For proteins in membranes, or proteins purified in the presence of detergents and/or lipids, the active site is sometimes surrounded by a hydrophobic mileau. The actual concentration that the binding site sees is then dependent on partitioning of the ligand between the water (where its concentration for Kd is determined) and the hydrophobic phase. Hydrophobic substituents could then result in higher concentrations at the binding site, and thus lower the apparent Kd, while having nothing to do with binding.
On 04/26/2018 10:50 AM, WENHE ZHONG wrote:
Dear Community, A little bit out of topic here. We are applying the structure-based approach to design compounds that can bind our protein target. We have synthesized a series of analogues based on the same scaffold with different substituents at one particular site. The most potent analogue (nM Kd) has a long alkyl chain substituent. We thought this hydrophobic substituent should have strong interactions with the target protein leading to nM range affinity. However, crystal structures show very weak densities for this substituent and no obvious interaction between the substituent and the target protein, suggesting that this long alkyl chain substituent is flexible without binding to the protein. This binding site is relatively negative charged according to the electrostatic potential analysis. So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain substituent can lead the compound to achieve nM affinity (>10-fold better than any other substituent) — in particular the binding site is not hydrophobic and no interaction is found between the substituent and the protein. Anything I have miss here that can increase the binding affinity without interacting with the target? Thanks. Kind regards, Wenhe
