Hey all, I was hoping some of you might have some insight into this topic.
I am a second year PhD student working to develop novel enzyme inhibitors. I have screened a large compound library and identified a number of enzyme inhibitors, the most potent of which all have a free thiol (-SH) group which could form a disulphide bond with a cysteine residue in the active site. This group is necessary for inhibition to be observed, substitution with an -NH2 or -OH results in no inhibition.
I am guessing this is a non-specific mechanism and therefore should be disregarded, but was hoping someone might be able to confirm this.
Any advice would be greatly appreciated!
Check whether a disulfide bond with the protein is actually formed by measuring a change in protein mass using mass spectrometry. If so, and if the protein has more than one accessible cysteine side chain, try to find out whether the disulfide bond is with the active site cysteine using peptide mapping MS. Test whether the inhibitor still works under reducing conditions that prevent disulfide bond formation.
Even if the mechanism of inhibition is specific and is due to disulfide bond formation, it may be of no use if the disulfide bond can not form in vivo because the target is cytoplasmic and therefore in reducing conditions.
The free thiol in the compound will probably be rapidly modified in vivo, preventing the drug from reaching the target.
If modification of an active site cysteine residue is a viable means of inhibiting the target, weak electrophiles such as Michael acceptors may be tried. However, the inhibitor still needs to have high affinity for the target without the covalent interaction, otherwise it will lack selectivity.
Captopril is an approved drug with a free thiol. So the thiol itself might not be an absolute no-go. However its mechanism of action is different from what you describe. In captopril, the thiol binds to a Zinc atom. It does not form a disulphide bond.
Adam's remark about cytoplasmic conditions is a good one. It is indeed probably a good idea to try replacing your thiol with another function that is commonly used for targeting Cys residues.
glutathion, Coenzyme M (Sodium 2-mercapto-ethanesulfonate, zofenoprilat drug are approved drugs with a free thiol group
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