Fragment-based Drug Design (FBDD) has emerged as one of the key strategies in drug design during the past two decades. FBDD hinges on the fact that fragments, as opposed to complete ligands, are easier to optimize and study since they possess lesser molecular complexity and have fewer binding interactions.
When fragments are optimized to bind to parts of a protein's active site, they can gain powerful binding affinity by being linked together. Usually fragments are relatively weak binders, and connecting them with linkers can provide orders of magnitude improvements in the free energy of binding. The reason for this affinity increase is usually stated to be entropic. The rationale is that when two fragments are linked together, the entropic cost that they would have to pay were they to separately bind is already paid for by the linker. Thus the combined free energy is much more favorable than the pairwise free energy. The increase in free energy is quantified by a number called the "linking coefficient", where a value of less than zeo indicates enhanced binding relative to the fragments.
However, such an analysis assumes that the contribution to the binding process from other factors is minimal, and the entropic advantage is the only major player in the affinity increase game. But as with protein-ligand interactions, the picture is more complex. There can be unfavorable enthalpic contributions from the fragments losing favorable binding interactions upon being constrained by linkers. There can sometimes be favorable enthaplic interactions from new contacts between the ligand and protein. And there can be enthalpic and entropic contributions from the linker itself. Thus, dissecting the factors that go into the free energy upon fragment linking is like the classic conundrums of physical organic chemistry which I encountered in college, where controlled experiments are deviously hard, and changing one factor inevitably changes another (when does it not?).
An ideal system for studying the contribution of entropy to FBDD would be a system of two fragments binding to a protein which can be linked together by a single bond and which retain all their existing contacts with the protein upon being linked. Such systems would admittedly be hard to design, but a group of Italian researchers has come up with a neat system linking two very simple fragments, a hydroxamic acid and a benzenesulfonamide, with a single bond.
The fragments and resulting molecule inhibit matrix metalloproteinase (MMP), a key enzyme implicated in cancer and inflammation. The authors perform x-ray crystallographic and isothermal calorimetry (ITC) to investigate the thermodynamics and binding of the individual fragments and their linked counterpart to MMP. They demonstrate that the two fragments preserve their binding modes even when linked together and observe a rather large free energy enhancement of almost 4 kcal/mole on fragment linking.
This is a nice case where a careful analysis and dismissal of other factors points accurately to entropy as the main contributor to enhanced binding of linked fragments.
Borsi, V., Calderone, V., Fragai, M., Luchinat, C., & Sarti, N. (2010). Entropic Contribution to the Linking Coefficient in Fragment Based Drug Design: A Case Study Journal of Medicinal Chemistry DOI: 10.1021/jm901723z