12 February 2024

Fragment screening across the proteome, noncovalently

Last week we discussed methodological improvements to industrialize covalent fragment screening across the proteome. While I’m a huge fan of covalent binders, their noncovalent counterparts are the vanilla ice cream of FBLD: also tasty and much more common. Back in 2017 we described how “fully functionalized fragments,” or FFFs, could be used to screen noncovalent fragments in cells. A new paper in Nat. Chem. Biol. by Christopher Parker and collaborators at Scripps and BMS further optimizes the approach.
 
FFFs contain, in addition to the variable fragment, a photoreactive group (often a diazirine) and an alkyne tag. When exposed to light the photoreactive group can react with nearby proteins and the alkyne tag can be used to fish out the proteins. In the new paper the researchers started with a dozen FFFs.
 
One challenge, which we discussed in 2021, is that the FFFs may react with many sites on a given protein. During analysis, a protein is typically digested into peptides for mass spectrometry. If a FFF reacts at several sites on a peptide the resulting spectra will be “chimeric” and more difficult to characterize.
 
The researchers developed methods to take these chimeric spectra into account when searching for sites of modification. The approach, called Dizco (for diazirine probe-labeled peptide discoverer) can identify three times as many peptides as standard approaches, as well as more detailed information on sites of modifications. 
 
Two pairs of FFF probes consisted of enantiomers, and these showed differential labeling across the proteome, consistent with specific molecular recognition. The researchers also confirmed binding of a few FFF probes to several proteins using a cellular thermal shift assay (CETSA).
 
In all, the probes modified 3603 peptides on 1669 proteins. The sites of modification were then mapped onto predicted or modeled three dimensional structures of the proteins. Importantly, and consistent with the 2017 work, most of the labeled sites were near predicted pockets. The researchers confirmed this for four proteins by showing that FFF probe binding could be competed by adding ligands known to bind to the pockets.
 
Next, the researchers docked (using AutoDock) their FFF probes onto 175 proteins (108 from structures in the Protein Data Bank and 67 from AlphaFold structures). They found that the docking experiments recapitulated the experimental data, and in fact often placed the diazirine tag near the protein residues found to react. Strikingly, and in another step forward for in silico approaches, docking against structures from AlphaFold was nearly as effective as those from the protein data bank.
 
As the researchers conclude, “we identified many binding pockets that have no reported ligands… these probes may serve as leads for further optimization.” It will be fun to see how far they go.

No comments:

Post a Comment