Therefore, there must always be a balance between signal window (Iversen et al., 2006) and desired inhibitor modality when choosing a substrate concentration. As well, one has to be careful to ensure that the amount of substrate turned-over is kept low when optimizing the signal window of the assay to ensure the identification of weak inhibitors (Inglese et al., 2007) (Figure 4). It is important to keep in mind that while running the assay at high conversions (>80%) may greatly improve the

signal window, such high levels of conversion will lead to weaker IC50s (Wu et al., 2003). Finally, substrate concentrations can be limited by the ability of the substrate to dissolve in the assay buffer, limiting the top concentration possible, and the types of inhibitors that can be identified. Solubility limitations should manifest themselves as a poorly fit Ruxolitinib purchase Michaelis–Menten

curve, Ku-0059436 molecular weight which result in an uncharacteristic plateau of the rate and can also result in a drop in enzyme activity at high concentrations due to substrate aggregates which decrease the concentration of substrate below the solubility limit. The physical properties of substrate molecules should be considered when adapting a biochemical assay to HTS. Because of the large number of compounds that need to be screened, automation using robotic systems is often employed. Automated protocols can involve leaving reagents for extended periods of time under conditions where the substrate is sup-optimal for stability, ultimately leading to degradation of the substrate over time. In addition, substrate molecules could interact poorly with the tubing and surfaces involved in the automation of dispensing Exoribonuclease assay plates for HTS. Stability

tests should be performed early on in the assay optimization process to identify stability effects which might occur on the HTS system and modifications made to address any issues that are identified. Many enzymes require cofactors for structural integrity or that assist in the reaction of substrate to product. A cofactor can remain unchanged during the reaction, or may cycle through various states during the reaction cycle. However, by definition the cofactor is not consumed in the reaction, and instead returns to its original state, able to participate in the reaction over and over again. Cofactors can be tightly bound, never truly dissociating from an enzyme or they can be transient, binding and dissociating in equilibrium. Common cofactors include metals (a zinc ion bound at the active site; coordinating magnesium; iron which exists in various redox states for catalysis) and organic compounds (FAD/FADH2 involved in hydride transfer; PLP in transamination reactions).