Macrocyclization can increase binding affinity by up to 3-fold per rotatable bond constrained in the macrocycle ring. This effect results from restricting the conformations available to the compound to a subset that includes the bound conformation, reducing the energetic cost of adopting the bound conformation. To gain these benefits, it is important that the conformation that is enforced upon the molecule through macrocyclization does not hold the molecule rigidly in a way that differs significantly from the bound conformation. In such cases, adopting the bound conformation will cause strain in the ligand that will weaken binding. For this reason, initial macrocycle designs sometimes do not exhibit significantly higher affinity than their acyclic precursor, and subsequent optimization of the linker length, geometry, and rigidity can be required to achieve a balance between pre-organization and strain that more fully captures the benefits of cyclization.