Magic Methyl and SBDD

As sites have closed down, we have seen a fair number of paper come out describing work at various sites.  Today's paper is another of these, from Roche, Nutley.  The target is Tankyrase, blogged previously here.  

This group started with a biochemical screen of their in house fragment library (here for analysis of their library) against both TNKS1 and 2.  This screen resulted in two compounds: 1. a pyranopyridone and 2. a benzopyrimidone (that looks like the first published TANK inhibitor).  

Figure 1.  Fragments identified from biochemical screen.

They were able to co-crystallize 1 with the TANKS2 and then were able to model 2's binding based upon the known inhibitor.  This pleasingly revealed both similarities and differences in the binding.  the main recognition elements are largely the same.  A big difference is that the phenyl maintains hydrophobic interactions that the cyano group does not. 

Figure 2.  TANKS2 X-ray structure with 1 (orange carbons) and 2 (yellow carbons). 

This lead to the obvious decision to merge the two fragments leading to fragment 3. This compound was reasonably potent (320 nM), however it should moderate to high clearance in an in vivo PK study. 

Compound 3. 

Then of course, the medchem kicks in aiming to decrease cLogP and increase solubility by focusing on two areas: the fused phenyl ring and the isopropyl side chain. This work was able to achieve their goals, but they also stumbled on a "magic methyl" (9) which improved potency 100 fold.  

9.  Magic methyl on fused phenyl ring.

This magic methyl works by sliding into a little pocket defined by three tyrosine residues. 

Figure 3.  Magic methyl binding mode. 

The t-butyl alcohol derivative of 9 had excellent properties: reasonable solubility, excellent permeability, stopped axin degradation in cells in a dose dependent manner, prevented mRNA production of beta-catenin dependent genes, and in mouse had satisfactory in vitro activity and PK profile.  

This is an excellent example of FBLG.  The magic methyl does exist and X-ray is a highly enabling technology. 

Uninteresting GPCR Fragment Work...meant as a Compliment!!

There are certain movies that when they are on TV, I can't not watch.  I call these Broken Leg Movies (as in if I were laid up with a broken leg what would I watch).  As I have said, Road House is one, Apollo 13 another.  Its about America's blase attitude towards the amazing feat of putting men on the moon.  It takes a potential horrific tragedy (For those off you who haven't seen it, let me say (**Spoiler Alert**) don't worry it has a happy ending.) in order for America to care about men in space.  Which of course is in direct contrast to Pigs in Space (with Swedish Subtitles)! 

One of the field changing technologies is Heptares' STAR technology (for creating stabilized, soluble GPCRs).   We have discussed it often on this blog.  Well, they are back with another paper, this time working the voodoo they do so well on a Class C GPCR.  Negative allosteric modulation of the mGluR has the potential for significant medical impact in a variety of diseases.  In a relatively well trod drug space (there have been several molecules in late stage trials), an issue appears to be the acetylenic moiety in these drugs (which appears to be manageable).  So, non-acetylenic molecules would be desireable.  

To attempt to ligand this molecule, they screened 3600 non-acetylenic fragments using a radio-labeled assay.  This is in contrast to previous work where they used SPR. From this screen, 178 fragments were tested in concentration-response curves leading to "a number of promising" hits, including the compound shown below. 

Cpd 5.  pKi=5.6, LE=0.36

This compound was advanced using the tools you would expect (especially from Heptares): modeling, X-ray crystallography, medchem, and so on.  The final molecule is an advanced lead with excellent mGluR selectivity and in vivo activity, clean tox, and so on. 

This is excellent work, but "yawn".  I think it might be interesting to hear why they went with the radioligand approach, as opposed to SPR.  You could quibble that 5 is too big to be a fragment, but really?  Papers like this are uninteresting, we know its going to work.  The science is excellent, but I want to see the triumph out of tragedy.  Not here.  I want to congratulate Heptares for making an achievement like this paper perfectly uninteresting.  And I mean uninteresting as the very best of compliments. 

ATAD2 Again...Now with a good tool.

Epigenetics is big.  We keep on beating that drum.  Just to prove it, today's paper is on a target we have talked about before: ATAD2.  That previous paper was unsatisfying: leading to my summary: "if you throw enough fragments at a target you can find a few that bind."  Today's entry  from GSK has produced the first micromolar inhibitors of ATAD2.  

