Back in 2017 we
observed with
characteristic subtlety that “heavy metals suck.” That post described a
hit-finding campaign which foundered when the apparent activity of the
fragments turned out to be due to contaminating zinc. A new
paper in
J. Med.
Chem. by Thomas Gerstberger, Peter Ettmayer, and colleagues at Boehringer
Ingelheim (BI) describes a similar story, along with suggestions of how to
avoid being misled.
BI had a collaboration with FORMA
Therapeutics that entailed screening roughly 1.7 million compounds against ten
targets using biochemical and cell-based assays. The effort resulted in
chemical probes against BCL6 and SOS1 and a clinical compound against the
latter. Another target was the activated (GTP-loaded) form of KRAS
G12D.
Of the 6917 hits from the primary AlphaScreen assay, 1535 gave dose-response
curves and passed various counter screens. Of these, 87 representative compounds
were tested in
STD NMR and
thermal shift assays. Only seven confirmed by STD
NMR, but these did not confirm by
SPR or crystallography.
In parallel, the researchers were
successfully using FBLD to develop inhibitors of KRAS
G12D, which we
wrote about
here. Some of the fragment hits were structurally similar to those
from the HTS screen, and further searching of the FORMA library led to fairly
potent (high nanomolar or low micromolar) hits in the AlphaScreen assay. Two of
these even yielded crystal structures, though despite their chemical similarity
to one another they bound to the protein in completely different orientations.
Unfortunately, follow-up work
“revealed erratic structure-activity relationships,” and upon resynthesis the compounds
were much less active. At this point the researchers became suspicious, and
analyses of the original samples showed they contained >20,000 ppm of
palladium contamination. Furthermore, PdCl2 itself turned out to be
a low micromolar inhibitor in the assay.
Metals are frequently used as catalysts
or reagents in organic synthesis and can be difficult to completely remove during
purification. Worse, their presence is often not detectable using standard
purity assessments such as HPLC and NMR. Particularly in the case of fragments,
which are expected to have low affinities, a small amount of metal contaminant
could give a reasonable-looking but misleading signal in an assay.
To avoid this problem in the
future the researchers developed a Metal Ion Interference Set, or MIIS,
consisting of a dozen different metal ions and other salts, all soluble in DMSO
so as to be compatible with typical screens. The MIIS is now routinely screened
before initiating HTS campaigns, and the results of 74 assays are summarized in
the paper. Pd2+, Au3+, and Ag1+ are
particularly nasty, often giving IC50 values < 1 µM, but every
metal gave IC50 values < 10 µM in at least two assays.
Biochemical assays such as AlphaScreen or TR-FRET were more susceptible to
artifacts, with 20.9% showing IC50 < 10 µM, while biophysics
assays such as mass spectrometry were better behaved, with only 2.3% showing IC50
< 10 µM. Cellular assays were also surprisingly robust, with 6.3% showing IC50
< 10 µM.
This is a nice paper showing that
even a massive screen may produce no useful chemical matter. Soberingly, the
fact that some of the fragments gave reasonable-looking crystal structures even though the functional activity came from metal contaminants is a salutary
reminder that just because you have a crystal structure of a bound ligand
doesn’t mean you have a viable starting point.
Forewarned is forearmed, and the
MIIS appears to be a valuable tool for assessing assay sensitivity to metal
ions, which are all too often lurking invisibly in compound samples.
