05 October 2015

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. 

01 October 2015

Aggregation alert

Practical Fragments has quite a few posts about PAINS, or pan-assay interference compounds. In part this reflects their sad prevalence in the literature, but it’s also fair to say that they are easy targets because many are readily recognizable.

But not all artifacts are so easily spotted, as discussed in a new paper just published in J. Med. Chem. by John Irwin, Brian Shoichet, and colleagues at the University of California San Francisco (see also here for Derek Lowe's excellent summary).

The researchers took on one of the most insidious problems, compound aggregation, in which small molecules form colloids that bind to and partially denature proteins, causing false positives in all sorts of assays. This can happen even at nanomolar concentrations of compound, and is all the more problematic at higher concentrations used in fragment screening and early hit to lead optimization. In many cases aggregates can be disrupted or passivated by including nonionic detergents such as Triton X-100 or Tween-80, but not all assays tolerate detergent, and some aggregates form even in the presence of detergent.

Worse, all sorts of molecules can form aggregates, including many approved drugs. Previous attempts to try to predict which molecules will aggregate have not been very successful. Colloid formation is essentially a phase transition, and like other such transitions (crystallization, for example) it is fiendishly difficult to predict what molecules will do this under what conditions. But if we can’t predict from first principles which molecules will form aggregates, can we at least draw empirical lessons?

The researchers assembled a set of >12,600 known aggregators and put together a very simple model that assesses how similar a molecule of interest is to one of these aggregators (using Tanimoto coefficients, or Tcs). Aggregators have a wide range of physicochemical properties, with ClogP values from -5.3 to 9.8, but 80% have ClogP> 3.0. The team hypothesized that a molecule sufficiently similar to a known aggregator – and also somewhat lipophilic – would have a higher probability of being an aggregator than a molecule chosen at random.

To test this idea, the team took  a batch of 40 molecules and tested them for aggregation. Among those most similar to known aggregators (Tc ≥95%), 5 of 7 molecules were confirmed as aggregators. This fell to 10 of 19 for the next set (Tc 90-94%), 3 of 7 after that (Tcs 85-89%) and only 1 of 7 for the least similar (Tcs 80-84%). Thus,Tc ≥85% was chosen as the cutoff.

Next, the researchers examined molecules that had been reported as active in some sort of biological assay, and found that 7% were ≥85% similar to a known aggregator and had ClogP> 3. Ominously, this rate is an order of magnitude greater than the number of commercially available compounds that also fit these criteria. More damning, most of this enrichment has occurred since 1995, when high-throughput and virtual screening really went mainstream. In other words, the past couple decades have seen a sizable enrichment of potential aggregators in the literature.

All of this is fascinating, but what really makes this paper significant is that the researchers have made all their primary data available, and also built a simple to use website called “Aggregator Advisor”. Just draw your molecule or paste a SMILES string to generate a report. For example, entering gossypol tells you that this molecule has previously been reported as an aggregator. (With two catechol moieties, it’s also a PAINS.) Perhaps not coincidentally, it shows up in more than 1800 publications.

Of course, as the researchers note, “just because a molecule aggregates, under some conditions, in the same concentration range as it is active, does not establish that its activity is artefactual.” Indeed, 3.6% of FDA-approved drugs are known aggregators. Still, particularly if your hit has only modest activity (0.1 µM or worse), similarity to a known aggregator should at least make you cautious.

The researchers are at pains to emphasize that their model is “primitive and subject to false negatives and false positives.” Thus, any hits need to be tested to see if they behave pathologically in any given assay. More importantly, a molecule that comes up as a negative should not be presumed to be innocent.

All these caveats aside, Aggregator Advisor is very easy to use. It’s certainly worth running the next time you find an interesting molecule – whether in your lab or in the literature – particularly if there was no detergent in the assay.

28 September 2015

NMR poll!

Among fragment-finding techniques, nuclear magnetic resonance (NMR) ranks near the top. Protein-detected methods, like HSQC/HMQC-based SAR by NMR, helped usher in fragment-based drug discovery as a practical endeavor. More recently, ligand-detected methods such as line broadening (or CPMG), STD, and WaterLOGSY appear to have gained the edge. There are also more boutique methods, such as ILOE and spin labeling. And of course, some people proudly embrace their fluorine fetishism.