As noted previously, ATAD2 is "undruggable" or at least VERY difficult to find chemical matter against.  To add to the difficulty,  the BET activity needs to be minimized.  With that in mind, they set a high threshold of activity (pIC50 greater than7) and 100 fold selectivity against BRD4 (a representative BET domain).  The ATAD2 site is more polar and flexible than BET.  The authors felt that this would be exploitable to create selective molecules.  To address ATAD2 they started with Ac-K mimics from previous BET work.  They supplemented this with diverse cores not represented.  One such array (which I read as libraries, somebody correct me if I am wrong) was based on the cpd 1,

Cpd 1. 

which is similar to the chemotypes discussed last year.   A crystal structure of 1 was solved, confirming that it bound as expected.  

Additional arrays were made around this core and tested in a TR-FRET assay.  30,000 compounds gave a 0.25% hit rate.  Confirmation was performed by HSQC NMR.  A subset of compounds interacted at the Ac-K site based upon comparison to compounds with known binding modes.  In this case, the peak that shifted upon binding were the same.  I would like to know if this was by visual inspection of spectra or if it was accomplished using PCA, or similar method.  It probably doesn't matter, but intrigues the NMR jock in me.

In rounds of medchem and X-ray confirmation, they were able to drive the potency against ATAD2 to the single digit micromolar.  The ligand efficiencies were maintained right around 0.30. Compound 57 (R=4-Me) and 60 (R=4-OMe) had the "best balance of ATAD2 and BET activity".  These compounds were also active in a cell-based assay known to be sensitive to BET inhibitors.  However, there is no selectivity.  ATAD2/BET pIC50 for 57 was 1.1 and 60 was 1.0. So, despite the selectivity threshold they developed, these compounds are not selective.  Despite that, I think this paper shows that the aphorism Undruggable =Undone is true.

The Value of DSF

Science is based upon incremental advances of previous work.  A year ago, Dan blogged about worked on BioA.  The key take home from that work was that a hydrazine fragment ended up destabilizing the target by 18C.  It ended up being, as expected, a reversible, SAM-competitive inhibitor with modest potency.  As Dan concluded:

This is a very nice paper, and it will be fascinating to try to understand how the fragments so effectively destabilize the protein despite binding tightly, and how this translates into inhibition. The researchers suggest that finding ligands that destabilize proteins could be generally useful for turning off proteins.

In this paper, the same group is back (This work was also presented at DDC in San Diego in April). Interestingly, they seemed to have abandoned the hydrazine.  Taking the same approach (DSF-Xray-ITC) they identify different fragments (2% hit rate from a 1000 screened).  9 were stabilizers (average of +3.8C) and 12 were destabilizers (average of -13.8C(!)).  5 fragments were able to be crystallized by soaking, co-crystallization was able to add one more structure (Figure 1).  Interestingly, the calorimetry showed that only F5's binding is strongly, enthalpically driven.

Figure 1.  Crystallographically Confirmed Fragment Hits

The authors make several interesting observations:

• Little correlation between magnitude of Tm shift and confirmation by crystallization
• Stabilizing and destabilizing compounds were confirmed by Xray
• No correlation between magnitude of the Tm shift and calorimetry determined Kd.
• Conformational flexibility in the target active site need to be taken into account.

This is not surprising to me; I have seen/heard this many times.  What does this mean for DSF in general? 

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. 

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.

When a Lead is a Lead

As we keep on saying, epigenetics is big.  So, today we present another paper on an old friend, BRD4.  This paper is a follow up from previous work where they used docking and X-ray to find the thiazolidinone fragment hit that was elaborated as shown below (Figure 1), but with potency in the single digit micromolar in vitro and double digit in cellulo. 

Figure 1.  Previous work from these authors.

In this work, they continue developing this scaffold investigating the reversed sulfonamide(Figure 2)

Figure 2.  Reversed Sulfonamide

which had significantly improved activity.  Cyclo-aliphatic rings showed increases in potency, but with larger rings also decreasing ligand efficiency.  Aromatic rings decreased potency and larger groups (rings with linkers) were not tolerated at all.  

The crystal structure of the cyclopentyl derivative was solved and was seen to have a different binding mode from the original fragment.  In this case, the WPF shelf is NOT the major binding site for the compound.  In the end, they ended up with

Figure 3.  End Result of this study.  

