30 June 2014

Fragments vs Dengue virus helicase and methyltransferase

Dengue fever is a disease whose nastiness is hinted at by its common name, “breakbone fever”. The eponymous virus relies on two viral proteins, a helicase (Hel) and a methyltransferase (MTase), for replication. In a recent paper in Antiviral Research, Karine Barral and coworkers at Aix-Marseille Université use fragment-based methods to tackle both of these proteins.

The researchers used the 500 compound Maybridge fragment library (2009 edition) and performed a biophysical cascade, starting with a thermal shift assay at 2 mM of each fragment. Hits were defined as fragments that stabilized the protein by at least 0.5 °C; there were 36 hits against Hel and 32 against MTase, with 6 in common.

Both Hel and MTase are amenable to crystallography, so the hits against each protein were soaked into crystals. Unfortunately, none of these yielded structures for Hel. Critics of thermal shift assays could argue that this is yet another example of false positives, a possibility the researchers consider. That said, 11 of the fragments inhibited Hel by at least 25% in biochemical assays (at 2 mM fragment), though none inhibited greater than 50%.

The results against MTase were more salubrious: 7 fragments produced structures, for a success rate of 22%. Interestingly, these fragments bound to 4 distinct sites on the protein. Two different enzymatic assays were used to test all the fragment hits, and many of them showed activity in one or both. A few fragments – including 5 of those characterized crystallographically – were sufficiently active that IC50 values could be determined, though these were mostly millimolar.

My one quibble is that the authors state that “to our knowledge, only one example of random FBS has been conducted on an RNA virus target.” This ignores some beautiful work from both Roche and Astex on Hepatitis C, another virus that carries its genome as RNA. Still, it is fair to say that fragments could play a larger role in tackling infectious diseases.

At the last CHI FBDD conference, Rod Hubbard noted that, as more academics enter the fragment field, we will see more publications describing fragment hits against tough targets. The next steps – taking a fragment to a nascent lead – are often harder to resource in an academic environment. Still, it’s good to see these initial studies. At the very least, they go some way to addressing the question of whether the targets are ligandable.


Ewa said...

could you provide references for " the work from both Roche and Astex on Hepatitis C"

thanks, Ewa

Dan Erlanson said...

Hi Ewa,

"Roche" and "Astex" both have hyperlinks that will take you to blog posts about the articles, which are:

J. Med. Chem., 2013, 56 (7), pp 3115–3119
DOI: 10.1021/jm4002605


Nature Chemical Biology 8, 920–925 (2012)

Anonymous said...

There are plenty of HCV NS5B fragment-based discovery stories out there too.