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 >> >> >> > > > > > >
