, 2008) in 1536-well microtiter plates (Cassaday et al., 2007). Ensuring that the enzyme assay is performed under acceptable conditions of enzyme and substrate concentrations to make the assay sensitive to modulators of the enzyme activity is a primary

consideration for enzyme assays. However, there are several artificial mechanisms by which compounds can interfere with the enzyme assay (Thorne et al., 2010) and in many cases there are methods to directly test for these interferences (Figure 8). These include compound aggregation which non-specifically INK-128 inhibits the enzyme, enzyme inactivation mediated by a by-product from the compound sample, and direct interference with the assay signal (McGovern et al., 2003). Compounds that aggregate to form large (>100 nm) colloidal particles can sequester the target enzyme and prevent interaction with the substrate leading to inhibition (Figure 8A). These Dabrafenib chemical structure aggregates are not precipitates of the compound which could be spotted by the presence of a “cloudy” solution, but instead these are colloids which give the appearance of a clear solution and therefore specific tests are required to detect the presence of such compound aggregates. A hallmark of this effect is that the inhibition

is sensitive to non-ionic detergents such as TWEEN or Triton (0.01–0.1% can relieve the inhibition) the IC50 curves can show steep Hill slopes, and the IC50 varies with enzyme concentration. As well, the same compounds often inhibit a completely different enzyme with essentially the same potency (β-lactamase has been used as a counter-screen, Feng et al., 2007). Not all aggregates act the same way with different enzymes so one needs to specifically test for this mode of interference using the methods

listed above. Recently, a compound was identified in an HTS which activated procaspase-3 and subsequent examination showed that the nature of the activation was due the formation of a nanotube by the compound which sequestered the proenzyme to the surface, increasing the local concentration or possibly modifying the conformation buy Metformin leading to activation (Zorn et al., 2011). Certain compounds, for example ortho-quinones, in the presence of common reducing reagents such as DTT can undergo a redox reaction which leads to generation of peroxide ( Thorne et al., 2010) that inactivates the enzyme ( Figure 8B). The hallmark of this effect is that the inhibition is relieved when the DTT concentration is reduced (<1 mM) or removed from the assay or a weaker reducing reagent such as Cys is used. A high-throughput colorimetric assay using horse radish peroxidase has also been developed to directly test for compounds which produce hydrogen peroxide through redox cycling ( Johnston et al., 2008). As mentioned briefly above for the SPA format, some compounds may absorb light at the wavelength in which the assay signal is generated.