19 March 2018

Industrializing native MS: hundreds of fragments against dozens of targets

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.

Ultimately I would take any of these molecules with a huge dose of caution. That said, there are lots of interesting molecular structures in here, so if you’re looking to jump-start a program against malaria while exploring new chemistry, it may be worth digging into the data.


Unknown said...

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.

Dan Erlanson said...

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.