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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:
[log in to unmask]"> 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) <[log in to unmask]> wrote:


Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG <[log in to unmask]> 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