If you’re tasked with developing proteolysis targeting chimeras (PROTACs), you know how critical it is to find the right method to characterize binary and ternary complexes. You need a method that resolves the limitations presented by methods like SPR, FRET, and AlphaScreen with mass⁠-⁠independent and in⁠-⁠solution measurements and that doesn’t require long and complicated assay development.

Top roadblocks faced during affinity screening of binary and ternary complexes and how they are overcome by Dianthus

The immobilization of the secondary complex makes studying the ternary complex very complicated

This is because when you immobilize the binary complex — as you do with SPR — its stability is often compromised while you try to assess its affinity for your third component.

 

Solution

With Dianthus, measurements are done in solution under controlled equilibrium conditions, so your binary complex stays stable when you characterize the ternary complex.

Difficulty detecting binding events with SPR when small molecules are involved

You want to measure affinities with small molecules like warheads — the component of a PROTAC molecule that binds to the protein of interest (POI) — or E3 ligase ligands. If your method relies on significant mass changes upon binding, getting the assay to work will require a lot of assay development.

 

Solution

Measurements of molecular interactions with Dianthus are mass-independent, so you use the same technology from primary screening of fragments to affinity maturation of lead candidates for your degraders — like the interaction between warhead and POI, E3 ligands, and E3 ligases, and later between PROTAC and POI and PROTAC and E3 ligase.

Challenges presented when you work with covalent ligands

Screening for covalent ligands with SPR is too labor-intensive and expensive since sensor chips can’t be regenerated and reused. This problem is encountered more often, as the interest in developing covalent ligands (both reversible and irreversible) is growing due to their enhanced engagement with their targets and increased cellular uptake.

 

Solution

Screening a large number of covalent ligands is never a concern since each molecular interaction is measured separately in solution

Biochemical assays require complex and long protocols

If you use proximity-based biochemical methods like AlphaScreen and TR-FRET you need to perform multiple and carefully optimized incubation steps. Additionally, these methods require not just one, but two different fluorophores to label the POI (TR-FRET) or to label donor or acceptor beads (AlphaScreen)

 

Solution

Characterization of binary and ternary complexes only requires two steps, just one fluorophore to label the POI or E3 ligase, and no beads.

Access to limited amount of sample and compounds

You will need a lot of your sample and compounds to complete a screening campaign with methods like SPR or FRET

 

Solution

With Spectral Shift you will spend less of your POI or small molecule ligands because you’ll do less assay development and use also less during screening

Finding a method that handles fragment screening and fragment growth

Fragment libraries are becoming increasingly popular to identify binders that will then be grown into warheads. This approach needs a biophysical method that, unlike SPR or NMR, easily handles a broad range of affinities and low molecular weight fragments.

 

Solution

With Dianthus, you identify hits from fragment libraries with confidence because measurements are mass-independent and detect a broad range of affinity strengths — ideal for hit identification and also when you grow fragments into warhead molecules that show tighter affinities.

Learn about the affinity screening platform that overcomes PROTAC development challenges

 

Discover Dianthus

Develop warheads into full PROTAC molecules with mass-independent measurements

To screen for small molecule warheads, you need a screening method that is mass-independent to reliably measure their affinities for the protein of interest (POI). Additionally, you want the same method to measure the affinity between the POI and the warhead once it’s assembled into a fully assembled PROTAC molecule.

Dianthus measures the interaction between warhead JQ1(+) and BRD4 (POI), and the interaction between dBET6 and BRD4 — a BRD4-specific PROTAC developed with JQ1 (+) that has almost twice the molecular weight of JQ1(+). Using Spectral Shift, the data clearly shows that the affinities are practically the same, making dBET6 a PROTAC candidate worth pursuing.

Data from a collaboration with Aurelia Bioscience, a Charnwood Molecular company.

a) Graph shows the emission fluorescence ratio 670/650 vs. ligand concentration for the two binary complexes JQ1(+) – BRD4 and dBET6-BRD4

Characterize molecular interactions

in binary complexes with high-quality data

High-quality binding assays to assess the interaction of PROTAC molecule SD-36 — a potent and selective degrader of STAT3 that has shown anti-tumor activity in preclinical models — with STAT3 and with the CRBN ligase were established using Spectral Shift. The affinity in the low nM range was determined with high-quality data, as shown by S/N above 30 for both binary complexes.

Graph 1 shows the fraction bound vs. ligand concentration for the binary complex of PROTAC SD-36 and target STAT3. Graph 2 shows the emission fluorescence ratio 670/650 vs. ligand concentration for the binary complex of PROTAC SD-36 and ligase CRBN

Measure a high-affinity ternary complex interaction with a covalent degrader

When you have to measure interactions that involve covalent ligands, measuring in solution makes it less complicated versus a method that calls for immobilization. Spectral Shift easily shows that covalent PROTAC molecule LC-2 has an affinity 80 times stronger for the E3 ligase complex VCB when it’s forming a binary complex with target KRAS — ternary complex, blue trace — than when it binds to VCB alone — binary complex, purple trace. The tighter binding translates into a high cooperativity value (α) and confirms why LC-2 shows great promise as a KRAS degrader.

a) Graph shows the fraction bound vs. ligand concentration for the binary complex VCB-LC2 and for the ternary complex LC2-KRAS + VCB

See how NanoTemper tools have been helping scientists develop protein degraders like PROTAC and RIBOTACs

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