Surface-plasmon resonance (SPR)
is among the most popular methods for finding fragments. However, as we have
noted, SPR can be very prone to operator error and misinterpretation. In a recent (open
access) SLAS Discovery paper, U. Helena Danielson (Uppsala University)
and a who’s-who team of biophysicists from across Europe provide experimental
strategies for screening difficult proteins.
The researchers chose five
different proteins, some of which were screened in two or three different forms
for a total of nine protein constructs. Six of these were screened against
their FL1056 library, a custom-built 1056-member library which include molecules
from the FragNet program. The library includes a number of “three dimensional” molecules
as assessed by principal moment of inertia (PMI). The other library, FL90, is
a small set of commercially available fragments we highlighted here.
Before screening compounds
against proteins, the researchers conducted a “clean screen.” This involved
injecting fragments (at 500 µM each) over the sensor surface using the same
buffer that would be used in the actual screen to pre-identify fragments that
stick to the surface. This typically disqualified about 1% of fragments, though
for one set of conditions the number was closer to 3%.
That work done, the researchers
turned to the actual screens. After proteins were immobilized on the sensor
chips, the fragments were typically screened at a single concentration of 250 µM
each. The threshold for the initial hit cutoff was set low,
often around 10% of the library, to minimize false negatives. Subsequent follow-up studies at varying
concentrations were used for confirmation. This led to a significant winnowing,
with the final number of confirmed hits between 0.5 and 7% of the library.
The proteins themselves were intentionally
chosen to present various difficulties. Acetylcholine binding protein (AChBP, which
we wrote about here) forms a large (125 kDa) pentameric complex with multiple
binding sites. Lysine demethylase 1 (LSD1) is a multidomain, cofactor-dependent
protein that requires a partner protein, CoREST, for activity. LSD1 was
screened in the presence or absence of CoREST. Farnesyl pyrophosphate synthase
(FPPS, which we wrote about here) is a target for cancer and osteoporosis, and
the microbial forms are targets for trypanosomiasis drugs. Human as well as Trypanosoma
cruzi and Trypanosoma brucei proteins were screened. Protein
tyrosine phosphatase 1B (PTP1B, which we recently wrote about here) is a difficult
enzyme with a couple allosteric sites. The C-terminal region is intrinsically
disordered, and the protein was screened with or without this region. Finally,
human tau is both intrinsically disordered and prone to aggregation. As we
noted earlier this year it is of interest due to its potential
role in Alzheimer’s disease.
Happily, hits were identified
against all the proteins, some with ligand efficiency values above 0.5 kcal/mol
per heavy atom. The chemical structures for selected hits are shown, and the researchers
do appropriately caution that validating them using orthogonal (non-SPR)
methods is essential before further studies.
I do wish the researchers had
noted whether shapely hits were enriched or depleted among the confirmed hits.
To my eye most seemed fairly flat, and some seemed dubiously PAINS-like,
including an eyebrow-raising dinitro-catechol. Nonetheless, the paper is a nice
summary of multiple SPR campaigns. If you’re about to embark on one yourself,
it is worth carefully perusing.
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