluation as the potential medicines of the future. Since the early part of this century, millions of novel, low-molecular-weight compounds have been screened for potentially useful medicinal properties. These have originated primarily from two sources: (1) products generated by synthetic organic chemists working in the major pharmaceutical drug houses; and (2) natural sources - plant, animal and microbial. As the demand for better and safer drugs has grown, so too has pressure for improved synthetic approaches to drug design and nqore sophisticated screening techniques to identify potential products. Both these methodologies have benefited greatly from advances in molecular biology, which have led to the identification o f molecular targets underlying the pathology of many diseases. The Human Genome Project (HGP) will uncover many more genetic abnormalities, an understanding of which may ultimately be exploited in rational drug discovery. The pressure to find novel small-molecule lead compounds to develop as thera-

P. Bevan, tl. Ryder and I. Shaw are' at Xenova Ltd, 2 4 0 Bath Road, Slouyh, Berkshire, U K S L I 4EF. © 1995, Elsevier Science Ltd

0167 - 7799/95/$9.50

peutics, as well as the opportunity to carry out such research, is set to increase dramatically. A major role fbr synthetic organic chemistry exists, particularly where advances in computer-aided molecular design (CANID) have given renewed impetus to attempts to design new drugs rationally. Rational design implies de novo design, synthesis and testing o f a small number o f specific molecules that have the potential to interact with the target. Assays for such purposes need not be as robust as those designed for mass screening. However, CAMD depends heavily on an existing knowledge base (for example, knowledge of the structure of a receptor or of the active site of an enzyme), or on surrogate models of the molecular system both from the biological and chemical standpoint. The computational approaches to drug design that are currently available and some of the results obtained using them are described in Box 1. An alternative to CAMD for identifying smallmolecule lead compounds is high-throughput screening (HTS). However, to be successful, efficient and cost-effective, HTS has to be viewed as a TIBTECH MARCH 1995 (VOL 13)

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reviews Box 1. Computational approaches to drug design Computational approaches such as GRID (Ref. 1), ALADDIN (Ref. 2), DOCK (Ref. 3) and LUDI (Refs 4,5) use knowledge of receptor sites to design potential new lead compounds, or to search small-molecule three-dimensional databases for complementary small molecules that might serve as new drug leads. Success stories are beginning to emerge. Both Abbott (Chicago, IL, USA) (Ref. 6) and DuPont Merck (Glenolden, PA, USA) (Ref. 7) have reported the computer-aided design of C2-symmetric, orally bioavailable HIV proteinase inhibitors. The DuPont Merck compounds (for example, Compound 1, DMP 323) are particularly notable for being nonpeptidic. DMP 323 is reported to be in Phase I clinical trial. 0

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Compound 1: DMP 323 De novo synthesis of inhibitors of thymidylate synthase has been described by Agouron Pharmaceuticals (La Jolla, CA, USA). The compound was designed and generated from knowledge of the structure of the active site of thymidylate synthase and use of the GRID programme (Ref. 8). Optimization of the structures using iterative analysis of the enzyme-inhibitor crystal structure yielded Compound 2, which is currently undergoing clinical trials as an anticancer agent (Ref. 6).

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high-technology process, drawing on the most recent understanding of disease mechanisms, while simultaneously using the most advanced technologies available in bioassays, robotics, computation, and data handling. Nevertheless, in the final stages, the HTS approach still relies on synthetic organic chemistry and CAMD to optimize the structure of the lead compound (Fig. 1). In recent years, HTS has made massive strides and vast libraries of compounds have been evaluated as sources of new potential lead compounds. However, the success of this approach has brought with it the problem of identifying sources of new compounds for screening. In the remainder of this article, recent advances in HTS technology and potential sources of molecules TIBTECHMARCH]995 (VOL 13)

to feed such screens, including using microbes as a source of natural products, are discussed. The relative merits of screening versus de novo synthesis are shown in Table 1.

