20 August 2014

Like the Elves to Valinor?

There are some perqs to being a consultant.  I go to work most days in my jammies, I can work from anywhere with WiFi, and get to work with great people.  I also have a lot of freedom with what I can say/do/write.  Back in April at the CHI Drug Discovery Chemistry meeting, I ran into an editor from an ACS publication.  Now, if you have ever been at a conference with me you know I am a social butterfly.  New face, let's chit chat.  This editor was very nice and after some social niceties, I asked who is refereeing your journal?  This was a general query having to do with some poor quality fragment papers recently, and before I even saw this!  She was polite and asked what do you mean?  I said, well I blog on fragments and we come across crap in JMedChem and the like all the time.  Like this.  I think at this point she obviously was thinking of me like this http://3.bp.blogspot.com/-_Vv1dw3xm-w/TzwEGm5D-YI/AAAAAAAAFAA/eROp1AEvGTo/s1600/basement2.jpg.  Then I mentioned that the blog was Practical Fragments and her attitude changed.  So, this blog has "street cred"!!  She then asked me if I would write a Viewpoint on the future of FBDD.  Sure, I said.  This could be fun.  

At the same time, I was preparing a talk for the Zing FBDD conference.  So, I decided to make that talk (that's a link to SlideShare, I hope it works) a demo for the Viewpoint. Give it a read, its short and sweet (at least I think).  

For those of you without 10 minutes to spare, here are the take home points from the Viewpoint:
  • Fragonomics is a key component of most (all?) hit generation processes
  • It is a fully mature field.  The current debates amount to quibbling about details.
  • It has no future as a stand-alone field.  But there are still challenges for it to tackle.
  • Medchemists no longer rule the field.  
"The age of the medchemist is over; now is the time of the biophysicist." 
 This got some serious push back at the conference, and I expect (hope?) it will here too.  Of course, I am paraphrasing this. I am not suggesting that medchemists make like the Elves and sail off to Valinor.  They are still incredibly important and can still play a role in early hit generation.  However, the focus, thanks to Fragonomics, is vastly different.  A chemically trained biophysicist can run a fragment-based hit generation project and you don't have to have engage the most precious resource (medchem) until well into the process.  This is a good thing.  

Well, I have planted my flag.  Now its time for you all to weigh in.

18 August 2014

248th ACS National Meeting

The Fall ACS National Meeting was held in my beautiful city of San Francisco last week, and a number of topics of interest to Practical Fragments were on the agenda.

First up (literally – Sunday morning) was a session on pan-assay interference compounds (PAINS) organized by HTSPAINS-master Mike Walters of the University of Minnesota. Mike developed his interest in PAINS like many – from painful experience. After screening 225,000 compounds against the anti-fungal target Rtt109, he and his group found several hits that they identified as PAINS, but not before spending considerable time and effort, including filing a patent application and preparing a manuscript that had to be pulled. One compound turned out to be a “triple threat”: it is electrophilic, a redox cycler, and unstable in solution.

Mike had some nice phrases that were echoed throughout the following talks, including “subversively reactive compounds” and SIR for “structure-interference relationships,” the evil twin of SAR. To try to break the “PAINS cycle” Mike recommended more carefully checking the literature around screening hits and close analogs (>90% similarity). Of course, it’s better if you don’t include PAINS in your library in the first place.

Jonathan Baell (Monash), who coined the term PAINS back in 2010, estimated that 7-15% of commercial compounds are PAINS, and warned that even though PAINS may be the most potent hits, they are rarely progressable, advice that is particularly needed in academia. For example, the majority of patent applications around the rhodanine moiety come from academia, whereas the majority of patent applications around a more reasonable pharmacophore come from industry. Jonathan also warned about apparent SAR being driven by solubility. Finally, he noted that while it is true that ~6.5% of drugs could be classified as PAINS, these tend to have unusual mechanisms, such as DNA intercalation.

As we discussed last week, anyone thinking about progressing a PAIN needs to make decisions based on sound data. R. Kip Guy (St. Jude) discussed an effort against T. brucei, the causative agent of sleeping sickness. One hit from a cellular screen contained a parafluoronitrophenyl group that presumably reacts covalently with a target in the trypanosome and was initially deemed unprogressable. However, a student picked it up and managed to advance it to a low nanomolar lead that could protect mice against a lethal challenge. It was also well tolerated and orally bioavailable. Kip noted that in this case chemical intuition was too conservative; in the end, empirical evidence is essential. On that note he also urged people to publish their experiences with PAINS, both positive and negative.