So what’s your favorite flavor? Now's your chance to weigh in on our latest poll (on the right). The first question asks whether you use NMR, and the second asks which methods you use. PLEASE ANSWER BOTH QUESTIONS - the free version of Polldaddy doesn't track individuals, so we need the answer to the first question to know the total number of respondents.

And, as always, your comments are welcome.

22 September 2015

Crystallography as a primary screen: the case of HisRS

X-ray crystallography plays a starring role in fragment-based lead discovery. But, with a few exceptions, it is rarely the primary screen. One of these exceptions was reported recently in Acta Crystallogr. D by Wim Hol and coworkers at the University of Washington, Seattle.

The researchers were interested in the histidyl-tRNA synthetase (HisRS) from Trypanosoma cruzi, the parasite that causes Chagas disease. They had previously constructed a library of 680 fragments in pools of 10, designed such that the fragments within each pool had different shapes to facilitate crystallographic screening. Crystals of HisRS•His (ie, the enzyme in complex with its histidine substrate) were soaked with the pools, such that the final concentration of each fragment was 1.5 mM. Fifteen of these pools showed new electron density, and these in turn yielded 15 different fragment hits when the pools were deconvoluted. Two additional fragments from the deconvolution process showed weak density, though these were not further pursued.

Strikingly, all 15 fragments bound to the same site, a site not observed in the absence of the fragments. This is a narrow groove described by the researchers as a “document sleeve,” and in fact all the hits are single six-membered aromatics or double aromatics with few or no aliphatic substituents. Most of the fragments are also quite small, with the majority having just 8 or 9 non-hydrogen atoms. Although all the fragments bind in roughly the same plane, there is considerable variation in the positions of substituents, and some of the fragments appear to bind in multiple orientations.

Next, the researchers tested their hits in orthogonal assays. Only one fragment showed thermal stabilization of the enzyme, and only three showed any inhibition in a functional assay (at most 39% at 2 mM of fragment). Thus, these are very weak binders.

The fragment-binding pocket is a few Ångstroms away from the histidine substrate, making linking the two ligands feasible. In preparation for this step, the researchers acetylated the amino group of the most potent fragment. This caused little change in the functional activity, but crystallography revealed that the fragment’s binding orientation had flipped around, such that the acetamide group pointed away from the histidine and towards a cysteine residue. Attempting to turn lemons into lemonade, the researchers added electrophiles to try to interact with the cysteine residue. Some of these molecules had measurable IC50 values (on the order of 1 mM), and crystallography of one of these showed covalent bond formation between the fragment and the targeted cysteine. This cysteine residue is found in the T. cruzi enzyme but not the human version, and indeed these molecules appear to be more active against the parasitic HisRS.

This is a nice example of fragment screening by crystallography that illustrates one of its main challenges: crystallography is capable of detecting extremely weak binders that may prove difficult to advance. Still, the researchers have taken some promising initial steps, and it will be fun to see what they come up with.

21 September 2015

Monday Morning Non-Blogging

I know you all come here looking for pithy breakdowns of recent publications in the fragment world.  Today Dan and I are both in Boston at CHI's Discovery on Target conference.  We are teaching our fragment course today, which is really focused on PPIs and Biophysics for this crowd. So, blogging will be limited this week (maybe). 

16 September 2015

Fragments vs ERK2

Extracellular-regulated kinase 2 (ERK2) is one of just two known substrates of the kinases MEK1 and MEK2, themselves the subjects of considerable clinical efforts to treat cancer. In a paper just published online in Bioorg. Med. Chem. Lett., Daniel Burdick and colleagues at Genentech describe how they have used FBLD to tackle ERK2.

A library of just 635 fragments was screened against the protein using STD NMR, yielding 54 hits, and SPR, yielding 78 hits. Thirteen of these came up in both assays, and compound 1 had the second-highest ligand efficiency. Not surprisingly, X-ray crystallography revealed that this purine binds to the hinge region of the kinase. The electron density also showed something else binding nearby, which the researchers interpreted as an imidazole molecule left over from the protein purification. Thus, they set out to grow their fragment in this direction.