Technological developments in high-throughput screening The multidisciplinary nature of HTS brings opportunities for great technological creativity, but also poses significant challenges. The most important challenge (discussed in greater depth below) is maintaining a ready supply of materials for the screen, whether from proprietary chemical collections or from natural sources. The second challenge is the need to design robust and reliable screens with a capacity for handling 10 6 or more samples per annum from a variety of sources. Assay systems using whole cells, tissue homogenates, membrane preparations or soluble recombinant proteins have been developed. The identification, cloning and expression of genes encoding particular proteins provides an opportunity for developing highly specific assays that target individual receptor subtypes or enzyme isoforms9. In addition, companies such as Oncogene Science Inc. (Manhasset, NY, USA) have developed a range of whole-cell, transcription factor/reporter-gene assays that enable very-highthroughput screening. The technology for immortalizing specific cell-line constructions for use in screening operations is also improving 1°. The assays used in HTS can be relatively conventional, such as enzyme-linked immunosorbent assay (ELISA), radio-immunoassay (ILIA), and receptor/ binding and enzyme-substrate assays; however, recent technological advances, such as the scintillation proximity assay from Amersham (Amersham, UK) and time resolved fluorescence (DELFIA ®) from Wallac Oy (Turku, Finland) are now being used. The availability of microtitre plates with specific binding coating (Nunc, Wiesbaden, Germany), glass-fibre filters incorporated into microtitre plates (Canberra Packard, Pangbourne, UK), solid scintillation materials (Canberra Packard and Wallac Oy) and, most recently, the incorporation of scintillant material into microtitre plates (Wallac Oy and Canberra Packard), have provided additional opportunities for a number of new assay formats to be developed. In many cases, the use of such assay formats can remove the necessity to separate bound from free radioactivity, reduce the number of processing steps and restrict the use of scintiUant material to a minimum. The time resolved fluorescence technology from Wallac Oy and potential developments in lanthanide chemistry in this area, such as delayed fluorescence resonance energy transfer (DEFILET), could increase assay sensitivity, reduce the amount of radioactivity used and improve the performance of homogeneous assays where the kinetics of interaction could be studied. Once a suitable, scientifically valid, assay design has been determined and validated thoroughly as a robust screening method that is compatible with the chosen

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reviews input materials, the third challenge is to operate the screen rapidly and cost effectively. Cost containment has been achieved, in part, by miniaturizing assays so that they can be performed in microtitre plates. Increasing the level of miniaturization is being explored now that microtitre plates with 384 wells (well capacity of 75 bd) are available. In addition, new methodologies incorporating assays on silicon wafers are also being considered (1Lef. 11). The use of automated instrumentation minimizes costs attributable to human resources and, for the past five to six years, such equipment has been available and is able to handle most procedures used in modern microtitre-plate assays. The choice of robotic work-stations for liquid handling is considerable, ranging from single, to multiple and 96 probes, with optional facilities for using pipette tips. These instruments have reached a very high level of technological sophistication and accuracy. Similar instrumentation for automating washing steps and read-outs, and which incorporates stacking facilities, is also available. The direct measurement of radioactivity in microtitre plates is possible with instruments such as the TopCount TM from Canberra Packard and the MicroBeta TM from Wallac Oy. A significant development in the level of automation is now underway with the integration of these instruments with pick-andplace robotics. The introduction of robotics will enable assays to be carried out without operator intervention. This will enable human staff to focus on tasks such as data analysis and interpretation and will further reduce the operational costs of actual screening. Assay reproducibility also tends to benefit from welldesigned automated screening procedures. However, robotic technology is, as one might expect, expensive to acquire and install. Nevertheless, the speed of screening, and the minimal operator input required, can be extremely cost-effective over time. The fourth challenge to HTS relates to data handling. Screens of this type inevitably generate a massive amount of information in a relatively short time, requiring the development of databases to enter, store and retrieve data. Developments in computation, particularly the widespread availability of the low-cost network PC and the Microsoft ® Windows operating environment, have created the opportunity to develop new tools for processing screening data to extract the maximum information possible. In terms of data analysis, the simplistic calculation of percentage inhibition and the assignment of arbitrary cut-off values do not do justice to the information that is available. The application of more sophisticated methods such as co-variance analysis, cross-screen analysis, artificial intelligence, neural networks and virtual reality can be developed and applied to these large information databases. The National Cancer Institute (NCI) (Bethesda, MD, USA), for example, has applied neural networks in their cancerscreening programmes to predict mechanisms of

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action from compound structures 12. In the future, it may be possible to pre-screen data banks of information on compounds, natural products and combinatorial libraries (see below) through trained neural networks in oMer to prioritize samples for testing, an approach that may be considered as the incorporation of a rational element into random screening. Sources o f chemical diversity for high-throughput screens

