Receptor-based assays in screening for biologically active substances lan Shaw X e n o v a Ltd, Slough, UK Molecular biology has identified new receptors and tigands which are deregulated in diseases such as cancer and autoimmune conditions and which provide rational targets for therapeutic intervention. Advances in instrumentation and methodology make it possible to screen large numbers of samples in simple receptor-ligand binding assays in the search for drug candidates. Caution must be exercised in the interpretation of data derived from such assays.This is particularly pertinent to the recently characterized receptors, such as the cytokine receptors, as we do not fully understand the relationship between the receptor type and the linkage of receptors to the appropriate or inappropriate second messenger systems that are used in the experimental screening protocols and the disease state. Current Opinion in Biotechnology 1992, 3:55-58
Introduction Over the last two decades, receptor-based assays have been used in the drug discovery process to aid structure-activity relationship studies to increase etficacy, to assist in validating rational drug design and to elucidate the mechanism of action and selectivity of known drugs. Such studies have both improved upon and produced novel drugs for use in man. The widespread use of receptor*based assays has also defined receptor subtypes and refined receptor-ligand interaction theory. As new ligands have been identified, receptor theory has been modified and extended to accommodate the new findings. Modem receptor theory has been reviewed in an excellent article by Williams [1 o*] and therefore will not be covered in detail in this article. Traditional drug-discovery receptor-based assays use standard pharmacological techniques, such as tissue preparations and organ baths, to measure a functional response invoked by a compound in the presence of a known ligand. Whilst these techniques produce excellent quality data, they are labour intensive, time consuming and have a limited capacity to test large numbers of samples. The advent of simpler techniques, such as radiolabelled ligand-binding assays, has provided the opportunity for more cost effective drug discovery with a rapid tumaround of results. Most importantly, these techniques enable large numbers of compounds to be tested in the search for drug candidates, particularly where no lead molecules exist. This review will focus on the use of the simple radiolabelled ligand-binding assay, as applied to membrane
bound receptors, in the search for novel lead compounds. A second focus will be the enormous advances in techniques and instrumentation that can be applied to these receptor-based assays.
Receptors and ligands of interest The explosion of knowledge about disease mechanisms generated by the widespread use of molecular biology has identified many new receptors and their endogenous ligands as therapeutic targets. Consequently, a considerable effort is being made in the drug discovery industry to capitalize on this understanding of disease processes. Although the biotechnology industry has focused on proteins as therapeutic entities, proteins have several constraints, such as poor oral bioavailability, short half life and metabolic instability, that limit their therapeutic uses. Non-peptidyl mimetics of these receptor-ligand interactions are therefore required to fully capitalize on modern knowledge in order to produce new orally-active drugs. Recently characterized receptors and ligands include the cytokines and haemopoietic factors [2-*], the adhesion receptors of the immune system [3°], and receptors present in the endothelium that control its many functions [4*]. Complex protein-carbohydrate and protein-carbohydrate-protein interactions [5.] have yet to be incorporated fully into classical receptor theory. For example, little is known about the influence of the interactions between fibroblast growth factor and glycosaminoglycans prior to receptor occupancy [6°],
Abbreviations IL--interleukin; TNF~tumour necrosisfactor.
© Current Biology Ltd ISSN 0958-7669
55
56
Analytical biotechnology Such interactions must be understood if a receptor-based screening method is to be meaningful.
