Drug Discovery
purity analysis, is essential. Regardless of the bio- physical technique employed for screening, the fragments must be soluble in aqueous media at high enough concentrations for weak binding interactions to be measured. Poor fragment solu- bility can also compromise the robustness of the screening data through aggregation and promiscu- ous inhibition34. Although solubility is related to lipophilicity, other factors affecting solubility are difficult to model, therefore experimental determi- nation of fragment solubility is critical.
Experimental solubility measurement Practical methods for the determination of aque- ous solubility of fragment molecules are not well described in the literature. A new, high-through- put solubility measurement protocol was devel- oped for the Maybridge Ro3 libraries, using a Stem Clarity Solubility Station with IR transmis- sion measurement giving a ‘soluble’ or ‘insolu- ble’ result for each fragment at 200mM DMSO, 5mM aqueous pH 7.5 phosphate buffer (con- taining 2.5% DMSO) and 1mM aqueous buffer (containing 0.5% DMSO). The cut-off transmis- sion value below which a compound was deemed to be insoluble, was validated by visually exam- ining the sample tubes in a 1,000 subset35. A correlation of 96.9% was achieved between the transmission result and the visual result. The full set of 4,000 Ro3 compliant fragments, pre-fil- tered using the parameters detailed in Figure 1, were subjected to the solubility measurement protocol. The percentage of insoluble com- pounds found in each MW range at concentra- tions of 5mM and 1mM in aqueous buffer is shown in Figure 2. There is a clear correlation between increasing MW and poor solubility and this data lends more credence to the view that ‘smaller is better’, when selecting fragments for inclusion in a library.
Assessing fragment binding using biosensor technology
Figure 3: Pre-screening control tests in SPR. (A) Control compounds are tested for binding to the primary target, as well as a reference surface and a control protein, such as a secondary target. (B) Activity and selectivity test. Responses and binding isotherms for concentration series of two control compounds demonstrate both immobilised targets are active and selectively bind one control compound or the other. (C) Stability test. Over several hours, repeated analyses of the controls tested at one concentration reveal a gradual loss in activity for one target
Once developed, the quality of a fragment library can be put to the test by screening it against targets. Structural methods, such as NMR and crystallography, are commonly used to identify the relative positions of, and specific contacts between, a fragment and its target. While these methods can provide high-resolution detail about the binding interface, they require relatively large amounts of reagents, have limit- ed throughput, and often do not provide insight into the strength of a binding interaction. In contrast, label-free interaction analyses, such as
54 Drug Discovery World Winter 2011/12
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