Get to Know Your Compounds

One of the first fragment screens I was ever involved with had RNA as the target (this is back when people did anti-bacterial research).  Because of that, I always try to write about targets, not proteins when referring to generic things we wish to find ligands for.  Nucleic acids have secondary and tertiary structure, just like proteins, and thus have ligandable pockets.  We have covered RNA as a target previously.  Well, we get to add another paper to the list.

In this paper from researchers at Goethe-Universität Frankfurt am Main present their results on HIV Tat-TAR.  This target was discussed over three years ago.  I am not particularly impressed with the compounds or the work (even though it included RNA NMR).  I was more impressed with comment made in the paper which hint at the kind of compound understanding we often cite as lacking from academic papers.

First, let's get to the guts of the paper.  They have been trying to identify ligands to this target for years, yielding nanomolar affinities but limited specificity.  Moving to fragments when all else fails, they decided to utilize very simple fragments: benzenes and amines, amidines, or guanidines able to be protonated at physiological pH.  Their fragments were screened in a fluorescent Tat-TAR-peptide assay.   Figure 1 shows the compounds tested.

Figure 1.  Compounds tested.  IC50s shown in parentheses. 

Cpds 1-6 were inactive, but cpd 7 looked promising...at first glance.  As the authors state:

"However, the IC50 value of this compound improves steadily when aqueous solutions are kept under air.  This effect was also found with other compounds, for example tetraaminoquinazoline 23.  The electron rich heterocycles in particular have the tendency to produce false positive results, presumably by forming positively charged oligomers."

I presume that they ran the assay and a few days later went back to re-test the compounds and saw anomolous results.  What really strikes me is that with a 7 mM IC50 upon retesting they saw a number sufficiently different (and that they trusted) to flag it.  They also note that such compounds must be carefully recrystallized and fresh powders ONLY used.   

The rest of the story is not nearly as interesting.  They performed 1H NMR titrations and 2D NOESY to confirm that these compounds are binding to the RNA.  They do some SAR and re-discover cpd 22, a known Tat-TAR inhibitor, that has already been patented.

 

So, what do we learn here? Understanding your fragments and their potential liabilities in your assay is crucial. 

Fragments vs PKCθ, selectively

There are more than 500 protein kinases in the human genome, many of which have been tackled with fragments – sometimes all the way to the clinic. Within the universe of kinases, the dozen different isoforms of protein kinase C (PKC) provide an interesting challenge. For example, PKCθ is important in T cell signaling and thus has potential for treating autoimmune and inflammatory disease, but one needs to steer clear of isoforms important for heart function, such as PKCα. In two recent papers in J. Med. Chem., Dawn George and collaborators at AbbVie, WuXi, and Inventiva describe their efforts towards this goal.

The first paper starts as many fragment stories do: a high-throughput screen had come up largely empty. The AbbVie (née Abbott) team had previously constructed a collection of fragment-sized kinase hinge binders, and after screening ~250 of these at 300 µM in a fluorescence (TR-FRET) assay they selected compound 1 because of its structural novelty and ligand efficiency.

Modeling suggested multiple possible binding modes. Crystallography for PKCθ is difficult, but the researchers were able to obtain a structure of the compound bound to a different kinase, FAK. This suggested introducing a positively charged moiety to target an aspartic acid residue in PKCθ, leading to the more potent compound 15a. Additional optimization led ultimately to compound 41, which had moderate potency in cell-based assays, good pharmacokinetics, and 74-fold selectivity against PKCα. The compound also showed activity in a mouse arthritis model, but only at high doses, and was toxic at a slightly higher dose.

That’s where the second paper picks up. The researchers thought that by improving the pharmacokinetics they could lower the dose of the compound required, thereby reducing the potential for off-target effects. By now they had been able to obtain co-crystal structures of some of the more potent compounds bound to PKCθ, which confirmed the proposed binding mode and also gave additional ideas as to how to proceed. Extensive medicinal chemistry ultimately led to compound 17l, with low nanomolar biochemical and cell-based activity as well as good pharmacokinetics.

Unfortunately, this compound did not give stellar efficacy results in the mouse arthritis model. Also, this compound and several others appeared to be toxic to mice, with effects ranging from lethargy to seizures to death. The compounds were screened against panels of kinases and other receptors to try to find the source of these effects, but all to no avail; the compounds were fairly selective. This one-two punch of limited efficacy and unpredictable toxicity led to the termination of the PKCθ program.

