When Fragments don't deliver...

In the olden days (1980s), during the cold war, Russia was "a riddle wrapped in a mystery inside an enigma".  Kremlin Watching was serious and important thing. When I write up papers, I do the same thing but trying to figure out what the actual story is.  We all know a lot more happened than is written down in 10-20 pages of an article.  This paper has me really doing it; so follow along.

Tuberculosis is a scourge caused by a mighty nasty bug.  People have been using fragments to try to combat it for a long time: 2009 and 2014: targeting pantothenate synthesis and biotin synthesis. AstraZeneca join the party (just as Entasis spins out) with this paper.  In it, they describe their NMR fragment screen combined with a HTS biochemical screen targeting thymidine synthesis.  All the TK inhibitors are TMP or thymidine analogs.  The HTS of 120,000 compounds lead to multiple 1-30 uM active site binding (confirmed by HSQC NMR) inhibitors.  Compound 1

Cpd 1.  3.6 uM, 0.46 LE, 3.54 LLE.  

Figure 2.

was chosen as the basis for the hit to lead campaign.  Modeling suggested that the pyridone core is a thymidine mimic (Figure 2). This novel core allowed to reach sub micromolar potency within 10 compounds of the original hit.  The pyrimidine core was also potent, but not as much as the pyridone.  Pyranones were inactive, as was any other group but the cyano at the 2 position. Crystallography was a key to verifying the binding mode of the compounds.  One point of this is that verified means within 1 A of the predicted pose.  SAR led to the fused pyridinone, a 2 nM inhibitor, which nonetheless had no cellular activity.  The propose that this is due to the ionic nature of the compound, but ureas, amides, and sulfonamides did not afford the desired activity. 

Figure 3.  Fused Pyridinone showing X-ray Contacts

So, as is becoming a very common theme in fragments, they decided to use fragments to try to discover an alternate scaffold.  Using TROSY (HSQC for big proteins), they screen 1200 fragments in pools of 6.  Those fragment hits, termed FRITs which is a first for me (I think I like it.), with a LE greater than 0.25 were followed up by X-ray crystallography.

Figure 4.  Napthyridinone FRIT.  590 uM, LE=0.3. 

Figure 4. shows the best FRIT and its crystal contacts.  Combining this with the knowledge from the cyanopyridinone series, a virtual library was created and docked.  Hidden in their description, it appears that the library was passed by real chemists to prioritize the cpds.  Kudos.  With very limited SAR, they achieved significant potency (Figure 5), but still without cellular potency. 

Figure 5. 200 nM, LE=0.34.  

But, WAIT, this series wasn't advanced any further because the cyanopyridinone was in "advanced lead generation".  Why, you ask?  Well, the oxidized form of Cpd 1 had exhibited moderate cellular activity.  While they don't say it, I would imagine that this means that in doing the analytical work on the compound they found a portion that had oxidized, cleaned it up, and then tested the "bad" part.  I would love to know if this is how it happened.  I would hate to learn they had planned on an oxidized compound all along.

So, on to sulfone and sulfoxides of Cpd 1.  Knowledge from the cyanopyridinone series was used to select appropriate substituents, which seems to indicate a timeline of how things happened or a "we've got nothing left to try" issue.  Again, I would love to know which.  Both the sulfones and sulfoxides showed cellular activity with increase in IC50.  And again X-ray showed that the binding mode was retained, with the sulfoxide adjacent to Arg95.  This then caused them to go back and look at the cyanopyridinones again and realize that the sulfone/sulfoxides might have just the right physicochemical properties.

I think this is a really good paper, and hopefully indicates that more work on this target and with these series are coming.So, I don't know if the fragments failed, or if something better came along.  I would think the latter, but it could be the former.  Again, I would love to know.