13 August 2014

Intentionally dirty fragments

Practical Fragments has tried to publicize the dangers of pan-assay interference compounds, or PAINS. These compounds show up as nuisance hits in lots of assays. So what are we to make of a new paper in Curr. Opin. Microbiol. by Pooja Gopal and Thomas Dick, both at the National University of Singapore, entitled “Reactive dirty fragments: implications for tuberculosis drug discovery”?

As the researchers point out, several approved anti-tuberculosis drugs are fragment-sized and hit multiple targets; they are “dirty drugs”. For example, isoniazid (MW 137, 10 heavy atoms), is an acylhydrazide that is metabolically activated and forms an adduct with an essential cofactor, causing havoc to the pathogen. Ethionamide (MW 166, 11 heavy atoms), a thioamide, works similarly. The fact that these molecules are so small probably allows them easier passage through the microbe’s rather impermeable cell membrane, and the fact that they hit multiple targets may make it more difficult for the organism to develop resistance. The researchers conclude:
The success of small dirty drugs and prodrugs suggests that fragment-based whole cell screens should be re-introduced in our current antimycobacterial drug discovery efforts.
While it is true that many antimicrobials do have reactive warheads, and it is also true that there is a huge need for new antibiotics, I worry about this approach. Not only is there an increased risk of toxicity (isoniazid in particular has a long list of nasty side effects), it can be very hard to determine the mechanism of action for these molecules, complicating optimization and development. As evidence, look no further than pyrazinamide (MW 123, 9 heavy atoms). Despite being used clinically for more than 60 years, the mechanism remains uncertain.

Fragment-based lead discovery is typically more mechanistic: find an ideal molecule for a given target. Indeed, much of modern drug discovery takes this view. Gopal and Dick propose a return to a more phenomenological, black-box approach. This may have value in certain cases, but at the risk of murky or worse misleading mechanisms.

If you do decide to put PAINS into your library, you might want to read a new paper in Bioorg. Med. Chem. by Kim Janda and collaborators at Scripps and Takeda. They were interested in inhibitors of the botunlinum neurotoxin serotype A (BoNT/A), which causes botulism.

Since BoNT/A contains an active-site cysteine, the researchers decided to pursue covalent inhibitors, and the warheads they chose, benzoquinones and napthoquinones, are about as PAINful as they get. However, in contrast to other groups, they went into this project with their eyes wide open to the issue of selectivity and examined the reactivity of their molecules towards glutathione. Reaction with this low molecular weight thiol suggests that a compound is not selective for the protein. Not surprisingly, selectivity was generally low, though a few molecules showed some bias toward the protein.

The researchers also tried building off the benzoquinone moiety to target a nearby zinc atom, and although they were able to get low micromolar inhibitors, these no longer reacted with the cysteine; apparently when the ligand binds to zinc, the protein shifts conformation such that the cysteine residue is no longer accessible.

To return to the premise of Gopal and Dick, there can be a therapeutic role for dirty molecules. The fact that dimethyl fumarate is a highly effective blockbuster drug for multiple sclerosis calls for a certain degree of humility. However, if you do decide to pursue PAINS, you should do so in full awareness that your road to a drug – not to mention a mechanism – will likely be much longer and more difficult.


Dr. Teddy Z said...

I also think the past is not prologue. Just because there are drugs on the market that were approved 50,60 years ago does not mean they would be approved today. Here is an interesting take on whether isoniazid would be approved or not: http://www.fda.gov/downloads/Drugs/ScienceResearch/.../ucm076755.pdf

Claudio said...

I read with interest this article guys and I agree with you that we should be cautious using this approach. But I'm happy to see this could be applied to fragments too. I worked on the so called TCIs (targeted covalent inhibitors)in a project on a kinase target at Cellzome (now GSK) looking for reactivity vs glutatione and it worked beautifully, obtaining potent and selective compounds. For people interested I suggest to read this article: NATURE REVIEWS DRUG DISCOVERY, 2011, 10, 307-317