Sources of receptors and ligands Many sources of receptors can be used in radioligandbinding assays. Ideally, the receptor should be of human origin and be derived from 'disease tissue' or a relevant cell-type, as there are reports of receptor changes in disease states. For example, natural soluble tumour necrosis factor (TNF) receptor derived from cancer patients binds TNFc~ better than TNFI3, but the relevance to the disease state is unknown [7°]. The interleukin (It)-I receptor has two forms, type I, which is present on T cells and fibroblasts and type II on B cells and myeloid cells. The ligands, IL-10c and IL-113 have different affinities for these receptors, but the relationship of these differences to particular disease states is unclear [8.]. Thus there are obvious practical problems in obtaining quantities of the ideal receptor for use in a screen. Ideally, the particular shortcomings of the receptor used in a screen must be understood and complemented by the judicious selection of additional secondary assays. A second key issue that relates to the use of a receptor for screening purposes is the form in which the receptor is used in the assay. Receptors can be obtained in a number of ways: as pure recombinant proteins, as pure solubilized proteins (from cells or tissues), as cell membrane preparations, as receptors cloned into an expression system, or as receptors present on the whole cell where the integrity of the receptor depends on the viability of the host cell during the assay. The merits of each receptor source as a screening tool must be addressed in the context of present knowledge of the integrity of the receptor derived from that source. Very little is known in detail about the three-dimensional structure of receptors. Many receptors, particularly the G-protein linked receptors, are present in the cell membrane as complex seven-helical transmembrane proteins. A model of this conformation has been elucidated from studies on the membrane protein, bacteriorhodopsin [9"], which shares many similarities with receptors of pharmacological interest. The information derived from these studies highlights the complexity of receptor structure and illustrates that the precise orientation and conformation of the protein is crucial to the binding of the ligand and therefore the resultant agonist or antagonist activity of compounds. Minor perturbations in conformation will radically change the expression of binding domains. Consequently, the value of solubilized receptors, where the phospholipid (cell membrane) is replaced by detergent, becomes questionable. Williams [1"'] also questions the value of using re~eptor sources in which the receptor has been cloned into a 'foreign' cell, such that it may be linked to an inappropriate second messenger system or not linked at all. The effect of such abnormal environments on receptor-ligand interaction is unknown. Formal occupancy theory states that formation of the receptor-ligand complex is reversible, the response
evoked is proportional to the number of receptors occupied, and the biological response is equilibriumdependent. The IL-1 receptor and its two endogenous ligands, IL-ltz and IL-I~, pose problems for conventional receptor theory, particularly when they are used in a receptor-ligand binding assay. The affinity (0.2-2 x 1010 M - 1) and association and dissociation rate constants (2.8 x 1 0 8 M - l m i n - 1 and 2.1 x 1 0 - 2 M - 1 respectively) o f IL-1 [3-binding are unusual as the association rate is relatively typical for a polypeptide hormone but the dissociation rate is very slow [10]. Thus, under the experimental conditions of a radioligand binding assay, equilibrium cannot be achieved, presenting obvious problems in the interpretation of data. In spite of all of the potential problems in data interpretation and validity of receptor source, receptor-ligand binding assays provide one of the most cost effective techniques with which to discover non-peptidyl agonists or antagonists of novel receptor -ligand interactions. The obvious question is whether or not the technique can be successful. At present there are very few reports of non-peptidyl agonists or antagonists that interfere with these complex protein-protein interactions. One notable exception is the discovery of a non-peptide antagonist of angiotensin II [11°].
Sources of molecules for screening In one successful strategy for drug discovery, particularly where no lead molecules are known, libraries of chemical structures are screened for novel pharmacophores. Chemical banks numbering hundreds of thousands of compounds are present in most major pharmaceutical companies. Williams [1 °° ] however, indicates that the structure diversity of these chemical banks is often limited because of the inclusion of simple chemical substitution analogues of key compounds. Natural products provide an alternative source of molecules of almost unlimited structural diversity. Natural product libraries can be derived from microorganisms, marine organisms or plant materials. Screening of natural products, particularly those derived from microorganisms, has provided useful drugs, such as antibiotics and cytotoxic anticancer drugs. Notable examples include: the cholecystokinin receptor antagonist, asperlicin, follow-up structure activity relationship studies of which have resulted in the benzodiazepine derivative MK 329 entering a phase U clinical trial as a gastrointestinal agent; cyclosporin, FK 506 and rapamycin, which are selective immunosuppressant agents: the hypotensive agent doridosine; and the antineoplastic agents Am-C, Ara-A and taxol. The success rate of these progmmmes was influenced substantially by the diversity of the organisms screened and the number of samples screened. These factors, particularly the numerical capacity of a screen, place severe constraints o n the type of assay that can be used to detect lead molecules. There have been several significant technological advances in receptor-ligand binding assays, which open up numerous opportunities to design cost-
Receptor-based assays Shaw
effective, scientifically valid screens. From the foregoing discussion o f receptor-based assays, however, the drug researcher must be aware o f the numerous theoretical pitfalls in using a 'simple' receptor-ligand assay, particularly when applied to recently characterized receptors.
in alternative assay techniques such as second messenger systems, gene transcription and translation events. Techniques to measure these events, particularly as high capacity screens, are in their infancy.