There were a scattering of nice fragment talks and posters. Doctoral student Jonathan Macdonald (Institute of Cancer Research) described how very subtle changes to the imidazo[4,5-b]pyridine core could give fragments with wildly different selectivities. I was particularly tickled by his opening statement that he didn’t need to introduce the concept of fragment-based lead discovery in a general session on medicinal chemistry – another indication that FBLD is now mainstream.

Chris Johnson (Astex) told the story of their dual cIAP/XIAP inhibitor, a compound in preclinical development for cancer. As we’ve mentioned previously, most IAP inhibitors are peptidomimetics and are orders of magnitude more potent against cIAP than XIAP. Astex was looking for a molecule with similar potency against both targets. A fragment screen gave several good alanine-based fragments, as found in the natural ligand and most published inhibitors, but these were considerably more potent against cIAP. They also found a non-alanine fragment that was very weak (less than 20% inhibition at 5 mM!) but gave a well-defined crystal structure. The researchers were able to improve the affinity of this by more than six orders of magnitude, ultimately identifying compounds with low or sub-nanomolar activity in cells and only a 10-fold bias towards cIAP. This is a beautiful story that illustrates how important it is to choose a good starting point and not be lured solely by the siren of potency.

Alba Macias (Vernalis) talked about their efforts against the anti-cancer targets tankyrases 1 and 2 (we’ve previously written about this target here). In contrast to most fragment programs at Vernalis, this one started with a crystallographic screen, resulting in 62 structures (of 1563 fragments screened). Various SPR techniques, including off-rate screening, were used to prioritize and further optimize fragments, ultimately leading to sub-nanomolar compounds.

The debate over metrics and properties continued with back-to-back talks by Michael Shultz (Novartis) and Rob Young (GlaxoSmithKline). Michael gave an entertaining talk reprising some of his views (previously discussed here). I was happy to see that he does agree with the recent paper by Murray et al. that ligand efficiency is in fact mathematically valid; his previous criticism was based on use of the word “normalize” rather than “average”. While this is a legitimate point, it does smack of exegesis. Rob discussed the importance of minimizing molecular obesity and aromatic ring count and maximizing solubility, focusing on experimental (as opposed to calculated) properties. However, it is important to do the right kinds of measurements: Rob noted that log D values of greater than 4 are essentially impossible to measure accurately.

Of course, this was just a tiny fraction of the thousands of talks; if you heard something interesting please leave a comment.

13 August 2014

Intentionally dirty fragments

Practical Fragments has tried to publicize the dangers of pan-assay interference compounds, or PAINS. These compounds show up as nuisance hits in lots of assays. So what are we to make of a new paper in Curr. Opin. Microbiol. by Pooja Gopal and Thomas Dick, both at the National University of Singapore, entitled “Reactive dirty fragments: implications for tuberculosis drug discovery”?

As the researchers point out, several approved anti-tuberculosis drugs are fragment-sized and hit multiple targets; they are “dirty drugs”. For example, isoniazid (MW 137, 10 heavy atoms), is an acylhydrazide that is metabolically activated and forms an adduct with an essential cofactor, causing havoc to the pathogen. Ethionamide (MW 166, 11 heavy atoms), a thioamide, works similarly. The fact that these molecules are so small probably allows them easier passage through the microbe’s rather impermeable cell membrane, and the fact that they hit multiple targets may make it more difficult for the organism to develop resistance. The researchers conclude:
The success of small dirty drugs and prodrugs suggests that fragment-based whole cell screens should be re-introduced in our current antimycobacterial drug discovery efforts.
While it is true that many antimicrobials do have reactive warheads, and it is also true that there is a huge need for new antibiotics, I worry about this approach. Not only is there an increased risk of toxicity (isoniazid in particular has a long list of nasty side effects), it can be very hard to determine the mechanism of action for these molecules, complicating optimization and development. As evidence, look no further than pyrazinamide (MW 123, 9 heavy atoms). Despite being used clinically for more than 60 years, the mechanism remains uncertain.

Fragment-based lead discovery is typically more mechanistic: find an ideal molecule for a given target. Indeed, much of modern drug discovery takes this view. Gopal and Dick propose a return to a more phenomenological, black-box approach. This may have value in certain cases, but at the risk of murky or worse misleading mechanisms.

If you do decide to put PAINS into your library, you might want to read a new paper in Bioorg. Med. Chem. by Kim Janda and collaborators at Scripps and Takeda. They were interested in inhibitors of the botunlinum neurotoxin serotype A (BoNT/A), which causes botulism.

