- Email: [email protected]
Probing protein surfaces for `hot spots': a new frontier
FORUM
Europe would be excluded if the Directive was adopted in its present form (draft dated 29 August 1997). D. Vandergheynst (European Commission, Brussels, Belgium) provided a progress report. The Directive was still at a draft stage, with some of the language still in need of further refinement. The new Article 5(3) introduces the need to disclose the function of a (partial) sequence in an application. There was concern among delegates that this may be setting a higher standard than the present need to satisfy industrial applicability under Article 57 of the EPC. The need for Article 11, the so-called ‘farmer’s privilege’, was questioned by B. Nash (Monsanto Services International, Brussels, Belgium), who said that it would be contrary to the Trade-related Aspects of Intellectual Property agreement. Furthermore and in practice, seed companies were unlikely to take legal action against individual farmers for patent infringement. H. Kolb (Hoffman-Eitle, Munich, Germany) identified two recent important decisions from the German courts that could provide important precedents elsewhere in Europe. In ‘Clinical Trials II’9, the court broadened the ‘experimental-use exemption’ to encompass clinical trials involving a known patent-protected medicament that were used to obtain
Letter
Probing protein surfaces for ‘hot spots’: a new frontier here has been a tremendous burst T of progress over the past few years in streamlining the drug-discovery process and augmenting its technological capabilities1. Programmes in the pharmaceutical industry are now being geared up to exploit highthroughput screening of smallmolecule combinatorial libraries against the validated targets emerging from research in functional genomics and proteomics. Clearly, the process by which leads are generated and elaborated into drug candidates has been accelerated, with the expectation that drug pipelines will be easier to fill in the future.
198
new knowledge, even if there was a commercial objective underlying the activity. In ‘Oberflachenaktives Material’10, the court considered for the first time infringement of a ‘product-by-process’ claim that included the term ‘obtainable by’. The use of a different process does not necessarily avoid such a claim. Furthermore, if the process featured in the product claim imparts a specific characteristic to the claim product, this characteristic had to be read into the claim. N. Byrne (Queen Mary College, London, UK) assessed the EU Directive on the Legal Protection of Databases. This directive has created a new sui generis right to protect the ‘maker’ of a database against unauthorized extraction and/or reutilization of the database’s content, as well as harmonizing copyright protection for the selection or arrangement of the contents of a database and its structure. It would, however, seem that some of the concepts and terms used in the directive did not fit well with the types of database used in genomics research. A. N. Nielson (Forrester, Norall and Sutton, Brussels, Belgium) invited the audience to consider whether the ‘Magill’ judgement and the ‘essential facilities’ doctrine of the EU had any relevance to biotechnology patents. Such patents had the potential to give
However, the full potential for developing intervention strategies depends not only on screening vast numbers of compounds against a validated target but also on exploiting the full panoply of mechanisms and loci available for direct attack on a specific target protein, in a focused way. Despite numerous advances in structural biology, enabling the visualization of proteins in considerable geometrical detail, drug strategies continue to centre on endogenous ligand-binding sites; strategies designed to capitalize on the possibilities for intervention along the entire protein surface have been slow to evolve.
rise to a dominant position that might be challenged by a third party seeking to enter the market, especially if certain aspects were not being fully exploited by the patentee. The EU Commission was understood to have already received complaints using such arguments. The meeting proved to be of great value in clarifying various issues of concern to those involved with biotechnology patents, in particular, the very negative attitude of the Patent Offices towards the patentability of ESTs, as well as the perennial topics of claim breadth and sufficiency.
