Reducing the conformational freedom of a flexible ligand, for example by macrocyclization, can result in an increased binding affinity by lowering the energetic cost of adopting the conformation required for binding to the protein receptor. However, this affinity increase will only be fully realized if the resulting macrocycle, in its unbound state, is constrained in the correct conformation to complement the binding site on the protein so that the compound can bind without strain. If macrocyclization instead locks the compound into a conformation significantly different from the optimal bound conformation, binding to the protein target will require the ligand to assume enthalpically costly strain which will proportionately reduce the binding affinity. Due to this interplay between conformational pre-organization and strain, it can be difficult to discern the extent to which each of these two factors are influencing affinity. The conformations available to a molecule can be represented in terms of a potential energy well in which the lowest energy conformations correspond to a set of minima at the bottom of the well, and the barriers that separate these minima correspond to the kinetic barriers for interconversion between the low energy conformational states (Figure ). In solution, a flexible molecule will exist as an ensemble of these low energy conformations. Other conformations of substantially higher energy will also exist, but these will be scarcely populated at equilibrium. As illustrated in Figure , a non-macrocyclic molecule with multiple rotatable bonds will typically have many energetically accessible conformations. When this acyclic ligand binds to its protein target there will be negligible strain provided that the bound conformation is among the many low energy states available to the compound. If this compound is cyclized, thereby restricting rotation around certain bonds, the number of low energy conformations available to the compound will substantially decrease because conformations incompatible with the ring structure become disallowed. If the optimal bound conformation is among those still available to the cyclized compound, cyclization will be expected to increase the binding affinity because the entropic cost of assuming the bound conformation has been reduced (Figure ). On the other hand, if the bound conformation is not among the available low energy state for the cyclized compound, to bind to the target site the compound must adopt a strained, higher energy conformational state.