This is compound is potent (albeit not super potent), ligand efficient, with cell-based activity,  selectivity, and good PK properties.   What I really like is that final sentence of the conclusion:

a promising BRD4inhibitor and a useful lead for further anticancer drug development.

Spinach affects the Water

People often ask what a fragment is.  I like to paraphrase Justice Potter and say that it is like pornography; it is in the eye of the beholder.  I am not one for hard and fast rules as to what a fragment should be.  But, I also have a definite opinion what a fragment is NOT.  To me, what a fragment should be is easily described: relatively unadorned molecules.  I have a whole set of rules as to what the substituents should look like (coined the Zartler Optical Filter or ZOF by a cheeky comp chem friend).  In this paper, a group from Merck Serono decide to probe exactly what role the spinach on fragments play.  

Specifically, they deconstructed a TIE2 inhibitor (Figure 1) into its core hinge binding motif (Figure 2). 

Figure 1.  Crystal Structure of the Intact Inhibitor

This hinge binding motif has the advantage in that "decoration" can be introduced at the 4 or 8 position (Figure 2) as well as giving three donor/acceptor moieties. 

Figure 2. 4-Amino-8H-pyrido[2,3-d] pyrimidin-5-one (compound 1)
as core hinge binding motif.

They determined crystal structures for this molecule and four related fragments (Figure 3)

Figure 3.  Fragments for this study.

and then went to town on them with in silico methods to study the roles of water.  In one of those "gotta love it" moments, they classified the waters as "happy" or "unhappy", depending on whether they have positive or negative free energy, respectively.

So, what do we learn?  First, changes in the decoration leads to different binding modes.  In this case, they conclude that replacement of different water molecules leads to differences in binding modes.  Well, not surprising.  But, I think this is part of a trend, studying water and how fragments affect them, and vice versa.  In fact, the authors suggest using WaterMap could help to rationalize the roles of waters.  So, are we entering a brave new world of experimental verification of in silico predictions?

A COMT Tease...

S-adenosyl-methionine (SAM) is a hot molecule; you could probably make a good living selling it these days.  SAM-transferases of all types are "hot" targets, especially in epigenetics.  However, one current target is COMT, or catechol-O-methyl transferase.  COMT lives in a far different space than the epigenetics one, neurodegeneration.  There are several current Parkinson's Disease treatments based upon catechol, but as you would expect, there is toxicity associated with these.  

So, a team at Takeda decided to go after SAM-competitive molecules.  To this end, they screened 11,000 fragments using a enzymatic assay @100 uM.  52 hits (>15% inhibition) were found for a 0.15% hit rate.  They note this is a very low hit rate for what appears to be a very ligandable pocket. They then used LC-MS/MS and SPR to remove reactive moieties and non-SAM competitive molecules.  This led to compounds (4-6) and SAR by Corporate Collection (7). 

They followed up on these four compounds with DSF, STD-NMR, and X-ray.  They were able to co-crystallize 5 with mouse COMT.  This is the first (reported) structure of COMT with a SAM-competitive molecule. 

They mention that they took a "build up" approach, but I presume that is for for future papers. 

Halogen Hydrogen Bonding...Designable or Not?

The use of brominated fragments for X-ray screening is well known; it was the basis for former company SGX (now part of Lilly).  The purported advantage of brominated fragment is that you can identify the fragment unambiguously using anomolous dispersion.  In this paper, they are focused on using fragments to identify surface binding sites on HIV protease.  Prior work has focused on creating a new crystal form (complexed with TL-3, a known active site inhibitor) that has four solvent accesible sites: the exosite, the flap, and the two previous identified sites.  They took 68 brominated fragments and soaked these crystals: 23 fragments were found.  However, most of these actives were uninteresting.  Two compounds were found to be interesting, one bound in the exosite and one in the flap site. 

So, what's interesting in this paper?  Well, they (re)discover that brominated fragments can bind all over with a variety of affinities.  However, the bromine allows you to unambiguously identify those fragments through anomolous dispersion.  This is NOT interesting.  They discover that although it is a subject of much debate lately: specific interactions of the ligand with the target dominate the "bromine interaction".  This IS interesting.  They do not discuss this in much detail, but their grand extrapolations of this method to general applicability I don't buy. 

 I think the key take away from this paper is whether the halogen hydrogen bond undesignable and just a subject of serendipity? 