References 1 2 3 4 5 6 7 8
9 10
T939/92; OJ EPO 1996 309 148USPQ681 Sup. Ct 1996 26USPQ2d 1529 Fed Cir. 1993 34USPQ2d 1210 Fed Cir. 1995 41USPQ2d 1172 Board of Patent Appeals and Interferences T694/92; OJ EPO 1997 408 Biogen Inc. v Medeva plc (1997) RPC 1 The Regents of the University of California v. Eli Lilly and Co., Appeal No. 96-1175, Fed Cir. 1997, decided 22 July 1997 XZR 68/94, 17 April 1997 LG Dusseldorf 4 0 265/95, 6 August 1996
Chris Connell SmithKline Beecham, New Frontiers Science Park, Third Avenue, Harlow, Essex, UK CM19 5AX. (E-mail: [email protected])
To be sure, known ligands tend to concentrate at a small number of sites, and are often competitive with physiological substrates and effector molecules. It is, however, well known that sites other than endogenous ligand-binding sites of proteins can be exploited in the development of tight-binding small molecules, as shown by numerous examples of noncompetitive antagonists of receptors. Also, a recent study of the structural basis of the noncompetitive inhibition of the enzyme glutathione reductase by a xanthene inhibitor highlights the point that significant disruptions in substrate binding can be induced by compounds binding at sites that do not overlap with substrate-binding sites2. It is also noteworthy that specific complementaritydetermining regions of antibodies and their inhibitory effects can be mimicked by small molecules, which
Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0167 – 7799/98/$19.00. PII: S0167-7799(98)01177-9
TIBTECH MAY 1998 (VOL 16)
FORUM
provides a rationale for targeting various epitopes along the protein surface with such entities3. Furthermore, ample precedents exist for reagents that label nonessential residues of enzymes, yet profoundly alter the catalytic activities of the enzyme4. This phenomenon has been clearly demonstrated by sitedirected-mutagenesis studies of enzymes, in which the labelling of specific polar groups abolishes activity, whereas the corresponding substitutions of aliphatic for polar groups have little effect on activity. In such examples, steric effects imposed on the labelled groups are presumed to account for enzyme inactivation. Given the structural framework of proteins (which shows them to be composed of mobile loops, flaps and hinges, and to contain channels that provide access to functionally important binding sites, thereby rendering them vulnerable to blockade at numerous sites), a broader view of intervention strategies in therapeutics is necessary. For example, it seems intuitively obvious that for aspartyl proteases like HIV protease, preventing the free movement of the ‘flap’, which undergoes displacements as large as 7 Å upon binding its ligands, is likely to alter the protease activity of these enzymes significantly5. Conceptually, it is useful to regard enzymes as machines that perform chemical reactions upon binding their substrates6, and to consider receptors as machines that trigger functional events upon binding their ligands. Indeed, if proteins are complex molecular machines with critical mobile parts, there should be many ways to interfere with their functions. Implicit in this view is a paradigm shift for drug targeting that has important functional consequences. In the case of enzymes, the shift is from the narrow point of focus of the active site as the enzyme’s Achilles’ heel to the broader notion of the holoenzyme functioning as a chemical machine, with its attendant vulnerabilities.
Potentially, there is a rich fallout emanating from such a shift in perspective. The implications for theory and experiment are significant. How does one predict and discover which sites (‘hot spots’) render a target protein inactive when occupied? (In fact, the first steps towards the development of a crystallographic method that allows mapping of the binding surface of a protein by solving its crystal structure in a variety of organic solvents have recently been reported7.) How can these sites be exploited to the fullest extent in lead discovery and optimization? A better understanding of the chemical and kinetic mechanisms of noncompetitive and uncompetitive enzyme inhibition should emerge. Improvements can be anticipated in our ability to achieve ground-state affinity labelling of receptors, which has often proved to be elusive. Most importantly for therapeutic intervention, the discovery and identification of several hot spots along a protein’s surface would create multiple options for controlling protein activity with diverse small-molecule drugs. Having a number of smallmolecule leads that differ in their loci of action should enable the researcher to create series of useful compounds, and to develop each series independently according to the structural dictates of the corresponding target sites. This is an important advantage and allows problems associated with a particular series to be circumvented, especially when a specific site of action may be associated with toxicities through homologies to other proteins. In principle, the ability to pinpoint hot spots along a protein’s surface can be developed in reversible and irreversible formats. The aforementioned approach3, involving complementarity-determining regions of antibodies, provides a reversible basis for targeting epitopes along the protein surface with small molecules. A strategy founded on the use of affinity-labelling combinatorial libraries involves the use of irreversible tags or labels. In this system, a target protein
is incubated with affinity-labelling libraries consisting of molecules with variable affinity groups, each linked to an identical labelling entity. The expectation is that library members bond rapidly only to complementary sites on the protein with nearby reactive groups, events that can be monitored by kinetic assays. The site of action of a library member that rapidly and irreversibly binds to its target can be determined in a straightforward manner by enzymatic digestion of the labelled protein and sequencing of the labelled peptides using a combination of high-performance liquid chromatography and mass spectrometry. Such bonding combinatorial libraries have been produced8,9 and provide a powerful means of probing the protein surface globally for hot spots. Drug discovery and drug development are ‘numbers games’ in which the odds are heavily weighted against success. Only a fraction of potential intervention sites are likely to be uncovered by current screens. Sensitive probes that are capable of revealing novel binding sites offer a basis for diversifying risk by significantly expanding the opportunities for blocking protein functional activities.
