I have 100+ small inorganic molecules that have been predicted to inhibit the APT1 enzyme (lysophospholipase 1, citosolic). In order to verify the inhibition in-vivo I would need to do 100+ ELISA assay, that are very expensive and time consuming.
I was hoping that I could find a quick (and cheap) in-vitro (or in-vivo) assay to verify at least the binding of the drugs on the APT1 protein and possibly its inhibition.
Any suggestion would be much appreciated!
Cheers
Carla
Firstly, an ELISA will not help you answer your question. It will tell you how much lysophospholipase 1 there is in a sample, but not whether it was inhibited by the compound.
Secondly, an in vitro enzyme assay is preferable to a cell-based assay to address your question. The reason is that several factors in cells can affect whether a compound inhibits a cytoplasmic enzyme. The intracellular concentration of the compound is affected by its physico-chemical properties, by the extent to which it is a substrate of efflux pumps and cellular degradatory enzymes, and by any tendency it might have to concentrate in acidic subcellular compartments.
So you should check the relevant literature to identify an assay for lysophospholipase catalytic activity, preferably with a purified protein.
Hi Maria, it looks like you have a problem here. The main thing is to eliminate weak binders.
There are a number of solutions here and many may be impractical:
1) run `100 gel lysates, (Native Page) cut them up and probe them for your enzyme. acivity, if you have an elisa activity assay will make this a bit easier.
2) Plate cell lysates on elisa plates and see which inhibitors work best.
Go Swans!
You can also do it computationally, which is cheaper. Assuming the 3D structure of cytosolic lysophospholipase 1 is known (go to PDB), you can try to dock each of the 100+ small inorganic molecules on it and determine the ones with the best docking parameters (affinity to the enzyme). If the 3D structure is not available, you can probably query UniProt for binding sites using the amino acid sequence, although this route is less reliable than 3D docking.
Here is a paper describing a fluorescence-based assay for phospholipase activity.
https://www.ncbi.nlm.nih.gov/pubmed/8129724
The fatty acid detection reagent ADIFAB is commercially available.
http://www.ffasciences.com/product2.html
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