29 July 2015

Novel Assay (SPR) Leads to Novel Allosteric Binding Site (nAChR)

I love this blog.  But sometimes it drags.  Finding new articles to blog about can be hard, there seems to be waves.  So, I love it when someone points one out to me.  This article was sent to me by someone associated with it.  We don't always blog those articles people point out to us, but this one has all the hallmarks: high visibility journal, good target, and "innovative" in the approach.  So, let's dive in and see what we have here.

Nicotinic receptors (nAChR) are pentameric, ligand-gated ion channels.  nAChR mutations are implicated in a wide range of neurological disorders and are the targets for a wide range of current drugs.  It is important to note that most of these drugs work through an allosteric site distant from the agonist binding site.  Most high resolution, structural data is from homologous molluscan Acetylcholine binding proteins.  The orthosteric site is located at an interface of a "principal" and "Complementary" subunit.  Ligand-binding induces conformational changes in the ligand binding site which are coupled to the ion opening.  There is little information on structural implications at allosteric sites.  The authors decided to address this unmet need with a chimeric human α7 ligand binding domain and AChBP which has 71% sequence similarity to the native protein, compared to 33% for the most commonly used target (Aplysia).  

The authors used SPR against a target with a blocked orthosteric site.  They did this one of two ways: 1. pre-incubation with a high affinity orthosteric ligand or 2. mixing each fragment with an orthosteric ligand of lower affinity.  This second approach (Figure 1) allows detection of fragments not competing for binding to the orthosteric site and were therefore potential allosteric ligands.  
Figure 1.  To distinguish allosteric binders from competitive binders using SPR spectroscopy we perfused each fragment alone (green triangle) or in combination with the competitive antagonist d-tubocurarine (black circle). In the case of an allosteric binder, the response units observed for the mixture of fragment + d-tubocurarine is close to the sum of fragment alone + d-tubocurarine alone (blue dashed line). No competition exists because the fragment and d-tubocurarine bind at distinct sites. (C) In the case of a competitive binder, the response units for the mixture of fragment + d-tubocurarine is lower than the sum of fragment alone + d-tubocurarine alone because both compounds compete for binding at the same site. (D) Example traces for fragment 4, which was identified as one of the allosteric binders in this study.
 In screening 3000 fragments, they found 300 putative allosteric binders.  Follow up, including dose-response, led to 24 fragments being selected for co-crystallization.  Crystal trials were set up using blocked nAChR and fragments that were soluble at 5-10mM; yielding in the end 5 crystal structures.  All five proved to be allosteric binders in three separate locations, including one never observed before (top pocket).  The most potent of these fragments had a IC50 of 34 uM, while the least potent was 400 uM. 
Figure 2.  Allosteric Binding Sites
This is a really nice piece of work.  I think the SPR assay is really clever and I would expect that many people will now be taking a similar approach to discovering allosteric sites in their targets.

27 July 2015

Fragments vs DDR1/2: a chemical probe

Our last post was about the utility of chemical probes: small molecules with sufficient potency and selectivity to be able to address specific biological questions. A recent paper in ACS Med. Chem. Lett. by Chris Murray and colleagues at Astex describes an excellent example of finding a new probe for the discoidin domain receptors (DDR1 and DDR2). Previous publications had suggested a role for these receptor tyrosine kinases in certain types of lung cancer, but some of this work had relied on non-selective inhibitors.

The researchers started with a thermal shift assay of their 1500 fragment library against DDR1, followed by crystallographic screening, resulting in around 50 fragment-protein complexes. Not surprisingly most of the fragments bound in the hinge-binding region of the kinase, but around 10 bound in the so-called “back pocket”, with the protein in the inactive DFG-out conformation. As the researchers point out, it is rare to see fragments binding here.

Compound 1 was of particular interest. At first glance, it's not impressive: with 18 heavy atoms and MW > 250 Da, it is on the large end for an Astex fragment, and it had low activity in a biochemical assay. However, its binding mode revealed potential areas for improvements, and the methylene was an unusual feature in a back-pocket binder.

The first step in improving affinity was to add a hinge binder. This was done with the aid of an in-house program called AstexMerge, based on the program BREED, which superimposes a set of ligands. The user chooses a starting molecule, and the program tries to merge that with other molecules while taking account of bond angles and distances. This process led to the design of compound 2, and a few tweaks quickly led to compound 4.

Although compound 4 was potent against DDR1 and 2 and showed good cell-based activity in a phosphorylation assay, it did potently inhibit some other kinases too, most notably c-kit. That problem was fixed with further medicinal chemistry, notably addition of a methyl group to the previously mentioned methylene and replacement of the urea, leading to compound 9, which was potent, selective, and showed good pharmacokinetics in mice.

Although compound 4 was not completely selective, it was more so than some of the previously described molecules, so the researchers tested it in lung cancer cells and found that, despite the fact that it inhibited DDR2 phosphorylation, it showed no effect on cell proliferation. Thus, “the project was halted in favor of more attractive targets.”

Clearly the researchers didn't start out trying to disprove the role of DDR1/2 in squamous cell lung cancer, but their efforts will save others from pursuing the same course. The publication introduces some attractive chemical probes for interrogating the biology of these receptors; hopefully one of these molecules will be added to the Chemical Probes Portal as an alternative to the less-selective probes that have been used in previous studies. Who knows, perhaps someone will find another indication for which DDR1/2 inhibitors are just the ticket.

22 July 2015

Introducing the Chemical Probes Portal

Chemical probes can be incredibly powerful reagents for understanding biology. A potent, selective, and cell-active modulator of a specific protein can be invaluable for figuring out what that protein actually does. Fragment-based methods can be effective at identifying these tool compounds, as we've described here and here.

Unfortunately, good chemical probes are difficult to discover, and scientists are left struggling with suboptimal reagents that hit multiple targets, often through pathological mechanisms. This leads to "pollution of the scientific literature," in Jonathan Baell's memorable phrasing. Despite our occasional PAINS Shaming, high-profile articles in C&EN and Nature, and even a dedicated blog, the problem continues. What is to be done?