The range of assay technology and instrumentation that is currently available is such that very large,

Table 1. The advantages and disadvantages of screening versus rational design in the search for new drug leads Approach Pros

Cons

Screening Immediate applicability to virtually any molecular target No structural information needed Track record of success

Limitedby quality of chemical diversity

Structurebased rational design

No chemical limitations

Specificity can be designed-in Reagents not required for design stage

Empirical; can be timeconsuming Many false 'hits' need to be rejected Not all targets amenable to this approach - needs detailed structural information Limited by computational algorithms Limited track record of success

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reviews Box 2. Combinatorial chemistry for the synthesis of peptides and nonpeptides Libraries of resin-bound peptides can be generated on beads by the 'split-synthesis' technique. Each bead carries a single peptide species. Beads carrying peptides with the desired activity are identified by bioassay, and isolation of the active bead and subsequent microsequencing reveal the peptide sequence 14. The size of the library is, for practical reasons, limited by bead volume, which is determined by the amount of peptide required for microsequencing. Library size can, however, be increased by using smaller beads (carrying a smaller amount of peptide) by modifying the technique. Each bead is linked by orthogonal chemistry to a unique oligonucleotide identifier. Once the bioactive beads have been identified, polymerase chain reaction (PCR) is used to amplify the attached oligonucleotide identifier, which can then be sequenced to reveal the identity of the peptide carried by the bead. Automated synthesizers have been developed for generating this type of library15. Resin-bound peptides can only be assayed by a limited range of bioassay formats; however, solution-based libraries can be used with all assay formats. R. Houghten has pioneered the use of such solution-based libraries: the most potent compounds are identified by iteratively screening mixtures, where one amino acid residue is defined at each iteration 16. Biological methods have also been described for the generation of peptide libraries displayed on the surface of bacteriophages or on plasmids17,18. The application of solid-phase nonpeptide synthesis to the production of combinatorial libraries is at a seminal stage. The N-substituted glycine oligomer technology (Compound 3) developed by Chiron Corp. (Emeryville, CA, USA) (Refs 19,20) generates compounds that have peptide backbones and lack chiral centres, and the N-substitution greatly reduces the vulnerability to proteolysis. R3

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In this technique, the compounds are synthesized in individual wells, and the number of compounds that can be generated by this technique is in the hundreds rather than thousands as new synthetic methodology has to be developed each time the template structure is changed. Therefore, at present, this technology may be best used for the rapid optimization of a structural class of compounds, rather than the identification of a novel lead.

TIBTECHMARCH1995 (VOL13)

Box 3. Advantages and disadvantages of combinatorial synthetic methods Pros

Cons

• Millions of peptides can be made simultaneously and tested as mixtures

• In general, peptides do not make good leads

• Eleganttechniques foridentifying active compounds have been developed

• Small- molecule (nonpeptide) combinatorial libraries are at an early stage of development

• Peptide libraries containing thousands of compounds have been reported

• Any combinatorial library made with current technology will be based on a limited number of molecular templates and thus will have low diversity density

cost-effective mass-screening operations that are capable of handling >1 × 10 (' samples per annum can be implemented. The continuous supply of novel compounds is, therefore, critically important for justifying and maintaining such high-throughput screens. These are likely to be provided from: (1) existing chemical libraries, (2) the generation of combinatorial chemistry, or (3) natural sources. Chemical libraries Traditionally, libraries of proprietary compounds have provided the largest source of chemicals for mass screening-major pharmaceutical companies can have upwards of 500 000 compounds in their collections. The chemical structures in these libraries tend to reflect the research areas of the individual companies, and hence the diversity of chemical template within these collections has been said to be restricted. In practice, it is rather difficult to judge the true diversity of any compound collection. However, one distinct advantage of screening this type of collection is that most of the members are small molecules and have a good chance of possessing bioactivity of potential therapeutic value. In addition, structureactivity relationships may be obvious from the primary screen, as many close analogues could be screened for the required activity. This will enable decisions to be made regarding the exploitability, novelty and structure-function divergence at an early stage. A major difficulty for HTS operators is gaining access to the compound collections. This requires willingness on the part of the company and the ability to organize access. To overcome this problem, many companies have bilateral agreements allowing access to each other's libraries for screening purposes. Alternatively, some organizations have addressed this