References 1 Persides, A. (1997) Nat. Biotechnol. 15, 1409–1411 2 Savvides, S. N. and Karplus, P. A. (1996) J. Biol. Chem. 271, 8101–8107 3 Saragovi, H. U. et al. (1991) Science 253, 792–795 4 Jordan-Starck T. C. and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17919–17923 5 Miller, M. et al. (1989) Science 246, 1149–1154 6 Williams, R. J. P. (1993) Trends Biochem. Sci. 18, 115–117 7 Allen, K. N. et al. (1996) J. Phys. Chem. 100, 2605–2611 8 Krantz, A., Hanel, A. and Huang, W. (1997) Patent number PCT/US97/00269 9 Krantz, A., Hanel, A. and Huang, W. (1997) Patent number PCT/US97/16435
Allen Krantz B.Therapeutics, 350 Sharon Park Drive, Menlo Park, CA 94025, USA.
The Forum section of TIBTECH is a platform for debate and analytical discussion of newly reported advances – whether in the research literature or at conferences. It includes Workshops, Meeting reports, and Letters to the Editor. If you would like to contribute, please contact the Editor.
TIBTECH MAY 1998 (VOL 16)
199
Europe would be excluded if the Directive was adopted in its present form (draft dated 29 August 1997). D. Vandergheynst (European Commission, Brussels, Belgium) provided a progress report. The Directive was still at a draft stage, with some of the language still in need of further refinement. The new Article 5(3) introduces the need to disclose the function of a (partial) sequence in an application. There was concern among delegates that this may be setting a higher standard than the present need to satisfy industrial applicability under Article 57 of the EPC. The need for Article 11, the so-called ‘farmer’s privilege’, was questioned by B. Nash (Monsanto Services International, Brussels, Belgium), who said that it would be contrary to the Trade-related Aspects of Intellectual Property agreement. Furthermore and in practice, seed companies were unlikely to take legal action against individual farmers for patent infringement. H. Kolb (Hoffman-Eitle, Munich, Germany) identified two recent important decisions from the German courts that could provide important precedents elsewhere in Europe. In ‘Clinical Trials II’9, the court broadened the ‘experimental-use exemption’ to encompass clinical trials involving a known patent-protected medicament that were used to obtain
Letter
Probing protein surfaces for ‘hot spots’: a new frontier here has been a tremendous burst T of progress over the past few years in streamlining the drug-discovery process and augmenting its technological capabilities1. Programmes in the pharmaceutical industry are now being geared up to exploit highthroughput screening of smallmolecule combinatorial libraries against the validated targets emerging from research in functional genomics and proteomics. Clearly, the process by which leads are generated and elaborated into drug candidates has been accelerated, with the expectation that drug pipelines will be easier to fill in the future.