Yesterday, a team of 53 authors from 46 academic and industrial organizations published a Commentary in Nature Chemical Biology entitled "The promise and peril of chemical probes" (see here for excellent coverage in Nature, here for Science's take, and here for In the Pipeline). This provides a good working definition for a chemical probe. According to the Structural Genomics Consortium, a chemical probe for epigenetics targets must have:

  • Potency < 100 nM against the desired target
  • >30-fold selectivity vs related targets
  • On-target cell activity < 1 µM

It should also be profiled against a larger panel of potential off-targets, and a related inactive compound (such as a stereoisomer) should be available as a control.

After discussing examples of high-quality probes, the researchers turn their attention to what they term – rather charitably – "probes of lesser value:"
The continued use of these probes poses a major problem: tens of thousands of publications each year use them to generate research of suspect conclusions, at great cost to the taxpayer and other funders, to scientific careers and to the reliability of the scientific literature.
The authors then go on to describe best-practices. For example, even high-quality probes can give spurious results when used at high concentrations. As Paracelsus recognized five centuries ago, the dose makes the poison.

All of this is important, but as the authors acknowledge, it's been said before. What really differentiates the Commentary is the simultaneous launch of a companion web site, the Chemical Probes Portal. Its creators hope that this will lead to vigorous community discussion around questions such as:

Is there a probe for my target protein?
Which ones should I use?
How should I use this probe properly?
Is this probe suitable for use in animal models?

Currently the Portal lists just seven probes with links to references and descriptions of selectivity, solubility, and the like. All of these are “good probes,” but hopefully this will expand: the paper itself discusses the shortcomings of molecules such as staurosporine, chaetocin, obatoclax, and gossypol, and including them in the portal with detailed warnings would be valuable for the scientific community.

I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments. Practical Fragments will check back in a year or so to see how the site is doing. In the meantime, probe cautiously!

15 July 2015

Covalent Inhibitor of KRas

So, Ras is big.  We keep on talking about it.  And sometimes we talk about the same work repeatedly.  This recent paper from AZ is a publication of work we have talked about here and here.  This follows on closely to work done by Vanderbilt and Genentech.  Those two papers were done using NMR and this one took a X-ray approach.  The AZ folks were taking a different approach to this PPI: stabilization of the interface.  They took 1160 fragments in pools of 4 and screened against HRas (homolog)-catalytic domain of SOS stable complex.  There were able to identify 3 bindings sites on HRas-SOS (Figure 1):
Figure 1.  HRas-SOS Complex.  HRas (Green), SOS (Blue), A: SOS binding site  (gold) (same as Vanderbilt), B SOS-Hras Interface binding site (Red) (same as Genentech), and C HRas covalent binding site (black). 
Site A was the same site identified by the Vanderbilt group  Site B was the same as identified as Genentech.  However, the AZ compounds bound to both proteins at the interface.  Their initial hope was to use this site to stabilize the Ras-SOS interface.  Both of the fragments binding to these sites had their affinity determined by TROSY-HSQC NMR.    However, they were not potent enough to elicit a biological effect, which was not unexpected.  After several rounds of chemistry, they were not able to improve these fragments significantly, or even show that they actually stabilized the interface.  

Looking at the growing covalent literature, they hypothesized that an irreversible inhibitor may be the only way to inhibit GTPase activity, especially considering the pM affinity of GTP for Ras.  They identified Cys118R (conserved between HRas and KRas) as a potentially reactive sidechain proximal to the GDP binding site on Ras.  To go after this site covalently, AZ assembled a 400 fragment covalent library (Figure 2) and screened it by mass spectrometry.
Figure 2.  Chemotypes represented in AZ 400 fragment covalent library.
They chose the N-substituted maleimide was deemed "ideal"; other warheads were either insufficiently reactive or overly reactive.  Covalent modification of Cys118R by a fragment partially occludes the nucleotide binding site and potentially prevents the reorganization of the Cys118R loop, thus locking it into the catalytically inactive Ras-SOS complex.  Interestingly, their covalent compounds only inhibited catalytically activity when pre-incubated with Ras-GDP-SOS.  This supports the hypothesis that Cys118R becomes more accessible during SOS-mediated nucleotide exchange.  

This paper brings together several topics which I think are becoming hot: covalent fragments, mass spectrometry, and K-Ras

13 July 2015

Fragments vs BTK: metrics in action

Sometimes the discussions over metrics, such as ligand efficiency, can devolve into exegesis: people get so worked up over details that they forget the big picture. A recent paper in J. Med. Chem. by Chris Smith and (former) colleagues at Takeda shows how metrics can be used productively in a fragment-to-lead program.

The researchers were interested in developing an inhibitor of Bruton’s Tyrosine Kinase (BTK) as a potential treatment for rheumatoid arthritis. This is the target of the approved anti-cancer drug ibrutinib, but ibrutinib is a covalent inhibitor, and the Takeda researchers were presumably concerned about the potential for toxicities to arise in a chronic, non-lethal indication. Many of the reported non-covalent BTK inhibitors are large and lipophilic, with consequently suboptimal pharmacokinetic properties. Thus, the team set out to design molecules with MW < 380 Da, < 29 non-hydrogen atoms (heavy atoms, or HA), and clogP ≤ 3.

The first step was a functional screen of Takeda's 11,098 fragment library, all with 11-19 HA, comfortably within the bounds of generally accepted fragment space. At 200 µM, 4.6% of the molecules gave at least 40% inhibition. Hits that confirmed by STD NMR were soaked into crystals of BTK, ultimately yielding 20 structures. Fragment 2 was chosen because of its high ligand efficiency, novelty, and the availability of suitable growth vectors.
Close examination of the structure suggested a fragment-growing approach. Throughout the process, the researchers kept a critical eye on molecular weight and lipophilicity. This effort led through a series of analogs to compound 11, with only 24 heavy atoms and clogP = 1.7. This molecule is potent in biochemical and cell-based assays and has excellent ligand efficiency as well as LLE (LipE). Moreover, it has good pharmacokinetic properties in mice, rats, and dogs, with measured oral bioavailability > 70% in all three species. Finally, compound 11 shows efficacy in a rat model of arthritis when dosed orally once per day.

