26 April 2012

And a Reviewer Responds

A recent paper in J. Med. Chem was reviewed on this blog.  It was also noted by In the Pipeline as one of the worst papers ever published in that journal in a long time.  Today, both ITP and PF received an email from one of the reviewers of the paper, Dr. Elizabeth Howell. ITP has a post about this and a correspondence from the main author of the paper here.  I am reproducing her entire email to us below:
Dear Teddy Zartler and Derek Lowe,

I was made aware of your blogs on the Bastien et al. paper (April 18th, How do these things get published? &April 17th, Sometimes your compound sucks). I am an enzymologist and I was one of the reviewers of the paper. I would like to respond to your comments.
I have worked on DHFR since the 1980s. You may perhaps be unaware, but there are 2 different types of DHFRs. The chromosomal DHFR is the target of many drug design efforts and is the more well-known enzyme. A protein conferring resistance to trimethoprim, typified by R67 DHFR, was identified in the 1970s. This R-plasmid encoded DHFR (a type II DHFR) has an entirely different scaffold than the chromosomal DHFRs. I wonder if you thought the Bastien et al. paper was describing drug design efforts for the chromosomal DHFR and this misunderstanding is at the root of your concern? For example, Dr. Zettler [Ed: Zartler] used the term “mutant DHFR” in his post. Is an apple the same as a mutant orange? To me, R67 DHFR and chromosomal DHFR are like apples and oranges. While these 2 DHFRs catalyze the same reaction, they have different protein folds, different oligomerization states, different active sites (R67 has a single active site pore while chromosomal DHFR has a typical binding cleft) and the enzymes use different transition states (endo vs exo).
FYI, I am attaching a pdf file [Ed:shown below as two jpgs] showing the 2 different structures and a Table comparing the binding constants and active site volumes of the 2 enzymes (emailed to Drs. Lowe & Zartler).
A few observations about R67 DHFR (I am happy to provide citations supporting these data if requested):
R67 DHFR is a homotetramer and each monomer has a fold that is related to an SH3 domain.
The R67 tetramer has exact 222 symmetry and a SINGLE active site pore.
For each cofactor (or substrate) binding site in R67 DHFR, there must be four related sites from the symmetry. However a total of 2 ligands bind per pore, either 2 substrates, 2 cofactors or 1 molecule of each. Only the latter is productive and results in catalysis.
The classical chromosomal DHFR inhibitors (trimethoprim & methotrexate) do not inhibit R67 DHFR very well (Ki values of 150mM and &500mM respectively).
The above observations (& others) lead to the hypothesis that R67 DHFR has a promiscuous binding surface that can accommodate both ligands. The 222 symmetry imposed on the single active site pore of R67 DHFR is utilized by Bastien et al. in their drug design process as they construct "dimeric" ligands. Again, I wonder if the bloggers did not realize a different DHFR was targeted, as for example, Dr. Lowe says “The authors string these things together into huge dimeric molecules, apparently because they think that this is a good idea, but they get no data to support this hypothesis at all.” The hypothesis of Bastien et al regarding symmetry related binding sites comes from the previous crystal structures of the R plasmid enzyme, by stoichiometries derived from time resolved fluorescence and ITC binding studies and by the ability of R67 to catalyze a transhydrogenase reaction (albeit weakly).
With regard to the bloggers comment on electon density interpretation, It is the 222 symmetry that makes it difficult to deconvolute electron density of crystal structures describing bound complexes in R67 DHFR , please see the attached figure of the symmetry problem in this enzyme.
In regards to my review of the paper, I read the paper carefully and wrote 2 pages of comments in my initial review. I also read the revised manuscript. In my opinion, the paper was a first step in the design of an inhibitor that would target this unusual binding site and have some specificity for the R67 DHFR vs. the chromosomal DHFR. To my knowledge, this is the first drug design effort to target R67 DHFR activity. I stand by my decision, the paper is worthy of publication.
Sincerely, Liz Howell, Professor, Biochemistry, Cellular & Molecular Biology Dept., University of TN, Knoxville, TN

4 comments:

Pete said...

It is extremely rare to see either an author or a reviewer of an article featured in a blog respond to the blogger. I believe that it is without precedent to have both respond and I think this is a very healthy development for science. It is especially brave for the reviewer to break cover and shed the cloak of anonymity.

I occasionally write blog posts that are critical of literature. In these posts I will generally read the article even more carefully than if I were reviewing for a journal.

Dr. Teddy Z said...

I agree with Chris Swain on ITP and Pete that this is a very good thing. I admit, like Derek, that I missed the implications of R67 DHFR as such a novel target, but I also think it is a red herring. It does not affect my opinion of the fatal flaws of the paper whether it was on trypsin or Alien Dinosaur Protease 1. The FBDD component of the paper was horrible and that was what I slammed. Notice in both the reviewer's and lead authors responses they do not defend the FBDD aspect, but rather what target it was against. I stand behind my comments on the paper. Bad science against a great target still makes bad science.

Dan Erlanson said...

I too would like to commend the author and reviewer for jumping into the fray. Having these discussions in real time and in the open is valuable not just for the participants but for many outside observers.

Over at In the Pipeline Derek has a new post that I think nicely summarizes some of the different points of view.

Pete said...

I particularly liked Chris Swain's comment on Derek's second post:

"I agree the online discussion about the article is great, but it does need to be more tightly linked to the original publication. No reason why the original publishers can't allow public comments."