Fragments vs viral protein EBNA1
Epstein-Barr virus (EBV) infects more than 90% of adults. In most cases it remains latent, but even then it expresses genes that cause cellular proliferation, which can lead to cancer. In fact, up to 2% of human cancers are caused by the virus. In a recent paper in Sci. Transl. Med., Troy Messick, Paul Lieberman (both at the Wistar Institute) and a large group of collaborators (including Teddy Zartler) take aim at this pathogen.
The researchers focused on the viral protein EBNA1, a DNA-binding protein essential for viral replication as well as host cell survival. They started with a virtual screen of 1500 fragments from Maybridge, and then did fragment soaking of the top 100 hits. Happily, this resulted in structures of 20 fragments in four separate sites on the protein. Less happily for modelers, none of the fragments bound as predicted – more grist for the “crystallography first” argument.
A dozen fragments bound in a deep hydrophobic pocket, and most of them contained an acidic moiety that made hydrogen bond contacts to conserved threonine and asparagine residues that normally contact the DNA backbone. Merging two of these fragments led to VK-0497, which disrupts binding of EBNA1 to DNA at sub-micromolar concentrations. Crystallography confirmed that it bound as expected. The molecule contains a potentially unstable pyrrole, and replacing this with an indole and growing led to molecules such as VK-1248, with high nanomolar activity in the DNA-binding assay. Additional biophysical techniques including SPR, ITC, and 2-dimensional (HSQC) NMR confirmed binding for this and related compounds.
The carboxylic acid moiety likely reduces permeability across cell membranes, and indeed the compounds showed no activity in cells. However, methyl esters were found to be rapidly cleaved by intracellular esterases, and these prodrugs were tested in a variety of assays.
The prodrugs inhibited proliferation of EBV-positive human cells but had no effect on non-infected cells. The prodrugs also reduced expression of both viral and host proteins. More importantly, they inhibited tumor growth in xenograft models using four different cancer cell lines, two of which were patient-derived. Prolonged dosing over as long as eight weeks showed a sustained effect, which is reassuring in terms of drug resistance. Finally, the molecules could effectively be combined with existing drugs or radiation.
There is still much work to be done, not just in terms of potency but also further pharmacokinetics, pharmacology and toxicology. And as the researchers acknowledge, xenograft models are regrettably poor surrogates for humans. Still, this is an interesting approach, and hopefully further work will be done on this series, or at least the target.