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reviews a

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issue by the creative integration of diverse technology to automate compound storage, access, weighing, solubilization and dispensing. This has been achieved, for example, by Wellcome Research Laboratories (Beckenham, UK) and The Technology Partnership

Ltd (Melbourn, UK) (Ref. 13). The system, termed 'haystack', uses robotics and automation with intelligent sample handling. This sets a good precedent, but needs to be extended to many other commercial compound collections. TIBTECHMARCH1995(VOL13)

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reviews Natural a

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Combinatorial chemistry Combinatorial chemistry (see Box 2) may be defined as the use of semi-automated and often miniaturized methods to prepare large series of synthetic analogues (often as mixtures), which may then be screened for activity. Technological advances in this area now enable combinatorial libraries of thousands or even millions of molecules to be generated for testing. This approach was born and matured in the laboratories of peptide chemists for the generation of peptide libraries (Ref. 22). However, peptides rarely represent ideal lead candidates. Their poor oral absorption and metabolic instability mean that a nonpeptide ~nimetic based on the structure of the peptide often has to be developed in order to exploit the desirable properties. Nevertheless, screening peptide libraries can rapidly provide structure-activity relationships, providing valuable information on which to base a non-peptide-mimetic synthesis programme. Although producing lead compounds by this route may be relatively rapid, the optimization process is often slow and inefficient and is a serious rate-limiting step in drug discovery. Furthermore, the extension of combinatorial approaches to the generation of other types of nonpeptide molecule is still in its infancy, although progress is being made (Box 2). The various pros and cons of the 'combinatorial' approach are shown in Box 3. TIBTECHMARCH1995(VOL13)

products

Random screening of chemical libraries and natural products can offer many opportunities for the discovery of new, clinically relevant molecular templates. Welldocumented examples of lead compounds identified by the natural-product approach to screening are cyclosporin, mevinolin, asperlicin and avermectins. The complex and varied structures of these compounds also illustrate the point that only natural-products screening could have discovered them. However, by current standards, these compounds were discovered by relatively low-throughput random screening techniques. It is important to recognize, however, that the natural-product approach often involves the testing of complex mixtures of chemicals derived from a range of natural sources. As such, the mixture may contain an unknown, but potentially large, number of compounds, requiring particular skills for generating samples of natural product of sufficient quality to be compatible with modern biological-assay technology (Ref. 23). Xenova (Slough, UK) specializes in the discovery of natural-product leads by combining various technologies. Applied microbiology and high-technology fermentation systems, and a highly automated HTS capability, provide the platform to service most therapeutic areas and targets. Currently, a screening rate >1 )< 106 samples per annum is being achieved; in the period between May 1989 and June 1994, 186 biologically active structures were isolated, 61 of which are novel (Fig. 2a). O f these 61 novel compounds, 47 arising from 24 different drug-discovery projects targeting four major disease areas, have been selected for further development. The majority were isolated from fungi with a smaller contribution from actinomycetes and plants (Fig. 2a). Additional analysis of the data, in terms of the distribution of producer organisms by geographical origin, shows a genuine pan-global distribution and serves to illustrate the importance of accessing different habitats for source materials (Fig. 2b). An analysis of the compounds themselves (Fig. 2c) also demonstrates that the molecular-weight distribution closely overlaps the molecular-weight range of marketed drugs. These data serve to highlight both the productivity and utility of this type of screening operation, but cannot illustrate the diversity. This may best be achieved by considering two examples of compounds isolated in screens carried out by Xenova. The first compound (Compound 6; Fig. 3a), interacts very specifically at the y-amino butyric acid (GABAA)-receptor and has potential use in the treatment of anxiety and epilepsy. Structurally, Compound 6 is very far removed from the benzodiazepines and [3-carbolines. It is also obvious from the novelty of the structure that this compound could only have been discovered from natural-product screening. The second compound (Compound 7; Fig. 3b) is an inhibitor of plasminogen activator inhibitor (PAI-1) and therefore of potential therapeutic use in

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reviews thromboembolic disorders. While it may be argued that this structural class could have been identified from screening chemical banks, it was discovered by natural-product screening. It is a straightforward target for synthesis, thereby enabling rapid lead optimization. From the above, it is clear that natural sources offer an enormous diversity of raw material, and great variety and novelty in the structures isolated. Furthermore, the previous cost limitations to mounting a large-scale screening operation have largely been overcome by the considerable advances in assay design, assay technolog'y, automation and computational developments in databases and analytical tools.