198
new knowledge, even if there was a commercial objective underlying the activity. In ‘Oberflachenaktives Material’10, the court considered for the first time infringement of a ‘product-by-process’ claim that included the term ‘obtainable by’. The use of a different process does not necessarily avoid such a claim. Furthermore, if the process featured in the product claim imparts a specific characteristic to the claim product, this characteristic had to be read into the claim. N. Byrne (Queen Mary College, London, UK) assessed the EU Directive on the Legal Protection of Databases. This directive has created a new sui generis right to protect the ‘maker’ of a database against unauthorized extraction and/or reutilization of the database’s content, as well as harmonizing copyright protection for the selection or arrangement of the contents of a database and its structure. It would, however, seem that some of the concepts and terms used in the directive did not fit well with the types of database used in genomics research. A. N. Nielson (Forrester, Norall and Sutton, Brussels, Belgium) invited the audience to consider whether the ‘Magill’ judgement and the ‘essential facilities’ doctrine of the EU had any relevance to biotechnology patents. Such patents had the potential to give
However, the full potential for developing intervention strategies depends not only on screening vast numbers of compounds against a validated target but also on exploiting the full panoply of mechanisms and loci available for direct attack on a specific target protein, in a focused way. Despite numerous advances in structural biology, enabling the visualization of proteins in considerable geometrical detail, drug strategies continue to centre on endogenous ligand-binding sites; strategies designed to capitalize on the possibilities for intervention along the entire protein surface have been slow to evolve.
rise to a dominant position that might be challenged by a third party seeking to enter the market, especially if certain aspects were not being fully exploited by the patentee. The EU Commission was understood to have already received complaints using such arguments. The meeting proved to be of great value in clarifying various issues of concern to those involved with biotechnology patents, in particular, the very negative attitude of the Patent Offices towards the patentability of ESTs, as well as the perennial topics of claim breadth and sufficiency.
References 1 2 3 4 5 6 7 8
9 10
T939/92; OJ EPO 1996 309 148USPQ681 Sup. Ct 1996 26USPQ2d 1529 Fed Cir. 1993 34USPQ2d 1210 Fed Cir. 1995 41USPQ2d 1172 Board of Patent Appeals and Interferences T694/92; OJ EPO 1997 408 Biogen Inc. v Medeva plc (1997) RPC 1 The Regents of the University of California v. Eli Lilly and Co., Appeal No. 96-1175, Fed Cir. 1997, decided 22 July 1997 XZR 68/94, 17 April 1997 LG Dusseldorf 4 0 265/95, 6 August 1996
Chris Connell SmithKline Beecham, New Frontiers Science Park, Third Avenue, Harlow, Essex, UK CM19 5AX. (E-mail: [email protected])
To be sure, known ligands tend to concentrate at a small number of sites, and are often competitive with physiological substrates and effector molecules. It is, however, well known that sites other than endogenous ligand-binding sites of proteins can be exploited in the development of tight-binding small molecules, as shown by numerous examples of noncompetitive antagonists of receptors. Also, a recent study of the structural basis of the noncompetitive inhibition of the enzyme glutathione reductase by a xanthene inhibitor highlights the point that significant disruptions in substrate binding can be induced by compounds binding at sites that do not overlap with substrate-binding sites2. It is also noteworthy that specific complementaritydetermining regions of antibodies and their inhibitory effects can be mimicked by small molecules, which
Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0167 – 7799/98/$19.00. PII: S0167-7799(98)01177-9
TIBTECH MAY 1998 (VOL 16)
FORUM
provides a rationale for targeting various epitopes along the protein surface with such entities3. Furthermore, ample precedents exist for reagents that label nonessential residues of enzymes, yet profoundly alter the catalytic activities of the enzyme4. This phenomenon has been clearly demonstrated by sitedirected-mutagenesis studies of enzymes, in which the labelling of specific polar groups abolishes activity, whereas the corresponding substitutions of aliphatic for polar groups have little effect on activity. In such examples, steric effects imposed on the labelled groups are presumed to account for enzyme inactivation. Given the structural framework of proteins (which shows them to be composed of mobile loops, flaps and hinges, and to contain channels that provide access to functionally important binding sites, thereby rendering them vulnerable to blockade at numerous sites), a broader view of intervention strategies in therapeutics is necessary. For example, it seems intuitively obvious that for aspartyl proteases like HIV protease, preventing the free movement of the ‘flap’, which undergoes displacements as large as 7 Å upon binding its ligands, is likely to alter the protease activity of these enzymes significantly5. Conceptually, it is useful to regard enzymes as machines that perform chemical reactions upon binding their substrates6, and to consider receptors as machines that trigger functional events upon binding their ligands. Indeed, if proteins are complex molecular machines with critical mobile parts, there should be many ways to interfere with their functions. Implicit in this view is a paradigm shift for drug targeting that has important functional consequences. In the case of enzymes, the shift is from the narrow point of focus of the active site as the enzyme’s Achilles’ heel to the broader notion of the holoenzyme functioning as a chemical machine, with its attendant vulnerabilities.
