Native mass spectrometry (MS) is a direct binding assay in
which fragment binding to a target is detected when the complex is ionized and
“weighed” in high vacuum. The technique is less commonly used than others, and there is
some debate as to how well it works. A paper just published in ACS Infect. Dis. by Ronald Quinn and
collaborators at Griffith University, the University of Washington, and the
University of Toronto provides some encouraging data.
To demonstrate just how high-throughput native MS could be,
the researchers started with 79 different proteins. These were all from Plasmodium falciparum, one of the main
organisms that causes malaria. The proteins were chosen based on their size
(< 50 kDa, for easier MS analysis) and likely importance for the parasite.
Of these, 62 gave a good signal-to-noise ratio by native MS and were screened.
The researchers used an existing fragment library
of 643 natural products; we highlighted an earlier version of this library in
2013. Of these, 602 molecules met the strict criteria defined in that design,
with MW < 250 Da but with other properties more relaxed than rule of three
guidelines. The library also contained significantly fewer aromatic rings than conventional
fragment libraries and was more “three dimensional,” as assessed both by PMI
and Fsp3.
Fragments were screened in pools of 8 at 5-400 µM each, with
protein present at 1-20 µM; final ratios were 5:1 to 20:1. Hits were judged
qualitatively as strong, medium, or weak, and the researchers estimate that
strong and medium binders have dissociation constants < 100 µM.
Just over half of the proteins (32) had at least one hit,
and a total of 96 fragments came up as hits. Importantly, many of these were
selective: 48 fragments bound just one target, while another 18 bound just two
(fragments that hit more than 6 proteins were considered promiscuous and
excluded from further analysis).
Similarly to what has been done with NMR and thermal-shift assays, the researchers suggest that native MS can be used to assess
ligandability. This is an appealing suggestion, though the researchers do not
correlate MS-assessed ligandability with other methods such as SPR or
high-throughput screens.
Conventionally, the next step would be to confirm binding
with orthogonal techniques. Instead, the researchers took the rather bold move
of testing fragment hits against the parasite directly. Remarkably, 79 of the
fragments were active at 100 µM, with 13 having IC50 values < 45
µM.
A major strength of this paper is the disclosure of all the
hits against all the targets. Not only does this allow others to confirm the
results, it also provides starting points for further studies. So what do the
fragments look like? Many of them are somewhat PAINful – we previously
mentioned the promiscuity of one of their compounds, securinine. Although this
molecule only hits two proteins in their panel, previous research has found
that native MS can give high false-negative rates. Moreover, even if a molecule is truly inactive against
a few dozen proteins, that doesn't mean it won’t hit many of the
thousands of other proteins in a live protozoan.
2 comments:
I'm curious about when we can say a fragment is promiscuous. Here they say 5 fragments bound to 5 proteins and 7 bound to 6 proteins. Then the authors say that binding to 6 proteins means they are promiscuous. I know it's relative to their screen, but are there more defined parameters that people have used in the past to declare a fragment as promiscuous? Perhaps low affinity combined with binding multiple proteins?
Promiscuity at the start (assuming you don't have better options) doesn't necessarily mean med chem won't increase the specificity.
Hi Daniel,
You raise good points - promiscuity is really a subjective concept, as evidenced by the fact that three clinical compounds against different kinases have all been derived from the same fragment. I don't think there are hard and fast rules, and certainly if you have good structural information I wouldn't worry. The more difficult case is where you don't have a structure, such as in the case of PrATs , which seem to show up as frequent hits but are difficult to move forward.
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