Although compound 11 is selective over the closely related kinase LCK, unfortunately it is a double digit nanomolar inhibitor of oncology-related kinases such as TNK2, Aurora B, and SRC, which would probably be unacceptable in an arthritis drug. Nonetheless, this study is a lovely example of fragment-growing guided by a strict commitment to keeping molecular obesity at bay.

08 July 2015

Merging Fragments for Matriptase

We often talk about methods here: how to screen, how to prosecute those actives, and everything in between.  This is one of those what you do with the actives posts.  In this paper, a group from Aurigene and Orion present their results on Matriptase.  There have been multiple reported matriptase inhibitors, small molecule and peptide based.  Previous work from this group showed compounds that were active in cell-based migration and invasion assays and in mice with tri-substituted pyridyls and benzene compounds.  For this work, they take a SBDD approach:  "structure divulges a trypsin-like S1 cavity, a small hydrophobic S2 subpocket, and a solvent exposed spacious S4 region."

In screening benzamidine fragments (MW less than 300) they found 2 actives, ~80uM (Figure 1).
Figure 1.  Benzamidine screening actives
These were modeled in to the active site and obviously the amidine moiety went into S1.  Cpd 1's benzene moiety went nicely into S4 while 2's piperidyl went into S1'.  S4 easily accepted the more hydrophobic napthyl instead of phenyl and then they decided to see if the napthyl compound and 2 could be "linked" and the beta carbon. [So, my first quibble here is that this is not really a linking approach; this is fragment merging. Linking involves modeling, SBDD, and discovery of different linkers.  Its very difficult to do without specialized methods.  What they did here was see huge spatial overlap of compounds and voila, "we can add something right here".]  Well, not surprisingly, this worked.  They describe their SAR around each pocket to pick the compounds to merge, go read it if that interests you. The did crystallize the merged compound and it confirmed the modeling.  The final compound showed activity in cell-based assays and in mice.  That's good.  

This work can be summarized as follows: if you have significant spatial overlap you have a very good chance of merging disparate moieties.  So, two things bother me here.  First, the actives 1 and 2 are mighty big for fragments (more than 22 HAC).  That's fine, tomato...to-mah-to.  The final compound ends up pretty honking big too (37 HAC).  What is really bothersome, at least to me, is the LE.  Both actives start well below 0.2 (for a protease!) and they never improve on it. Now, Pete may disagree, but metrics have a place in FBDD.  Does the LE metric in this case tell us anything? 

06 July 2015

Fragments vs 53BP1

As we’ve noted (repeatedly), epigenetics is big. However, much of the focus has been on bromodomains, which recognize acetylated lysine residues. In a paper published earlier this year in ACS Chem. Biol., Lindsey James, Stephen Frye and collaborators at the University of North Carolina, the University of Texas, the Mayo Clinic, and the University of Toronto describe their efforts on a protein that recognizes methylated lysine residues (a Kme reader).

The protein 53BP1 is involved in DNA repair and could have anticancer potential. It recognizes a dimethylated lysine sidechain within a histone protein, so the researchers screened a set of molecules containing amines to mimic this moiety. They used an AlphaScreen assay, with each compound at 100 µM. This does not appear to have been a library of fragments (and unfortunately the number of compounds screened was not stated), but the most notable hit was the fragment-like UNC2170.


Although the affinity was modest, it was quite selective for 53BP1, showing no activity up to 500 µM against 9 other Kme readers. Since AlphaScreen assays can be prone to false positives (the original PAINS compounds were identified in this assay), the researchers tested their compound using ITC, which gave a dissociation constant of 22 µM, in good agreement with the AlphaScreen assay, though with unusual stoichiometry (more on that later).

Thus encouraged, the researchers set off to optimize their hit. Initially they tried modifications around the amine, but even changes as subtle as adding or removing a methyl group killed activity. Attempts to rigidify the propyl linker were also unsuccessful, and shortening it or lengthening it failed too. Replacing the amide with a sulfonamide or amine abolished activity. Most substitutions around the phenyl ring also gave dead compounds, though the bromine atom could be replaced with similarly hydrophobic moieties such as iodine, isopropyl, or trifluoromethyl. Many other analogs were made too, all to no avail. Though the text is measured, the frustration is palpable.

Ultimately the researchers were able to solve the crystal structure of the compound bound to 53BP1, which produced a surprise: one molecule of UNC2170 binds to two molecules of protein, making interactions with each. This explains the stoichiometry seen in the ITC data. It also explains the intolerance to substitutions, as “the ligand is encircled by both proteins,” with no room for modifications.

Happily, UNC2170 is highly cell permeable and non-toxic, and does show some modest activity in cell-based assays. Hopefully the researchers will ultimately find more potent compounds, though this may require a different approach. Indeed, another Kme reader also proved to be quite challenging, but was amenable to fragments. It would be fun to see whether an explicit fragment screen produces more tractable starting points against 53BP1.

01 July 2015

Updated: fragment events in 2015 and 2016

It is hard to believe that the year is already half over, but there are still important events coming up, and 2016 is already starting to take shape!

2015

August 11-13: The OMICS Group is holding a conference entitled Drug Discovery & Designing in Frankfurt, Germany, with FBDD listed as a conference highlight.

December 15-17: More than 40 presentations. 8 countries. 3 days. One event:
The first-ever Pacifichem Symposium devoted to fragments.

The Pacifichem conferences are held only once every 5 years in Honolulu, Hawaii to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, New Zealand, and the US. Registration is now open!

2016

February 21-24: Zing conferences is holding its inaugural Structure Based Drug Design Conference in Carlsbad, California. This looks like a cousin of last year's Caribbean meeting, so it should be a lot of fun.

