Fragment screening across the proteome, industrialized

Last week we discussed covalent fragment screens against isolated enzymes, which can be very effective. But screening in cells or cell lysates preserves proteins in a more physiological environment and allows many proteins across the proteome to be screened simultaneously. In 2016 we wrote about covalent screens in human cell lysates which identified fragment hits for 758 cysteine residues in 637 proteins. Mass spectrometry techniques have improved since then in terms of both speed and sensitivity, as illustrated in a new Cell Chem. Biol. paper from Steve Gygi, Qing Yu, and collaborators at Harvard Medical School and Biogen. (Disclosure: Steve Gygi is on the Scientific Advisory Board of my current company, Frontier Medicines.)

 

The approach is called TMT-ABPP, or tandem mass tag activity-based protein profiling, and it involves multiple improvements to previous methods, some of which Steve discussed at the Discovery on Target meeting last year. Covalent fragments are added separately to cell lysate aliquots, after which a desthiobiotin iodacetamide (DBIA) probe is introduced. If a given site on a protein has reacted with a fragment, it will not be available to react with the DBIA probe.

 

Next, proteins are digested to peptides and labeled with TMT (tandem mass tag) reagents, which allow multiple samples (18 in this case, either individual fragments or DMSO-only controls) to be combined for simultaneous analysis. Peptides functionalized with the DBIA probe are captured on streptavidin resin while those that had previously reacted with a covalent fragment will not stick to the resin and be lost. Peptides eluted from the resin are then analyzed by mass spectrometry. The “competition ratio” between treated and untreated lysate gives a measure of how strongly a given site on a given protein is labeled by a fragment.

 

Multiple other tweaks, such as capturing proteins using magnetic beads and using a special type of mass-spectrometry (high-field asymmetric waveform ion mobility spectrometry, or FAIMS), further streamline the process to a 96-well plate format, with each well containing a mere 10-20 µg of cell lysate, as much as 100-fold less than earlier approaches.

 

The researchers benchmarked TMT-ABPP using three reactive “scout fragments,” including compound 1 from last week’s post. Collectively they identified 6813 cysteine residues hit by one or more of the scouts.

 

To demonstrate throughput, the researchers next screened 192 fragments, a third of which were acrylamides while the rest were chloroacetamides. Even with two controls for every 16 samples, this only required 12 injections on a mass spectrometer and resulted in hits against 38,450 cysteines, about 50-fold more than the 2016 paper. Proteins that were more highly expressed were better represented, as were proteins with known reactive cysteine residues, such as thioredoxins. Surprisingly though, surface-exposed cysteine residues were only slightly enriched over more buried cysteines.

 

The researchers also applied TMT-ABPP to five well-characterized covalent molecules, including the mutant KRAS^G12C inhibitor ARS-1620, which we wrote about here. In addition to the G12C site of KRAS, several other proteins were also liganded, including adenosine kinase (ADK). The researchers confirmed that ARS-1620 inhibited ADK in an enzymatic assay.

 

As the researchers note, “proteome-wide profiling of thousands of compounds remains a formidable challenge, both technically and financially.” This paper reveals how to significantly reduce the costs. By using such approaches, it is possible to build a catalog of fragment ligands for thousands of proteins. Doing so with a well-curated library could enable rapid fragment-to-lead campaigns.