It’s not often that bloggers get responses from the authors of the articles that they’ve featured and it’s much rarer still for a reviewer of a manuscript to break cover and join the discussion. However, that is exactly what happened when an article on (Trimethoprim-resistant, Type II R67) Dihydrofolate Reductase got coned in the searchlights at Practical Fragments. Soon it had also been Pipelined as well and the matter would normally have ended there. At this point both the corresponding author and one of the reviewers of the manuscript got in touch with both Teddy and Derek, which prompted a follow up post at Practical Fragments and two in the Pipeline ( 1 | 2 ). I have a particular interest in the relationship between journals and blogs and thought it would be a good idea to see what all the fuss was about.
The article describes a fragment screen (using a biochemical assay) against the target and the synthesis and characterisation of two inhibitors based on one of the fragment hits. None of the IC50 values measured for the fragment hits was less than 1.7mM and I was curious why the authors ran the assay at 30 x Km for NADPH since this (presumably) meant that fragments needed to be screened at higher concentrations. Although many in the FBDD community are dismissive of the use of biochemical assays for fragment screening, I believe that in some situations they do represent a viable way forward, but only if appropriate precautions are taken. The authors do hint at attempts to deal with interference (e.g. reducing path length) but I’d like to have seen some evidence that they were fully aware of the problem and had taken the appropriate steps to recognise and, if necessary, correct for interference. Some discussion of intracellular co-factor and substrate concentrations would also have been helpful since it would help to place the IC50 and Ki values for the structurally elaborated compounds in the appropriate context. Some readers may have encountered this issue when comparing assay results for ATP-competitive kinase inhibitors using different ATP concentrations.
Putting my concerns about the assay aside, I do have some issues about how the fragments were selected for assay. We screen fragments to ask questions about the structural requirements for binding and you don’t always need a large screening library in order to ask good questions. Once you’ve got hits, it’s important to follow these up with carefully chosen analogues to build SAR that can (hopefully) be mapped onto synthetically elaborated structures. This is especially critical when the hits are weakly active by a biochemical assay and neither crystal structures for bound fragments nor an orthogonal assay are available. Given the results presented in Figure 1 and Table 1, I was really surprised that they didn’t assay the unsubstituted 2-naphthoic acid. While assaying fragments 4a and 4c represents a step in the right direction for following up fragment 4, I’d be looking for something along the lines of the following scheme from researchers who were thinking in terms of molecular recognition:
In my view, the authors didn’t demonstrate any real insight in the way that they selected the compounds for their fragment screening library or how they explored fragment SAR. Although they had a protein structure available for the target, they don’t seem to have used this for selection of fragments for screening. The execution of the fragment screen and its follow up would be less of an issue had the authors managed to get crystal structures for fragment-protein complexes or discovered a series of highly active compounds with interesting SAR that killed bacteria. However, all the authors really have to show for having run this fragment screen is two inhibitors with two dianionic inhibitors of low ligand efficiency. I couldn’t help wondering why linking fragments led to such a catastrophic loss of ligand efficiency. I also couldn't help wondering why all the ligand efficencies were negative and in the wrong units since you'd have expected the reviewers (there were at least two of them) to pick this sort of thing up.
The process by which the authors used the output of the fragment screen to ‘design’ the elaborated molecules is especially deserving of comment. The imidazole carboxylic acid was selected as a starting point for elaboration because it was more selective with respect to the human enzyme. Personally, I’d be wary of placing too much weight on selectivity given the possibility of interference in these assays but I’d certainly be prepared to put my concerns to one side if the elaboration process had led to some really active compounds that were indeed selective. The idea of linking two carboxylic acids appears to have been inspired by the structure of Congo Red. Dyes typically need to stick to the organic material that they are used to color and in the early days of high throughput screening, we’d see a lot of dyes like this coming up as active. I was surprised that the authors chose to link from the carbon between the imidazole nitrogen atoms because the fragment with a methyl substituent at this position showed no inhibition at 10mM and this is precisely the sort of result that is invoked to support the assertion that fragment 4 is selective with respect to the human enzyme. However, I’d like to get back to the structure of Congo Red...
Congo Red is actually a pretty rigid beast and can only exist in extended conformations with each half of the molecule tending to coplanarity. If you’re going to use Congo Red as a template for design then it really would be a good idea to check that your designed molecules really do overlay with the template. It really isn’t rocket science. At this point, it really should be mentioned that the authors had got to their inhibitors without any of what I’ll call geometric modelling. This might have been more acceptable if the compounds had been more potent, more numerous and actually killed bacteria. Reading this article caused me to dream up new efficiency metric for medicinal chemistry journal articles in which the sum of the pIC50 values for all the compounds in the article is divided by the number of pages in the article...
