Earlier this year we highlighted
an analysis of rings found in drugs. Thiazoles are tied for thirteenth place, occurring
in at least 30 drugs. (A substructure search in DrugBank pulls up 49.) They pack a lot of
diversity into just 5 heavy atoms, with a nitrogen atom capable of acting as a hydrogen
bond acceptor as well as a sulfur atom. But they can also be tricksy: an
analysis several years ago found that 2-aminothiazoles are over-represented as
hits in fragment screens but are often not advanceable. A new open-access paper in ACS
Med. Chem. Lett. by Rok Frlan and collaborators at the University of
Ljubljana confirms and broadens these conclusions.
The researchers assembled a library
of 44 fragment-sized 1,3-thiazoles and five 1,3,4-thiadizaozles. These were
then screened at 0.5 or 0.625 mM against four unrelated enzymes in biochemical assays.
Two of the enzymes contain catalytic cysteine residues, and these had high hit
rats: 14 hits for the SARS-CoV-2 3CLpro enzyme and 26 for the E.
coli MurA enzyme. In contrast, MetAP1a had only 3 hits, while DdlB had
none. Are any of these hits real?
None of the compounds had been
classified as PAINS, and aggregation was deemed unlikely for all but one compound
based on chemical searches and the presence of detergent in the assays for MurA,
DdlB, and 3CLpro. One compound also seemed to interfere with the
fluorescent assays and was ruled a false positive. So far, so good.
However, 8 of the compounds
turned out to be unstable in aqueous buffer. Moreover, four compounds turned
out to be redox active in at least one of three different assays. Redox cycling
can generate reactive oxygen species, which inhibit cysteine-dependent proteins
nonspecifically.
Next the researchers tested to
see whether their fragments reacted with a small test thiol, 5-mercapto-2-nitrobenzoic
acid. Shockingly, 19 of them did, and most of these inhibited at least one of
the enzymes. Many of these contain potential leaving groups such as halogen
atoms, but some didn’t, leaving the nature of the reaction unclear. Still, the
results suggest that the fragments are more thiol-specific than protein-specific,
and so another potential source of false leads.
When the researchers retested the
ability of the fragments to inhibit the enzymes in the presence of the reducing
agent DTT, only one of the CLpro hits reproduced – and that was the
compound that showed fluorescence interference. The results were not quite so
bad for MurA, though many hits fell out.
Finally, the researchers tried to
correlate reactivity with quantum-mechanical calculations using several
different methods. Unfortunately, as they note, “no meaningful relationships
were observed.” Laudably, data for all the compounds are provided, so interested readers are
free to try their own analyses.
In the end it is not clear
whether any of the hits will be useful, but the high correlation between pathological
mechanisms and activity does not make one optimistic. As the first paragraph
above makes clear, this does not mean that thiazoles should be avoided. Indeed,
the researchers explicitly state that “we do not want to establish a general knockout
criterion to exclude thiazole or thiadizaole screening hits from further development,
but it is essential to evaluate their reactivity if they prove to be hits.” This
is where orthogonal biophysical methods, such as crystallography, can distinguish
true hits from artifacts.
Posts
10 comments:
In Chem. Res. Toxicol. 2010, 23, 653–663 (https://doi.org/10.1021/tx900414g), we reported on the liability of Cyp-mediated epoxidation of thiazoles, with the R1 group attached to the resultant epoxide affecting the propensity for subsequent GSH-adduct formation.
Thanks Matthew. Lots of things can happen once Cyps bite into molecules, but what's scary about this paper is that these effects are being seen with pure compounds in simple biochemical assays.
Hi Dan, this looks like an interesting study. I’d expect electrophilicity to increase in the order thiazole < 1,3,4-thiadiazole < 1,2,4-thiadiazole and nucleophilic attack can potentially occur at either carbon or sulfur. Sulfur can also function as a chalcogen bond donor (just like halogen bonding but with S, Se…) and aza-substitution will tend to strengthen chalcogen bonds. It’s quite possible that thiazole/thiadiazole sulfur can interact with catalytic cysteine by functioning as a chalcogen bond donor. There’s some discussion (including a couple of references) of chalcogen bond donors in my HB donor perspective that might be helpful.
Hi Dan, Around 2016 we introduced a redox-QC method into the Astex fragment library selection procedure. This was done by adding dithiothreitol (DTT) into the standard aqueous buffer used for NMR-based solubility and stability measurements. The potential of a fragment for redox activity and redox-mediated catalysis could then be assessed by observing the loss of reduced DTT over the time taken for the fragment-stability measurement (a few hours). The most common redox-active hits found in the current library were thiadiazoles, with all amino-substituted thiadiazoles showing strong redox activity. One of the thiadiazole isomers was the worst offender, but I cannot remember which one. In some cases the thiadiazole also underwent significant decomposition, but it was not uncommon to see little change in the fragment, indicating a catalytic mechanism by which DTT was oxidised by O2 (and O2 reduced to active oxygen species), with participation of the fragment. The recommendation was to exclude aminothiadiazoles from the library and to exercise caution with any hits, particularly from the reactive isomer.
