As our poll last year demonstrated,
there is no shortage of methods to find fragments. But that doesn’t mean new approaches
aren’t welcome, particularly when they also apply to fragment growing. This is
the promise of a recent paper in J. Med. Chem by Thomas Kodadek and collaborators
at University of Florida Scripps and Deluge Biotechnologies. (Tom and first author
Isuru Jayalath also presented this at the DDC meeting earlier this year.)
The researchers were inspired by
the concept of avidity, the observation that multiple copies of a ligand bound to
a multiprotein assembly can form a more stable complex than monomeric ligands bound
to monomeric proteins. Could this phenomenon be exploited to find weak
fragments?
A previous DNA-encoded library
screen on streptavidin had identified 28 macrocycles, all of which contained
one of two closely related fragments. The affinity of the more potent fragment
came in at 706 µM using SPR. The researchers coupled this fragment to TentaGel
beads, 10 µm wide polystyrene spheres covered in polyethylene glycol (PEG)
chains terminated by amine groups. The PEG makes the beads water soluble. The
beads were soaked in a solution of fluorescently labeled streptavidin, washed, and
analyzed. Importantly, streptavidin exists as a tetramer, so each tetramer could
bind up to four bead-bound fragments.
Streptavidin bound avidly to the
beads, even when incubated at low (50 nM) concentrations. A control protein did
not bind, nor did streptavidin bind to beads modified with a negative control
fragment. Moreover, a monomeric version of streptavidin did not bind to the
beads, illustrating the importance of avidity. Finally, adding the natural ligand
biotin kept streptavidin from binding to the beads.
TentaGel beads have long been
used in combinatorial synthesis, so the researchers built a small library in
which the initial fragment was coupled to 48 carboxylic acids. These were then
incubated with labeled streptavidin, and some of the beads showed more intense
fluorescence, suggesting more protein binding. SPR analysis revealed that these
new molecules had improved affinity, with the best coming in at 90 µM as a
monomer. Thus, the primary screen can rank order affinities.
This is great for oligomeric
proteins, but what about the large number of targets that are monomeric? Many
recombinant proteins are expressed as fusions with glutathione S-transferase
(GST), which facilitates purification. Importantly, GST exists as a homodimer
in solution. The researchers screened a GST fusion of the oncology target Rpn13
against a small library of 94 fragment-coupled beads and found five hits. SPR
studies confirmed weak (KD > 2 mM) binding for two hits to pure Rpn13 (ie,
without the GST fusion), and this binding could be competed with a known peptide
ligand of Rpn13.
Screening beads in individual
wells is one thing, but to really increase throughput it would be nice to be
able to screen mixtures of different beads. To do so, the researchers developed
a photocleavable linker between bead and fragment. The linker also contained an
alkyne group that could be modified with a brominated imidazopyridinium moiety.
This tag is UV active, ionizes well, and the bromine’s unique isotopic
signature helps distinguish true hits from noise. Beads containing more than 50 different
compounds, including the two fragment hits we mentioned above, were incubated
with labeled streptavidin. Beads to which protein bound were separated by
fluorescence-activated cell sorting (FACS), clicked with the tag, cleaved from
the beads, and analyzed by mass spectrometry. Only the two known binders were
identified, demonstrating the specificity of the approach.
This is a neat paper well worth reading.
I particularly like the fact that the method can be done with minimal
equipment. I look forward to seeing how it works against more targets.
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