The purine moiety of compound 1 was not well-suited for growing towards the imidazole, and purines have also been picked over extensively by numerous groups, so the researchers used scaffold-hopping to develop compound 3. This turned out to have acceptable affinity and dramatically improved ligand efficiency. Growing led to compound 14, and structural characterization of a related molecule confirmed that the added heterocycle bound in the same region as the originally observed imidazole.

Next, the researchers grew in a different direction, ultimately leading to compound 39, with low nanomolar potency. Although no cell activity or selectivity data are reported, the authors note that the series underwent further optimization that will be reported in future.

This is a nice, concise description of fragment-based lead discovery and optimization that incorporates multiple biophysical methods, structure-based drug design and modeling, and creative medicinal chemistry. It is not clear whether targeting ERK2 has advantages over MEK or RAF, but work like this is precisely what is needed to generate chemical probes to answer this question.

14 September 2015

Is this still a thing? And why?

As regular readers here know, we often discuss metrics because everyone uses them.  Last year, agent provcateur Pete Kenny unleashed a broadside against those who defended metrics.  This seems to be  like the corpse flower that blooms once a year, stinks the place up, yet everyone runs to go see it.  Well, recently Pete posted in the LI group: "Ligand efficiency validated fragment-based design?"and asked whether or not people agreed with the statement.  This of course has inspired a wave of comments.  I disagreed with the statement, but not for the "metrics suck" argument.  I strongly urge people to go read the thread, unless of course you have something better to do.  

To me, this is not about the validity of metrics.  [Let me add here, that I prefer the "LEAN" metric (pIC50/HAC) because it can be done in your head on the fly.]  I think people have a good understanding of what they do, their limitations, and their strengths.  I disagreed with the statement because of the use of the word "validated".  In the development world, we talk about our assays very specifically: they are qualified or validated.  A validated assay is one that has been shown to be accurate, specific, reproducible, and rugged for the analyte in the concentration range to be measured.  Put plainly, this means that if you expect to measure analyte X at 5 uM, you have to show that for all samples it will be measured in you can identify it, measure 5 uM accurately, and do it every time.  That's a validated measurement.  When you are qualifying an assay, the bar is much lower.  An assay is considered qualified if it has been demonstrated to be "fit for purpose".  Fit for purpose means that it will do the job, but you haven't beat the sugar out of it to make sure it is "valid".  To me, ligand efficiency is fit for purpose of driving medchem decisions; it is qualified for that purpose, but not validated (N.B. I am not saying "not valid".) 

08 September 2015

Dry solutions for crystallography

To some, X-ray crystallography may be a rather dry topic. However, the process generally entails lots of liquids. In particular, the commonly used practice of crystal soaking entails transferring protein crystals to a new solution containing dissolved ligands, which is both tedious and can cause crystals to shatter or dissolve. A new paper by Jean-Francois Guiçhou at Université de Montpellier and collaborators in Acta Cryst. D aims to streamline the process, and so lower barriers for obtaining structural information that could guide drug design.

Rather than manually transferring crystals to new solutions, the researchers pre-coated crystallization plates with ligands and then grew protein crystals in them. They first dissolved the ligands, transferred these to the wells, and allowed the solvent to evaporate. Although they tested a variety of solvents, including acetone, tetrahydrofuran, ethanol, acetonitrile, 2-propanol, water, and DMSO, only the last two proved suitable; most of the rest wicked up the well, spreading over too large of a surface (though methanol has been used by Beryllium, née Emerald). DMSO is, of course, the most commonly used solvent for storing small molecules, and so should work for most ligands. DMSO is not very volatile, but only 1 µl was used per well, and putting the plates in a fume hood for a week left behind dry ligand.

To make things easier still, the researchers used special crystallization plates that could be put directly into an X-ray beam (in situ crystallography), further diminishing the amount of manipulation required. The technique was tested against four different proteins: the old standard hen egg-white lysozyme and the drug targets cyclophilin D, PPARγ, and Erk-2.

