Metallophilic fragments – or PAINS?

Several years ago we described fragment libraries designed to chelate metal ions. The idea is that these could serve as affinity anchors to target metal-containing proteins. However, in designing any fragment library, it is essential to avoid pan-assay interference compounds (PAINS), molecules that act through pathological mechanisms. A new paper in Bioorg. Med. Chem. Lett. by Amy Barrios and collaborators at the University of Utah and University of California San Diego illustrates one of these mechanisms.

The researchers were interested in a protein tyrosine phosphatase (PTP) called LYP. PTPs contain catalytic cysteine residues and are thus particularly prone to false positives caused by oxidation or adventitious metals such as zinc. The researchers screened a library of 96 metal-chelating fragments against LYP in the presence or absence of zinc to find fragments that could either rescue the enzyme or inhibit it, either cooperatively with zinc or on their own. Not surprisingly, they were able to find several fragments that could rescue LYP from added zinc, presumably by coordinating the metal and removing it from the active site.

The most potent inhibitor of the enzyme was 1,2-dihydroxynaphthalene, with an IC₅₀ of 2.5 µM. However, the researchers quickly discovered that it was time-dependent, showing greater potency the longer it was incubated with the enzyme. This is a classic sign of something funny going on, and the researchers realized that molecules like this can oxidize spontaneously. In fact, the oxidized product (1,2-naphthoquinone) is even more potent, and also time-dependent (IC₅₀ = 1.1 µM after 2 hours). Not only can napthoquinones directly modify cysteine residues, they can generate reactive oxygen species that can in turn modify cysteine residues – this very molecule was reported as doing so more than five years ago.

This is the kind of mechanism you want to avoid, and it is likely to rear its ugly head whenever compounds of this ilk are screened, particularly against a protein with an active-site cysteine. Hopefully this publication will serve as a warning to folks who may be using this screening library.

One could argue that this paper falls into the category of “you probably already knew this.” However, even if you knew it, many others likely did not. At the ACS meeting symposium on PAINS earlier this month, Kip Guy urged people to publish their PAINS stories. They may not be the sexiest papers, but if they inform others what to avoid they may be among the most useful.