Nucleophilic fragments: the other kind of covalent inhibitors

Covalent fragment-based lead discovery is becoming increasingly popular, spurred on by the rapid discovery and approval of sotorasib. In general, covalent inhibitors contain cysteine-reactive electrophiles, though efforts are also targeting other amino acid residues such as serine and lysine. In all these cases though, the fragment contains an electrophile, while the protein contains the nucleophile. A new paper in J. Am. Chem. Soc. by Megan Matthews and collaborators at University of Pennsylvania and Oberlin College turns things around.

 

None of the twenty standard amino acids are electrophilic, but some proteins do use electrophilic cofactors, such as pyridoxal phosphate. Moreover, some proteins undergo post-translational modifications which introduce a pyruvoyl (Pyvl) or glyoxylyl (Glox) group onto the N-terminus; these contain, respectively, an electrophilic ketone or aldehyde. As we wrote about here, aldehydes and ketones can react covalently with hydrazines, and the new paper shows that the kinetics of this reaction vary – as expected – with the nucleophilicity of the hydrazine.

 

Next, the researchers assembled a library of 17 fragment probes containing both a nucleophile as well as an alkyne that could be used for click chemistry. These probes were screened against cells for 30 minutes at 37 °C, the cells were lysed, labeled proteins conjugated to a dye, and the whole gemish run on a denaturing gel; the results showed a wide range of reactivities for the different probes.

 

To assess which proteins were reacting with which probes, the researchers turned to isoTOP-ABPP, a chemoproteomic method we previously wrote about here in the context of electrophilic fragments. (Chemical biologists are fond of abbreviations, and they call this new approach with nucleophilic fragments “reverse-polarity activity-based protein profiling”, or RP-ABPP.) Three probes, P11, P12, and P13, were found to modify 98, 60, and 16 proteins, respectively. Remarkably, despite their small size and common hydrazine nucleophile, only a single protein was labeled by all three probes.

 

Two of the proteins labeled by P11 include secernin-2 and -3 (SCRN2 and SCRN3). The functions of these proteins are unknown, though genome-wide studies have associated SCRN3 with several diseases.

 

The requirement for the probes to contain both an alkyne handle and a nucleophile increases complexity, and the researchers recognized that they could use the probes in competition mode against fragments lacking the alkyne. They assembled a set of 45 nucleophile-containing fragments and treated cell lysates with these, followed by treatment with probe P11, click chemistry to introduce a fluorescent dye, and gel electrophoresis. Hydrazine-containing fragments that inhibited the binding of P11 were found for SCRN2, SCRN3, and the protein AMD1. Some of these fragments showed EC₅₀ values less than 1 µM and were up to 25-fold selective for SCRN3 over SCRN2 despite the 54% sequence identity shared between the two proteins.

 

An orthodox medicinal chemist might sniff at the hydrazine moiety in these molecules, but it is worth noting that P12, P13, and P17 are all derived from approved drugs (carbidopa, hydralazine, and phenelzine; substructures colored blue).

 

The functional roles of Pyvl and Glox modifications in proteins are poorly understood, and whether modulating them will prove useful in treating diseases remains uncertain. But the best way to answer this question will be by inventing suitable chemical probes. This paper suggests that nucleophilic fragments may prove useful.