What we do in life, echoes in eternity (or the life of the patent)

Next week is the Drug Discovery Chemistry conference where Dan and I will be co-teaching our award-winning FBDD short course (or at least our mom's think it is great). We look forward to seeing any/all of you next week.  Blogging may be light next week, but we promise to give an update of the going-ons at the conference soon after.  

Kinases are fun, and those of us who have worked in them have probably all worked on the same ones.  I always loved the MAP family.  Why would I have a favorite kinase family?  Because of the cascading MAP kinases, like the one in this paper, Mitogen-activated protein kinase kinase kinase kinase 4 (that's a lot of kinase!).  But, unlike a lot of other kinases, there is no good tool compound.  So, using SPR, they decided to generate one. This paper is not particular interesting in terms of what they did, but rather it raises interesting questions. While the approach they describe is not novel, it is nice to see the data supporting them. 

They screened their 2500 fragment library against immobilized protein at 100 uM (single point).  225 hits were found with Kd ranging from 10 to 2000 uM (LE =0.24 to 0.59) for a 9% hit rate.This paper is about progressing this oxazole fragment 1: 

Based upon its structure and the wealth of kinase structure knowledge extant, they surmised it would be ATP-competitive and a hinge binder.  Based upon a binding model, the attempted to prosecute this fragment by "close-in" analogs and looking for groups that would extend farther into the hydrophobic pocket, but with MW less than 350 Da and clogP less than 3.5.  Exploring bi-aryl space resulted in 8:  

This compound had an activity of 143 nM and it was at this point that they decided to switch to the biochemical assay as their primary assay.  In the end, using X-ray focusing on LLE, they ended up deliveringa low molecular weight compound with favorable in vivo PK.  It also demonstrated a pathway functional response. 

This raises an excellent point, something I get asked frequently.  When do you switch from a biophysical assay to a biochemical one?  This maybe arguing semantics, but I think as more and more companies enter this arena these are exactly the things we need to discuss.  I think the switch happens when you feel comfortable, there is no hard and fast rule.  There is a difference in the SPR Kd and biochemical IC50 by more than 10x.  It is very important to note that they relied heavily on LE (-RTlnKd/HA) and LLE (pKd-cLogP), or pIC50 for biochemical assays.  But, it also raises the issue of correlation between SPR Kd and IC50.  I raise these socratically, and as maybe as a topics for discussion next week (or in July and September).   

Druggable is as Druggable Does; Or a Million Ways to use NMR

As we all know, the closure of sites is a bad thing for those of us in Pharma.  One very small silver lining is that this frees up a lot of very nice work to be published.  The former BI site in Laval has been closed for a year and we are still seeing great papers coming out.  In this one in JMed Chem,  LaPlante and co-workers tell us about their fragment efforts against HCV helicase. 

HCV has recently had drugs approved for its treatment, but as with any virus, different modes of treatment are important.  The ATP-dependent helicase activity is found in the C-terminal 2/3 of the NS3 protein. Helicase activity is straight forward to measure and there has been some success in terms of non-viral specific inhibitors.  The inhibitors found to date have been found to act through undesireable mechanisms, but with a wealth of structural information there is no reason why helicase is inherently undruggable.  With this information in hand, they decided to target site 3+4 (green sticks are DNA from the structure), near the most conserved residue W501.  The ATP-binding site is 1+2 for reference. 

 Their first approach was to screen the 1,000,000+ corporate compound collection.  As you would expect for a paper blogged about here, they failed to find anything interesting (all the inhibitors worked by undesireable modes).  So, on to the FBDD campaign, to save the day once more.  The used a "shotgun" approach with their fragment screen:

One source of compounds came from an earlier HTS where they rejected fragment-like molecules for lack of potency, additional HCS screening of in house fragment collection, commercial fragments were screened in an SPR assay, virtual screening, and NMR.  They had a stringent workflow aimed at producing quality compounds for X-ray.  [The in-house fragment collection was 1000 compounds.]  This, along with NMR, validated ligands that bound to site 3+4.  They note one particularly noteworthy problem: high false positive rates due to the high ligand concentrations needed for the assays.  This lead to aggregation, solubility, and promiscuity.  This lead them to implement specific assays designed to eliminate these compounds (two NMR papers published in 2013, ref 18). 