For lysozyme, the water-soluble fragment benzamidine was used, and the resulting structures showed the fragment binding in a similar manner as previously described. So did structures of PPARγ bound with the high affinity ligand rosiglitazone. Cyclophilin, though, was not as successful: of nine fragments attempted, only one produced a structure. In contrast, three fragments produced structures using conventional approaches. ‘Dry’ crystallization was more successful with two more potent (micromolar or better) cyclophilin ligands. Interestingly, dry crystallization succeeded with one ligand that had previously been characterized only by co-crystallization; even week-long soaking experiments had not worked.

Finally, Erk-2 was screened against 14 ligands designed as hinge-binders with low solubility in water. Crystals were obtained with five of the ligands, and four were large enough to generate good-quality structures.

Overall this seems like a convenient approach, though it does seem prone to false negatives. What do the crystallographers out there think – is this a practical solution?

03 September 2015

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.

01 September 2015

Polypharmacology for Kinases

We've been a roll with epigenetics and PPIs lately.  So, its a nice break when a kinase paper comes out.  But, in keeping with the theme of hard targets, today's paper is about a tyrosine kinase.  We've started to see more and more FBDD on TKs.  One problem is that TKs can acquire resistance to drugs, quickly eliminating their therapeutic usefulness.  One way around this is to use polypharmacology: "optimized inhibitory profiles for critical disease-promoting kinases, including crucial mutant targets."  In this work, they are targeting RET and VEGFR2 dual inhibitor using a in silico/fragment approach.  

Compound design was largely based upon homology modeling the "DFG-out" RET structure utilizing the VEGFR2 structure as a template.  Their Kinase Directed Fragments (KDF) are shown in Figure 1.
Figure 1.
Their fragment design rationale makes some interesting comments.  They state that a "hinge binding" fragment alone can aggregate at high concentrations needed to achieve activity in a biochemical screen.  So, their fragments have an additional moiety that interact with the lipophilic or ribose pocket.  
Accordingly, KDFs have larger molecular weights and are generally more active than the fragments contained in traditional libraries, permitting screening in the micromolar range.
I would say the first statement is conjecture and the second untrue.  17 heavy atoms is squarely in the regime of what people consider "fragment" sized.  I think instead the authors are using the wrong tool for the job.  Using a biochemical screen to find fragment actives is akin to hammering a nail with a screwdriver.  Sure, you can do it, but why would you?  

Rather expectedly, they identified compound 1 as a promising starting platform.  Of course, the criteria for selecting this compound are kept highly secret.  It did "effectively" inhibit RET at 100 (63%) and 20 uM (28%) in the presence of 190 uM ATP [Km for RET 12uM].  It had VEGFR2 activity of 59% at 100 uM. 
Add caption
Modeling allowed them to generate the compounds showed in Figure 2.
Figure 2. 
Pz-1 had activity less than 1 nM against RET, RET(V804M/L)[a gatekeeper mutant],  and VEGFR2.  This equipotency was also demonstarted in cell-based assays.  Against a panel of 91 other kinases at 50 nM, Pz-1 had significant activity against 7 others (TRKB, TRKC, GKA, FYN, SRC, TAK1, MUSK).  So, in the end using primarily modeling and a biochemical assay they were able to generate a polypharmacological TK inhibitor.  I leave it to those more well versed in the biology whether or not those 7 other kinases pose a potential problem.  I however would argue that they generated an agent with polypharmacology against 9 kinases not 2. 

24 August 2015

Fragment-Based Drug Discovery

This is the straight-to-the-point title of a new book published by the Royal Society of Chemistry, edited by Steven Howard (Astex) and Chris Abell (University of Cambridge). It is the second book on the topic published so far this year, and it is a testimony to the fecundity of the field that the two volumes have very little overlap.

