Anectodal evidence I have heard from colleagues working with things, which are immobilized is that the measured Kd value on the surface can be wildly different from what is measured in solution. A superbinder on a surface might not be as good in solution. There seems still a lot of debate why that is.
Cheers Christian On Fri, Apr 27, 2018 at 5:07 AM, WENHE ZHONG <wenhezhong.xmu....@gmail.com> wrote: > Hi Philippe, > > The affinity was measured by SPR where we immobilized the protein on the > chip. One thing I forgot to mention is that the association rate (kon) > shown in SPR experiment for this compound is faster (>10-fold faster) > compared to other analogues with similar koff. There is a pi-pi > interaction between the scaffold structure and the protein (tyrosine ring). > Is it possible that the hydrophobic substituent could facilitate the > formation of this pi-pi interaction but not necessary to involve in the > interaction? Thanks. > > Kind regards, > Wenhe > > On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC) < > p.du...@ibmc-cnrs.unistra.fr> wrote: > > > Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG < > wenhezhong.xmu....@gmail.com> a écrit: > > Just to be sure: how was the nM affinity evaluated ? By in vitro > measurements, or by obtaining an IC50 by tests on cells ? > Of course, if you are mentioning an IC50, you may have a measurement of > the efficacy of drug entrance in the cells, not just of specific binding to > your protein target. > Philippe D. > > 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 > > > > > > > > > > >