April 19-22: CHI’s Eleventh Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of this year's meeting here, here, and here; last year's meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

October 9-12: Finally, FBLD 2016 will be held in Boston, MA. This marks the sixth in an illustrious series of conferences organized by scientists for scientists, the last of which was in Basel in 2014. Surprisingly, this also seems to be the first dedicated fragment conference in Boston. You can read impressions of FBLD 2012FBLD 2010, and FBLD 2009.

Know of anything else? Add it to the comments or let us know!

29 June 2015

Crystallographic screening of soluble epoxide hydrolase

Last year we highlighted a paper from Yasushi Amano and colleagues at Astellas in which they performed fragment-screening on soluble epoxide hydrolase (sEH), a potential target for inflammation and hypertension. A new paper from the same group in Bioorg. Med. Chem. builds on that work and provides some interesting comparisons.

In the first paper, the researchers performed an enzymatic screen with fragments at high concentrations, resulting in a hit rate of around 7.3%, of which 126 of the 307 hits resulted in crystal structures. However, despite this bounty of hits, only 2 new scaffolds were found that bind to the catalytic triad of the enzyme.

Given the success rate with crystallography, the new paper focused on crystallographic screening as the primary fragment-finding method. The researchers chose 800 fragments from their in-house collection with molecular weights between 151-250 Da. To identify new scaffolds, fragments containing amide or urea moieties – known catalytic-site binders – were excluded. The fragments were then pooled into cocktails of 10 and soaked into crystals of sEH, with each fragment at a final concentration of 1 mM. X-ray diffraction data of the soaked crystals resulted in 8 hits. To ensure that nothing was missed, cocktails of the remaining 9 fragments from pools with a hit were retested, but nothing new came up. Although the researchers do not comment on the lower hit rate compared with the original screen, this could be because they were looking specifically for new scaffolds.

Despite the 1% hit rate, the fragments identified were quite interesting, with IC50 values ranging from 52 to 2200 µM. Most of the fragments formed hydrogen bonds to the catalytic triad, but the details differed from reported inhibitors. For example, several fragments contained secondary amines. Fragment 1 (cyan) in particular was well-positioned to reach into two sub-pockets on either side of the catalytic center, so 14 analogs were chosen for screening, resulting in molecules with significantly increased activity, such as compound 9 (magenta).
The crystal structure of compound 9 bound to sEH reveals that it binds in a similar manner as fragment 1. However, the added hydroxyl group is able to make new interactions that were unavailable to fragment 1, and the larger adamantyl group of compound 9 is able to make more hydrophobic interactions than the smaller phenyl ring.

This is a lovely illustration of the gains in both affinity and ligand efficiency that can be had by scaffold-hopping. It is also a nice example of using fragments to explore new chemical space. Finally, it is laudable that all the structural information is deposited in the protein data bank.

24 June 2015

One Fragment to Rule them All

Recently, I have been riffing on the ontology of FBDD.  FBDD has become so popular that we are now seeing appropriation of the term in many papers that don't really mean it.  So, I came across this paper.  Now, don't be fooled by the title, this is about fragments, the abstract promises me so.  Let me skip the science, which to my eyes is actually quite boring, and get right to the heart of their fragment case.  
How is this paper fragments you ask?  Well, this is not about scaffold hopping or innovative uses of fragments to develop SAR.  This is not about interesting approaches to screening.  It is most certainly not about in silico approaches.  This is most certainly about fragment library design.  We often discuss here the sizes of fragment libraries and what they should look like.  One important concept we often tackle here is how big should the libraries be and what size should fragments be.  More importantly we often discuss how much of chemical space a fragment library should cover.  This paper takes an anti-Reymond approach to address that question. 
The Reymond approach tries to determine how big chemical space is, what it looks like, and what portion of it is available.  The Anti-Reymond approach identifies what is available and validates its inclusion in a fragment library.  Here is the last sentence of this paper:
"These findings...verify the value of the benzamide fragment in drug design."
Now, I was worried that benzamidine was not a valuable fragment.  This paper has removed all doubt in my mind.  Now that is settled, we can go on an validate the other 165, 999, 999,999 other possible fragments. 

22 June 2015

Fragments vs P2X1

Four years ago we highlighted a paper in which researchers performed a fragment screen against ion channels. There have been other occasional reports, but for the most part this has been a quiet area. A new open-access paper in Neuropharmacology by Andrew Thompson and collaborators at Cambridge University, University of Bern, VU University Amsterdam, and Washington State University provides another case study.

The researchers were interested in the P2X1 purinergic receptor, which allows calcium ions to pass into cells when ATP binds. An antagonist could be a safe anti-clotting agent as well as a potential male contraceptive. However, the only reported inhibitors are freakish molecules like suramin.

The paper is heavily focused on assay development and validation, in this case using cells stably transfected with P2X1. These were loaded with a voltage-sensitive fluorescent dye: when the channel opens, fluorescence increases. (Control cells not expressing P2X1 do not behave this way.) By adding potential ligands first and then adding ATP, both agonists and antagonists could be identified.

The researchers screened 1443 fragments (from IOTA) at 300 µM each. Cell-based fragment screens are rare but not unprecedented. In this case, 46 hits were obtained, and these were retested at multiple concentrations; 39 hits showed dose responses. These were both agonists and antagonists, with EC50 values ranging from low micromolar to above 1 millimolar.

For confirmation, the researchers used a fluorescently labeled analog of ATP that binds to the P2X1 on transfected cells but not to cells that don’t express P2X1; the increased fluorescence of the cells could be visualized using confocal microscopy. Most of the fragment hits reduced the fluorescent signal, suggesting that they block ATP binding.

A structural analysis suggested that the hits are quite diverse, though annoyingly only a single fragment structure is provided. Still, these do look like useful assays, and the paper provides another successful example of fragment screening in a complicated cellular system.

