Overcome Roadblocks to PROTAC Characterization

Rely on Dianthus to overcome roadblocks in your PROTAC characterizations You have many methods to choose from to screen for warheads and ligase ligands, and to characterize ternary complex formation and cooperativity. But you’ve been held back by their limitations — they’re inadequate to study multimeric complexes and covalent ligands when immobilization is required, lack consistent high-quality data, and they’re unable to easily characterize interactions involving small and large molecules — so you’re ready for a method that resolves every one of them. Learn how with Dianthus. Discover how Dianthus solves 4 common problems Dianthus is a plate-based affinity screening platform that enables you to overcome the challenges presented by other biophysical methods. Avoid these common roadblocks with Dianthus and keep your PROTAC projects moving forward. Stability of your binary complex is compromised if you have to immobilize it to study the ternary complex Measure in solution under controlled equilibrium conditions, so the binary complex stays stable when you evaluate ternary complex affinities 1 It’s virtually impossible to completely remove a covalent analyte from a sensor chip during regeneration Evaluate molecular interactions in solution in separate wells, so your affinity assay is less complicated, stress-free, and more budget-friendly 2 Measuring affinities with small molecules like warheads is more difficult with other affinity methods Measurements are mass-independent, so you use Dianthus for primary screening of fragments, but also to screen for affinity maturation in PROTAC candidates 3 You only have access to limited amounts of target and ligands You’ll use less of your target protein, ligase, and ligands because you’ll do less assay development. And you’ll need less once you start running your affinity assays 4 Get data that agrees with the fit model currently used to study ternary complexes It’s hard to make decisions with data that can’t give you clear answers on whether hits are real, or binding affinities are precise. Dianthus consistently outputs highly reproducible measurements that align to fit the model that best represents the interactions in binary and ternary complexes. The interaction between target Brd2 and PROTAC MZ1 was measured and the data was in very good agreement with the fit model* to calculate the Kd (18.8 nM) and the cooperativity value (a = 9.3). From these parameters, the affinity constant of the ternary complex Brd2+MZ1+Ligase VCB was determined (Kd = 2.02 nM). 10-2 10-1 100 101 102 103 104 0.92 0.96 1.00 1.04 1.08 1.12 Ratio 670nm/ 650nm MZ1 (nM) Ternary complex BRD2 + MZ1 + VCB Binary complex BRD2 + MZ1 *Douglass et al J Am Chem Soc 2013, 135:6092-6099 Stress-free characterization of a KRAS ternary complex containing a covalent binder KRAS — the protein mutated in colorectal and lung cancers — is not only difficult to drug but its interactions with drug candidates are challenging to study because KRAS isn’t amenable to immobilization-dependent measurements with SPR. And if the interaction involves a covalent binder, your work got even more complicated and expensive because covalent analytes are virtually impossible to remove from the sensor chip surface during regeneration. With measurements in solution, Dianthus removes the stress from studying ternary complexes that involve a challenging target like KRAS and covalent PROTAC LC-2. The formation of ternary a complex between KRASG12C,PROTAC LC-2, and VCB was successfully studied in solution by labeling ligase VCB, which avoided disturbing finicky KRAS. 10-5 10-4 10-3 10-2 10-1 100 101 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Ternary complex KRASG12C + LC-2 + VCB Fraction Bound LC-2 (M) Kd = 4.4 nM S/N = 31.6 Reliably evaluate PROTAC interactions involving small and large molecules To develop effective PROTACs candidates, you need to characterize interactions involving small ligands — like warheads and ligase ligands — and larger molecules such as fully assembled PROTACs. If your affinity screening method relies on significant mass changes upon binding, you’ll spend a lot of time on assay optimization before you see any results. Measurements of molecular interactions with Dianthus are mass-independent, so you don’t need special tricks to successfully measure binding affinities with small and large molecules. Dianthus measured the two affinities between target protein BRD4 and warhead JQ1(+): for the interaction with the warhead alone and when the warhead is part of the fully assembled PROTAC dBET6. Data collected from a collaboration with Aurelia Bioscience, a Charnwood Molecular company. 10-10 10-9 10-8 10-7 10-6 10-5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Binary complex BRD4 + Warhead JQ1 (+) Binary complex BRD4 + PROTAC dBET6 Fraction Bound Concentration (M) JQ1(+) dBET6 Kd (nM) Molecular weight (Da) 51.8 457.0 58.7 841.4 Choose Dianthus for your PROTAC candidate characterization Get a plate-based and microfluidics-free affinity screening platform that easily integrates into any automated setup via gRPC framework. And with no regular maintenance, your projects don’t get delayed due to downtime. Dianthus is ready whenever you need it — non-stop, 24/7. BR-DI-PROTACS-062022-v2 nanotempertech.com/dianthus-for-protacs NanoTemper’s mission is to create biophysical tools for scientists to tackle their challenging characterizations