Drug Discovery
addition of protein stabilisers such as glycerol to improve target stability.
Screen the library. To increase throughput, frag- ment libraries are typically screened at one con- centration to first identify potential binders. With biosensors, screening is normally done between concentrations of 50 to 500µM. These concentra- tions are lower than those run in structural-based fragment screening studies, where a high percent- age of site occupancy is needed. Biosensors are capable of detecting binding at concentrations
that are well below the KD. The use of lower con- centrations helps reduce the number of false pos- itives and favours the selection of the higher-affin- ity fragments.
Identify the poorly behaved compounds. When reviewing the sensorgram response data, the first step in hit selection is to look at the fragments’ responses from a reference surface that has no pro- tein immobilised (Figure 4A, left panel). Plotting raw response points taken just after each com- pound test on the reference surface is used to iden- tify compounds that stick non-specifically or aggre- gate on the sensor surface itself (Figure 4A, right panel). Any compounds that show significant bind- ing to the reference surface should be omitted from further consideration. In the full 1,500-compound Ro3 library from Maybridge, only a few fragments bound to the reference surface or showed unusual injection response indicating aggregation. This low occurrence of poorly-behaved compounds is a result of the careful selection process the com- pounds have gone through in establishing this frag- ment library.
Check the control. The left panels of Figure 4B show the responses obtained for a fragment screen against two targets. Report-point trend plots (right panels of Figure 4B) of the processed response data from the surfaces are useful for visualising the behaviour of the assay over time. In this example, the binding of one control slowly decreased over the time required for this screen (Figure 4B, lower right panel). Across the panel of fragments, a majority show little or no binding to either target surface (which we would expect for a non-focused library), while a few fragments appeared to be promising hits.
Identify the selective binders using vs plots. To identify selective hits efficiently, the responses at the end of the binding phase from one target sur- face are plotted versus the responses from the other
Drug Discovery World Winter 2011/12 57
Figure 4: Biosensor-based fragment screening against two targets. (A) Raw responses from the reference surface reveal which fragments (highlighted by *) bind nonspecifically to the sensor surface. To easily identify the non-specific binders, the responses at 27 sec are plotted in a trend plot. (B) Double-referenced responses of the fragment library binding to two fragments. Responses at 15 sec are shown in the trend plots, with the replicate tests of the control compounds indicating the stability of the targets across the entire screen. (C) Versus plot of the responses from target 1 plotted against the responses from target 2. Blue and red indicate the replicate controls from each target surface and the orange and green indicate selective fragments chosen for follow-up analyses. The dashed line indicates fragments that bind similar to both targets
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80