17 June 2015

Fragments vs HIV Reverse Transcriptase - again

Some targets are so heavily studied that you would think there is nothing left to discover. HIV-1 Reverse Transcriptase (HIV-1 RT) is one of these, with 13 marketed drugs against it: half of all anti-HIV drugs. But as Gilda Tachedjian and collaborators at Burnet Institute, Monash University, the University of Pittsburgh, and the University of Melbourne show in a recent (and open-access) paper in Proc. Nat. Acad. USA, there are still new insights to be learned about this target.

The researchers started with an STD NMR screen of 630 Maybridge fragments, each at ~350 µM in pools of up to five. This gave 84 hits – a healthy 13% hit rate. However, when these were tested in a functional assay (RNA-dependent DNA polymerase activity, or RDDP) only 12 showed significant inhibition, of which 6 were better than 1 mM. Testing 14 related compounds led to 2 more hits, for a total of 8 fragments with IC50s from ~70-750 µM. However, one showed signs of aggregation in dynamic light scattering and was not further pursued.

Since HIV-1 RT has been the object of such intensive research, the team looked at the similarity of their fragments to known binders, including those from previous fragment screening. Surprisingly, their hits turned out to be quite distinct.

Next, the researchers looked at the effect of their fragments on the DNA-dependent DNA polymerase activity of HIV-1 RT, and happily found results similar to the RDDP assay above. The 5 most potent fragments were also tested against three clinically important mutants of HIV-1 RT, and while two of them showed reduced activity, the other three were either as potent or even more so. Testing these against unrelated polymerases revealed that they are not merely promiscuous inhibitors.

Of course, functional activity at high concentrations can have all sorts of causes, so the researchers performed a battery of careful enzyme kinetics experiments to ascertain the mechanisms. One fragment turned out to be competitive with respect to deoxynucleotide triphosphate substrate, even though it looks nothing like a nucleotide. Another is competitive with the DNA substrate. In other words, both these fragments operate through different mechanisms of action from clinically approved HIV-1 RT inhibitors.

One of the most potent fragments is a p-hydroxyaniline, which the researchers recognized as a PAINS compound (it can form reactive quinones). However, freshly prepared samples of this fragment were just as active as samples that had been stored in DMSO for months. Also, an analog without the ability to form a quinone was still active, albeit less so.

The p-hydroxyaniline fragment also showed activity in a cell-based assay. Just as with biochemical assays, cell-based assays are also susceptible to false positives, but the kinetics of viral inhibition were consistent with inhibition of HIV-1 RT rather than other other mechanisms. Further work on the compound may be merited; these are exactly the kinds of investigations necessary to decide if an interesting PAINS molecule is worth pursuing.

Unfortunately there is no crystallographic or detailed NMR structural information as to how these molecules actually bind. Previous work has identified multiple fragment binding sites on HIV-1 RT, so further work should eventually reveal how these molecules interact with the protein.

In the end this paper shows that, even in the absence of structure, it is possible to learn a great deal about how fragments inhibit an enzyme. It is also a useful reminder that fragment-based approaches can identify new types of inhibitors even for a target that has been intensively – and successfully – studied for decades.

15 June 2015

Natural Product Derived Fragments against MMP-13

I have been lucky to work on a lot of systems that very much interest me.  I, in particular, love metallo-proteins.  I worked on rubredoxin as a post-doc and when I moved into industry I worked on a slew of metalloproteins.  So, I love it now when I see papers on targets I used to work on.  This paper does exactly that while also letting me riff (later) on Natural-Product-Derived Fragments (NPDF). 

NPDF has a long history in FBDD having been discussed here, here, here, and so on.  Many vendors and some companies have NPDF libraries (whether they call them that or not).  However, these libraries have yet to be proven to be an efficient route for "discovering clinical drug candidates".  Lanz and Riedl set out to do this against MMP-13 (how many of your just said, yeah I worked on that target?).  All MMP-13 clinical candidates with strong ZBG (Zinc-binding groups) have failed.  They are aiming to develop a MMP-13 without a strong ZBG.  Of course, we have seen a LOT of work towards this goal: here, here, and here for example.  The authors propose that the use of NPDF prevents the problem of using fragments with "debatable biological properties".  This seems to the be the argument used by the NPDF people, since these fragments are found in nature they have desirable properties.  I have never bought this line of reasoning for a variety of reasons.  

To their end, the authors selected uracil as their starting NPDF for these reasons: good synthetic starting points, cis amide bonds, and its found in a variety of natural products (nucleic acids).  They docked it in the S1' non-zinc binding site and found a strongly conserved binding site. [For me, and I would imagine a whole lot of people, this fits in the "things you already knew" category.]  The uracil interacted with the NH an CO of Met232 via its cis amide bonds and "addresses" Lys228.  Several compounds were made from the uracil starting point (Figure 1):
Figure 1.  2: 5 uM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 20 uM. 3: 10 nM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 20 uM2: 5 nM vs. MMP-13, < 50% Inhib against 1,2,3,7,8,9,12, and 14 at 10 uM
So, in the end, they have created a potent and selective compound.  They did use a NPDF as a starting point.  Making these compounds is not something that bowls me over either for a Technical Difficulty score or Artistic Merit.   However, I would not go so far as to say that they have validated the NPDF approach.  I think to show that a generic approach works you need more than one (relatively well known) target with more than one (relatively well known) fragment. 

08 June 2015

Benchmarking native mass spectrometry

Mass spectrometry (MS) is one of the less common tools to find fragments. In the conceptually simplest approach (native mass spectrometry), you incubate your protein with a putative ligand and ionize the mixture. Fragment binding is detected by an increased mass for the complex, and the strength of binding by the ratio of heavier bound complex peak to protein peak. However, the liquid to gas phase transition is a big step, and often the complex does not survive. Aside from more specialized applications of MS (such as herehere, and here) there aren’t many published examples. A recent paper from Federico Sirtori and colleagues at Nerviano and Università degli Studi di Milano in Eur. J. Pharm. Sci. describes fragment screening by native MS in detail.