After a brief forward by Harren Jhoti (Astex) and a preface by the editors, the book opens with two personal essays. The first, by me, is something of an apologia for Practical Fragments and the growing role of social media in science (and vice versa). If you’ve ever wondered how this blog got started or why it keeps going, this is where to find out. The second essay is by Martin Drysdale (Beatson Institute). Martin is a long-time practitioner of FBDD, dating back to his early days at Vernalis (when it was RiboTargets) and he tells a fun tale of “adventures and experiences.”

Chapter 1, by Chris Abell and Claudio Dagostin, is entitled “Different Flavours of Fragments.” With a broad overview of the field it makes a good introduction to the book. There are sections on fragment identification, including the idea of a screening cascade, as well as several case studies, some of which we’ve covered on Practical Fragments, including pantothenate synthetase, CYPs, RAD51, and riboswitches.

The next two chapters deal with two of the key fragment-finding methods. Chapter 2, by Tony Giannetti and collaborators at Genentech, GlaxoSmithKline, and SensiQ, covers surface plasmon resonance (SPR). This includes an extensive discussion of data processing and analysis, which is critical for improving the efficiency of the technique. Competition studies are also described, as are advances in hardware, notably those from SensiQ. This is a good complement to Tony's 2011 chapter.

Chapter 3, by Isabelle Krimm (Université de Lyon), provides a thorough description of NMR methods, both ligand-based (STD, WaterLOGSY, ILOE, etc) and protein-based (mostly HSQC). The chapter does a nice job of describing techniques in terms a non-specialist can understand while also providing practical tips on matters such as optimal protein size and concentration.

Chapter 4, by Ian Wall and colleagues at GlaxoSmithKline, provides an overview of FBLD from the viewpoint of computational chemists. The chapter includes some interesting tidbits, such as the observation that fragment hits that yield crystal structures tend to be less lipophilic but also contain a smaller fraction of sp3 atoms and more aromatic rings. The researchers note that the current fashion for “3D” fragments is yet to be experimentally validated. They also include accessible sections on modeling, druggability, and integrating fragment information into a broader medicinal chemistry program.

The remaining chapters focus on specific types of targets. Chapter 5, by Miles Congreve and Robert Cooke (both at Heptares) is devoted to G protein-coupled receptors (GPCRs). This includes descriptions of how to screen fragments against these membrane proteins using SPR, TINS, CE, thermal melts, and competition binding. It also includes a detailed case study of their β1 adrenergic receptor work (summarized here). Congreve and Cooke assert that, although many of the GPCR targets screened to date have been highly ligandable, technical challenges only now being addressed have caused this area of research to lag about a decade behind other targets. They predict a bright future.

Rod Hubbard (Vernalis and University of York) turns to protein-protein interactions in Chapter 6. After describing why these tend to be more challenging than most enzymes and covering some of the methods for finding and advancing fragments, he then presents several case studies, including FKBP (one of the first targets screened using SAR by NMR), Bcl-2 family members (including Bcl-xL and Mcl-1), Ras, and BRCA2/RAD51. He concludes with a nice section on “general lessons,” which boils down to “patience, pragmatism, and integration.” As Teddy recently noted, this can lead to substantial rewards.

Allosteric ligands have potential advantages in terms of selectivity and addressing otherwise challenging targets, and in Chapter 7 Steven Howard (Astex) describes how fragments can play a role here. This includes how to establish functionality of putative allosteric binders, as well as case studies such as HIV-1 RT, FPPS, and HCV NS3. Astex researchers have recently stated that they find on average more than two ligand binding sites per protein, and this chapter includes a table listing these (including 5 binding sites each on bPKA-PKB and PKM2).

The longest chapter, by Christina Spry (Australian National University) and Anthony Coyne (University of Cambridge) describes fragment-based discovery of antibacterial compounds. After discussing some of the challenges, the authors report several in depth case studies including DNA gyrase, DNA ligase, CTX-M, AmpC, CYP121, and pantothenate synthetase, among others. At least one fragment-derived antibacterial agent entered the clinic; hopefully more will follow.