They then clustered the best hits into 9 chemotypes:

 They used an "Analog by Catalog" approach and soaked or co-crytallized the best compounds into crystals.  S6, S7, and S9 were not found to bind to helicase in the crystallization trials and were deprioritized.  S5 was found at Site 3+4, but also others.  S1-4, and S8 were found to bind solely to site 3+4 (12 examples shown overlain). The key feature of this is the compounds are centralized in a wide groove over W501.  The topology of the binding site (wide groove and small lipophilic pocket) meant that optimizing for potency could be challenging.

From this, they decided S2-S4 were the most promising.  In the end, the focused on the S2 indole series as the most promising.  The S2 stereotype 1

was found from an STD-NMR screen of 3 fragment per sample (300 uM fragment and 3.5 uM helicase).  They then, much to my heart's delight, they reached into the NMR cabinet for line broadening and competition experiments confirming it binds in site 3+4.  X-ray confirmed the binding mode, but potency was not improved with chemistry.  So back into the NMR cabinet they went: a methyl resonance assay, 

 15N TROSY showing peaks shifting upon addition of a derivative of 1, and 19F NMR!  OMG, how awesome is this?  

In terms of the chemistry, removing the Br does not change the potency, but did change the orientation of the compound in the binding site.  Further elaboration led to this compound 19 (3 uM and 0.23 LE):

It contains a nitro group, think what you may.  In order to confirm the binding affinity of the compound without immobilizing protein, they used the methyl resonances to do the titrations.  The two separate peaks they followed gave values of 32 and 28 uM (+/- 8).  Given the broadness of these peaks, I think this is a pretty decent assay, although it is an order of magnitude different than the biochemical Kd.  However, subsequent structural studies revealed that there is significant structural dynamic differences between pH 6.5 and 7.5.  ITC gave the same number (33 uM and enthalpy driven); however, the ITC had to be run at high compound concentration and a different pH.  They then went off the deep end and decided to use CD (I can't link to a previous post of using CD because we have never had a post where someone used it).  With a horrible assay (don't even get me started on near-UV CD as a readout of tertiary structure), they got reasonably close to the Kds determined by ITC and methyl-NMR.  

This is a very nice example of not being afraid of a target and using all available tools to advance hits against it.  It also shows the WIDE range of NMR experiments that can be used and that are easy and practical.  In terms of full disclosure, Steven LaPlante is a FOT (Friend of Teddy) and I have been working with him. 

Fragments in Australia

Last year we highlighted the first FBDD conference held in Australia. That meeting has now led to a dozen papers in the December issue of Aus. J. Chem. Many of the papers use the same fragment libraries, so this is a good opportunity to survey a variety of outcomes from different techniques and targets.

The collection of papers (essentially a symposium in print) starts with a clear, concise overview of fragment-based lead discovery by Ray Norton of Monash University. Ray also outlines the rest of the articles in the issue.

A well-designed fragment library is key to getting good hits, and the next two papers address this issue. Jamie Simpson, Martin Scanlon, and colleagues at Monash University discuss the design and construction of a library built for NMR screening. Compounds were selected using slightly relaxed rule-of-three criteria, and special care was taken to ensure that at least 10 analogs of each were commercially available to facilitate follow-up studies. Remarkably, of 1592 compounds purchased, only 1192 passed quality control and were soluble at 1 mM in phosphate buffer. The properties of the final library are compared with nearly two dozen other libraries reported in the literature; this is the most extensive summary I’ve seen on published fragment libraries. The paper also analyzes the results of 14 screens on various targets using saturation transfer difference (STD) NMR. As the researchers note, this technique is prone to false positives, and indeed the average hit rate of 22.5% is high, with only about 50% confirming in secondary assays. There is also a nice analysis of what features are common to hits, along with a list of the 24 compounds that hit in more than 90% of screens.

The other paper on library design, by Tom Peat, Jack Ryan and others (including Pete Kenny), discusses library design at CSIRO. The researchers started with 500 fragments commercially available from Maybridge and supplemented these with roughly the same number of fragments from a collection of small heterocycles that had been synthesized internally; additional “three-dimensional” fragments are also being constructed. At CSIRO the primary screening method appears to be surface plasmon resonance (SPR), in particular the ProteOn instrument that allows simultaneous analysis of six fragments against six targets. Eight of about ten targets have yielded confirmed hits. The researchers show examples of specific (good), nonspecific (probably bad) and ill-behaved (ugly) fragments.