Acknowledgements Significant
technological
advances
Advances in instrumentation over the last few years have enabled radioligand binding assays to be carried out in microtitre plates. Separation o f b o u n d radioligand from free radioligand, has been greatly facilitated b y the availability o f 96-well microtitre plate harvesters while the efficiency o f processing and quantitation o f radioactivity has been significantly improved by the development o f ]]-plate readers from LKB/Pharmacia and a microplate reader from Canberra Packard [12o-]. This technology, combined with automated pipetting w o r k stations, enables the researcher to complete 2000 conventional radioligand binding assays with the minimum a m o u n t o f handling and the minimal use o f scintiUant. As outlined by Butch [13"], further robotic instrumentation will not necessarily increase the capacity or efficiency o f this type o f assay. The ability to automate receptor-ligand binding assays has resulted in commercial enterprises such as Novascreen TM and the Drug Discovery Kits marketed b y NEN@DuPont. Nevertheless, the question o f whether o r not receptor-ligand binding assays are the m o s t appropriate tool for drug discovery remains unanswered. The most significant recent advance in receptor ligandbinding techniques has b e e n the scintillation proximity assay developed by Amersham International plc. The basis of this assay [14] is a b e a d containing a fluorophore to which the receptor of interest is bound. W h e n radiolabelled ligand binds to the receptor it excites the fluo r o p h o r e and emits a signal which can b e quantitated; u n b o u n d radiolabeUed ligand is not close e n o u g h to the bead to emit a signal. Thus, separation o f b o u n d ligand from free ligand is not necessary and the radioligand binding assay has been reduced to a few simple pipetting steps, which can readily be automated.
Future developments From this brief review of recent advances in techniques, it would appear that any receptor-ligand binding interaction can be reduced, in practical terms, to a few pipetting steps and that large numbers o f samples can be examined in a very short time. Obviously, attendant problems o f computerization of data handling must also be resolved in order to achieve success. It should b e clear, however, from the foregoing discussions that the drug researcher must challenge the validity o f receptor-ligand screens particularly when the 'new receptors' are used. The availability o f techniques to operate screens in a cost effective way does not in itself confer scientific validity to the assay. Perhaps the future for drug discovery residues
I would like to thank I Chicarelli-Robinson, M Moore and D Norris for their constructive criticisms and comments.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest oo of outstanding interest 1. Wmuais M: Receptor Binding in the Drug Discovery Proeo ces$. Med ICes Rev 1991, 11:147-184. Reviewof modern receptor theory and use of receptor binding assaysin drug discovery. Provides good insights into the application of receptorbased assays in drug screening. 2 MOOREMAS:Haemopoietic Growth Factor Interactions: In oo Vitro and In Vtvo Preclinical Evaluation. Cancer Surv 1990, 9:7-80. Revaewof cytokines, colony stimulating factors and their importance in disease. Excellent background reading which identifies key targets for potential drug intervention. 3. SPRINGERT& Adhesion Receptors of the Immune System. t Nature 1990, 346:425-434. Review of adhesion molecules that identifies drug targets. 4. VANEJR, HUGGARDEE, BOTTINGRM: Regulatory Function of • the Vascular Endothelium. N Engl J Med 1990, 323:27-36. Review of receptors on the endothelium that identi~ drug targets suitable for receptor-ligand screening programmes. 