Drug Discovery
References 1 Hajduk, PJ, Greer, J. Nat. Rev. Drug Discov., 2007, 6, 211. 2 Hubbard, RE, Murray, JB. Methods in Enzymology, 2011, 493, 509-531. 3 Chessari, G, Woodhead, AJ. Drug Discov. Today, 2009, 14, 668-675. 4 Congreve, M, Chessari, G, Tisi, D, Woodhead, AJ. J. Med. Chem., 2008, 51, 3661-3680. 5 Lipinski, CA, Lombardo, F, Dominy, DW, Feeney, PJ. Adv. Drug Deliv. Rev., 1997, 23 3-25. 6 Lipinski, CA, Lombardo, F, Dominy, DW, Feeney, PJ. Adv. Drug Deliv. Rev., 2001, 46 3-26. 7 Hann, MM, Leach, AR, Harper, GJ. Chem. Inf. Comput. Sci.; 2001, 41, 856-864. 8 Murray, CW, Rees, DC. Nature Chemistry 2009, 1, 187-192. 9 DegJarlais, RE. Methods in Enzymology, 2011, 493, 137-155. 10 Chen, IJ, Hubbard, RE. J. Comput. Aid. Mol. Des., 2009, 23, 603-620. 11 Whittaker, M, Hesterkamp, T, Barker, J. Europ. BioPharm. Rev., Autumn 2006. A Cohesive view of Fragments. 12 Hubbard, RE, Davis, B, Chen, I, Drysdale, M. J. Curr. Top. Med. Chem., 2007, 7 1568. 13 Blomberg, N, Cosgrove, DA, Kenny, PW, Kolmodin, K. J. Comput. Aided Des., DOI 10.1007/s10822-009-964-5. 14 Congreve, M, Carr, R, Murray, C, Jhoti, H. Drug Dis. Today, 2003, 8, 876-877. 15 Hajduk, PJ. J. Med. Chem,., 2006, 49, 6972-6972. 16 English, AC, Groom, CR, Hubbard, RE. Protein Eng., 2001, 14, 47-59. 17 Lepre, CA. Meth. In Enzym., 2011, 493, 219-237. 18 Leach, AR, Hann, MM, Burrows, JN, Griffen, EJ. Mol. BioSyst., 2006, 2, 429-446. 19 Butina, D, Gola, JMR. Chem. Inf. Comput. Sci., 2003, 43, 837-841. 20 Baurin, N, Baker, R, Richardson, C, Chen, I, Foloppe, N, Potter, A, Jorden, A, Roughley, S, Parratt, M, Greaney, P, Morley, D, Hubbard, R. J. Chem. Inf. Comput. Sci., 2004, 44, 643-651.
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surface plasmon resonance (SPR) biosensor tech- nology, have rapidly evolved as the methods of choice for screening fragment libraries upstream of structural analysis36,37. Biosensor technology’s advantages include its low sample consumption (typically 1-5µg of target to create a reaction surface and <5µL of each frag- ment (prepared at 10mM)) and relatively high throughput. A significant benefit of biosensor tech-
nology is the ability to determine both affinity (KD, equilibrium dissociation constant) and specificity. Establishing that a fragment binds in a stoichio- metric manner to the target is an unappreciated benefit of the methodology. Historically, biosensor technology was used to characterise protein/protein and antibody/antigen interactions because it was thought that the tech- nology lacked the sensitivity to be useful in small molecule analyses. Fortunately, over the past 10 to 15 years improvements in experimental design and data processing have been adopted through- out the user community to a point where small molecule analysis has become fairly routine. Additionally, advances in instrument hardware have improved both sensitivity and sampling throughput38. GE Healthcare’s Biacore 4000 platform can test four samples over four target surfaces at one time. Bio-Rad’s ProteOnXPR36 can measure six samples over six targets. ForteBio’s Octet384 can be configured with up to 16 sensor tips for higher parallel processing. Even plate based label-free systems such as SRU Biosystems’ BIND technology are being effective- ly utilised to triage fragments prior to screening on a target to identify poorly behaved (eg ‘sticky’ or insoluble) fragments. And, towards the next step in increased throughput, ICx Nomadics’ SensiQ can automatically dilute analytes to test a gradient of concentrations for each fragment within a 96- or 384-well plate, thereby combining preliminary screening and follow-up affinity test- ing into one assay39. Today’s sensor technology is readily capable of screening libraries of several thousand compounds. In addition, most biosensor platforms have the capability to screen each fragment in parallel against multiple targets. This means one can select for fragments that bind only to the target of inter- est. Identifying these selective hits is essential for a successful fragment screen. Novice users are sur- prised to see how often small molecules bind indis- criminately to proteins when the compounds are assayed at high concentrations. Fortunately, biosensor technology can be used to identify the selective compounds.
Basic steps in biosensor-based fragment screening Start with an active target. Since most biosensor technologies are essentially mass based, the bind- ing of low-molecular-mass analytes inherently pro- duces small changes in the biosensor binding response. The quality of the results is directly pro- portional to the quality of the starting material. Unlike enzymatic assays that can often be conduct- ed on material with low specific activity, biosensor analysis requires high activity to begin with.
Immobilise the target and control protein on the sensor surface. Biosensor analyses require that the targets be tethered to the sensing surface. For frag- ment screening, this is actually advantageous because the same sensor surface can be used to sam- ple many fragments. Proteins can be immobilised using a variety of chemistries ranging from amine-, carboxyl-, and thiol-coupling to capturing methods including biotinylation, poly-His-fusions and GST- fusions. The best practice is to try different coupling methods and select the one that retains the highest functional activity of the target and then mimic those conditions for the control protein. The con- trol can be unrelated to the target or something more specific to the target class, depending on the goals of the fragment screen. However, a successful fragment screen requires equal attention be paid to the control protein as to the target. As illustrated by the cartoon in Figure 3A, the control protein can even be an unrelated second target, which means the fragment library can simultaneously be screened against two targets.
Establish target activity and stability. Prior to run- ning a full fragment screen, a positive control com- pound is often used to confirm the immobilised target is active. It is possible to run a fragment screen without a positive control, but in those cases it is imperative to have good control protein sur- faces to help with the hit selection process. For enzymatic systems, substrates can be a good start- ing point for a control. The responses produced from concentration series of the control com- pounds confirm the targets are active enough to detect small molecule binding and the controls bind selectively to one target or the other, as well as reveal the range of signals one can expect to see for fragments (Figure 3B). To establish the stabili- ty of the target surfaces, the binding of control compounds are tested repeatedly over time (Figure 3C). In cases where the target rapidly loses activi- ty, many biosensor systems can support analysis at lower temperatures (for example, at 4˚C) and/or
Drug Discovery World Winter 2011/12
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