The researchers used the reliable model protein Hsp90, which was also used in a previous MS study and in benchmarking other techniques. One of the many benefits of Hsp90 is a wealth of well-characterized inhibitors with a range of affinities, and these were used to calibrate the technique. This turned out to be critical: beyond sample preparation itself (beware non-volatile buffer components), all kinds of parameters can be adjusted including various voltages, temperatures, vacuum strength, and ion source. Get one of these wrong and your non-covalent complex either fails to ionize or blows apart.

In addition to using published data on known compounds, the researchers ran both fluorescence polarization (FP) and surface plasmon resonance (SPR) assays to independently determine dissociation constants. Initially the results from MS (a Q-TOF) were quite different, but after optimization the team was ultimately able to find conditions that gave qualitatively as well as quantitatively similar results for ligands with affinities ranging from picomolar to ~100 micromolar.

Thus encouraged, the team embarked on a fragment screening campaign. The Nerviano fragment library consists of 1914 molecules mostly following the rule of 3, though halogenated fragments up to 380 Da are allowed as are compounds with up to 6 hydrogen bond acceptors. The fragments were run in mixtures of 5, with protein at 2.5 µM and each compound at the low concentration of 10 µM. Sample injection and data processing were automated, and the entire screen took 2 days and 2 mg of protein.

Given the low concentration of fragments, the researchers lowered the bar for potential hits, yielding 282 compounds. These were retested individually, yielding 146 confirmed hits that gave signals of 5.2-29.7% bound protein. This is a high hit-rate, particularly given that these binding levels suggest affinities in the 20-179 µM range. Indeed, only 5 fragments could be competed by a high-affinity binder, suggesting either that the others bind outside the active site or are non-specific (false positives). Regarding false negatives, Nerviano reported the results of an NMR fragment screen against Hsp90 last year, and 12 of 14 hits identified there could also be detected by MS. The other two were likely below the detection limit of the MS assay.

Unfortunately, the researchers do not discuss thermodynamics. In theory enthalpic interactions dominate over entropic interactions in the gas phase, but it is unclear whether any of the observed binders were strongly entropy-driven.

In the end, it appears that fragment screening by native MS is workable, but the sensitivity is probably lower than other techniques. Of course, increasing the ligand concentration would increase the sensitivity to weaker binders, but at the cost of more non-specific binding – which is already considerable. Also, Hsp90 is about the friendliest protein one can imagine. I would be reluctant to try this with a more challenging target that lacks good tool ligands. But if you want to give it a go, this paper provides a wealth of information for getting started. And if you have experience with native MS, please share it in the comments.

03 June 2015

Fragment Ontology

We here at Practical Fragments look for papers in the literature about fragments.  Typically, it is a Web of Science search, or I see something come in an alert that has "fragment" in the title.  Well, not everything with fragment in the title is not really about fragments as we typically think of them. So, I recently came across this paper titled : "Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins".  I decided to give the paper a good perusal, largely because one of the authors is from where I did my post-doc.

The authors are interested in making competitive inhibitors of carbohydrate recognition domains for the treatment of a variety of diseases.  The challenge with lectin inhibitors is that the native carbohydrate has relatively low affinity and are synthetically complex.  As you would think, you can use the carbohydrate for binding specificity and then add something more "drug-like" to increase affinity through other interactions.  This approach has been successful but require complex multistep syntheses.  In this paper, they decided to search for peptides which can synergize with carbohydrates rather than serving solely as a linker or standalone recognition element.  To do this, and increase throughput they used a genetically encoded library, phage display.  In short, they created a glycopeptide library of 10^8  molecules through derivatization of a peptide library with carbohydrate.  This approach allows the addition of different carbohydrates (targeting different lectins) with the same peptide library.  
Figure 1.  Library Screening Approach for Genetically Encoded Glycopeptide Libraries.
In this approach, the first library (Man-X7) is screened against the target and anti-target.  The second library (methyl-X7) and the third library (Ser-X7) are screened only against the target.  After the first round of panning, they identified a weak consensus of Man-[WYF]Y[SDEA].  These peptides were made and able to compete with ConA for ligand in SPR, the mannose was shown to be essential to activity, the specific peptide sequence was required for synergistic binding.  Further work showed that the final four residues of the peptide contributed minimally to binding, so they lopped them off.  

They then performed two more rounds of panning with Man-WY[D/E]-X7.  All of the hits from these rounds had single digit micromolar affinity and the glycan-proximal ligands are responsible for most of the affinity.  How did they know if this is actuallly binding to where they want it to?
Figure 2.  Man-WYD co-crystalllized with ConA. 
Figure 2. shows the crystal structure of Man-WYD.  The mannose moiety binds where it is expected.  However, the peptide is not binding in the remainder of the trisachharide binding site, but instead in a somewhat deeper cavity near Y12.  Additionally, a latent hydrophobic site is opened up through induced fit (asterisk), filled by the Y residue of the glycopeptide.  

This approach led to the discovery of a novel class of compounds which would not have been discoverable by "standard" approaches.  But, is this fragments?  In my eyes, fragments takes simple compounds and screen them against the target.  It then optimizes the actives as quickly as possible and does iterations.  A key component to FBDD is SBDD and identification of how the actives/hits bind.  To me, this approach adheres to all the tenets of FBDD.  We have seen super huge screening molecules before, so that should not be an issue. As I have said, FBDD is about small little things being screened effectively.  I think this paper shows it is more about how you think about your system. 

01 June 2015

Fragments vs MCL-1 revisited: on to low nanomolar potency

The protein MCL-1 binds to other proteins to protect cancer cells from apoptosis. Protein-protein interactions have historically been considered difficult, but as we’ve noted previously (herehere, here, and here, for example) fragments have been successfully deployed against this target. A recent paper in J. Med. Chem. provides the latest update from Stephen Fesik and co-workers at Vanderbilt University.

We last highlighted this program in early 2013, when the Fesik lab disclosed a series of mid-nanomolar inhibitors, such as compound 1, derived from fragment merging. In the new paper, they report compound 2 as another fragment identified in the original NMR screen.