What does the future hold? The demand for lead compounds to investigate the plethora of molecular targets identified by molecular sciences can currently best be met by mass-screening operations. Source materials will undoubtedly arise from combinatorial libraries, which will continue to develop and provide additional opportunities for lead discovery. However, natural-product screening still continues to provide the richest source of novel and often structurally complex compounds with exploitable biological activity. This approach has been made more attractive through technological advances in assay and screening methodology, which have made HTS operations a very cost-effective and viable option. We believe that the resources and human endeavour being applied to the Human Genome Project will identify an increasing number of molecular-target opportunities at an increasing rate. From this standpoint, the cost effectiveness and speed of HTS will make this technology central to the exploitation of this information and the generation of exciting and effective new medicines.

Acknowledgements The assistance of In& Chicarelli-Robinson and Trevor Twose in the compilation of this publication is greatly appreciated. References 1 Goodford, P. R. (1985)_J. Med. Chem. 28, 849-857 2 Van Drie,J. H., Weininger, D. and Martin, Y. C. (1989)J. Camput. Aided Mol. Des. 3, 225-251 3 Kuntz, I. D., Blaney, J. M., Oadey, S. J., Langndge, R. and Ferrin, T. E (1982)J. Mol. BiaL 16J, 269 288 4 B6hm, H-J. (I992)J. Comput. Aided ,Viol. Des. 6, 61-78 5 B6hm, H-J. (1992)J. Cornput. Aided ,Viol. Des. 6, 593-606 6 Greer, J., Erickson, J. W., Baldwin, J. J. and Varney, M. D. (19!)4) J. Med. Chem. 37, 1035-1054 7 Lain, P. Y. S. et al. (1994) Science 263, 380-384 8 Vamey, M. D. et al. (1992)ff. Med. Chem. 35, 663-676 9 Luyten, W. H. M. L. and Leysen, J. E. (1993) Trends Biate&nol. 11, 247 254 10 McLean, J. S. (1993) Trends Biatechnol. 1I, 232-238 11 Stylli, H.(1994) in The 1994 h2ternational Forum on Advances in Screening Technologies and Data Management, Semin. Abstr.

12 Weinstein, J. (1994) in The 1994 huemational Fornm on Advances in Screening Technala2ies and Data Management, Sernin. Abstr.

13 Harrison, W.J. and Powell, K. (1994) in The 1994 International Forum on Advances in Screening Technolo,~iesand Data Management, Semin. Abstr.

14 Lain, K. S., Salmon, E., Hersh, E. M., Hmby, V. J., Kazmierski, W. M. and Knapp, R.J. (1991) Nature 354, 82-84 15 Zuckennann, R. N., Kerr, J. M., Siani, M. A., Banville, S. C. and Santi, D. V. (1992) Proc. Natl Acad. Sci. USA 89, 4505-4509 16 Houghten, lk. A., Pinilla, C., Blondelle, S. E., Appel, J. R.., Dooley, C. T. and Cuervo, J. H. (1991) Nature 354, 84-86 17 Smith, G. P. (1985) &ience 228, 1315-1317 18 Scott, J. P,.. and Smith, G. P. (1990) Science 249, 380--390 19 Zuckermatm, R. N. et al. (1992) J. Am. Chem. Sac. 114, 10646-10647 20 Simon, R. J. et al. (1992) Proc. Natl Acad. Sci. USA 89, 9367-9371 21 De Witt, S. H., Kiely, J. S., Stankovic, C. J., Schroeder, M. C., Reynolds Cody, D. M. and Pavia, M. R. (1993) Proc. Natl Acad. &i. USA 90, 6909-6913 22 Geysen, H. M., Meloen, 1<. H. and Barreling, S.J. (1984) Prac. Natl Acad. &i. USA 81, 3998-4002 23 Yarbrough, G. G., Taylor, D. P., IZowlands, R. T., Crawford, M. S. and Lasure, L. L. (1993)J. Antibiotics 46, 535-544

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