5. ~ N K: Glycobiology: a Growing Field for Drug Design. • Trends Pharmacol Sci 1991, 12:265-272. Describes the numerous roles of carbohydrates in celi--cell recognition. 6. ROBINSONCJ: Multiple Receptors Found for the Growing • FGF Family. Trends Pbarmacol Sci 1991, 12:123--124. Describes the role of heparan sulphate in the binding of fibroblast growth factor to its receptor. 7. GATANAGA T, HWANGCD, KOHRW, CAPPUCCINIF, D.JCCIJA, • JEFFESEW, LEUTZR, TOMICHJ, YAMAMOTORS, GRANGERGA. Purification and Characterization of an Inhibitor (Soluble Tumor Necrosis Factor Receptor) for Tumor Necrosis Factor and Lymphotoxin Obtained from Serum Ultraffltrates of Human Cancer Patients. Proc Natl Acad Sci U S A 1990,
87:8781-8784. Describes differences in receptors in disease states. 8. DINARELI_O CA~ Interleukin-1 and interleuldn-1 antagonism. • B/ood 1991, 77:1627-1652. Describes differences in receptor types and their occurrence on different cell types. 9. FINDLAY J, EtaOPOULOSE: Three-Dimensional Modelling of • G Protein-Linked Receptors. Trends Phamna~l Sci 1990, 11:492--499. Describes relationships and similanties of seven-helical transmembrane protein receptors. 10. DOWERSK, URADLDI; The Interleukin-1 Receptor. lmmunol Today 1987, 8:46-50. 11. TIMMERMANS P, WONGPC, CHIUAT, FIERBHNWF: Nonpeptide • Angiotensin II Receptor Antagonists. Trends P h a r m a ~ l Sci 1991, 12:55-62.
57
58
Analytical biotechnolosy Describes the discovery o f a nonpeptide antagonist. 12 •.
KESSLERMJ. New Instrumentation Technology: Analyse 96 Radioactive Samples Simultaneously. Biotecbnology Int 1991, 251-256. Oudines new instrumentation for receptor-ligand assays. Describes the latest instrumentation that can be used to automate receptor-ligand screening programmes. 13. •
BURCH RM, KYLE DJ: Mass Receptor Screening for New Drugs. Pharm Res 1991, 8:141-147.
Provides practical and logistical informatton on running receptor.based assays. 14
BOSWORTH N, TOWERSP: Scintillation proximity assay. Nature
1989, 341:167-168.
I Shaw, Xenova Ixd, 240 Bath Road, Slough SL1 4EF, UK
Introduction Over the last two decades, receptor-based assays have been used in the drug discovery process to aid structure-activity relationship studies to increase etficacy, to assist in validating rational drug design and to elucidate the mechanism of action and selectivity of known drugs. Such studies have both improved upon and produced novel drugs for use in man. The widespread use of receptor*based assays has also defined receptor subtypes and refined receptor-ligand interaction theory. As new ligands have been identified, receptor theory has been modified and extended to accommodate the new findings. Modem receptor theory has been reviewed in an excellent article by Williams [1 o*] and therefore will not be covered in detail in this article. Traditional drug-discovery receptor-based assays use standard pharmacological techniques, such as tissue preparations and organ baths, to measure a functional response invoked by a compound in the presence of a known ligand. Whilst these techniques produce excellent quality data, they are labour intensive, time consuming and have a limited capacity to test large numbers of samples. The advent of simpler techniques, such as radiolabelled ligand-binding assays, has provided the opportunity for more cost effective drug discovery with a rapid tumaround of results. Most importantly, these techniques enable large numbers of compounds to be tested in the search for drug candidates, particularly where no lead molecules exist. This review will focus on the use of the simple radiolabelled ligand-binding assay, as applied to membrane
bound receptors, in the search for novel lead compounds. A second focus will be the enormous advances in techniques and instrumentation that can be applied to these receptor-based assays.
