09 October 2012

Fragments vs Hepatitis C NS3 protein

Hepatitis C is the target of numerous drug discovery programs, so it was only a matter of time before fragments were used to tackle it. In a paper just published online in Nature Chemical Biology, Harren Jhoti and colleagues at Astex Pharmaceuticals describe a particularly elegant example of fragment optimization against an unusual allosteric site.

The NS3 protein contains two functional domains, both of which are essential for viral function. Two drugs have recently been approved that target the serine protease domain, and the helicase domain has also been extensively studied. However, much of the effort has focused on truncated proteins consisting of only one domain without the other; in the cell, the protein remains intact and also complexes with another viral polypeptide, NS4a. It was this full-length protein complex that the Astex researchers went after.

In the full-length form of the protein, the protease is auto-inhibited by the C-terminus of the helicase domain, which binds in the active site of the protease domain. A crystallographic fragment screen identified fragments such as Compound 2 (below) that bind in a pocket near the protease active site, and the researchers wondered whether these might trap the protein in the inactive state. Compound 2 binds in a hydrophobic pocket and weakly inhibits the protease activity of the full-length protein. Initial optimization focused on improving hydrophobic contacts and restricting the conformational mobility of the molecules, leading to compound 4, with low micromolar activity. Further optimization to pick up additional polar contacts led to compound 6, with nanomolar biochemical and cell-based activity.

As is often (though not always) the case in fragment optimization, the optimized compound 6 shows a similar binding mode to the initial compound 2.

If the compounds truly act by keeping the NS3-NS4a protein complex in the “closed,” or auto-inhibited state, this should be detectable by various biophysical measurements, and in fact sedimentation velocity analysis, size exclusion chromatography, and dynamic light-scattering were all consistent with this mechanism.

The binding energetics of the identified molecules were also studied by isothermal titration calorimetry. In general, enthalpy played the major role in improving free energy of binding, with entropy playing an increasingly deleterious role as affinities improved. Though interpreting thermodynamic data is tricky, the contribution of enthalpy versus entropy is consistent with the molecules locking the protein into a single conformation, thereby decreasing its conformational freedom (and entropy).

This paper is a beautiful illustration of anti-reductionism: the compounds are not active against the isolated protease domain commonly studied; only by looking at the full-length protein complex could the allosteric site be identified. Molecules such as compound 6 should prove useful reagents for exploring, and ultimately preventing, hepatitis C replication.

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