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Dear all,
I have some doubts regarding interpretation of ATPase assay using P-gp expressed membrane (SOLVO-kit).
1) Is it possible to find out inhibition capacity of a test drug, as kit has mentioned ? Here what i found in literature that Cyclsporine which is P-gp ATPase inhibitor capable of showing efflux ratio >2.
2) What can we predict from the data if it reduces Verapamil stimulated P-gp ATPase Pi release in presence of Vanadate.
3) In Ritoavir study i found the efflux ratio 1.8 and inhibition of 87% ATPase activity at 20micM, so what would be the nature of the drug?
Thanks for your reply in advance.
Regards,
Rahul vats
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Dear Rahul,
There are some high throughput non cell based assays useful in early phases of drug discovery like ATPase assay, calcein assay, vesicular transport assay to assess the interaction with transporter. All these assays are complementary to each other and do not rely on the results obtain from single assay.
Data obtain from ATPase assay can only state either there is interaction between test article and P-gp (i.e. positive hits in test, in your case it is showing interaction) or not, but do not comment on the nature of interaction i.e. substrate or inhibitor. Ones you obtain positive hits in ATPase assay assess nature using other complementary assay system like calcein, vesicular transport or caco-2 cell line.
In ATPase assay to check the interaction there are two types of protocol one activation protocol and another inhibition protocol. Which one did you follow? Its better to use both protocol. The purpose of doing ATPase assay in presence and absence of vanadate is to get sensitive ATPase activity.
Hope it will be helpful.
Comments from experts are welcome.
Thanks and Regards,
Rhishikesh
Advinus Therapeutics, Pune, India.
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Dear Rhishikesh,
Thanks for your precious comment on ATPase assay.
For your information i used both the protocol (activation as well as inhibition). As a result in activation type -i got 21% relative ATPase activity for Ritonavir and in inhibition protocol i got a value 7.So can i use this 7 to convert in to % inhibition value (100-7=93% inhibition).
thus how should i interpret the result.?
1) Drug Ritonavir is interacting with P-gp as a substrate, results from activation protocol or
2) Drug Ritonavir is inhibitor of P-gp results from inhibition protocol?
Eagerly waiting for your expert comment
Regards,
Rahul Vats
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Dear Rahul,
Ritonavir is P-gp substrate as well as inhibitor. Beware about the drawback of ATPase assay; being an indirect assay it may not differentiate well between substrate and inhibitor and substrates transported at a lower rate may not affect the basal ATPase activity.
Regards,
Rhishikesh Advinus Therapeutics, Pune, India.
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