Receptors and ligands of interest The explosion of knowledge about disease mechanisms generated by the widespread use of molecular biology has identified many new receptors and their endogenous ligands as therapeutic targets. Consequently, a considerable effort is being made in the drug discovery industry to capitalize on this understanding of disease processes. Although the biotechnology industry has focused on proteins as therapeutic entities, proteins have several constraints, such as poor oral bioavailability, short half life and metabolic instability, that limit their therapeutic uses. Non-peptidyl mimetics of these receptor-ligand interactions are therefore required to fully capitalize on modern knowledge in order to produce new orally-active drugs. Recently characterized receptors and ligands include the cytokines and haemopoietic factors [2-*], the adhesion receptors of the immune system [3°], and receptors present in the endothelium that control its many functions [4*]. Complex protein-carbohydrate and protein-carbohydrate-protein interactions [5.] have yet to be incorporated fully into classical receptor theory. For example, little is known about the influence of the interactions between fibroblast growth factor and glycosaminoglycans prior to receptor occupancy [6°],
Abbreviations IL--interleukin; TNF~tumour necrosisfactor.
© Current Biology Ltd ISSN 0958-7669
55
56
Analytical biotechnology Such interactions must be understood if a receptor-based screening method is to be meaningful.
Sources of receptors and ligands Many sources of receptors can be used in radioligandbinding assays. Ideally, the receptor should be of human origin and be derived from 'disease tissue' or a relevant cell-type, as there are reports of receptor changes in disease states. For example, natural soluble tumour necrosis factor (TNF) receptor derived from cancer patients binds TNFc~ better than TNFI3, but the relevance to the disease state is unknown [7°]. The interleukin (It)-I receptor has two forms, type I, which is present on T cells and fibroblasts and type II on B cells and myeloid cells. The ligands, IL-10c and IL-113 have different affinities for these receptors, but the relationship of these differences to particular disease states is unclear [8.]. Thus there are obvious practical problems in obtaining quantities of the ideal receptor for use in a screen. Ideally, the particular shortcomings of the receptor used in a screen must be understood and complemented by the judicious selection of additional secondary assays. A second key issue that relates to the use of a receptor for screening purposes is the form in which the receptor is used in the assay. Receptors can be obtained in a number of ways: as pure recombinant proteins, as pure solubilized proteins (from cells or tissues), as cell membrane preparations, as receptors cloned into an expression system, or as receptors present on the whole cell where the integrity of the receptor depends on the viability of the host cell during the assay. The merits of each receptor source as a screening tool must be addressed in the context of present knowledge of the integrity of the receptor derived from that source. Very little is known in detail about the three-dimensional structure of receptors. Many receptors, particularly the G-protein linked receptors, are present in the cell membrane as complex seven-helical transmembrane proteins. A model of this conformation has been elucidated from studies on the membrane protein, bacteriorhodopsin [9"], which shares many similarities with receptors of pharmacological interest. The information derived from these studies highlights the complexity of receptor structure and illustrates that the precise orientation and conformation of the protein is crucial to the binding of the ligand and therefore the resultant agonist or antagonist activity of compounds. Minor perturbations in conformation will radically change the expression of binding domains. Consequently, the value of solubilized receptors, where the phospholipid (cell membrane) is replaced by detergent, becomes questionable. Williams [1"'] also questions the value of using re~eptor sources in which the receptor has been cloned into a 'foreign' cell, such that it may be linked to an inappropriate second messenger system or not linked at all. The effect of such abnormal environments on receptor-ligand interaction is unknown. Formal occupancy theory states that formation of the receptor-ligand complex is reversible, the response
evoked is proportional to the number of receptors occupied, and the biological response is equilibriumdependent. The IL-1 receptor and its two endogenous ligands, IL-ltz and IL-I~, pose problems for conventional receptor theory, particularly when they are used in a receptor-ligand binding assay. The affinity (0.2-2 x 1010 M - 1) and association and dissociation rate constants (2.8 x 1 0 8 M - l m i n - 1 and 2.1 x 1 0 - 2 M - 1 respectively) o f IL-1 [3-binding are unusual as the association rate is relatively typical for a polypeptide hormone but the dissociation rate is very slow [10]. Thus, under the experimental conditions of a radioligand binding assay, equilibrium cannot be achieved, presenting obvious problems in the interpretation of data. In spite of all of the potential problems in data interpretation and validity of receptor source, receptor-ligand binding assays provide one of the most cost effective techniques with which to discover non-peptidyl agonists or antagonists of novel receptor -ligand interactions. The obvious question is whether or not the technique can be successful. At present there are very few reports of non-peptidyl agonists or antagonists that interfere with these complex protein-protein interactions. One notable exception is the discovery of a non-peptide antagonist of angiotensin II [11°].
