Webinar highlights: Integrated fragment based approach reveals enzymatic inhibitors with potential therapeutic application

Keynote speakers Professor Antonio Macchiarulo and Dr Alice Coletti from the University of Perugia presented the webinar on behalf of NanoTemper Technologies.

Have you already profiled signalling pathway responses in an unbiased and quantitative manner?

It would be interesting to see real, global data showing changes in non‑catalytic signalling activity and to then contrast with (i) Epacadostat’s mechanism of action and/or (ii) degree of IDO1 functional modulation. We are indeed working on this. We are actually optimising a pharmacological assay that can provide us with a quantitative pharmacological parameter to characterise these compounds, and we will also compare these activity values with those from the actual drug candidates, including Epacadostat that are in clinical trials.

Do you think that biophysical methods are sensitive enough to detect the binding of the fragment to protein target?  The size of fragments is extremely small…

The size of the fragments is very small but biophysical methods such as MST and even SPR are so sensitive that minimal variation in their baseline is sufficient to reveal a change in the studied system. It’s broadly reported in the literature that they can very well detect weak interactions like those forming between proteins and fragments that show affinities around 100μM.

How does MST compare to other technologies such as NMR for FBDD?

MST presents a lot of advantages over NMR. We can use a very small amount of the protein and it has also allowed us to reduce the number of false positives due to aggregation, something common when you have to use a large amount of protein.

What is the risk of detecting false positives or false negatives at early stages with your approach?

With our approach this risk is reduced. We used the PAINS filter during the virtual screening campaign and we chose fragments with high solubility, which reduces the risk of aggregation. Finally, thanks to MST we can reveal quickly the presence of aggregation or precipitation to reduce false positives.

Did you analyse your inhibitor concerning AHR interaction?

So far, we have analysed our inhibitors for AHR transcription activities using cellular assays. We are also trying to develop a biophysical assay for AHR binding activity. In terms of AHR transcription activities, none of these fragments can activate the receptor.

Which cells did you use for your assays?

We used the p815 mouse mastocytoma cell line.

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