Hi Glyn, my understanding is that 1,2,4-triazole has been shown by crystallography to react with catalytic cysteine residues. I would expect electrophilicity and electron affinity of thiadiazole isomers to decrease in the order [1,2,5 > 1,2,4 > 1,3,4] and would anticipate an inverse relationship between these properties and polarity of the nitrogens (the B2012 and G2012 articles discuss polarity differences for oxadiazole isomers). With respect to aminothiadiazoles, it is just possible that the repulsion the between N3 and N4 lone pairs could nudge 2-amino-1,3,4-thiadiazole into the imine tautomeric form (I’ve seen these for sulfonamides that are N-substituted with heterocycles) although my CSD access has expired so I can’t check.
Hi Pete,
Thanks for the additional information - I will see if I can find out which thiadiazole isomers were tested in 2016. I have not seen the triazole-cysteine data (my literature access is also limited these days) but it reminded me that Astex had also observed a fragment-driven oxidation of the catalytic cysteine of protein tyrosine phosphatase 1B (PTP1B). The fragment responsible for the oxidation was not identified in the publication, but, under favourable conditions, the +1 oxidation state of sulphur in PTP1B could be reversibly trapped by intramolecular formation of a cyclic sulphenamide (see Nature 2003, 423, 773).
Perhaps I have been too hasty in thinking of these redox reactions as potential fragment-PAINs! In these days of quasi-reversible covalent drugs and drug-candidates, it is possible that a correctly tuned, (mildly) redox-active warhead could be used to catalyse specific, active-site oxidations of cysteine proteases and phosphatases. If so, information on promising chemotypes may already be present in many QC databases.
Correction: Suplhenyl = sulphur(0). People have been drummed out of the Inorganic Chemists Union for less! My Apologies.
Astex has kindly supplied the structures of the five strongly redox-active thiadiazoles that were identified in 2016.
Four were 1,2,4-thiadiazoles with one or more electron-donating substitutions: [5-amino], [3-methyl,5-amino], [3-hydroxy,4-amino], [3-hydroxy,5-thio]. One was a 3-hydroxy,4-amino-1,2,5-thiadiazole.
This may be of use when evaluating the mechanism of action of hits containing similar substructures.
Hi Glyn,
Thanks, this is really helpful, and I like the idea of observing DTT directly in NMR experiments.
Do you recall whether thiazoles showed redox activity? The paper did not include any 1,2,4-thiadiazoles, only thiazoles and 1,3,4-thiadiazoles.
As for mild oxidants as drugs, ebselen is an interesting little molecule that has gone into the clinic for various indications, though never approved.
Hi Pete,
I suspect you're correct about electrophilicity. Do you recall whether the the cysteine-reactive compounds contained a "proper" leaving group, or was there something more mysterious going on? For example, the paper reports 5-methylthiazol-2-amine as reactive, though not redox-active.
Hi Dan,
As far as I remember, no Astex thiazoles showed redox activity with DTT.
Redox effects of fragments were first noticed during our ligand-detected 1H-NMR screens, where the buffers contained DTT to maintain protein reduction. In a typical run lasting 12-24 hours, successive samples would show a progressive loss of reduced DTT, up to 50% by the final samples of a run. However in some samples no reduced DTT remained, even at much shorter times.
The drive to explore more fragment space while improving solubility has (understandably) led to the inclusion of many small rings with additional heteroatoms, so the risk of redox activity has increased. DTT (E0 = -330mV at pH7) is a significantly stronger reducing agent than mercaptoethanol or glutathione (E0 ~ -250mM at pH7). It also is able to carry out concerted reductions at two sites, so may have a kinetic advantage for some reductions. However, reactivity of a screening hit with DTT is a significant warning, particularly when the hit may progress into more complex assays.
I gave 3 examples of redox-active fragments in a presentation at the Novalix Biophysics meeting in 2017. These were (a) 5-trifluoromethoxy-1H-indole-2,3-dione, (b) 5,6,7,8-tetrahydro-1,8-naphthynidin-3-ol and (c) 1,3-diazinane-2,4,5,6-tetrone. The last is a particularly ugly compound that had somehow slipped through our pre-filters! There were several other redox-active compounds detected in the library but no clear chemotypes except the 1,2,4-thiadazoles. Also, in some cases, I suspected that the fragment was innocent and that the redox-activity may have been due to an undetected contaminant.
It is certainly an interesting area!
Post a Comment