Since BoNT/A contains an active-site cysteine, the researchers decided to pursue covalent inhibitors, and the warheads they chose, benzoquinones and napthoquinones, are about as PAINful as they get. However, in contrast to other groups, they went into this project with their eyes wide open to the issue of selectivity and examined the reactivity of their molecules towards glutathione. Reaction with this low molecular weight thiol suggests that a compound is not selective for the protein. Not surprisingly, selectivity was generally low, though a few molecules showed some bias toward the protein.

The researchers also tried building off the benzoquinone moiety to target a nearby zinc atom, and although they were able to get low micromolar inhibitors, these no longer reacted with the cysteine; apparently when the ligand binds to zinc, the protein shifts conformation such that the cysteine residue is no longer accessible.

To return to the premise of Gopal and Dick, there can be a therapeutic role for dirty molecules. The fact that dimethyl fumarate is a highly effective blockbuster drug for multiple sclerosis calls for a certain degree of humility. However, if you do decide to pursue PAINS, you should do so in full awareness that your road to a drug – not to mention a mechanism – will likely be much longer and more difficult.

11 August 2014

Measuring 3D Fragments

3D fragments is still a topic up for discussion/debate.  Of course, determining what is a 3D fragment is also open to debate.  As presented last year, nPMI seems to be the current "best" method.  FOB Chris Swain has done some neat analyses around nPMI and included it in his overview of commercial libraries.  Last year at the NovAliX Biophysics conference, Glyn Williams from Astex presented the "plane of best fit" (PBF) as a superior way to analyze 3D-arity.  At the recent Zing FBDD conference, Chris Murray presented the "plane of best fit" and argued again that it was superior.  So, of course, I asked Chris Swain if he had compared them, and he said, but wait a minute.  

Well, Chris was able to compile it with the help of his son, Matt (obviously the apple does not fall from the tree).  So, go check out his comparison of nPMI with PBF.  He ran 1000 fragments through both and concluded this: 
Whilst both descriptors are intended to provide information on the 3D structure of the molecule it looks like the PBF provides more granularity which may be particularly useful when looking at small fragments.
 So, I present this as a "go get 'em, folks!".  I am particularly interested to know if people are currently using PBF and if their results jibe with Matt's. 

04 August 2014

Hydrogen/Deuterium Exchange Mass Spectrometry (HDX MS)

Mass spectrometry does not come up frequently in the context of fragment-based lead discovery (though see here, here, and here). A new paper in Bioorg. Med. Chem. Lett. from Matthew Carson and colleagues at Lilly, with collaborators from Scripps, seeks to change that by describing a technique that can elucidate binding sites for fragment hits.

The researchers were interested in the vitamin D receptor (VDR), an osteoporosis target. Upon binding to ligands such as the D vitamins, this nuclear hormone receptor changes conformation and binds to another receptor, retinoid X receptor (RXR), to control gene expression. The biology quickly gets complicated, but suffice it to say that there is a need for ligands that behave differently than the D vitamins. Enter fragments.

The researchers assembled a collection of about 10,000 compounds, most of which had fewer than 23 non-hydrogen atoms. These were screened at 0.1 or 1 mM concentration in a fluorescence polarization binding assay, resulting in 417 hits. These were then tested in an AlphaScreen assay for compounds that would enhance or decrease binding to RXR (ie, agonists or antagonists). The screen came up empty for agonists. VDR is a member of the same family as the PPARs, for which fragment screens have delivered agonists, so this result was a bit disappointing. The researchers speculate that the fragments may not be large enough to induce the required conformational changes in VDR.

The researchers were more successful finding antagonists: 247 fragments with “lean values” > 0.25 (corresponding to ligand efficiencies > 0.35 kcal/mol/atom). About 2000 analogs of these were then tested, leading to more hits, some of which were quite potent, and 13 of which are shown in the paper. Although some of these look structurally reasonable, one is a toxoflavin-type molecule with a catechol attached that looks disconcertingly similar to a molecule I proposed as an April Fools’ joke. Presumably they are keeping the more interesting structures confidential.

The ultimate goal is to find agonists. Antagonists could potentially be grown into agonists, and to do so it would be helpful to know how they bind. Unfortunately, co-crystallography proved unsuccessful, so the researchers turned to hydrogen deuterium exchange mass spectrometry (HDX MS).

In HDX MS, a protein-ligand complex is exchanged into D2O for seconds to minutes, allowing exchangeable protons (such as those in the amide backbone of the protein) to exchange with deuterium. The reaction is stopped by lowering the pH, the protein is digested into individual peptides, and these are analyzed by mass spectrometry to assess the extent of exchange. If an amide makes a hydrogen bond in a highly structured region of the protein it will be less prone to exchange than if it is in an unstructured region of the protein. Therefore, if a ligand induces structural changes in the protein these should manifest themselves by altering the exchange rates, and if the crystal structure is known this provides a rough map of which regions of the protein are affected by ligand binding.