Next up is an excellent discussion of PAINS by Jonathan Baell (at Monash) and collaborators. Although Practical Fragments has covered this topic repeatedly (here, here, here, here, here, and here) it is a sad fact that more examples appear in the literature every day, so there is always something new to write about.

Fragment-finding methods make up the next several papers, starting with a nice overview of native mass spectrometry by Sally-Ann Poulsen at Griffith University. This paper covers theory, practical issues, and recent examples. Roisin McMahon and Jennifer Martin at University of Queensland, along with Martin Scanlon, describe thermal shift assays. In addition to highlighting a number of published examples, the paper also delves into some of the technical challenges and issues with false positives and false negatives, concluding with a nuanced discussion of how to deal with conflicting data.

The subject of conflicting data is central to the work of Olan Dolezal and Tom Peat, both of CSIRO, and their collaborators. They screened the protein trypsin against 500 Maybridge fragments using SPR. Unfortunately they couldn’t go higher than 100 micromolar without running into problems of solubility and aggregation, but even at this relatively low concentration they found 18 hits. X-ray crystallography validated 9 of them, and isothermal titration calorimetry (ITC) also validated 9, with 7 confirmed by all three techniques. (Incidentally, there are lots of great experimental details here.) Four of the SPR hits could not be confirmed by either ITC or X-ray, and 3 turned out to be false positives when repurchased and tested; in one case this appeared to be due to cross-contamination with a more potent compound. In general, the more potent compounds tended to be the ones that reproduced best, and solubility seemed to be a limiting factor for ITC. Despite the imperfect agreement of biophysical techniques, these were still superior to computational approaches on the same target with the same library. As they conclude:

It is gratifying to know (at least for these authors) that experimental data are still of enormous value in the area of fragment-based ligand design and that the modelling community still has a way to go before the experimentalists are put out to pasture.

But experimentalists should not get too cocky: the next paper, by Jamie Simpson and collaborators at Monash University, describes some of the things that can go wrong. An STD NMR screen of the antimicrobial target ketopantoate reductase (KPR) using the same Maybridge library of 500 compounds revealed 196 hits! The 47 with the strongest STD signals were then tested in a ¹H/¹⁵N-HSQC NMR assay, leading to 14 hits, of which 4 gave measurable IC₅₀ values in an enzymatic assay. Unfortunately, follow-up SAR was disappointing, and subsequent experiments revealed that aggregation was to blame: when the biochemical experiments were rerun in the presence of 0.01% Tween-20, only a single fragment gave a measurable IC₅₀ value. The researchers redid their STD-NMR screen in the presence of detergent, resulting in 71 hits, all of which were tested in the biochemical screen. This led to the identification of a new (and fairly potent) hit that had previously been missed. This nicely illustrates the fact that false positives are not just a problem in terms of wasted resources, they can also overwhelm the signal from true positives. The moral? Always use detergent in your assay!

The question of whether structure is needed to prosecute fragments has come up before, and the next paper, by Stephen Headey, Steve Bottomley, and collaborators at Monash University, addresses this question directly. The target protein, a mutant form of α1-antitrypsin called Z-AAT, unfolds and polymerizes in vivo, causing a genetic disease. The researchers used an STD NMR fragment screen of 1137 fragments to identify several hundred hits, and focused on those that bound to the mutant form of the protein rather than the wild-type. They then used a technique called Carr-Purcell-Meiboom-Gill (CPMG) NMR (which relies on line broadening when fragments bind to a protein) to confirm 80 hits, the best of which had a dissociation constant of 330 micromolar. If you’ve stuck through this post thus far you’ll recall that the Monash library was designed for “SAR by catalog”, and 100 analogs of this fragment were purchased and tested, leading to several new hits, one with a dissociation constant of 49 micromolar. Although there is still a long way to go, metastable proteins are tough targets, so this is a nice start.

The next paper, by Ray Norton at Monash University and collaborators, describes a fragment screening cascade against the antimalarial target apical membrane antigen 1 (AMA1). An initial STD NMR assay of 1140 fragments produced 208 hits, but competition experiments with a peptide ligand whittled this number down to 57 that confirmed in both STD and CPMG NMR assays. Of these, 46 confirmed in an SPR assay, and although most are fairly weak, some SAR is starting to emerge as new analogs are synthesized.