Sources of molecules for screening In one successful strategy for drug discovery, particularly where no lead molecules are known, libraries of chemical structures are screened for novel pharmacophores. Chemical banks numbering hundreds of thousands of compounds are present in most major pharmaceutical companies. Williams [1 °° ] however, indicates that the structure diversity of these chemical banks is often limited because of the inclusion of simple chemical substitution analogues of key compounds. Natural products provide an alternative source of molecules of almost unlimited structural diversity. Natural product libraries can be derived from microorganisms, marine organisms or plant materials. Screening of natural products, particularly those derived from microorganisms, has provided useful drugs, such as antibiotics and cytotoxic anticancer drugs. Notable examples include: the cholecystokinin receptor antagonist, asperlicin, follow-up structure activity relationship studies of which have resulted in the benzodiazepine derivative MK 329 entering a phase U clinical trial as a gastrointestinal agent; cyclosporin, FK 506 and rapamycin, which are selective immunosuppressant agents: the hypotensive agent doridosine; and the antineoplastic agents Am-C, Ara-A and taxol. The success rate of these progmmmes was influenced substantially by the diversity of the organisms screened and the number of samples screened. These factors, particularly the numerical capacity of a screen, place severe constraints o n the type of assay that can be used to detect lead molecules. There have been several significant technological advances in receptor-ligand binding assays, which open up numerous opportunities to design cost-
Receptor-based assays Shaw
effective, scientifically valid screens. From the foregoing discussion o f receptor-based assays, however, the drug researcher must be aware o f the numerous theoretical pitfalls in using a 'simple' receptor-ligand assay, particularly when applied to recently characterized receptors.
in alternative assay techniques such as second messenger systems, gene transcription and translation events. Techniques to measure these events, particularly as high capacity screens, are in their infancy.
Acknowledgements Significant
technological
advances
Advances in instrumentation over the last few years have enabled radioligand binding assays to be carried out in microtitre plates. Separation o f b o u n d radioligand from free radioligand, has been greatly facilitated b y the availability o f 96-well microtitre plate harvesters while the efficiency o f processing and quantitation o f radioactivity has been significantly improved by the development o f ]]-plate readers from LKB/Pharmacia and a microplate reader from Canberra Packard [12o-]. This technology, combined with automated pipetting w o r k stations, enables the researcher to complete 2000 conventional radioligand binding assays with the minimum a m o u n t o f handling and the minimal use o f scintiUant. As outlined by Butch [13"], further robotic instrumentation will not necessarily increase the capacity or efficiency o f this type o f assay. The ability to automate receptor-ligand binding assays has resulted in commercial enterprises such as Novascreen TM and the Drug Discovery Kits marketed b y NEN@DuPont. Nevertheless, the question o f whether o r not receptor-ligand binding assays are the m o s t appropriate tool for drug discovery remains unanswered. The most significant recent advance in receptor ligandbinding techniques has b e e n the scintillation proximity assay developed by Amersham International plc. The basis of this assay [14] is a b e a d containing a fluorophore to which the receptor of interest is bound. W h e n radiolabelled ligand binds to the receptor it excites the fluo r o p h o r e and emits a signal which can b e quantitated; u n b o u n d radiolabeUed ligand is not close e n o u g h to the bead to emit a signal. Thus, separation o f b o u n d ligand from free ligand is not necessary and the radioligand binding assay has been reduced to a few simple pipetting steps, which can readily be automated.