The 13 fragment hits were tested in HDX MS with a 200-fold excess of fragment to protein. Of these, 6 stabilized the protein, as assessed by decreased H-D exchange. The stabilized regions overlap with the regions stabilized by the natural ligand, vitamin D3, though the extent of the regions and degree of stabilization is considerably less, consistent with the fragments binding within a smaller footprint.

Of course, since we don’t have co-crystal structures, it is difficult to interpret the HDX data precisely. Still, it is nice that fragments can produce a signal in this type of assay. It will be interesting to apply this technique to better characterized systems to see how general it is.

30 July 2014

Fragments in the Caribbean

Last week saw the inaugural Zing FBDD conference in Punta Cana, Dominican Republic. Zing has been around only since 2007, and seems to focus on small conferences in exotic locales. The benefit is that they are able to attract high-profile speakers, as illustrated by the group photo below. However, in an era of shrinking travel budgets, getting approval to attend a conference at a resort is becoming a bit more challenging. That said, participants enjoyed nearly 30 presentations and great discussion – think of a Gordon Conference without the dorms, and breaks on the beach.

My favorite “equation” from the conference comes from Mike Serrano-Wu of the Broad Institute:
Undruggable = Undone
This was supported by some nice work on the anti-cancer target MCL-1, which makes a protein-protein interaction that was widely consider undruggable just a few years ago. An 19F NMR fragment screen gave a hit-rate of around 10%, leading eventually to low nanomolar leads. Fragment optimization was facilitated by a new crystal form of the protein that allowed the team to rapidly generate over a dozen protein-ligand co-crystal structures. Rumor has it that more details on this will be disclosed at FBLD 2014 in Basel in September (there are still a few openings available, but register soon.)

MCL-1 also figured heavily in talks by Andrew Petros (AbbVie, see also here) and Steve Fesik (Vanderbilt, see also here), who described cell-permeable molecules with high picomolar activity in biochemical assays. Steve also discussed programs against Ras and RPA, both also using SAR by NMR. As Mike Shapiro (Pfizer) pointed out in his opening presentation, one of the breakthrough ideas of SAR by NMR was to screen a library more than once per target, the second time in the presence of a first ligand to identify another. It is nice to see this strategy continuing to deliver against difficult targets, though preliminary results of our current poll (right hand side of page) indicate that linking is not necessarily easy.

One of the payoffs of doing fragment screens for many years on dozens of targets is a rich internal dataset. Chris Murray (Astex) mentioned that company researchers have solved close to 7000 protein crystal structures, more than a third of them with fragment ligands. A cross-target analysis found that hits tended to be more planar (ie, less “three-dimensional”, with apologies to Pete Kenny) than non-hits. This was particularly true for kinases; for six protein-protein interactions (PPIs) there was no correlation between shape and hit rate. Although defining complexity is difficult, Chris provided evidence that 3D fragments tend to be both larger and more complex.

Rod Hubbard (University of York and Vernalis) mentioned that Vernalis has determined more than 4000 protein crystal structures. Since 2002, 2050 fragments have been screened against more than 30 targets. Based on “sphericality” – the distance from the rod-sphere principle component axis – hits against kinases are marginally less spherical, while PPI hits reflect the shape of the overall library. So, despite the current push for more three-dimensional fragments, it remains to be seen whether this will be useful.

Jonathan Mason (Heptares) described how successful fragment approaches can be against membrane proteins such as GPCRs. Anyone who has worked on these targets will know that the SAR can be razor sharp, and their surfeit of structures is helping to explain this. For example, although many of the protein-ligand interactions appear merely hydrophobic, some displace high-energy water molecules, which can be revealed by crystal structures of both the free and bound forms of the protein. Displacement of high energy water molecules also helps to explain some “magic methyl” effects.

Fragment-finding methods were not neglected. Jonathan mentioned that, for the A2A receptor, SPR identified only orthosteric ligands, while TINS identified only allosteric ligands – the orthosteric ligands were actually too potent to be detected by this technique. John Quinn (Takeda, formerly SensiQ) and Aaron Martin (SensiQ) also discussed SPR, and in particular how variable temperature SPR analyses could be used to rank ligands based on their enthalpic binding, though as Chris Murray warned, this information can be difficult to use prospectively.

I also learned that a selective BCL-2 inhibitor from Vernalis and Servier has just entered into Phase 1 clinical trials. This has been the result of a long-running collaboration that has required creativity on the part of the scientists and patience on the part of management.

