Ligand-observed NMR is one of the
most popular fragment-finding methods. Among its strengths is the ability to
find extraordinarily weak fragments that most other techniques would miss.
However, common ligand-observed NMR methods such as STD are not quantitative:
they can tell you that a fragment binds, but not how tightly. In a new
open-access J. Med. Chem. paper Manjuan Liu and colleagues at the
Institute of Cancer Research provide an easy solution.
The approach is based on an NMR phenomenon
called transverse relaxation (see here), which describes how atomic nuclei
return to their ground state after being excited by a radiofrequency pulse in a
magnetic field. The transverse relaxation rate R2 for a given
nucleus depends on the tumbling speed of the molecule in which it is contained:
small molecules tumble rapidly and have small R2 values,
while larger molecules tumble slowly and have larger R2
values. When a small molecule binds to a protein its tumbling speed slows and
its R2 increases. The R2 values can be
measured experimentally using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence.
This is all fairly standard for
NMR spectroscopists, and in fact CPMG is widely used to find fragments. Liu and
colleagues proposed that, by measuring the change in R2 with changing
concentrations of small molecule, they would be able to extract the dissociation
constant (Kd). The theory gets a little hairy (14 equations), and
the analysis depends on non-linear regression curve fitting, but this is easily
done using modern analytical software. The technique is called R2KD.
The experiment itself is straightforward.
The ligand alone is prepared at two different concentrations; these are used to
determine the R2 values of the free ligand. Another eight
samples contain protein and various concentrations of ligand. The R2
values are measured and fit to an equation to extract the dissociation constant.
An initial test case with a known 50 µM ligand for the protein BCL6 was
encouraging, giving a Kd of 53 to 78 µM for four different protons
on the ligand. The accuracy could be further improved by using a “global fit”
with all the data rather than analyzing each NMR peak in isolation.
Next, seven ligands against three
proteins were analyzed using R2KD and compared with their literature values.
Here too the results were in agreement, mostly within a factor of two. The lower
limit for sensitivity is dependent on the NMR signal for the ligand; below
concentrations of about 20 µM the experiments become impractically long. The
upper limit is dictated by the solubility of the ligand. The researchers could
reliably measure dissociation constants around 1 mM and suggested that with a sufficiently
soluble ligand even weaker ligands could be measured.
The R2KD experiment requires that
the protein concentration be less than about 20% of the lowest ligand
concentration. (That said, protein concentrations up to 35 µM gave reasonable
results.) Preserving protein is usually a goal, so lower concentrations
(single-digit micromolar) are desirable from both a practical and theoretical
standpoint.
Finally, the researchers demonstrated
the application of R2KD to assess 10 fragment hits from a 1000-compound screen
against the E3 ligase complex CRBN/DDB1, one of the most popular targets for
PROTACs. The hits had dissociation constants ranging from 70 to 1200 µM, and
the R2KD values were similar to those found in a fluorescence polarization (FP)
assay, though for the most part the affinities from R2KD were higher. In
particular, two compounds with essentially no activity in the biochemical assay
came in at sub-millimolar by R2KD, which may speak to the insensitivity of the
FP assay.
Overall this is a lovely and, as befits this blog, practical paper,
and I hope R2KD becomes widely adopted. With a sweet spot for Kd values
of 10-1000 µM the technique fills an important niche: biochemical assays are
well-suited for tighter binders but less reliable at millimolar ligand
concentrations. As crystallography becomes increasingly popular as a primary screen, I could imagine R2KD being used to rank the resulting fragment hits.
4 comments:
Thats really neat! Nice application to get more out of an existing technology.
Reg the observation you make that this fills the 10uM-1000uM gap, is there a good review on biophysical techniques and the ranges they're best suited for? Or even an image? Looking for one as I prepare an introductory talk for undergraduate class
Hi Anonymous,
This is a nice 2020 review from Astex in Biochem Soc Trans. In particular, Figure 3 is exactly what you're looking for. Enjoy!
Esteban A Fridman, is there a relationship between the R2 used and the R2 used in PET analysis to obtain the BPnd?
Thxs
As a former NMR spectroscopist, I like this easy ligand-observed R2KD method for Kd values of 10-1000 µM. I wonder if this an alternative or will replace the NMR titration. I am sure R2KD will become widely adopted and have great applications in drug development.
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