I am working with a group of inhibitors that were obtained by scaffold hopping. However, I would like to clarify that they do not make up a QSAR series. Detailed inhibition kinetics performed with them shows different mechanisms of inhibition, i.e. non-competitive, uncompetitive and linear-mixed type. However, none of the inhibitors are competitive vis-a-vis the substrate.
I would also like to state here that at high inhibitor concentrations, I see a substrate inhibition like behavior ( In the absence of the inhibitor, there is no substrate inhibition seen). The Kis for the various inhibitors ( from global non-linear fits to the best respective models) are anywhere from 30 uM-90 uM. The IC-50s also range from 30 uM-100uM.
Are there any papers that can account for such inhibition kinetics? I would appreciate any help. Thanks!
The situation you describes sounds very complicated. The first thing I would want to establish is that these are specific, stoichiometric inhibitors, as opposed to nonspecific inhibitors (aggregators, denaturants, detection artifacts, solubility artifacts). This is a frequent problem with "lead-like" inhibitors, especially fairly low-potency ones such as you are working with. There are several enzymatic and biophysical approaches to establish this. If they are specific, I would also want to know whether these inhibitors are time-dependent because that would be a problem for interpretation of steady-state kinetic data.
Hi Adam,
Thank you for your response. To answer your questions systematically,
1) The inhibition seems very specific according to the criterion elaborated below. I screened around 10 different compounds in each scaffold class that are biosynthetic precursors of the compounds that inhibit ( in total I have screened about 30 different compounds for the 3 different scaffolds that show inhibition). Unfortunately, any restrictions on the degrees of freedom about the central bond by means of introducing an additional ring or any substitutions that are not either hydrogen bonding donors or acceptors on the flanking benzene groups, abolishes the inhibition totally indicative of highly specific inhibition.
2) There seems to be no aggregation, either with the compound alone or with the compound in the presence of the two substrates that I employ for my enzyme. These experiment were done at both low and high concentrations of the inhibitor and the substrates and their combinations thereof. I neither observed a soret peak corresponding to the pi-pi stacking at 400 nm nor did I see a scattering band at around 600 nm, indicative of aggregation, as assesed by wavelength scans on UV-Vis spectrophotometers.
3) The compounds do not interfere with the assay read-outs as assessed by estimation of the extnction coefficient for the substrate and product in the absence and presence of the inhibitor.
4) The solutions appears clear visually precluding the possibility of any precipitation too.
5) Moreover, all the inhibiting compounds show binding to the protein with reproducible thermal shifts as assessed by DSF.
6) No, I do not see any time-dependence of inhibition with any of these compounds. The absorbance vs Time curves are all linear, albeit lesser in slope in the presence of inhibitor than "no-inhibitor" control.
I would appreciate any help that you can provide or any literature that you have come across that can help me understand this problem. Thanks!
It seems to me, intuitively, that an uncompetitive inhibitor might result in the appearance of substrate inhibition, under the right circumstances, because the binding of the substrate is a prerequisite for binding of the inhibitor. I don't have direct experience with this situation, however.
This should not occur for a non-competitive (allosteric) inhibitor, the binding of which is unaffected by the binding of substrate.
As for the rest of your question, perhaps other people have seen examples of different compounds in the same series having different modes of inhibition. I have not, but my experience has been with competitive inhibitors.
certainly it will do. even a substance can show different mechanism of regulator for a receptor (enzyme) under different conditions.
see my papers: A thermodynamic and theoretical view for enzyme regulation.
another paper of others may be helpful: "Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli"
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