Irreversible fragments

Last year we highlighted a paper in which fragments were attached to a “warhead,” a chemical group that could form a reversible covalent bond with cysteine residues in proteins. These fragments were then screened against a kinase to identify nanomolar inhibitors. In a new paper in J. Med. Chem., Alexander Statsyuk and coworkers at Northwestern University describe a similar approach using irreversible fragments. (See here for In the Pipeline’s take.)

As we noted last year, one of the nice things about using reversible warheads is the fact that you can run experiments under thermodynamically-controlled conditions. Indeed, this was the idea behind Tethering (here and here), in which a library of disulfide-containing fragments is allowed to equilibrate with a cysteine-containing protein; if a fragment has inherent affinity for the protein, the disulfide bond will be stabilized towards reduction and can be identified using mass spectrometry. (Full disclosure: I am an inventor of Tethering, and my company, Carmot Therapeutics, Inc., has an exclusive license to the intellectual property covering this and other technologies.)

Irreversible warheads operate at least partly under kinetic, rather than thermodynamic, control. For example, if all the fragments are extremely reactive, the protein will react with whichever fragment it happens to encounter first, regardless of whether the fragment has any inherent affinity for the protein.

Acrylamide moieties are able to form irreversible bonds to cysteine residues and are even starting to be found in drugs. In 2012, researchers at Imperial College London tested an acrylamide-containing analog of one of the (disulfide) hits from the original Tethering paper. This successfully labeled the target protein thymidylate synthase (TS), while several other acrylamide-containing molecules did not, and the fragment was selective for TS over two other enzymes with active-site cysteine residues.

Regardless of whether your warhead is reversible or not, it is important that different members of the library have similar reactivities: if the inherent reactivities of the fragments are different, it will be difficult to distinguish inherent binding energies from chemical reactivities. One of the problems with acrylamides is that subtle changes in chemical structures can have dramatic effects on intrinsic reactivities. The new paper compares rate constants of two acrylamides reacting with a low molecular-weight thiol, and finds that one reacts 2,000-fold faster than the other. The researchers tested three other classes of electrophiles – vinylsulfonamides, aminomethyl methyl acrylates, and methyl vinylsulfones – and found that these had narrower ranges of reactivity. They chose acrylates and built a set of 100 acrylate-modified fragments. Happily, when 50 of these were tested for reactivity, there was only a 2.4-fold difference between the most reactive and the least reactive members.

These 100 fragments were tested in pools of ten against the classic cysteine protease papain using mass spectrometry, and three hits were identified. Enzymatic assays revealed that these three hits were also irreversible inhibitors of the protein with respectable activities (k_inact/K_i = 0.46-1.2 M^-1s^-1), while a member of the library that did not label in the mass spectrometry assay was a weaker inhibitor (k_inact/K_i = 0.037 M^-1s^-1). Moreover, the papain hits did not label three other enzymes containing active-site cysteines, though these enzymes could be modified with other fragments in the library.

In the case of Tethering, the disulfide linker was a means for finding fragments; when these fragments were developed further, the disulfide linker was replaced. However, given the renewed interest in covalent drugs, some warheads might be able to be retained. It will be fun to see how these types of strategies develop.