NMR-based structural information of this molecule bound to 15N, 13C double labeled MCL-1 revealed a similar binding mode as the previous series, and merging the molecules led to the low nanomolar compound 34, with impressive ligand efficiency. This compound was also >1700-fold selective for MCL-1 over the related protein BCL-xL and >250-fold selective over BCL-2.

Although compound 34 did show activity in cell lysates, the authors note that it is unlikely to be potent enough to show unambiguous activity in cellular assays. Indeed, researchers at AbbVie and Genentech have recently reported MCL-1 inhibitors that show picomolar activity in biochemical assays but only high nanomolar to low micromolar activity in cells.

Still, this is another nice illustration of the power of fragments – combined with a healthy dose of medicinal chemistry – to tackle a difficult target. Notably, the researchers didn’t have to turn to super-sized fragments. Moreover, the best molecule shown is well within Lipinski space, and there are plenty of avenues for further optimization. It will be fun to watch this story progress.

27 May 2015

Stopping Virulence...One Fragment at a Time.

The best way to not get an infectious disease is vaccinate.   Streptococcus pneumoniae is repsonsible for a million deaths world-wide every year.  For Streptococcus pneumoniae, there are a numbers of vaccines on the market.  These vaccines are bacterial polysaccharides either naked or conjugated to a protein.  They are highly effective, but don't cover all serotypes (there are ~100).  And sometimes a novel serotype arises.  So, if you do get infected treatment is key.  Beta-lactams are the first line of defense, but multi-drug resistance is on the rise, so alternate forms of treatment are needed. Targeting virulence factors has become a recent line of research.  Pneumococcal surface antigen A (PsaA) is strictly conserved surface-exposed lipoprotein expressed by all known pneumococcal serotypes and is essential for colonization and pathogenesis.  PsaA is an integral part of an ATP-binding cassette(ABC) transporter protein complex known as the PsaBCA permease, which is involved in manganese (Mn2+) transport across the bacterial cell membrane. (See there's always a metal involved in cool biology.)  This makes PsaA a good target for pneumococcal infections.  In this paper, a group from down under presents their results using fragments to target PsaA.

They custom built a fragment library (via outsourcing) ~1500 fragments.  This struck me as unusual, if not unique.  Typically, academics make their own or just buy one off the shelf.  I would love to hear why this path was chosen.  In the SI, they do say they used "relaxed" Ro3, but the only relaxation seems to be on the MW.  Have other academics gone this route?  I would love to know more (you can be anonymous in the comments, hint hint).  These were docked into the PsaA metal binding site (Figure 1) based on 3D shape and electrostatic similarity. These were then scored using FlexX. 
Figure 1.  Structure of PsaA. 
The top 300 fragments were manually inspected and then subjected to a cluster analysis.  The 60 most diverse fragments were then tested in a competitive Zn-binding assay.  Zn is a irreversible inhibitor of PsaA and the assay uses this to test for compound binding.  10 of the 60 fragments exhibited greater than 15% inhibition at 100 microM.  Two of these compounds showed greater than 50% inhibition at 1mM (Cpd 15 and 58, Figure 2.)
Figure 2.  Fragments with greater than 50% activity at 1 mM.  Hydrogen bond acceptors are shown in red, H-bond donors in Blue.
So, with crystal structures available, the authors decided to inspect the docked poses rather than actually trying to obtain a structure of the fragments bound to the protein. So even though docked fragments can, and do tend to, keep their original locations, experimental data is key to confirming in silico predictions.  The made 31 compounds around 15, and one that replaced the p-nitro, o-methoxy phenyl with o-hydroxypphenyl was the best 15h (28 microM, pIC50/HAC=0.37).  To that end, they tried to soak apo-crystals with cpd 15h and were unsuccessful due to limited compound solubility and affinity for the target.  They did not attempt soaking compound 58, which they was unable to be further "optimized" with simple SAR.  Cpd 15h did have antimicrobial activity: significant growth inhibition at 180 ug/ml and total growth inhibition at ~800 ug/ml.  They did a further round of optimization.

This is an example of real FBDD approach, in contrast to just using the words.  However, I think this is really a MPU (minimal publishable unit).  If we are lucky, we can expect to see future papers coming out describing their success (or failure) against this target. 

25 May 2015

Charting new chemical space for kinase inhibitors

Since the advent of imatinib, kinase inhibitors have become a thing in drug discovery, with more than two dozen already approved. Indeed, kinases are the targets of more than a third of reported fragment-derived compounds to reach the clinic. Given that all 500+ human kinases bind ATP, you would think that the chemical space would be pretty well picked over by now. As Hongtao Zhao and Amedeo Caflisch at the University of Zurich show in a recent Bioorg. Med. Chem. Lett. paper, this is not the case.

The researchers started by extracting all 26,668 kinase inhibitors with MW < 600 Da and IC50 or Ki < 10 µM from the ChEMBL database; three quarters of these were better than 1 µM. These have been tested in aggregate against 367 kinases, of which 88 have more than 100 reported inhibitors!

The molecules were then deconstructed into 10,302 ring-containing fragments, such as benzene (7.1% of kinase inhibitors), 2-methylaminopyrimidine (3.5%) and N-methylmorpholine (2.3%), as well as more obscure structures. In fact, more than half (53%) of these fragments were not found within 7.5 million commercial compounds in the ZINC database. In other words, many fragments that form a part of known kinase inhibitors are not represented among commercial compounds, despite many vendors offering “kinase inhibitor libraries”.

What about the reverse question, analyzing commercial molecules for new kinase inhibitors? The researchers focused on possible “hinge-binding” fragments – those that have at least one hydrogen bond donor and one acceptor in close proximity to one another so as to be able to interact with a conserved region of kinases. Not surprisingly, more than half of the fragments (5681) found by deconstructing the kinase inhibitors fit this description. More interestingly, 196,904 potential hinge binders resulted from deconstructing the ZINC compounds, of which only 1% had been reported as kinase inhibitors.