Another antimicrobial target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), is the subject of a paper by James Swarbrick at Monash and collaborators. An initial STD NMR screen gave an unnervingly high hit rate (notice any themes emerging?), so 2D ¹⁵N-HMQC experiments were performed on 750 Maybridge fragments, yielding 16 hits. Competition experiments using CPMG NMR and close analyses of the chemical shifts suggested that these fragments bind in the substrate binding site, and SPR confirmed binding for some of the fragments.

Finally, Martin Drysdale of the Beatson Institute highlights some of the success stories of FBDD, including clinical compounds, and ends with a call for shapelier fragments.

All in all this is a great collection of papers, particularly for those relatively new to the field. It will be fun to revisit some of these projects in a few years to see how they’ve progressed.

Undruggable? Pshaw, Fragments can do it.

Bromodomains, as well as other epigenetic targets, are hot right now.  This paper adds to the growing library of fragment success against bromodomains.  As in any nascent field, some of the targets do not have a known biological role.  BAZ2B (bromodomain adjacent to zinc finger domain protein 2B) is one of these.  BAZ2B is interesting compared to the 41 other bromodomains where there is structural information.  Its KAc binding pocket is smaller than other bromodomains (92-105 Angstrom vs 130-220 Angstrom for the other bromodomains and lacks features of BET bromodomains, like the ZA channel and the hydrophobic groove adjacent to the WPF motif.  

BAZ2B (Left), BRD2-BD1 (Right)

Due to these structural differences, the strategies that have been applied successfully to other bromodomains will not transfer to BAZ2B; thus, it considered one of the least druggable bromdomains.  Hence, fragments to the rescue!  

They screened 1300 commercially available (and thus Voldemort Rule compliant) with an alpha screen with hits being defined as 50% activity at 1mM.  10 compounds were identified and confirmed using STD, Waterlogsy, and CPMG NMR experiments (0.8% hit rate).  

(The same library was screened against BRD2-BD1 and CREBBP and had hits rates of 1.8% and 6.1% respectively.)  The ten fragments were then soaked into BAZ2B crystals yielding structures for 1,3,6 and KAc. 

a) KAc, b) 1, c) 3, d) 6

Fragment 6 had poor solubility, so direct ITC was not possible, instead a competition ITC study was used and yielded a 65 uM Kd.  They attempted to optimize this fragment from the 1 position of THgammaC.  All of these modifications resulted in worse affinity.  They then attempted to replace the Chlorine with aryl substituents (based on modeling results).  These compounds showed some improvement in solubility, but no significant improvements in affinity.  They then tried to change the electronic properties of the aromatic substituent.  EWG showed the expected reduction in affinity, but EDG did not show an increase in affinity.  

The most ligand efficient fragments (7, 8, and 10) all contained thioamides, so they synthesized thioamide and thiourea analogs of fragment 6.  Both of these molecules did not bind to the protein. Finally, they attempted to merge 3 and 6 putting the KAc mimetic on the scaffold of 6 . 

This urea containing compound (40) showed improved solubility, and 8-fold reduction in Kd, and a corresponding increase in ligand efficiency. 
This paper is a nice story of using fragments, structural biology, and modeling to generate useful compounds as tools.  I think it also points to "druggability" being a useless term when it comes to fragments.  Archimedes may have wanted a lever long enough, but for me, I just want fragments diverse enough.

Fragments vs hematopoietic prostaglandin D2 synthase

Prostaglandins are modified fatty acids involved in myriad biological processes. The enzyme hematopoietic prostaglandin D2 synthase (H-PGDS) converts prostaglandin H2 to prostaglandin D2 and is a potential target for inflammatory disorders. In an article just published online in MedChemComm, Gordon Saxty and co-workers at Astex and collaborators at GlaxoSmithKline describe how they used fragment-based methods to develop orally available inhibitors of this enzyme.

The researchers started by screening their fragment library against crystals of H-PGDS, resulting in 76 fragment hits, some of which were quite potent (sub-micromolar). Two are described in the paper, with most of the focus being on Fragment 6, which wasn’t the most potent but did produce an interesting conformational shift in the protein. Also, although H-PGDS typically binds lipophilic molecules, the researchers were intrigued to observe that the polar pyrazole moiety made two hydrogen bonds to the protein.

