Dear Wenhe,
A thought came to mind after having read all the other threads, for
which I generally agree.
An alkyl chain on a molecule (charged? hydrophilic?, you mention a
negatively charged binding site) will most likely not lead to micelle
formation as the cmc of the object will be most likely higher than the
amount you use in solution, especially at uM concentrations. But the
alkyl chain nevertheless creates a lot of entropy, it doesn't like being
in the water. How long is the alky chain? if it is 8-9-10 or even 11
carbons, it is likely to be not hydrophobic enough to want to burry the
side chains into a micelle, and be very exchangeable in solution, yet
not happy to be there. Binding onto a surface would reduce entropy,
resulting in a better kon?
You could try ITC, you will have access to detlaH and deltaG of binding,
and by comparing with your other molecules maybe something would come up?
please correct me if I'm wrong.
All the best
Vincent
On 27/04/2018 05:07, WENHE ZHONG 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)
<[email protected] <mailto:[email protected]>>
wrote:
Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG
<[email protected] <mailto:[email protected]>>
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
--
Vincent Chaptal, PhD
Institut de Biologie et Chimie des Protéines
Drug Resistance and Membrane Proteins Laboratory
7 passage du Vercors
69007 LYON
FRANCE
+33 4 37 65 29 01
http://www.ibcp.fr