There is much more to tell – for example Teddy's extended metaphor of the Silk Road (this one, not this one!) – but in the interest of space I’ll stop here. Feel free to comment if you were there (or even if you weren’t!)

20 July 2014

Poll: fragment linking and growing

A seminal paper in the fragment field is the 1996 SAR by NMR report in which two fragments were linked together. In theory, linking fragments can give a massive improvement in affinity beyond simple additivity, but in practice this is rare. The challenges of linking were not obvious in the early days, and led to much hair-pulling. Indeed, partially for this reason, Teddy has asserted that the 1996 paper is not just the most impactful paper in the field but also the most destructive.

Nonetheless, there are successful examples of linking, particularly for challenging targets (such as here and here). So how often does it really work?

Our latest poll has two questions: one on fragment linking, the other on fragment growing (see sidebars on right side of page). Tell us whether, in your experience, fragment linking didn’t work at all, worked marginally (ie, perhaps a modest boost in potency), worked OK (perhaps additivity), or worked well (synergy). You can vote multiple times, so if you’ve worked on multiple projects with different outcomes, please vote early and often. We’re asking the same questions for fragment growing since these two strategies are often compared.

Admittedly the categories are somewhat fungible: one person’s “OK” may be another person’s “well,” and some may see merging where others see linking. Still, hopefully we’ll get enough votes to discern some trends.

16 July 2014

You Probably Already Knew This...

Academics can spend time and resources doing, and publishing, things that people in the industry already "know".  This keeps the grants, the students, the invitations to speak rolling in.  It also allows you to cite their work when proposing something.  This is key for the FBHG community.  There are many luminaries in the FBHG field, and we highlight their work here all the time. Sometimes, they work together as a supergroup.  Sometimes, Cream is the result.

Brian Shoichet and Gregg Siegal/ZoBio have combined to work together.  In this work, they propose to combine empirical screening (TINS and SPR) with in silico screening against AmpC (a well studied target).  They ran a portion of the ZoBio 1281 fragment library against AmpC.  They got a 3.2% active rate, 41 fragments bound.  6 of these were competitive in the active site against a known inhibitor.  35 of 41 NMR actives were studied by NMR; 19 could have Kds determined (0.4 to 5.8 mM).  13 fragments had weak, but uncharacterizable binding; 3 were true non-binders. That's a 90% confirmation rate.  34 of 35 were then tested in a biochemical assay.  9 fragments had Ki below 10 mM.  Of the 25 with Ki > 10mM, one was found to bind to target by X-ray, but 25A from the active site.  They then did an in silico screen with 300,000 fragments and tested 18 of the top ranked ones in a biochemical assay.  

So, what did they find? 
"The correspondence of the ZoBio inhibitor structures with the predicted docking poses was spotty. "  and "There was better correspondence between the crystal structures of the docking-derived fragments and their predicted poses."
So, this isn't shocking, but it is good to know.  This is also consistent with this comment.  So, the take home from this paper is that in silico screening can help explore chemical space that the experimentally much smaller libraries miss.  To that end, the authors then do a a virtual experiment to determine how big a fragment library you would need to cover the "biorelevant" fragment space [I'll save my ranting on this for some other forum].  Their answer is here [Link currently not working, so the answer is 32,000.]

14 July 2014

Getting misled by crystal structures: part 4

A picture is worth a thousand words, but words can mislead as easily as inform. So it is with crystal structures, as Charles Reynolds discusses in the July issue of ACS Med. Chem. Lett. We’ve touched on this issue before (for example, here and here), but this is a nice update.

He starts with a cringe-worthy catalog of horrors found in the protein data bank (pdb):

Just to give a few examples: 1xqd contains three planar oxygens as part of a phosphate group; 1pme features a planar sulfur in the sulfoxide; 1tnk, a 1.8 Å resolution structure, contains a nonplanar tetrahedral aromatic carbon as part of a substituted aniline; and 4g93 contains an olefin that is twisted nearly 90° out of the plane.

Of course, with 100,000 structures, it is inevitable some dross will slip through, but Reynolds argues that around a quarter of all co-crystal structures contain errors so severe that they could lead to misinterpretations.

Why is the situation so dire? Reynolds suggests a number of reasons. First, there’s the push for quantity over quality: fully refining a structure may not be as valued as solving a new one. Second, small molecules comprise only a small portion of the overall structure and thus make minimal contributions to the metrics crystallographers use to assess quality during refinement. Third, with the exception of very high resolution structures, the quality of the electron density maps are such that properly placing the small molecule requires a fair bit of modeling. This challenge is complicated by the fact that most crystallographers were not trained as chemists and thus may not immediately recoil from a tetrahedral aromatic carbon atom. Also, much of the off-the-shelf software used for refining structures is not optimized for small molecules.