These two papers reveal yet again that fragment-based lead discovery is often just the beginning of an arduous medicinal chemistry journey that can lead a long way from Valinor. The final destination here proved to be a dead end, but at least a useful one: it shows that PKCθ is certainly not a straightforward target for arthritis, or perhaps any indication. Kudos to the researchers for publishing this story so other scientists will not have to take the same journey. And at the very least, this compound is a useful probe for dissecting the biology of PKCθ.

Not Every Clam will Hurt You

I grew up on a wonderful little island called Jamestown (although technically it is Conanicut island and the town is Jamestown).  It was a great place to grow up, especially because in the summer we lived walking distance to the beach.  One of the very cool things about the beach is that it has an awesome sand bar that pokes up at low tide.  That was the most fun part of the beach to me.  One of the things we did was stand on the sand bar and dig our feet into it.  You scrunch your toes into the sand until you hit something hard.  Then you excavate with your toes around it.  If you got a foot or so down (this took some patience) and got lucky you would find a quahog.  Thems is good eating.  Many an hour was spent doing this and bringing home dinner.  Sometimes, you found something hard and started more excavation around it...and WHAM!  Not a quahog, but a razor clam instead! There goes your day as blood starts gushing out of your foot stuck in a foot of mud.  You can come up with a different approach to find the clam, but you still get hurt by razor clams.  Eventually, you give up digging for clams with your feet because you hit one too many razor clams and you get your clams from Zeek's Creek Bait Shop.

We here at Practical Fragments have a great job, we get to pontificate on fragment papers.  As most people know, its Good Cop (Dan)/Bad Cop (Me) by and large.  It works for us and the blog gets read by more than our mothers.  But this is an opinion blog, and as everyone knows (G-rated version): Opinions are like belly buttons, everyone has one.  We welcome contrary opinions, sometimes even try to provoke them.  Dan and I do very little coordinating for this blog beyond the "I will have something for Monday".  So, when we both find something that bothers us, well that's worth discussion.  One topic in particular Dan and I have found is PAINS (Pan Assay INterference Compounds).

PAINS are gaining traction as things to avoid in screening collections; there's even a Facebook page.  The literature is pretty clear as to what these are and why they are bad.  In my eyes, I am fine just removing them all from my screening deck and being done with them.  In fragment space, there is MORE than enough other compounds that I don't worry about missing whatever chemical space they live in.  However, as I have often said, a fragment is like pornography, the viewer knows it when they see it.  As you may know, I am not one for hard and fast rules.  If you want to keep PAINS-like compounds in your collection, fine by me.  BUT, you must be aware that they are PAINS.  You must know that they must be kept to a higher standard of evidence, you must do more controls, etc. And of course, if you are making tools then it doesn't matter if it is a PAIN (Dan and I disagree here.) 

So, Practical Fragments gadfly Pete Kenny has a post up at his (recently renamed blog) about PAINS.  In it, he takes Practical Fragments to the woodshed over PAINS, even though his main point is about how we make decisions on data.  He starts his commentary by pointing to this post and calling it a "vapid rant".  As noted in the comments to my post Pete immediately took exception to it believing the burden of proof should be on the blogger to demonstrate the guilt of the compound(s) in question.  He also cites this post as one that should be wary of calling something crap or pollution.  He then goes in to the ontogeny of PAINS and raises some points:

• PAINS study is irreproducible because structures and targets are not revealed
• Only 6 HTS campaigns were analyzed when 40+ were available
• All screens used Alpha-Screen, so this may not be very "PAN"

He then goes deep into the actual structure of rhodanines and how some are good, or less bad.  I think Pete has lost the forest for the trees, or shrubs.  Its not that there are probably some rhodanines that are NOT bad actors; but we know many are, and these require a higher level of confirmation than other compound classes.  Not every clam you dig for is going to slice your foot open, but when enough do, but after enough do, you change your approach.

Fragments vs HSP90: Nerviano’s turn

HSP90, an oncology target, is one of those proteins that seems tailor-made for fragments: it has an active site with a predilection for small molecules, it’s easy to work with, and it crystallizes readily. Indeed, at least two fragment-derived molecules targeting this protein have advanced to Phase 2 clinical trials. In a recent Bioorg. Med. Chem. paper, Elena Casale, Francesco Casuscelli, and colleagues at Nerviano describe their efforts against this target.

