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Opinion: 2006, a year of champagne

Stephen Cusack, Head of EMBL’s site in Grenoble, looks back on the early days of his collaboration with Anacor developing anti-infectives targeting leucyl-tRNA synthetase

Stephen Cusack, Head of EMBL’s site in Grenoble. PHOTO: EMBL

By Stephen Cusack

It all started at the 21st International tRNA Workshop in Bangalore in December 2005 where I first met Dickon Alley, Head of Discovery Biology at Anacor Pharmaceuticals in Palo Alto.

Anacor was founded to discover, develop and commercialise novel small-molecule therapeutics derived from its boron chemistry platform. Anacor scientists had screened a library of benzoxaborole compounds and shown that one of them (AN2690) was a potent and broad spectrum anti-fungal, whose target they showed to be the fungal leucyl-tRNA synthetase (LeuRS). This enzyme is essential for the incorporation of the amino acid leucine into newly synthesised proteins. However the LeuRS sometimes mistakenly uses the wrong amino acid, but remarkably, has a built in mechanism called ‘editing’ to correct such errors before any damage is done.

For many years, my lab had been determining crystal structures to understand how bacterial LeuRS works, including its editing function. Hence the meeting with Dickon in Bangalore, facilitated by Susan Martinis, from the University of Illinois, another biochemist working on LeuRS editing with whom we were already collaborating. We were all excited at the idea that structural biology might help understand how AN2690 functions.

Things moved fast and 2006 turned out to be an incredibly exciting year of discovery, the fruits of a highly interactive collaboration between the Anacor chemists and biochemists and my group.

100 mgs of AN2690 was shipped to Grenoble in February 2006. On 21st March 2006 at 12.13pm I emailed Anacor:

Dear All, Champagne all round! Anya (Yaremchuk) and I got the data yesterday. We have a beautiful 2 Å structure with the compound and AMP! It reacts with AMP to make a post-transfer substrate-like compound (transition state?) in the editing site with the AMP mimicking the terminal adenosine 76 of tRNA. So I guess you guessed right about the mechanism! Stephen

Dickon replied:

Congratulations that is excellent news!!! It was such good news the chemists productivity dropped dramatically this morning as they were thinking about all the possible chemical mechanisms that could go on in the editing active site.

Then at 16:22 pm the same day I sent the following mail:

Dear Dickon, I have just analysed the data we collected yesterday on the tRNA complex crystals soaked with the compound. These data are only at 3.5 Å… but the compound is clearly there covalently attached to the ribose in the editing site!!!! (more champagne).

These first structures, done in collaboration with my colleagues Anya Yaremchuk and Michael Tukalo, although of the bacterial LeuRS, not the fungal enzyme, still told us the essentials about how the compound inhibits leucyl-tRNA synthetase. By binding in the amino acid pocket of the editing site (where mischarged isoleucine would bind), AN2690 can react, via its boron atom, with the acceptor end of the tRNA. The covalent adduct formed this way almost irreversibly blocks the tRNA on the enzyme, thus preventing normal enzyme function.

2006 turned out to be an incredibly exciting year of discovery, the fruits of a highly interactive collaboration between the Anacor chemists and biochemists and my group.

In parallel, my student Elena Seiradake was working on the editing domain from the fungal pathogen Candida albicans (the LeuRS editing domain is significantly different between eukaryote and prokaryote LeuRS, although the mechanism of action does not change) and, with the help of Weimin Mao from Anacor, quickly made progress. On the 17th October 2006 I emailed Anacor:

Dear Dickon, For the third time … champagne (you owe me a lot of bottles!). Crystals of Candida editing domain diffract to better than 2 Å resolution (even though they are only 10 microns thick) and we have a dataset at 2.2 Å. Stephen.

The bubbly reply came back on the 18th October 2006:

Dear Stephen, Excellent news!  We will have to make it a Jeroboam or Mathusalem of Californian sparkling wine. The last couple days I went to a couple of sparkling wine vineyards, Chandon and Tattinger. There is also Freixenet and Mumm vineyards here as well.  What is your preference? Fantastic. Dickon.

Later on 21st November 2006 I wrote:

Dear Dickon, As of today, the Candida editing domain with AN2690-AMP is solved! Stephen.

And he replied:

Dear Stephen, Wow!!!!!! and Congratulations, this is excellent news!!!!  This will help us get better antifungal compounds as we are at the moment still stuck with AN2690 as our best antifungal compound. Sounds like we will need an entire vat or pipeline of champagne to celebrate at this rate!!!!! Dickon.

Meanwhile, my postdoc Thibaut Crépin succeeded in obtaining crystals of the complete E. coli LeuRS-tRNALeu complex that diffracted to exceptionally high resolution (2 Å). In November 2006 I emailed again:

Dear Dickon, This should have the whole company jumping up and down! Basically Audrey (Zhou) got it right! Stephen.

Thibaut’s beautiful high resolution structure of the E. coli LeuRS-tRNALeu complex was bound to a new benzoxaborole called AN3017. This compound had been designed by Anacor modellers/chemists based on the crystal structure with AN2690 and comparison with a previous structure with an amino acid bound in the editing active site. They predicted that addition of an amino-methyl group at the 3-position of the benzoxaborole should be rewarded by the formation of three extra hydrogen bonds with the protein, since it would mimic the interaction made by the amino group of the amino acid. They were quite right and the compound AN3017 is consequently considerably more potent against bacterial LeuRS than AN2690.

we will need an entire vat or pipeline of champagne to celebrate at this rate!

A manuscript entitled “An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site” was published in June 2007 in Science. AN2690 received FDA approval in 2013 and is now on the market as Kerydin® (tavaborole), a topical treatment of onychomycosis (toe nail fungus). The amino-methyl group at the 3-position, designed into AN3017 on the basis of the original co-crystal structures, has been maintained in all subsequent benzoxaboroles targeting leucyl-tRNA synthetase that have been developed. Lots more crystal structures done by Thibaut and then Andrés Palencia, another postdoc in my lab, were crucial in guiding development of new benzoxaboroles specifically tailored to target Gram-negative bacteria (AN3365, published  2013, entered Phase II clinical trials) and M. tuberculosis (AN6426, published 2016, a closely related compound is expected to go into clinical trials in 2017). AN6426 was also shown to be active against the apicomplexan parasites Plasmodium and toxoplasma (published 2016).

However nothing quite matched the excitement and fun that we had that first year!

In May 2016 Anacor was bought by Pfizer for a reported 5.2 billion dollars.

Tags: Crystallography, Cusack, Grenoble, opinion, Partnerships, Structural Biology

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