04 May 2015

Sloppy science

As regular readers may have discerned, I’m favorably disposed to most of the papers I highlight. They may have flaws or inconsistencies, but, with rare exceptions, I generally just ignore particularly problematic publications. Last year Teddy introduced the term PAINS-shaming to draw attention to – how shall we phrase it? – less salubrious specimens. Building on this alliterative theme, today’s post is about sloppy science. A fundamental tenant of sound science is to consider alternate explanations for results. Ignore this at your peril.

An example was published in J. Cancer Prev. The researchers were interested in a mutant of isocitrate dehydrogenase 1 (IDH1), a hot cancer metabolism target. They screened 500 fragments in a functional spectrophotometric assay, with each fragment present at the very low concentration of 5-10 µM. One of these inhibited the mutant protein by 80% – pretty impressive for a fragment. Until you look at the structure: 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one (shown below).

Fifty years ago, researchers showed that this chemical class (isothiazolinones, also called isothiazolones) could react with thiols, like this:

Isothiazolinones have been categorized as PAINS, though they do not show up in the original computational filters. However, Pete Kenny has (repeatedly) stated that having a dubious structure should not automatically disqualify a compound from further investigation, so what else do we know about isothiazolinones?

Well, there’s this paper, which concludes a discussion of isothiazolinones by stating:
We could not develop these into useful compounds and ultimately the structure–activity relationship (SAR) was uninterpretable. Most insidiously, there were encouraging aspects of sharp SAR as there always are with these PAINS, but this is eventually overwhelmed by flat and nonsensical SAR. Unpredictable nonspecific cytotoxicity was manifest. We found our compounds to be rapidly reactive with thiols under assay conditions.
Of course, one could argue that this is anecdotal. But then there’s this paper, with the unambigious title “Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCA”. The first line of the abstract states:
Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides.
The researchers go on to demonstrate this using both small molecules and proteins, and some of the compounds they investigate are structurally quite similar to the hit here.

So in all likelihood the fragment described in the most recent paper reacts with one or more cysteine residues in IDH1, of which there are several. It is notable that the researchers conducted their assay in the absence of added thiol reducing agents, so modification of the cysteines would effectively be irreversible under their assay conditions.

What we have here is the re-identification of a known thiol-reactive molecule without any acknowledgement or apparent awareness that the molecule is reactive. I have no problem with covalent inhibitors, but I do have a problem with a generically reactive molecule being touted “for a future lead development”, as the researchers state in the abstract. It took me just minutes to track down the references above, and the fact that neither the researchers nor the reviewers did so is inexcusable.

Granted, this paper is not published in a high profile journal, and the easiest response would be to ignore it. It is certainly not the only one of its kind. Doing so, however, implicitly endorses sloppy science. This paper will undoubtedly pad the resumes of the authors. Highlighting its problems will hopefully make others wary of wasting time with this new "selective inhibitor."


Anonymous said...

Cysteine proteases are in my experience quite sensitive to false positive hits, so one has to be careful. Redox cycling compounds can also cause problems: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027197

Anonymous said...

It does perhaps beg the question as to why these compounds would be in a library in the first place? I can't think of a reason why you would want a thiol reactive probe in a fragment screening collection.

Anonymous said...

Also see

So, the issue has been known for quite some time.

Dan Erlanson said...

Anonymous (2:17 PM) brings up an excellent point. The library used in this case was the Maybridge Ro3 library, though it is not clear when it was purchased. Hopefully this compound has since been removed from this library!

Dr. Teddy Z said...

There are two points I want to make. Despite what Pete thinks, I am not about removing every potential bad actor from a fragment library. Having a thiol reactive probe could be useful if you are trying to generate a tool for that target. But, you have to know that it is a potential red flag and all the liabilities it would bring. In this case, they really don't seem to have that understanding.
Secondly, I and my partners have been working with Maybridge to better curate the current version of the Ro3 library (email for details as to what I am doing for them). In particular, we have put the entire 30,000 fragment library through a comprehensive PAINs filter and identified those. It is a rather small number, so based upon that, I would guess that this library is an older, less curated version.

Anonymous said...

A bit late to this... but leaving aside the potential for isothiazolone Cys-promiscuity, it's interesting that the compound only seems to have activity against IDH1 carrying the R132H mutation and not WT IDH1... despite as you say, carrying multiple cysteines.
Perhaps a bit more to it than pan-Cys reactivity in this example. But the point about "sloppy science" is generally noted.