Chapter 9, by Iwan de Esch and colleagues at VU University Amsterdam, focuses on acetylcholine-binding proteins (AChBPs), both as surrogates for membrane-bound acetylcholine receptors and as well-behaved model proteins on which to hone techniques (see for example here, here, and here). Since AChBPs have evolved to bind fragment-sized acetylcholine, these proteins can bind tightly to small ligands; 14-atom epibatidine binds with picomolar affinity, for example, with a ligand efficiency approaching 1 kcal mol-1 atom-1.

And Chapter 10, by Chun-wa Chung and Paul Bamborough at GlaxoSmithKline, concisely covers epigenetics. Bromodomains are well-represented, including a table of ten examples (see for example here, here, here, here, here, and here). Happily, although some of these projects started from similar or identical fragments, the final molecules are quite divergent. However, the authors note that much less has been published on histone-modifying enzymes, such as demethylases and deacetylases, perhaps reflecting the challenges of achieving specificity with what are often metalloenzymes.

Finally, this is the 500th post since Teddy founded Practical Fragments way back in the summer of 2008. Thanks for reading, and special thanks for commenting!

19 August 2015

Caveat Emptor...or marketing does not always tell you whats really in the package.

In case you missed it, I spoke at the ACS on Sunday.  It was in a computational session looking at designing libraries and I am pretty sure I was the only non compchemist.  It was about all the problem compchemists have caused in library design.  My talk was even live tweeted by Ash (@curiouswavefn) and was well received.  So, looking at the next paper in my queue, its a computational-focused paper.  So, After spending several hours on a Sunday listening to compchemists, have I softened?  

This paper is the subject of today's post.  It is an extension of this paper which describes their virtual screen.  From a 2 million compound virtual screen, they tested 17 compounds in vitro leading to 2 micromolar compounds.  This paper is the story of the most potent of the two micromolar compounds.  The target is CREBBP, which is another in the long line of epigenetic targets.  Compound A was one of the original in silico actives that was tested.  Three analogs were obtained and tested (B-D).
Figure 1.  Original active, A.  Analogs B-D.  Common structural motif is shown in blue.
Compound B was the most potent and become the focus of their optimization efforts. Of course, my eyes are drawn to that potential michael acceptor, but the authors dismiss it based upon their docking results: the only alkylatable residue in the area of its putative binding is well buried.  It is a moot point anyway because they were able to replace it with a isopthalate group and increase potency by 5x, 0.9uM (Compound 6).  Interestingly,the potency of 6 is different depending on the assay used: 0.8 um in a competition binding assay and 8.7uM in a TR-FRET assay.
Figure 2.  Compound 6
This compound was crystallized and showed that the predicted binding mode was correct. 

They then performed some gobbledy-gook MD calculations (finite-difference Poisson, warning PDF) in order to evaluate the electrostatic contribution of the polar contribution to binding of 6 and 7.  Compound 6 had more favorable electrostatic interactions (0.8 kcal/mol) than 7, which had more favorable van der Waals interactions (1.4 kcal/mol).  With this crucial information AND the crystal structure in hand, they then explored additional chemical space.  

Despite the authors' claim, I don't think they actually improved the potency significantly.  Compound 6 is 8 uM in the TR-FRET assay and the best compounds they claim are 1 or 2 uM.  I really have to call monkeyshines here.  They use the different assays interchangeably, yet never explain the one is used for what purpose.  Its cherry picking values.  When talking about selectivity, they switch to using thermal shift values.  And we all know the value of that.  So, I find it hard to believe their "most potent" this or "selectivity" that. The title of the paper includes "nanomolar", but that is only in one assay.  That's like saying I can run a 6 minute mile, since I did it once under optimum conditions.  Honestly, my typical times (WAY back when) were more 8:30 miles.  That honesty in data reporting.  Since they obviously had access to different assays, why weren't all compounds run in one, or optimally both.  I don't see that the MD calculations had any positive impact.  Maybe its the heat, but this paper is a not a sham, but definitely full of deceptive advertising.

17 August 2015

Fragments vs IAPs: resisting the affinity Siren

In 2011, Mike Hann decried “addiction to potency”. Indeed, newcomers to fragment-based methods often have to undergo a psychological shift to work with low affinity binders. But, as we asked last year, how weak is too weak? In a paper just published in J. Med. Chem., Gianni Chessari and colleagues at Astex may have set a new bar.