Digging into the data more deeply, the researchers classified hinge binders as monocyclic, bicyclic, and multicyclic. This analysis revealed that the overlap between kinase inhibitors and commercial compounds was particularly low for multicyclic fragments. This intuitively makes sense: medicinal chemists often turn to ring construction to fix all manner of problems, both pharmaceutical and IP-related, so the under-representation in commercial compounds is likely because medicinal chemists introduce rings into simpler starting molecules. Also, from a molecular complexity standpoint, multicyclic ring systems may be less likely to bind to a wide variety of proteins than simpler monocyclic fragments.

More than five years ago Practical Fragments highlighted a paper from Abbott describing their efforts to generate novel hinge binders. As this and related analyses show, there is still plenty of chemical space left to explore and exploit.

21 May 2015

Just Because its called "Fragment-Based"...

When my parents were young and just starting out (the late 60s) they needed a vacuum cleaner.  So a vacuum cleaner salesman came to the house eager to make the sale.  This was the era of the Space Race, plastics, and so on.  So, it was cool to be associated with this.  The eager young vacuum cleaner salesman showed my parents the fine, sleek design of the vacuum cleaner (it was a ELECTROLUX).  It was long and sleek, looking like a rocketship (or a dachshund).  It came with a lot of nozzle attachments.  One in particular was shaped to be very narrow, and get in between the couch and wall for example.   He was particularly proud of this piece: the AEROspace tool.  He even wrote it down as such.  You must have a "AEROspace" tool for your vacuum.  It was an example of great marketing, associate yourself with something very popular to make something mundane appear special.     

So, this paper comes along from Moffatt Cancer Center and USF targeting ACK1 (aka TNK2).  This paper purports to have a "innovative fragment approach" (mix and match).  I love novel approaches to libraries.  So, let's dive in. There is a good deal of work that has been done with ACK1 by Amgen, OSI/Astellas, and others.  Dasatinib and Bosutinib also show activity against ACK1 also.  Based upon all of this previous work and the knowledge of the pyrimidine core they decided to approach the target as laid out in Figure 1.
Figure 1.  Library Design Approach
So, this paper doesn't interest me, although they do come up with some potent compounds, from a what they discovered aspect, rather from a more philosophical aspect. What does it mean to do fragments?  This harkens back to the Safran Zunft challenge.  To me, FBDD is about using simple, small molecules.  Pyrimidine series 9 does not fit any definition of a fragment (Cpd 8 would, but it was never tested AFAIK).  What they did was identify a variety of fragments which would be inputs for creating a small library of lead-like compounds.  However, for this to be "Fragment-based" I would think that they would tested each individual component and prioritized chemistry based upon that.  Or maybe they could have made R3=H.  They don't report Ligand Efficiencies (cue Pete Kenny).  This is simply not "Fragment-based" anything.  Nor, do I think this approach is novel.  Nor do they explain how this is novel.  
So, I think we have entered the time when anything that uses a fragment in the design process is fragment based.  Based on this line of thinking, Nicolaou's total synthesis of Taxol is "Fragment-based". Beware those talking the talk, but not walking the walk.

18 May 2015

Predicting protein ligandability and conservation of fragment binding modes

Say you have a protein target, and you want to know whether you will be able to find small molecules that bind to it. A fragment screen can give you a good idea as to the likelihood of success: if you find lots of different fragments with high affinities (say, better than < 0.1 mM), your protein is likely to be highly “ligandable.” On the other hand, if you get very few fragments, and most of them are weak (> 1mM), be prepared for a slog.

Of course, it would be even better if you didn’t have to do a physical screen at all, and two recent papers show how a computational approach may be sufficient. The first, by Dima Kozakov, Sandor Vajda, and their collaborators at Boston University and Acpharis is a detailed how-to guide in Nature Protocols. The second, in Proc. Nat. Acad. Sci. USA by Dima Kozakov, Adrian Whitty, and Sandor Vajda and their collaborators at Boston University, Northeastern University, and Acpharis, addresses some interesting questions about fragment binding.

The main program is called FTMap (also highlighted here); it and several related programs are accessible through a free web server. It is remarkably easy to use: just provide a protein data bank (PDB) ID or upload your own structure and away it goes.

The program works by docking a set of 16 virtual probes (such as ethanol, acetonitrile, acetamide – the largest molecule is benzaldehyde) against a protein and looking for “hot spots” where many fragments cluster. Previously the researchers demonstrated that known ligand-binding sites in proteins tend to be computational hot spots, where at least 16 probes bind. (Note that due to their small size, multiple probes of the same type – acetone, for example – can bind within the same hot spot simultaneously.) In other words,

The strongest hot spot tends to bind many different fragment structures, acting as a general “attractor.”

On the other hand, a hot spot with fewer probe molecules is unlikely to have enough inherent binding affinity to bind to ligands with low micromolar or better affinity.

A related program is called FTSite, which focuses on more thoroughly characterizing the best binding sites. Other programs allow for protein side chain flexibility, docking custom probes, or docking against ensembles of protein models such as generated by NMR structural methods.

The PNAS paper goes further to ask about ligand deconstruction. Specifically, why is it that when a larger ligand is dissected into component fragments, sometimes the fragments recapitulate the binding modes seen in the larger molecule, and sometimes they do not? The answer:

Because a substantial fraction of the binding free energy is due to protein-ligand interactions within the main hot spot, a fragment that overlaps well with this hot spot and retains the interacting functional groups will retain its binding mode when the rest of the ligand is removed.

The researchers support this assertion by examining eight literature examples in which structural information was available for fragments and larger ligands (some of which we’ve covered here, here, and here). In cases where the isolated fragments overlapped with 80% of atoms in probe molecules within a given hot spot, the fragment binding mode remained conserved. Also, these fragments tended to have high ligand efficiency values.

This is neat stuff, and it will be fun to see how general it is. I’m especially happy to see that all of the software is free and open access. Even though I’m hardly a computational chemist, I tried playing around with it and found it remarkably fast and easy to use. So if you have a protein with no known ligands, FTMap can find hot spots, and if they’re particularly promising, this should embolden experimental work.

13 May 2015

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.