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

REPLY

Lodola, Incerti, and Tognolini (1) have raised questions regarding the effective concentrations of inhibitors that we used and the specificity of inhibition described in our recently published article (2).

We thank Lodola et al. for pointing out that some spontaneous transformations of compounds 1 and 2 may be required for their inhibitory activity. In addition to the possible explanations already listed in our publication for the considerable differences in 50% inhibitory concentrations (IC₅₀s) measured in different laboratories, we agree that it would be appropriate to also include the effective concentration of the inhibitory substance.

We feel that it is important to reiterate the substantial body of evidence for the specificity of inhibition that is presented in our publication (2). The compound did not inhibit entry of a vesicular stomatitis virus (VSV) G-transcomplemented retrovirus at concentrations that potently inhibited entry of Kaposi's sarcoma-associated herpesvirus (KSHV). Binding of monoclonal antibody 6F8 to EPHA2 was not inhibited by the compound at concentrations that potently inhibited binding of gH-gL to EPHA2. The ability of the compound to inhibit the different interactions (EPHA2-ephrinA4, EPHA2-ephrin A5, and EPHA2-gH/gL) was shown to be directly related to the measured affinities; the inhibitory potencies toward binding and infection were correlated. Neither compound 1 nor compound 2 exhibited toxic effects in cell culture. Others have similarly shown the specificity for targeting of EPHA2 and EPHA4 by these compounds or their active products (3, 4). For example, Noberini et al. (3) found no inhibition of ephrin A5 binding to EPHA7, EPHB1, EPHB2, EPHB3, and EPHB4 by these compounds despite their potent inhibition of binding to EPHA2 and EPHA4.

It is erroneous to think that the IC₅₀ for a protein-protein interaction should be exactly reflected in the 50% effective concentration (EC₅₀) for inhibition of entry of virus into a cell, as suggested by Lodola et al. (1). A large number of additional factors will influence the ability of an inhibitor to block entry of a virus into a cell. For example, receptor density will be one crucial factor. The virus needs to engage only one receptor complex on the surface of a cell to gain entry. The higher the number of receptors on the surface, the greater the concentration of inhibitor that will be needed to block entry. In fact, the rank order of EC₅₀s in the three different cell lines used in our recent Journal of Virology article (14, 47, and 276 μM) (2) corresponds exactly to the EPHA2 mRNA levels in these same cells measured previously (5). Thus, receptor density along with a diverse array of other factors can explain the wide variation in EC₅₀s on different cell types in our publication.