Nonetheless, there is good software available that properly accounts for small molecules. Hopefully publicizing errors will encourage more crystallographers to use it. In the meantime, caveat viewor!

09 July 2014

EthR revisited: fragment growing this time

A few months ago we described a fragment linking approach against the protein EthR, a transcriptional repressor from Mycobacterium tubercuolosis responsible for resistance to the second-line tuberculosis drug ethionamide. In a new paper in J. Med. Chem., a different team led by Benoit Deprez and Nicolas Willand (Université Lille Nord de France and Institut Pasteur) describe work on the same target using fragment growing and merging.

The researchers started with a fragment (compound 3) they had previously made as part of an in-situ click chemistry effort. A thermal shift assay revealed that this compound marginally stabilized EthR. More convincingly, it displayed mid-micromolar inhibition of EthR binding to DNA, with respectable ligand efficiency.

Interestingly, when compound 3 was cocrystallized with the protein, it bound at two different locations within the binding site. (In the work we highlighted previously this year, a different fragment also bound at two sites, and in that case the researchers linked fragments bound at each site to create a tighter binder.) In the current paper, the researchers focused on fragment growing.

Compound 3 is a sulfonamide that can be readily constructed from amines and sulfonyl chlorides, and the researchers started by constructing a 976-member virtual library of larger sulfonamides. These were then screened in silico against the protein, and many of the top-scoring hits resulted from an isopentylamine building block (such as compound 4). Ten of these were made and tested, and indeed, compounds 4 and 8 were more effective than compound 3 at stabilizing EthR in the thermal shift assay. Moreover, not only did compound 8 show low micromolar activity in the DNA-binding assay (IC50 = 4.9 µM), it also showed low micromolar activity in sensitizing M. tubercuolosis to ethionamide (EC50 = 5.7 µM).

Crystallography of compound 8 bound to EthR revealed that the isopentyl substituent was binding in a hydrophobic part of the pocket, and adding a few fluorine atoms (compound 17) gave a satisfying increase in potency as well as solubility. Replacing the sulfonamide with an amide (compound 19) further improved potency.

The researchers also made a couple compounds in which a second copy of compound 3 was merged with compound 19, and although this approach did produce a compound with nearly the same potency, it was also larger and less soluble.

This team has been pursuing EthR for some time, and they were able to use information from previous structures both in the computational screening as well as in the optimization. In that sense, this is an example of fragment-assisted drug discovery. It is also another nice example of fragment work in academia.

07 July 2014

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? 

30 June 2014

Fragments vs Dengue virus helicase and methyltransferase

Dengue fever is a disease whose nastiness is hinted at by its common name, “breakbone fever”. The eponymous virus relies on two viral proteins, a helicase (Hel) and a methyltransferase (MTase), for replication. In a recent paper in Antiviral Research, Karine Barral and coworkers at Aix-Marseille Université use fragment-based methods to tackle both of these proteins.

The researchers used the 500 compound Maybridge fragment library (2009 edition) and performed a biophysical cascade, starting with a thermal shift assay at 2 mM of each fragment. Hits were defined as fragments that stabilized the protein by at least 0.5 °C; there were 36 hits against Hel and 32 against MTase, with 6 in common.

Both Hel and MTase are amenable to crystallography, so the hits against each protein were soaked into crystals. Unfortunately, none of these yielded structures for Hel. Critics of thermal shift assays could argue that this is yet another example of false positives, a possibility the researchers consider. That said, 11 of the fragments inhibited Hel by at least 25% in biochemical assays (at 2 mM fragment), though none inhibited greater than 50%.

The results against MTase were more salubrious: 7 fragments produced structures, for a success rate of 22%. Interestingly, these fragments bound to 4 distinct sites on the protein. Two different enzymatic assays were used to test all the fragment hits, and many of them showed activity in one or both. A few fragments – including 5 of those characterized crystallographically – were sufficiently active that IC50 values could be determined, though these were mostly millimolar.

My one quibble is that the authors state that “to our knowledge, only one example of random FBS has been conducted on an RNA virus target.” This ignores some beautiful work from both Roche and Astex on Hepatitis C, another virus that carries its genome as RNA. Still, it is fair to say that fragments could play a larger role in tackling infectious diseases.