The researchers started by identifying a fluorinated probe molecule that they could use in a Fluorine chemical shift Anisotropy and eXchange for Screening (FAXS) assay. This is an NMR-based competition method, in which fragments are screened to find those that displace a known ligand, in this case one that binds in the active site. A total of 1200 fragments were screened in pools of 10, each at the relatively low concentration of 50 micromolar. Nonetheless, 23 hits were found, four of which were characterized crystallographically bound to the protein.

Fragment 3 was among the more interesting, both because of its high ligand efficiency as well as its structural novelty. SAR-by-catalog failed to find anything better from 20 compounds tested, and initial fragment growing also proved disappointing. However, a closer inspection of the crystal structure (cyan) revealed the possibility of linking the fragment to the well-known HSP90 fragment resorcinol. This led to compound 8b, which binds about 5-fold more tightly. Crystallography revealed that the molecule (magenta) also binds as expected.

However, the team wisely chose to test synthetic intermediate 7h (in which the hydroxyl groups were still methylated) and this turned out to be even more active than the designed compound. Since the hydroxyls of the resorcinol are essential for binding in other lead series, the team solved the crystal structure of compound 7h (green) and was surprised to find that it binds in a completely different manner than compound 8b; the ligand essentially flips over.

This discovery led to a change in direction for medicinal chemistry, leading ultimately to the low nanomolar compound 12a. Unfortunately this molecule had only modest cell-based activity and was metabolically unstable.

This is a solid, nuts-and-bolts sort of story. Although it does not conclude with a clinical candidate, it does provide a useful window into how fragment-based methods are applied in industry. It is also a reminder to screen all your intermediates and to remember that even subtle changes to a molecule may have dramatic effects on its binding mode. Those surprising shifts can point the way to promising chemical space.

A Great New Tool....for what?

As has been noted here, frequently, is that in silico design of fragments is very hard, fraught with problems, and often leads to crap.  As was pointed out elsewhere recently, computational tools are getting more powerful, but still don't have chemical intuition leading to suspect structures.  I am assuming that computational scientists have heard the critiques because we are seeing better and better work, with more experimental verification.  Now, what about better structures?  In this paper from Kaken Pharmaceutical and Toyohashi University of Technology, the propose a way to do this.  

In silico tools can be divided into two classes, structure-based and ligand-based design (TOPAS and Flux are two examples of the latter).  These methods are based upon biological evolution: reproduction, mutation, cross-over, and selection.  Mutation and cross-over are vital for creating new chemical structures.  Mutation can be atom or fragment-based.  In a previous study by these authors, the atom-based method was used for the mutation, in which an atom is modified into another atom to explore the chemical space. The method often resulted in a lot of unfavorable structures that contained invalid hetero−hetero

atom bonds such as O−O and N−F. The fragment mutation approach avoids this problem, especially when the fragments are from known molecules (this assumes they were synthesized and thus could be again). This is one key to their approach: chemical feasibility is considered.

Figure 1.

The method (Figure 1) uses a known molecule to "navigate a chemical space to be explored." [I love this phrase, but immediately I think of this.]  The reference molecule is also used to generate the seed fragments (Figure 2), which can be rings, linkers, or side chains.  

Figure 2

 With a good set of seeds, connection rules, and so forth, the key is the mutation and cross-over events.  A parent molecule is randomly selected and then one of three operations occurs: 1. add a fragment, 2. remove a fragment, or 3. change a fragment.  For "Add Fragment", if the base fragment is ring, then a new linker, side chain, or ring is chosen.  If the base fragment is linker or side chain, then a ring is added. "Remove fragment" removes a terminal fragment.  "Replace fragment" is a fragment for fragment swap (Figure 3). The cross-over function is also shown in Figure 3. 

Figure 3

Then they used this protocol to design ligands against GPCR (AA2A and 5HT1A). 

Figure 4.

Figure 4 shows some of the results against AA2A.  They were able to generate a molecule that is very similar to a known active and because of the generation of the fragments these are all presume to be chemically feasible.  

 

So, my first complaint here is where's the experimental verification?  OK, this is not a medchem journal, but still...  I am not nearly as savvy as some of our regular readers, but I am completely missing the forest for the trees here.  This paper first struck me as pretty neat, but then the "neat-o" factor fell away and I was left asking "what is it for?"  To me, this would seem to be a patent-busting tool.  We need to generate a structure that is very similar to billion dollar compound A, but it cannot contain fragments X, Y, and Z.  Is this better than locking your favorite medchemists in a room with a few pads of paper?  I am not being flippant here.  If I am missing something, please let me know in the comments.

