On the Use of 2,5-Dimethyl-Pyrrol-1-yl-Benzoic Acid Derivatives as EPH-Ephrin Antagonists

LETTER

Since their discovery as EPH receptor inhibitors in 2008 (1), 2,5-dimethyl pyrrol-1-yl-benzoic acid derivatives 1 and 2 (Fig. 1) have been used as pharmacological tools aimed at elucidating the role of EPH kinases in pathological conditions (2, 3). Compounds 1 and 2 were originally found to block the binding of ephrin A5 to EPHA2 receptor with inhibitory potency in the low micromolar range, i.e., with 50% inhibitory concentrations (IC₅₀s) of 65 and 33 μM, respectively (1).

FIG 1.

Chemical structure of 2,5-dimethyl pyrrol-1-yl-benzoic acid derivatives 1 and 2.

In a recent issue of Journal of Virology, Hahn and Desrosiers (4) observed that 2,5-dimethylpyrrol-1-yl-benzoic acid derivatives were significantly less potent at preventing EPH-ephrin interactions than what was originally reported by Noberini et al (1). The IC₅₀ for inhibition of the ephrin A5 interaction with EPHA2 was estimated to be 1,683 μM for compound 1 and >2,000 μM for compound 2. To explain this considerable discrepancy, Hahn and Desrosiers (4) suggested that different assay conditions and reagent concentrations accounted for the inconsistency in the experimental IC₅₀s. However, in the same work, compound 1 surprisingly became 100-fold more active in a functional assay conducted in human umbilical vein endothelial cells, which naturally express the EPHA2 receptor. In this assay, the IC₅₀ of compound 1 decreased to 14 μM, suggesting the involvement of a biological pathway not correlated with the EPH-ephrin system or the formation of an unknown metabolite of compound 1 in cells characterized by an exceptional affinity for EPHA2.

Prompted by these controversial findings, we performed a search in the literature, and we found that the 2,5-dimethyl pyrrol-1-yl derivatives are classified as pan-assay interference compounds (PAINS), giving positive results in a large number of biological assays in which they were tested (5). More recently, the group of Prof. Elena Pasquale showed that a freshly synthetized compound 1 was not active on the EPH receptors (6). However, when exposed to air, compound 1 gained a brown color and, along with this transformation, it became able to inhibit the ephrin A5-EphA4 interaction with an IC₅₀ in the micromolar range. This finding suggested that compound 1 underwent chemical modifications, likely oxidation followed by polymerization, leading to the formation of an unknown chemical species active in the enzyme-linked immunosorbent assay (ELISA) (6).

The chemical processes occurring on 2,5-dimethyl pyrrol-1-yl-benzoic acids have been recently elucidated (7). Specifically, it has been demonstrated that 2,5-dimethyl pyrrol-1-yl-benzoic acids spontaneously form a mixture of polymers with an average molecular mass of 40 kDa (7). Furthermore, it has been proposed that these polymers are likely the chemical entities responsible for the promiscuous biological activity attributed to the 2,5-dimethyl pyrrol-1-yl class (7).

The inconsistency in the binding data reported by Hahn and Desrosiers reflects the difficulties encountered in manipulating reactive compounds in biochemical assays. As the biological activity of compound 1 is due to chemical modifications simply triggered by air and light (6), we believe that experimental evidence obtained with the use of these compounds should be critically reviewed and that, more generally, the use of highly unstable pharmacological tools should be avoided in biological assays.