At the last CHI FBDD conference, Rod Hubbard noted that, as more academics enter the fragment field, we will see more publications describing fragment hits against tough targets. The next steps – taking a fragment to a nascent lead – are often harder to resource in an academic environment. Still, it’s good to see these initial studies. At the very least, they go some way to addressing the question of whether the targets are ligandable.

26 June 2014

Who's Reviewing this Crap?

Dan and I teach a short course on FBDD; next chance to catch a version in October.  My favorite part of Dan's section is when he goes off on PAINS and the continuing pollution of the literature.  Rhodanines in particular get Dan's dander up.  I often come off as anti-academic because many of their "drug discovery" papers are crap.  Or they claim something is a lead without it being one.   I think it is time to start PAINS shaming these papers.  For those of you not intimately familiar with "shaming" it is very popular with dog shaming.  Well, here is our first PAINS Shaming.  This paper (pointed out by Matt Netherton at B-I, thanks Matt!) unabashedly points out the offensive molecule as a rhodanine. In the article, they point out:
What is particularly interesting about the most active species investigated here is that it has a structure that is very similar to that found in the drug epalrestat, an aldolase reductase inhibitor that is used to treat diabetic neuropathy, and is approved for clinical use in Japan, China, and India. This is encouraging because rhodanines as a class are known to often have activity in widely different assays, and indeed computer programs such as PAINS categorize, [our compounds] (as well as epalrestat) as possible “pan assayinterference compounds”. This can mean that the compounds cause false positives in assays, or that they may be multitarget inhibitors. In some cases multitargeting may be undesirable; however, in the context of anti-infective development, multitargeting is expected to increase efficacy as well as decrease the possibility of resistance development, both very desirable features.
  I am sorry, but this is exactly the Underpants Gnomes business plan. All I can say after reading this is who's reviewing this crap and saying it is all right?

23 June 2014

Metric poll results: plus ça change…

Of all the topics on Practical Fragments, none have generated as much Sturm und Drang as ligand efficiency (LE) and related metrics. But how are researchers actually using these? The results of our latest poll are in, and it appears that ligand efficiency is still widely used.
Lipophilic ligand efficiency (LLE or LipE) has made significant strides in the past 3 years, but despite the proliferation of other metrics none of them have really caught on. (Note that we didn’t ask about enthalpic efficiency or SILE in 2011.)

So at least for now – and while fully acknowledging that all metrics are simplifications – I think we can conclude that most readers of this blog find LE and LLE both useful and sufficient for their needs.

(Full technical disclosure: the 2011 poll was run in Blogger, but the polling feature is buggy and has a tendency to lose votes. This poll was run using Polldaddy, but unfortunately the free version does not reveal how many individuals voted. Since respondents could choose more than one metric, the number of voters is impossible to determine from the results. I tried to account for this upfront by including a “Please Click this Box” to tally individuals, but only 77 people did so, less than the 91 people who voted for LE. I thus had to guess at the total number of respondents. The most conservative choice was to add 91 (the largest single category) to the 8 respondents who use no metrics, giving 99 independent voters. Obviously this might overstate the apparent percentage of people who use any given metric, but as 86% of respondents used LE in 2011 the effect is probably small.)

18 June 2014

Fragment Events in 2014 and 2015 - midyear update

The year is nearly half over, but there are still some great events ahead, and 2015 is already shaping up nicely!


July 19-22: Zing conferences is holding its first-ever Fragment Based Drug Discovery in Punta Cana, Dominican Republic. In addition to the amazing location there are more than two dozen great speakers, so definitely check this one out - there is still time to register by June 30.

August 10-14: The 248th ACS National Meeting will be held in the cool gray city of San Francisco, and there will be several presentations on fragments as well as what I believe will be the first session on PAINS.  

September 21-24: FBLD 2014 will be held in Basel, Switzerland. This marks the fifth in an illustrious series of conferences organized by scientists for scientists, the last of which was in San Francisco in 2012.  I believe this will also be the first major dedicated fragment conference in continental Europe. You can read impressions of FBLD 2010 and FBLD 2009. Also, if you're new to the field (or looking for a refresher) Rod Hubbard, Ben Davis, and I will teach an introductory workshop on Sunday, September 21.

October 8-10: CHI's 12th Annual Discovery on Target takes place in Boston, where it looks like there will be several talks relevant to readers of this blog. And on October 7, Teddy and I will be teaching a short course on targeting protein-protein interactions, which naturally includes fragments.


February 17-18: Newly added! 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 series organized by RSC-BMCS. You can read impressions of Fragments 2013.

