/By Natalie Gath

Comparison of InCELL Hunter™ vs InCELL Pulse™ for Analyzing Compound-Target Engagement

Whether you’re looking to screen a library of drug candidates for binding to a particular target protein, checking drug cell membrane penetration, or intending to confirm the mechanism of action of your favorite small molecule prior to moving forward with drug development, measuring compound-target engagement is a crucial tool.

Two Assay Platforms for Increased Target Coverage

Eurofins DiscoverX’s portfolio has two reliable, easy-to-use assay platforms – InCELL Hunter and InCELL Pulse – for use in measuring target engagement. These two assay types have similar underlying principles, but with some crucial differences that suit them for different targets. Ultimately the specific assay type needs to be determined empirically during assay development for each target. The following information will seek to help you understand the differences in these assay formats.

InCELL Hunter and Pulse assays all function based on the fact that engagement of a target with a compound leads to protein stabilization, regardless of the intended pharmacological mechanism of action (MOA) of the compound (activation, inhibition, etc.). This allows these assays to be used for target proteins without a known downstream readout/functional assay and with compounds that may not have a known MOA. It also allows for assay of multiple compounds with multiple MOAs within the same assay – a single assay can function for agonists, antagonists, allosteric modulators, and more.

How do the InCELL Assays Work?

InCELL Target Engagement assays are based on the Enzyme Fragment Complementation (EFC) technology. Briefly, this technology uses two components, an enzyme donor (ED) and an enzyme acceptor (EA) which are fragments of the β-galactosidase enzyme. Alone, these fragments are inactive. Together, they complement and form active β-galactosidase, which produces detectable chemiluminescence upon exposure to a substrate.

In the InCELL assay formats, cells expressing the target protein in an ED tagged form are incubated with test compounds, and the EA is applied as part of the detection reagents. If the ED is present after compound incubation, i.e. has not been degraded, then a chemiluminescent signal is produced.

Overall, the two InCELL assay format subtypes include:

InCELL Hunter Stabilized Compound-Protein Complex Assay Format
InCELL Pulse Assay Format

Please refer to the InCELL Target Engagement Landing Page to learn more about each assay format.

Choosing the Right Assay Platform

InCELL Hunter assays tend to work best with proteins that have a relatively high turnover rate (or short half-life) in the cell, and which are not ordinarily stable for long periods on their own. As such, it is best suited for cytosolic or nuclear proteins that are maintained at a certain steady-state level by cellular machinery. For example, cell cycle proteins are typically well suited to assays using an InCELL Hunter approach.

InCELL Pulse assays, on the other hand, do not require the target protein to have such a high turnover status. Instead, these assays work best with proteins that are thermally labile, and which will degrade or denature when exposed to heat, forming aggregates that render the EFC tag inaccessible. InCELL Pulse assays tend to be slightly more sensitive and applicable to a wider range of target proteins, and during assay development, trying the InCELL Pulse format first is typically recommended.

In many cases with a new target we may not be able to predict (or correctly predict) which InCELL assay type will work out better. However, both InCELL Hunter and Pulse assays use the same cells – cells which express the target protein tagged with a component of the EFC readout system. Therefore, testing which assay type will work best is a simple matter of changing the assay procedure from the Hunter to Pulse workflow (and back again), without the need for time-consuming re-engineering of the cell line. The table below highlights key differences between the two assay formats.

ASSAY COMPARISON	InCELL Hunter	InCELL Pulse
Assay Principle	Protein Stability (half-life)	Protein Thermal Stability
Heat Step	No	Yes
Ligand Modification Required	No	No
Assay Completion Time	~30-40 hours	~26 hours
Target Protein Modification Required	Yes (Target Protein) No (Destabilized Partner)	Yes
Requires High Protein Turnover	Yes	No
Requires Heat-labile Protein	No	Yes
Works with Soluble Proteins	Yes	Yes
Works with Membrane Embedded Proteins	Yes, some	Yes, some

Challenges of InCELL Assays

Membrane-embedded proteins can be a more difficult target type for either variation of the InCELL assay. These proteins often do not cycle via rapid degradation and production but are regulated via transport, for example to the endosome and back to the membrane (though this is not universal). Membrane-embedded proteins are also typically more stable to heat exposure, and even if denatured, may not be able to clump together to form the denatured protein aggregates that allow the InCELL Pulse assays to give the proper readout. This does not mean that this assay type can never work with membrane-embedded proteins – but it can be a somewhat challenging target type to develop. However, there is proof of concept and success stories with membrane proteins, and if your protein of interest is membrane-embedded, it may still be quite possible to create an InCELL assay.

As an example, we have successfully built InCELL assays with two membrane-embedded proteins as part of our Custom Assay Development program. The first is a polytopic multi-pass (non-GPCR) membrane protein, and the second is a single-pass membrane protein.

In addition, a variant application of InCELL Hunter, the destabilized-partner-protein readout, works on another type of target – a chaperone or modifier proteins that bind to a partner protein and stabilizes it or protect it from the degradation machinery. This is a slightly more narrow class of targets, but this assay format can be very useful for these otherwise difficult to assay targets.

Overall, InCELL Hunter and Pulse are powerful assay types to measure compound-target engagement, confirm drug cell membrane permeability, measure and rank drug potencies, screen hits, and much more on a wide variety of target protein types.

InCELL Success Stories: Assayed Target Types