Choosing fragments and assays

One of the advantages of running lots of fragment screens is that it generates lots of data that you can mine for general trends and insights. Astex and Vernalis have both done this; in a paper just published online in J. Biomol. Screen. Peter Kutchukian and (former) colleagues at Novartis provide their own meta-analysis of 35 fragment screening campaigns.

The Novartis fragment library consists of 1400 fragments with molecular weights ranging from 102 to 306 Da and logP values from -2.19 to 3.9. This library has been screened against dozens of targets using a variety of different methods. The researchers looked at the hit rates and used Bayesian methods to try to answer three broad questions.

What makes a fragment amenable for fragment-based screening?

Many people have found that some fragments hit many targets while others hit none, and the results here are no different. Over a set of 20 targets, only 37% of fragments came up as a hit, as opposed to the 54% that would be expected if the odds of hitting a target were the same for all fragments (and using the hit rates actually observed). Correspondingly, some fragments hit more targets than expected. Indeed, 1.4% hit six or more, which is orders of magnitude more than would be expected by chance. Given justifiable concerns about artifacts, one might be tempted to dismiss these hits, but the researchers found that these frequent hitters turn out to be more likely to generate crystal structures than other active fragments. In other words, these are privileged fragments (think 7-azaindole).

Do these privileged fragments have anything in common? Previous work from Astex and Vernalis has suggested that fragment hits tend to be slightly more lipophilic than non-hits, and this trend is all the more apparent here. In fact, fragments that hit more than five targets had a median logP of 2.47 versus 1.45 for fragments that hit just a single target. Promiscuous fragments also tended to be slightly larger than other fragments, in contradiction to the molecular complexity hypothesis. They also tended to have more aromatic bonds, fewer rotatable bonds, and higher solubility.

How do hits from different fragment screening technologies and target classes compare with each other?

Do different target classes find different sets of hits? An analysis of substructures identified in hits against various target classes suggests the answer is yes. Certain substructures are preferred by kinases, for example, while other substructures are preferred by serine proteases. This suggests that building fragment libraries specific to a target class may be productive, though certainly not essential.

Regarding screening technologies, the researchers examined both biophysical (for example NMR, SPR, and DSF) as well as biochemical (such as fluorescence) assays. In general, the hit rates were similar for different technologies, with two exceptions. In SPR, a number of fragments nonspecifically interacted with the surface of the chip, giving a higher number of false positives. On the other hand, DSF gave an anomalously low hit rate, and on closer inspection the researchers found that about 1% of the fragment library appeared to denature proteins.

Interestingly, there was less overlap of hits between biophysical methods and biochemical methods than among biophysical methods or among biochemical methods. In other words, hits from an NMR (biophysical) screen were less likely to be found in a fluorescence (biochemical) screen than in an SPR (biophysical) screen. This is similar to the results of a previous study, though not stated explicitly there.

What is the best way to pair FBS assay technologies?

Given this finding, the researchers suggest that, to find the greatest number of hits, it is best to pair a biochemical method with a biophysical method. Of course, this assumes that the goal is to find as many hits as possible, but these may come at the expense of false positives. Still, if you’re going after a tough target, you want to find every possible hit you can. And if you are more interested in weeding out false positives than finding every viable hit, choosing fragments that hit in both a biochemical and a biophysical assay is probably a good starting point.

This is a fascinating paper and contains far more data than can be practically summarized here. It will be fun to see whether similar analyses, from different organizations, come to similar conclusions. 

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?

Fragments in the clinic: 2015 edition

It’s been two years since Practical Fragments updated its list of fragment-derived compounds in the clinic, and since then there have been some nice developments. The table below starts from the previous list and also includes everything new we've managed to find. As before, this includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). Drugs reported as still active in clinicaltrials.gov, company websites, or other sources are in bold, and those that have been discussed on Practical Fragments are hyperlinked to the most relevant post.

