I have some enzyme inhibitors that have showed good IC50 and Ki values, but I doubt they are NON-specific inhibitors since their structure consisted of three aromatic systems with only three polar groups. My guess is that they just bind the enzyme everywhere, probably including the active site with hydrophobic interactions.
So what kind of experiments are able to tell without doubt, whether my inhibitors are specific (only bind the active site) or non-specific (bind elsewhere and change the active site structure to achieve inhibition)?
Thanks
I would start by testing the enzyme against increasing concentrations of the inhibitor in order to characterize the inhibition in terms of simple models (eg., competitive, non-competitive, acompetitive inhibition, etc.). If the inhibitor and substrate are mutually exclusive, they might be binding to the same site; if not, the effect should be indirect, suggesting the binding sites are different. Of course these assays are not conclusive; it should be complemented with other tests like using fluorescent probes (these usually have aromatic rings that might have the same behavior as you inhibitor), or perhaps your inhibitor can quench the intrinsic fluorescence of a tryptophan residue near the active site, etc. but that depends on your enzyme's biophysical properties. Probably the ultimate answer would only be given by protein crystallography, through solving the structure of the inhibited enzyme.
Hi Javier, thanks for the reply. The inhibition curves look normal, just like any other competitive inhibitors, and tests with fluorescent probes have also been done.
I wonder if this could be verified by using a known bona fide inhibitor (proven by crystallography) that is far stronger than my inhibitors as a reference. If my inhibitors are NOT able to bind the enzyme in the presence of the reference inhibitor, it can be concluded that they are competing for the same active site.
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Samo Turk has already given you a brief introduction to PAINS and other papers. Generally aggregators have Kis in the low-mid micromolar range, although they can be lower. If your protein has a co-factor then you can try spectroscopic methods on the co-factor. e.g. FAD fluorescence or heme circular dichroism. Competition with a more potent ligand has also been mentioned, but you need a method of differentiating between which if the two ligands is biinding at a given time (i.e. for spectroscopic methods instead of crystalographic ones). If you are lucky enough to have a tryptophan near the binding site, then tryptophan polarisation fluorescence might be another method.
Additionally, if you know the interacting residues, you can remove them via mutation and see if the kd of the inhibitor changes, although you will often need a method of detection not based on inhibition of product formation such as spectroscopy or calorimetry.
Also, the idea that 3 aromatic groups and 3 polar groups can't form a protein-specific interaction i dont feel is correct. There are certainly enough pharmacophores that can be generated by such molecules such that a particular pharmacophore (perhaps yours) is specific to a particular protein. This will especially be the case the more rotatable bonds you have (e.g. biphenyl vs naphthalene).
Hmmm, re reading your question, it seems as though you are only after active-site vs alosteric binding? Performing a matrix of varying substrate and inhibitor concentrations will achieve this. Non-linear regression can then be used to define the specific form of inhibition. Remember to regress against all forms of inhibition equations, not just one, to make sure it can be defined as a single type (might be mixed inhibition after all). Also, if you have more than one substrate (e.g. an oxygenase enzyme) then you should repeat the experiment varying a single substrate at a time. inhibitors can often be competitive with one substrate and non-competitive with another.
Hi Yun. Is your inhibitor in a solvent and have you excluded its effect on the enzyme?
In addition to the +/- detergent suggestion to check to see if aggregation is in play, you might want to simply check for selectivity against a few other related and unrelated enzymes.
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