This is probably a good point to note that the authors do not appear to have made any real use of the crystal structure of the target either to select fragments or in the design of the elaborated molecules. Therefore, I was somewhat surprised to see over two pages of the article devoted to docking study of the inhibitors. They appeared to have some difficulty in reproducing the (crystallographically determined) binding mode of the NADPH ligand using the docking software so a reader could legitimately ask why she or he should believe the proposed binding modes for the other ligands. One common feature of the predicted binding modes is that the molecules are ‘folded into a U shape’ and you can see these in Figure 4. Bending the molecules like these into ‘U shapes’ costs energy and many (most) docking programs don’t account for conformational energy costs particularly well. Also, Congo Red (which appears to have provided the basis for the dicarboxylic acid inhibitors) can’t bend into a ‘U shape’ so if these inhibitors really are binding in the proposed manner then they can’t really be functioning as the mimics of Congo Red that they were 'designed' to be. Also the authors note that some other researchers have proposed ‘a linear mode of binding’ (whatever that means) in which the molecules do not bend into ‘U shapes’. I couldn’t help wondering why they’d not bothered to dock Congo Red...
Neither of the inhibitors showed a measurable effect on bacteria even when tested at 1mM and the authors suggested ‘that these compounds do not penetrate into E. Coli’. This may well be the case but potency also needs to be seen in the context of the physiological concentrations of both substrate and co-factor. The authors claim selectivity for the compounds 8 and 9 with respect to human DHFR although the maximum test concentrations used were both less than ten-fold above the relevant IC50 values for the R67 enzyme.
The authors presented cytotoxicity data for compounds 8, 8a, 8b and 9 and noted that all except 8b showed weak cytotoxicity. It’s worth noting that the IC50 (34 µM) for 8a in the cytotoxicity assay is almost two-fold lower than the IC50 for 8 against the target enzyme. Compounds 8 and 9 may only be weakly cytotoxic but they still appear to kill the 3T6 cells in the cytotoxicity assay more efficiently than they kill bacteria. Apparently 8b has some antiproliferative effects against a number of parasites although it is not clear what relevance this has to R67 DHFR inhibition since this compound shows no measurable activity against this enzyme..
That brings me to the end of my review of this article and it’s now time to sum up. The authors conclude their article by stating:.
‘This work provides inspiration for the design of the next generation of inhibitors.’.
and I believe this statement is as inaccurate as it is immodest. I thought that the execution of the fragment screen, in particular the exploration of fragment SAR, was weak. The ‘design’ didn’t hang together (e.g. linking benzimidazole from C2 where methylation led to decrease in inhibition by fragment) and may have been overly-influenced by some of the authors’ interest and previous experience in the synthetic chemistry of linked benzimidazoles. The docking studies appeared to have been done after the compounds had been ‘designed’ and synthesised and in my view, contributed absolutely nothing. I suggest the authors, reviewers and editor of this article all read (and make sure that they understand) the recent J. Med. Chem. guidelines on computational medicinal chemistry. All of this would have been less of an issue if the authors had actually discovered a novel series of R67 DHFR inhibitors that actually killed bacteria. However, the two compounds, which they optimistically describe as a class, that they’ve discovered are unexciting from the pharmaceutical perspective. Perhaps if they'd pursued the fragments a bit more imaginatively and done some real design they might have come up with something more exciting that was actually anti-bacterial. However, that is speculation and I believe that the Journal's readers and subscribers deserve better than this.
To be quite honest, I wouldn't normally review an article like this and only did so because of the reaction(s) generated by the original blog posts and my interest in the relationship between blogs and journals. A specialist scientific journal needs to shape the debate that drives its field forward and create an environment in which articles are discussed publicly. The authors of those articles should be active participants in the discussion. I'll finish with one of the comments from 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.'
Earth calling Journal Editors, the ball's now in your court and we'd all really love to hear what you've got to say.
Bastien, Ebert, Forge, Toulouse, Kadnikova, Perron, Mayence, Huang, Eynde & Pelletier, Fragment-Based Design of Symmetrical Bis-benzimidazoles as Selective Inhibitors of the Trimethoprim-Resistant, Type II R67 Dihydrofolate Reductase. J. Med. Chem., 2012, 55, 3182–3192 | DOI.
Mayence, Pietka, Collins, Cushion, Tekwani, Huang & Eynde, Novel bisbenzimidazoles with antileishmanial effectiveness Bioorg. Med. Chem. Lett. 2008, 18, 2658–2661 | DOI
Stahl & Bajorath, Computational Medicinal Chemistry. J. Med. Chem., 2011, 54, 1–2 | DOI.