Well that’s an acronym soup!
SAR by NMR was the first practical fragment-finding method, and over the years
Practical
Fragments has covered
lots of other techniques. Small-angle X-ray scattering,
or
SAXS, has not been among them. As the name suggests, this technique uses
X-rays, typically produced at a synchrotron. However, unlike conventional
crystallography, it doesn’t require crystalline material. Instead, proteins in
solution are analyzed to provide information on their size and shape. The
resolution is too low to assess small molecule binding, but suitable for observing
dimerization or changes in conformation.
Time-resolved SAXS, or TR-SAXS, examines
SAXS over time in response to a trigger. For example, you can rapidly add a
ligand to a protein and watch for changes in conformation. And HT simply means high
throughput. A recent
Nature Chemical Biology paper from Chris Brosey,
John Tainer, and collaborators at the University of Texas MD Anderson Center, Lawrence
Berkeley National Laboratory, University of California Santa Cruz, and University
of Arkansas for Medical Sciences Little Rock describes structure-activity
relationships by time-resolved high throughput small-angle X-ray scattering (TR-HT-SAXS).
The researchers were interested
in apoptosis-inducing factor (AIF), a mitochondrial protein with potential implications
for cancer and other diseases. AIF normally exists as a monomer in complex with
an FAD cofactor. Binding of NADH causes reduction of FAD to FADH- and
concomitant dimerization of the protein. Could fragments do the same, allowing
dimerization on demand?
A library of 2500 fragments
purchased from
Life Chemicals was screened at 0.75-1.5 mM against the AIF-FAD
complex using differential scanning fluorimetry (
DSF), and those that raised or
lowered the temperature by more than 1.7 ºC were further characterized by
microscale thermophoresis (
MST). This led to 32 binders and 7 negative controls,
or molecules that did not confirm either by DSF or MST. (Side note: although
many people discount compounds that give
negative thermal shifts, the natural
ligand NADH lowers the melting temperature of AIF by a whopping 10.8 ºC.)
Next, the fragment binders and
negative controls were screened at 0.5-1 mM by TR-SAXS. Intense X-rays cause
reduction of the FAD cofactor, but in the absence of NADH or other ligands the
AIF protein remains monomeric. However, some fragments did cause dimerization of
the protein during TR-SAXS. Interestingly, these fragments were structurally
related to one another. Subsequent crystallography revealed that they bind
where NADH normally binds and make some of the same interactions to induce protein
dimerization. The paper includes much more detailed characterization, including
mutagenesis, spectroscopic, and protein crosslinking experiments to further
understand the mechanism.
TR-SAXS is an interesting addition
to our toolbox of biophysical methods suitable for fragment screening. It does
have some disadvantages, such as the need for large amounts of protein at high
concentrations: 67 µM in this case. Also, the “HT” may be somewhat
aspirational, with a current throughput of 100-200 compounds per synchrotron
shift. Finally, the technique is probably best suited to well-characterized
proteins where SAXS data can be carefully modeled. With these limitations in
mind, it will be fun to see how generally TR-SAXS finds fragments that alter the conformation and multimerization
of proteins.