Drug	Company	Target
Approved!
Vemurafenib (PLX4032)	Plexxikon	B-Raf(V600E) inhibitor
Phase 3
ABT-199	Abbott	Selective Bcl-2 inhibitor
LEE011	Novartis/Astex	CDK4 inhibitor
MK-8931	Merck	BACE1 inhibitor
Phase 2
AT13387	Astex	HSP90 inhibitor
AT7519	Astex	CDK1,2,4,5 inhibitor
AT9283	Astex	Aurora, Janus kinase 2 inhibitor
AUY922	Vernalis/Novartis	HSP90 inhibitor
AZD3293	AstraZeneca/Astex/Lilly	BACE1 inhibitor
AZD5363	AstraZeneca/Astex/CR-UK	AKT inhibitor
Indeglitazar	Plexxikon	pan-PPAR agonist
Linifanib (ABT-869)	Abbott	VEGF & PDGFR inhibitor
LY2886721	Lilly	BACE1 inhibitor
LY517717	Lilly/Protherics	FXa inhibitor
Navitoclax (ABT-263)	Abbott	Bcl-2/Bcl-xL inhibitor
PLX3397	Plexxikon	FMS, KIT, and FLT-3-ITD inhibitor
Phase 1
ABT-518	Abbott	MMP-2 & 9 inhibitor
ABT-737	Abbott	Bcl-2/Bcl-xL inhibitor
AT13148	Astex	AKT, p70S6K inhibitor
AZD3839	AstraZeneca	BACE1 inhibitor
AZD5099	AstraZeneca	Bacterial topoisomerase II inhibitor
DG-051	deCODE	LTA4H inhibitor
IC-776	Lilly/ICOS	LFA-1 inhibitor
JNJ-42756493	J&J/Astex	FGFr inhibitor
LP-261	Locus	Tubulin binder
LY2811376	Lilly	BACE1 inhibitor
PLX5568	Plexxikon	kinase inhibitor
(RG-7129)	Roche	BACE1 inhibitor
SGX-393	SGX	Bcr-Abl inhibitor
SGX-523	SGX	Met inhibitor
SNS-314	Sunesis	Aurora inhibitor
Undisclosed	Vernalis/Servier	Bcl-2 inhibitor

The current list contains more than 30 clinical-stage drugs but is certainly incomplete, particularly in Phase I. If you know of any others (and can mention them) please leave a comment.

Fragment events in 2015

Happy 2015! Lots of exciting events coming up this year - hope to see you at one!

Newly Added! The American Chemical Society is organizing a series of FREE Thursday webinars on Drug Design and Delivery. They all look fun; readers of this blog may be particularly interested in Designing Better Drug Candidates (January 29, by Paul Leeson), Fragment-Based Drug Design Strategies (March 19, by yours truly), PAINS (May 28, by Jonathan Baell), and X-ray Crystallography in Drug Discovery (July 30, by Jon Mason and Miles Congreve).

February 17-18: SELECTBIO is holding its Discovery Chemistry Congress in Berlin, Germany, with a number of talks on fragment-based lead discovery.

March 22-24: The Royal Society of Chemistry will be holding Fragments 2015 in Cambridge, UK, the fifth in the illustrious series organized by RSC-BMCS. You can read impressions of Fragments 2013 and Fragments 2009.

April 21-23: CHI’s Tenth Annual Fragment-Based Drug Discovery will be held in San Diego. You can read impressions of last year's meeting here and here, the 2013 meeting here and here, the 2012 meeting here, the 2011 meeting here, and 2010 here. This will be the ten-year anniversary, and it looks like a great lineup of speakers. Also, Teddy and I will be teaching our short course on FBDD over dinner on April 22.

June 9-12: NovAliX will hold its second conference on Biophysics in Drug Discovery in Strasbourg, France, and is currently accepting abstracts. Though not exclusively devoted to FBLD, there is lots of overlap; see here, here, and here for discussions of the 2013 event.

August 11-13: The OMICS Group is holding a conference entitled Drug Discovery & Designing in Frankfurt, Germany, with FBDD listed as a conference highlight. I confess I've never heard of this group, so if anyone has attended one of their events please leave a comment.

December 15-20: Finally, I'm helping to organize the first ever Pacifichem Symposium devoted to fragments in Honolulu, Hawaii. The Pacifichem conferences are held every 5 years and are designed to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, Korea, New Zealand, and the US. There is lots of activity in these countries, and since travel to mainland US and Europe is onerous this should be a great opportunity to meet many new folks - in Hawaii no less! Abstract submissions are now open, and we're giving preference to people and organizations that have not presented before.

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