The researchers wanted to develop leads against inhibitor of apoptosis proteins (IAPs). The BIR3 domains of proteins such as cIAP1 and XIAP bind to and block the action of caspases and other proteins, allowing cancer cells to survive. Several groups have developed molecules that block these protein-protein interactions, usually by starting with an endogenous peptide inhibitor. However, most of these bind preferentially to cIAP1, and some evidence suggests that a balanced antagonist may have advantages.

The BIR3 domain is quite small, just 11.8 kDa, making standard ligand-observed NMR screening difficult. Instead, the researchers looked at line broadening and chemical shift changes of protein protons with δ < 0.4 ppm or 9.8 – 10.4 ppm on addition of fragments. Anticipating very weak binders, they used 0.2 mM protein and 10 mM fragments (in mixtures of two). A total of 1151 fragments were screened, of which 100 had been computationally preselected.

Among the best hits were those containing an alanine residue; one of these had mid-micromolar affinity and reasonable ligand efficiency. However, these bound preferentially to the BIR3 domain of cIAP1, in common with other reported inhibitors that also contain an alanine. In contrast, compound 1 appeared to have more balanced activity. Its affinity was risibly weak, but crystal soaking led to interpretable electron density, and also suggested that adding a suitably positioned methyl group could fill the small hydrophobic pocket normally occupied by the alanine side chain. The resulting compound 5 showed measurable – and balanced – activity against both proteins.

Next, a small virtual library was constructed in which the pyrrolidine was replaced with substituents to both improve affinity and create a scaffold for reaching into the P4 pocket. Thirty compounds were made, but disappointingly none of these had significantly improved affinity. Undeterred, the researchers obtained crystal structures of some of them bound to XIAP-BIR3 and performed careful modeling. The phenyl ring of compound 7 binds in a region of the protein with an electronegative potential, and by simply adding electron withdrawing substituents the affinity could be improved by >50-fold. Further growth ultimately led to compound 21, with nanomolar potency against both XIAP and cIAP1, though with a preference for the latter. This compound also showed on-target activity in cell-based assays as well as activity in mouse xenograft models.
It would have been easy to overlook compound 1; indeed, it took rather strenuous efforts to find it. Yet, comparing the structures of compound 1 and 21 bound to XIAP-BIR3 reveals that the initial fragment maintains its position and binding interactions in the elaborated molecule. This is a clear example that, with persistence and creativity, it is possible to advance even the weakest of fragments. The researchers note in the conclusion (and have reported at conferences) that they were able to optimize this series to low nanomolar inhibitors against both targets. Whether or not this leads to a drug, it does look like another candidate for a useful chemical probe.

12 August 2015

Silver Ain't Bad

For those of you who have been reading this blog for a while, you are familiar with the "Fragments in the Clinic" posts, Jan2015, Jan2013, and Jan2010.  Two of  those slowly making its way through the pipeline is navitoclax, ABT-263, and venetoclax, ABT-199.  Today Abbvie and Genentech announced that it had met its end point in phase II trials.  The companies plan to file for approval by the end of this year.  At that point we will then have TWO compounds approved from fragments.  Congratulations to all of those folks who have worked on this over the years!!!

10 August 2015

Fragments vs PAK1 – allosterically

Some of our recent posts have discussed the use of fragment-based approaches to discover selective new chemical probes. Continuing this theme, a paper from Alexei Karpov and colleagues at Novartis in ACS Med. Chem. Lett. describes a success story against the kinase PAK1.

The six p21-activated kinases (PAKs) have been implicated in a variety of indications, from cancer to neurodegenerative diseases. Unfortunately, most reported inhibitors are not sufficiently selective to elucidate the biology. The researchers were particularly interested in PAK1, against which they performed a fragment screen (no details of methods or libraries are provided). One of the more interesting hits was fragment 1. Although a bit chunky (24 heavy atoms, MW = 328 Da) with only modest ligand efficiency, it is structurally unusual for a kinase binder.