Structure-guided optimization of Fragment 6 led to Fragment 8 with a modest improvement in affinity, and fragment growing led to Compound 9, with a satisfying 400-fold boost in affinity. In one of those “nice to have” problems, this compound was actually too hydrophilic (ClogP < 1), but increasing the lipophilicity slightly led to Compound 10 (AT24111 / GSK2696124A), which has low nanomolar potency and oral bioavailability in both mice and rats. The compound also blocked prostaglandin D2 production in mice when dosed orally.

This is a concise but elegant paper, and it is impressive that the researchers managed to maintain or improve ligand efficiency throughout optimization. Of course, all the molecules contain a pyrazole moiety, which is a privileged pharmacophore for kinases, so it will be important to carefully assess selectivity.

Finally, you may recall that Astex was acquired by Otsuka earlier this year. Whenever an acquisition happens there is always the worry that the acquired company will be decimated or shuttered entirely. Happily, this doesn’t seem to be the case here. In fact, Astex actually seems to be expanding: I recently saw a full page job advertisement from them in Nature, and as of this morning their website lists 9 openings, with several in research. Hopefully the honeymoon lasts a long time!

Fragments against PPI Hot Spots

Protein-Protein interactions are important to so many physiological processes.  There is mounting literature examples of utilization of fragments to block PPIs.  In this paper, Rouhana et al. show how they approached the PPI of Arno and ARF1, ADP-ribosylation factor (part of the RAS superfamily). Arno is part of the brefeldin A-resistant GEFs and share a 200 amino acid domain called SEC7.  SEC7 interacts with ARF through insertion of ARF switch regions into hydrophobic regions of SEC7.  This interaction is interesting from a ligand design standpoint is very interesting because it does not involved an alpha-helix inserting into the partner's hydrophobic groove.  Rather SEC7 has a rather large interface denoted by "hot spots". 

The figure shows their "innovative" FBDD strategy.  First, a Voldemort Rule compliant library was screened in silico.  Since in silico screening is not typically used for fragment screening (but becoming more common) they imposed some initial rules: docking site is small (1-2 residues!), hot spots defined by interaction energy (>1kcal/mol from alanine scan), and very strict selection criteria.  3000 fragments from the Chembridge library were screened.  33 molecules were selected and 40 random fragments chosen as negative controls. 

This was followed by a fluorescence assay (2mM fragments) to test their computational results, just as I say you should do.  Promiscuous binders were removed, not by using detergent, but using protein polarization to directly detect interaction with the target.  This seems like over-complexation of an assay, but without knowing the details of system there may be a very good reason for this approach. 

Compounds 1-4 were identifed as inhibitors (35%, 16%, 38%, and 23% inhibition at 2mM respectively) from each of the "hot spots".  I think it is interesting that these compounds were predicted to have affinities of 10uM or better from the docking.  To me, that just illustrates that predicted affinites are rediculous.  Why do people even report them?  Compound 1 had a Kiapp of 3.7mM which is a LEAN of 0.12!  These were then compared to the PAINS list and 3 is "ambiguous".  Compounds 5 and 6 were chosen as negative controls.  SPR confirmed the binding of 1,2, and 4, but at less than stoichiometric binding levels (the assay was run at 250uM).  3 could not be confirmed as a binder.  Does this mean anything for ambiguous PAINS? 

STD NMR was then used to confirm binding.  In a nice departure, they actually talk about conditions they used: 10 and 30uM ARNO with 0.1mM and 1mM compounds at 32 and 12C.  30uM ARNO with 1mM fragments @12C was what worked (33x fold excess fragments). Confirming the SPR, compounds 1, 2, and 4 were shown to bind, while "ambiguous" 3 had some binding. Finally, compounds were soaked with fragments 1, 2 and 4.  This led to crystal structures which could then be used for more model building, compound design, etc.  This led to the following compound (1.61mM KiApp, LEAN = 0.13) (the methoxy derivative of 1) for further analysis:

By and large, this is a well done, thoughtful work.  They really understand how to setup and interpret STD-NMR. However, these compounds are really atom inefficient.  Is that a consequence of the type of interaction they are inhibiting?  As a fragment, there is nothing wrong with it. 

[Quibble: The authors claim that this is an innovative approach, but I am not seeing it.  They claim their in silico screen first then following up by biophysical techniques is the innovation. ] 

Supplemental Information here.