Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that offer a promising therapeutic strategy against hard-to-treat diseases. Unlike traditional small molecule inhibitors, developing PROTACs presents unique challenges — particularly the characterization of binary and ternary complexes.
This guide highlights how an innovative, plate-based, and automation-ready affinity screening solution helps you overcome roadblocks in your PROTAC characterization projects.
Download this guide to discover a platform that helps you:
- Characterize ternary complexes, covalent binders, or small molecules without immobilization
- Take in-solution and mass-independent affinity measurements
- Reduce sample consumption while maintaining exceptional data quality
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
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
Measuring affinities with small molecules
like warheads is more difficult with other
Measurements are mass-independent,
so you use Dianthus for primary screening
of fragments, but also to screen for affinity
maturation in PROTAC candidates
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
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
Ratio 670nm/ 650nm
Ternary complex BRD2 + MZ1 + VCB Binary complex BRD2 + MZ1
*Douglass et al J Am Chem Soc 2013, 135:6092-6099
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
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
Ternary complex KRASG12C + LC-2 + VCB
= 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
Binary complex BRD4 + Warhead JQ1 (+) Binary complex BRD4 + PROTAC dBET6
Molecular weight (Da)
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.
NanoTemper’s mission is to create biophysical tools
for scientists to tackle their challenging characterizations