Potentially, there is a rich fallout emanating from such a shift in perspective. The implications for theory and experiment are significant. How does one predict and discover which sites (‘hot spots’) render a target protein inactive when occupied? (In fact, the first steps towards the development of a crystallographic method that allows mapping of the binding surface of a protein by solving its crystal structure in a variety of organic solvents have recently been reported7.) How can these sites be exploited to the fullest extent in lead discovery and optimization? A better understanding of the chemical and kinetic mechanisms of noncompetitive and uncompetitive enzyme inhibition should emerge. Improvements can be anticipated in our ability to achieve ground-state affinity labelling of receptors, which has often proved to be elusive. Most importantly for therapeutic intervention, the discovery and identification of several hot spots along a protein’s surface would create multiple options for controlling protein activity with diverse small-molecule drugs. Having a number of smallmolecule leads that differ in their loci of action should enable the researcher to create series of useful compounds, and to develop each series independently according to the structural dictates of the corresponding target sites. This is an important advantage and allows problems associated with a particular series to be circumvented, especially when a specific site of action may be associated with toxicities through homologies to other proteins. In principle, the ability to pinpoint hot spots along a protein’s surface can be developed in reversible and irreversible formats. The aforementioned approach3, involving complementarity-determining regions of antibodies, provides a reversible basis for targeting epitopes along the protein surface with small molecules. A strategy founded on the use of affinity-labelling combinatorial libraries involves the use of irreversible tags or labels. In this system, a target protein
is incubated with affinity-labelling libraries consisting of molecules with variable affinity groups, each linked to an identical labelling entity. The expectation is that library members bond rapidly only to complementary sites on the protein with nearby reactive groups, events that can be monitored by kinetic assays. The site of action of a library member that rapidly and irreversibly binds to its target can be determined in a straightforward manner by enzymatic digestion of the labelled protein and sequencing of the labelled peptides using a combination of high-performance liquid chromatography and mass spectrometry. Such bonding combinatorial libraries have been produced8,9 and provide a powerful means of probing the protein surface globally for hot spots. Drug discovery and drug development are ‘numbers games’ in which the odds are heavily weighted against success. Only a fraction of potential intervention sites are likely to be uncovered by current screens. Sensitive probes that are capable of revealing novel binding sites offer a basis for diversifying risk by significantly expanding the opportunities for blocking protein functional activities.
References 1 Persides, A. (1997) Nat. Biotechnol. 15, 1409–1411 2 Savvides, S. N. and Karplus, P. A. (1996) J. Biol. Chem. 271, 8101–8107 3 Saragovi, H. U. et al. (1991) Science 253, 792–795 4 Jordan-Starck T. C. and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17919–17923 5 Miller, M. et al. (1989) Science 246, 1149–1154 6 Williams, R. J. P. (1993) Trends Biochem. Sci. 18, 115–117 7 Allen, K. N. et al. (1996) J. Phys. Chem. 100, 2605–2611 8 Krantz, A., Hanel, A. and Huang, W. (1997) Patent number PCT/US97/00269 9 Krantz, A., Hanel, A. and Huang, W. (1997) Patent number PCT/US97/16435
Allen Krantz B.Therapeutics, 350 Sharon Park Drive, Menlo Park, CA 94025, USA.
The Forum section of TIBTECH is a platform for debate and analytical discussion of newly reported advances – whether in the research literature or at conferences. It includes Workshops, Meeting reports, and Letters to the Editor. If you would like to contribute, please contact the Editor.
TIBTECH MAY 1998 (VOL 16)
199