Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - jamclay (Chemical Tools to Probe the Role of Bromodomains in the Parasite Trypanosoma cruzi)

Teaser

Recently the development of small molecule bromodomain ligands has been the focus of significant attention from the medicinal chemistry community. The bromodomain and extra C-terminal domain (BET) family of bromodomain-containing proteins (BCPs) have emerged as important...

Summary

Recently the development of small molecule bromodomain ligands has been the focus of significant attention from the medicinal chemistry community. The bromodomain and extra C-terminal domain (BET) family of bromodomain-containing proteins (BCPs) have emerged as important therapeutic targets for cancer and inflammation therapy, and focus has now turned to developing ligands for non-BET BCPs, to determine their function and therapeutic potential.

Bromodomains are epigenetic reader proteins that bind to acetylated lysine (KAc) residues, these fundamental interactions play a key role in regulation of important transcriptional protein-protein interactions that regulate the expression of certain genes.

Here we report the design, synthesis, and biological evaluation of a range of potent and selective ligands for the CREBBP bromodomain, which is a key transcriptional co-activator.

Objective 1. Our initial hit molecule contained a metabolically unstable dihydroquinoxalinone acetyl-lysine mimicking \'head group\' and an aryl amide. The acetyl-lysine mimicking \'head group\' was developed into a more metabolically stable benzodiazepinone it\'s ring expanded head group containing an additional CH2 reduces degradation, as oxidation of dihydroquinoxalinone gives an aromatised quinoxalinone with reduced activity.

Objective 2. Although improving metabolic stability introduction of the benzodiazepinone caused the molecules internal hydrogen bonding network to change, a reduced affinity binding conformation now formed in solution. The internal hydrogen bonding in the presence of the benzodiazepinone head group was a hinderance. Thus we re removed the aryl amide and replaced it with an E-alkene a classical amide bio-isostere.

Work performed

Substituting an amide for an E-alkene (a classical amide isostere) into our CREBBP bromodomain probe molecule, resulted in an almost 4-fold increase in binding affinity from 390 nM (amide) to 100 nM (E-alkene) by ITC. Furthermore substituting the amide for an alkyne, which removes the hydrogen bond but is not an amide isostere, increases the affinity relative to the amide but to a lesser extent than the isosteric E-alkene. This demonstrates that it is necessary to both remove the hydrogen bond from the molecule and retain its geometry to maintain potency for the target bromodomain of CREBBP.
In order to engage in ligand development we draw information from a number different of techniques including protein and small molecule crystallography, 1H-NMR, waterLOGSY, differential scanning calorimetry (thermal shift) and isothermal calorimetry (ITC).
Furthermore we use a diverse array organic transformations such as β-amino acid synthesis, amidations, hydroborations and Suzuki reactions to stereo-selectively synthesise our target molecules.

Final results

Progress beyond the state of the art - Our work developing bromodomain inhibitors has lead to some innovative simple solutions to observing the effect hydrogen bonding has on a ligand’s binding conformation and solution structure.  Our work shows that through thorough interrogation of weak intramolecular interactions by meticulous building of specific molecules to probe our hypotheses about these interactions enables improvement of ligand selectivity and potency.

Based on the project’s progress we believe that our methods will lead to a significant improvement in the way the field thinks about intramolecular interactions and identifies the presence of hydrogen bonds in their molecules.
Details of the socio-economic impact will be forthcoming following our publication disseminating the results of our investigation and the techniques we used and developed. However it is already clear that the simplicity of our methods lend them selves to adoption as standard procedures for the wider field of medicinal chemistry.

Website & more info

More info: http://conway.chem.ox.ac.uk.