Future developments From this brief review of recent advances in techniques, it would appear that any receptor-ligand binding interaction can be reduced, in practical terms, to a few pipetting steps and that large numbers o f samples can be examined in a very short time. Obviously, attendant problems o f computerization of data handling must also be resolved in order to achieve success. It should b e clear, however, from the foregoing discussions that the drug researcher must challenge the validity o f receptor-ligand screens particularly when the 'new receptors' are used. The availability o f techniques to operate screens in a cost effective way does not in itself confer scientific validity to the assay. Perhaps the future for drug discovery residues
I would like to thank I Chicarelli-Robinson, M Moore and D Norris for their constructive criticisms and comments.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest oo of outstanding interest 1. Wmuais M: Receptor Binding in the Drug Discovery Proeo ces$. Med ICes Rev 1991, 11:147-184. Reviewof modern receptor theory and use of receptor binding assaysin drug discovery. Provides good insights into the application of receptorbased assays in drug screening. 2 MOOREMAS:Haemopoietic Growth Factor Interactions: In oo Vitro and In Vtvo Preclinical Evaluation. Cancer Surv 1990, 9:7-80. Revaewof cytokines, colony stimulating factors and their importance in disease. Excellent background reading which identifies key targets for potential drug intervention. 3. SPRINGERT& Adhesion Receptors of the Immune System. t Nature 1990, 346:425-434. Review of adhesion molecules that identifies drug targets. 4. VANEJR, HUGGARDEE, BOTTINGRM: Regulatory Function of • the Vascular Endothelium. N Engl J Med 1990, 323:27-36. Review of receptors on the endothelium that identi~ drug targets suitable for receptor-ligand screening programmes. 5. ~ N K: Glycobiology: a Growing Field for Drug Design. • Trends Pharmacol Sci 1991, 12:265-272. Describes the numerous roles of carbohydrates in celi--cell recognition. 6. ROBINSONCJ: Multiple Receptors Found for the Growing • FGF Family. Trends Pbarmacol Sci 1991, 12:123--124. Describes the role of heparan sulphate in the binding of fibroblast growth factor to its receptor. 7. GATANAGA T, HWANGCD, KOHRW, CAPPUCCINIF, D.JCCIJA, • JEFFESEW, LEUTZR, TOMICHJ, YAMAMOTORS, GRANGERGA. Purification and Characterization of an Inhibitor (Soluble Tumor Necrosis Factor Receptor) for Tumor Necrosis Factor and Lymphotoxin Obtained from Serum Ultraffltrates of Human Cancer Patients. Proc Natl Acad Sci U S A 1990,
87:8781-8784. Describes differences in receptors in disease states. 8. DINARELI_O CA~ Interleukin-1 and interleuldn-1 antagonism. • B/ood 1991, 77:1627-1652. Describes differences in receptor types and their occurrence on different cell types. 9. FINDLAY J, EtaOPOULOSE: Three-Dimensional Modelling of • G Protein-Linked Receptors. Trends Phamna~l Sci 1990, 11:492--499. Describes relationships and similanties of seven-helical transmembrane protein receptors. 10. DOWERSK, URADLDI; The Interleukin-1 Receptor. lmmunol Today 1987, 8:46-50. 11. TIMMERMANS P, WONGPC, CHIUAT, FIERBHNWF: Nonpeptide • Angiotensin II Receptor Antagonists. Trends P h a r m a ~ l Sci 1991, 12:55-62.
57
58
Analytical biotechnolosy Describes the discovery o f a nonpeptide antagonist. 12 •.
KESSLERMJ. New Instrumentation Technology: Analyse 96 Radioactive Samples Simultaneously. Biotecbnology Int 1991, 251-256. Oudines new instrumentation for receptor-ligand assays. Describes the latest instrumentation that can be used to automate receptor-ligand screening programmes. 13. •
BURCH RM, KYLE DJ: Mass Receptor Screening for New Drugs. Pharm Res 1991, 8:141-147.
Provides practical and logistical informatton on running receptor.based assays. 14
BOSWORTH N, TOWERSP: Scintillation proximity assay. Nature
1989, 341:167-168.
I Shaw, Xenova Ixd, 240 Bath Road, Slough SL1 4EF, UK
