Covalent fragments vs RAB27A/B: towards a chemical probe

Last week we highlighted an example of adding a warhead to a fragment-derived, non-covalent protein binder to generate a covalent inhibitor. This is probably the most common method for identifying covalent ligands. But as we noted in 2019, the reverse “covalent-first” approach can be just as effective. A recent open-access paper in J. Am. Chem. Soc. by Elena De Vita, Edward Tate, and collaborators at Imperial College London and University of Nottingham provides another success.

 

The researchers were interested in the closely related proteins RAB27A and RAB27B, two of the more than 60 members of the Rab family, which is itself part of the large Ras superfamily. RAB27A/B are potential antitumor and antiviral targets, but the only reported chemical probe, Nexinhib-20, sets off PAINs alerts.

 

The researchers had previously found two non-conserved reactive cysteine residues, C123 and C188, near the co-called effector binding region, a shallow pocket involved in protein-protein interactions essential for RAB27A/B function. They screened a library of 960 acrylamide-containing fragments, each at 500 µM, in an assay that detects free thiols. Versions of RAB27A in which either C123 or C188 was mutated to a serine were used so the researchers could assess whether compounds were selective for one cysteine. They also screened the compounds against glutathione, the abundant cellular thiol present in all mammalian cells, and focused on those compounds that were at least five-fold more reactive against the protein than against glutathione. The best compound was IMP-1704, which modified C123 much more efficiently than C188.

 

A crystal structure of IMP-1704 bound to RAB27A confirmed that the compound binds in the “WF pocket” of the effector region, with the phenyl ring displacing a tyrosine residue and a sulfonamide oxygen atom making a hydrogen bond to a lysine residue. The researchers also obtained a crystal structure of IMP-1704 bound non-covalently to a C123A mutant of RAB27A and found that it binds in a similar manner. In other words, IMP-1704 does not require the acrylamide warhead to bind the protein, though with no reported affinity it might be quite weak.

 

The crystal structures suggested a fragment growing strategy from the pyrrolidine ring, leading to IMP-1712, which modified the protein twice as rapidly. Interestingly, the enantiomer of this compound was inactive. Further SAR and fragment-growing ultimately led to the most potent compound in the paper, IMP-2552. As with IMP-1712 the enantiomer was inactive, providing a useful negative control

 

Although the covalent efficiency of IMP-2552 is relatively modest, the researchers used an analog with a clickable alkyne probe to assess what proteins it binds to in cells. RAB27A was one of the top protein hits, and engagement with RAB27A could be competed with the non-tagged IMP-2552. Coimmunoprecipitation experiments demonstrated that IMP-2552 could block interaction with an effector protein, while the enantiomer was inactive. IMP-2552 was also active at 10 µM in a phenotypic assay, while the enantiomer was not.

 

Importantly, the researchers also profiled Nexinhib-20, the previously reported inhibitor. In contrast to IMP-2552, Nexinhib-20 reacted with both C123 and C188 and also reacted very rapidly with glutathione, suggesting generic rather than specific reactivity. Moreover, in the phenotypic assay at 10 µM, “Nexinhib-20 showed profound cytotoxicity and cell death.” Hopefully these findings will make people reconsider before using Nexinhib-20 in their assays, or when reading conclusions drawn from using this compound.

 

Overall this paper is a nice example of screening for covalent ligands and optimizing them. It is also another application of covalent fragment screening applied to a GPTase; we’ve written previously about KRAS. In contrast to KRAS and several other covalent programs, the initial hit here is rule-of-three compliant.

 

And although improvements in potency and selectivity are still needed to formally designate IMP-2552 a chemical probe, the molecule appears to be the best we currently have, particularly when used with its negative control.