A particularly interesting and well-characterized example of macrocyclization-induced affinity increase in a kinase inhibitor is the PI3K/mTOR/PIM inhibitor 7 recently reported by Martinez-Gonzalez et al. The authors addressed the issue directly by dissecting the optimized macroyclic inhibitor in several different ways and assessing the activity of these different acyclic congeners against each of the three kinase targets in in vitro assays. The results of the most instructive dissections are shown in Figure . Specifically, comparison of 8 with 7 showed that macrocyclization had a negligible effect on potency towards mTOR, gave roughly a 10-fold increase in potency towards PI3K, but increased potency towards PIM1 by >500-fold. The alternative dissection of 7 to give the acyclic 9 gave largely similar results. Given that 7 contains no atoms or groups that are not also present in the acyclic 8, and molecular modeling showed these compounds to share similar binding modes, essentially the entire affinity difference for PIM1 can presumably be attributed to conformational pre-organization in the macrocycle, amounting to 3.8 kcal/mol of binding energy. In PI3K and mTOR, this entropic advantage must be offset by ~2.4 and ~3.8 kcal/mol of strain, respectively, in the bound complex. The strong target dependency of the affinity enhancement highlights that small differences in the shape and composition of the kinase binding site can have a large impact on the amount of strain present in the bound state, and thus on binding affinity, consistent with the general experience that optimizing macrocycle affinity and selectivity typically requires substantial empirical SAR work.