In fact, crystallography revealed that the fragment binds not in the active site at all, but rather in an allosteric site adjacent to the ATP-binding site, with the so-called DFG-loop in the “out” conformation. Not surprisingly, this molecule was more active against the inactive form of the enzyme. Despite its binding mode, it did appear to be ATP-competitive, perhaps because the DFG-out conformation of the protein is not able to bind ATP.

Aficionados of GPCRs will not be surprised to learn that fragment 1 – a dibenzodiazepine – bound several with high affinity, but the researchers used structural information both to ablate this off-target activity as well as improve affinity for PAK1, resulting ultimately in compound 3. This molecule was completely selective for PAK1 when tested at 10 µM concentration against a panel of 442 kinases, as well as against a panel of 22 other potential off-targets. Unexpectedly, it was even >50-fold selective against the closely related PAK2. It also displayed good permeability and acceptable solubility, though the stability could be improved.

Compound 3 blocked PAK1 autophosphorylation in cells, but was only modestly effective at blocking proliferation of a pancreatic cancer cell line with high levels of PAK1. As it turns out, all known cancer cell lines that are dependent on PAK1 also seem to use PAK2, so perhaps this chemical probe is too selective. Nevertheless, it will be useful to help disentangle the overlapping roles of these two kinases.

More broadly, this is a nice illustration of the selectivity achievable with allosteric kinase inhibitors. Indeed, as we recently noted in a comment to a post earlier this year, Novartis has put at least one allosteric kinase inhibitor into the clinic.

05 August 2015

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? 

03 August 2015

Fragments and HTS vs BCATm

One of the themes throughout this blog is that fragments are useful not just in and of themselves, but as part of a broader tool kit, what Mark Whittaker referred to as fragment-assisted drug discovery, or FADD. A nice example of this has just been published in J. Med. Chem. by Sophie Bertrand and colleagues at GlaxoSmithKline and the University of Strathclyde.

The researchers were interested in mitochondrial branched-chain aminotransferase (BCATm), an enzyme that transforms leucine, isoleucine, and valine into their corresponding α-keto acids. Knockout mouse studies had suggested that this might be an attractive target for obesity and dyslipidemia, but there’s nothing like a chemical probe to really (in)validate a target. To find one, the researchers performed both fragment and high-throughput screens (HTS).

The full results from the fragment screen have not yet been published, but the current paper notes that the researchers screened 1056 fragments using biochemical, STD-NMR, and thermal shift assays. Compound 1 came up as a hit in all three assays, and despite modest potency and ligand efficiency, it did have impressive LLEAT. The researchers were unable to determine a crystal structure of this fragment bound to the protein, but STD-NMR screens of related fragments yielded very similar hits that could be successfully soaked into crystals of BCATm.

The HTS also produced hits, notably compound 4, which is clearly similar to compound 1. In addition to its increased biochemical potency, it also displayed good cell activity. Moreover, a crystal structure revealed that the bromobenzyl substituent bound in an induced pocket that did not appear in the structure with the fragment, or indeed in any other structures of BCATm.

The researchers merged the fragment hits with the HTS hits to get molecules such as compound 7, with a satisfying boost in potency. Interestingly, the fragment-derived core consistently gave a roughly 10-fold boost in potency compared to the triazolo compounds from HTS. Comparison of crystal structures suggested that this was due to the displacement of a high-energy water molecule by the nitrile.

Extensive SAR studies revealed that the propyl group could be extended slightly but most other changes at that position were deleterious. The bromobenzyl substituent was more tolerant of substitutions, including an aliphatic replacement, though this abolished cell activity. Compound 61 turned out to be among the best molecules in terms of potency and pharmaceutical properties, including an impressive 100% oral bioavailability and a 9.2 hour half-life in mice. Moreover, this compound led to higher levels of leucine, isoleucine, and valine when mice were fed these amino acids.

This is a lovely case study of using information from a variety of sources to enable medicinal chemistry. Like other examples of FADD, one could argue as to whether the final molecule would have been discovered without the fragment information, but it probably at least accelerated the process. More importantly, molecules such as compound 61 will help to answer the question of whether BCATm will be a viable drug target. 

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.