April 21-23: CHI’s Tenth Annual Fragment-Based Drug Discovery will be held in San Diego. You can read impressions of this year's meeting here and here, last year's meeting here and here, the 2012 meeting here, the 2011 meeting here, and 2010 here. As this will be their ten-year anniversary, I think they're planning something big!

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

December 15-20: Finally, the first ever Pacifichem Symposium devoted to fragments will be held 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 will open in January.

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

16 June 2014

Irreversible fragments

Last year we highlighted a paper in which fragments were attached to a “warhead,” a chemical group that could form a reversible covalent bond with cysteine residues in proteins. These fragments were then screened against a kinase to identify nanomolar inhibitors. In a new paper in J. Med. Chem., Alexander Statsyuk and coworkers at Northwestern University describe a similar approach using irreversible fragments. (See here for In the Pipeline’s take.)

As we noted last year, one of the nice things about using reversible warheads is the fact that you can run experiments under thermodynamically-controlled conditions. Indeed, this was the idea behind Tethering (here and here), in which a library of disulfide-containing fragments is allowed to equilibrate with a cysteine-containing protein; if a fragment has inherent affinity for the protein, the disulfide bond will be stabilized towards reduction and can be identified using mass spectrometry. (Full disclosure: I am an inventor of Tethering, and my company, Carmot Therapeutics, Inc., has an exclusive license to the intellectual property covering this and other technologies.)

Irreversible warheads operate at least partly under kinetic, rather than thermodynamic, control. For example, if all the fragments are extremely reactive, the protein will react with whichever fragment it happens to encounter first, regardless of whether the fragment has any inherent affinity for the protein.

Acrylamide moieties are able to form irreversible bonds to cysteine residues and are even starting to be found in drugs. In 2012, researchers at Imperial College London tested an acrylamide-containing analog of one of the (disulfide) hits from the original Tethering paper. This successfully labeled the target protein thymidylate synthase (TS), while several other acrylamide-containing molecules did not, and the fragment was selective for TS over two other enzymes with active-site cysteine residues.

Regardless of whether your warhead is reversible or not, it is important that different members of the library have similar reactivities: if the inherent reactivities of the fragments are different, it will be difficult to distinguish inherent binding energies from chemical reactivities. One of the problems with acrylamides is that subtle changes in chemical structures can have dramatic effects on intrinsic reactivities. The new paper compares rate constants of two acrylamides reacting with a low molecular-weight thiol, and finds that one reacts 2,000-fold faster than the other. The researchers tested three other classes of electrophiles – vinylsulfonamides, aminomethyl methyl acrylates, and methyl vinylsulfones – and found that these had narrower ranges of reactivity. They chose acrylates and built a set of 100 acrylate-modified fragments. Happily, when 50 of these were tested for reactivity, there was only a 2.4-fold difference between the most reactive and the least reactive members.

These 100 fragments were tested in pools of ten against the classic cysteine protease papain using mass spectrometry, and three hits were identified. Enzymatic assays revealed that these three hits were also irreversible inhibitors of the protein with respectable activities (kinact/Ki = 0.46-1.2 M-1s-1), while a member of the library that did not label in the mass spectrometry assay was a weaker inhibitor (kinact/Ki = 0.037 M-1s-1). Moreover, the papain hits did not label three other enzymes containing active-site cysteines, though these enzymes could be modified with other fragments in the library.

In the case of Tethering, the disulfide linker was a means for finding fragments; when these fragments were developed further, the disulfide linker was replaced. However, given the renewed interest in covalent drugs, some warheads might be able to be retained. It will be fun to see how these types of strategies develop.

11 June 2014

Liberte, egalite, fraternite! with Lemonade

Ligand efficiency has a long and glorious history in FBDD, discussed in depth.  Most recently, it was the subject of a LONG discussion.  Since its inception, FBDD has tried to fit in, be like the cool kids as it were.  "Regular" medchem had the Rule of 5, so Astex gave us the Rule of 3, aka the Voldemort Rule.  The principles of FBDD just make sense, but overturning entrenched dogma is hard.  So, simple metrics were devised to explain to people why smaller is better.  Groups tried to create better and more predictive metrics.  Well, we can add lipophilicity, or log D, or make it empirical.  The more complicated, obviously the better it is.  The search for a GUM (Grand Unifying Metric) is ongoing.  

Then Schultz had to come along and assault the Bastille.  So, to the walls Men!  Defend the King! And so he was, but there was no peace.  But trouble was brewing and now another assault has come.  Peter Kenny and his compatriots have taken another shot at the King, with the completely unprovocative title: "Ligand Efficiency Metrics Considered Harmful".  The paper reviews current LEMs (Ligand Efficiency Metrics), and proposes a better approach.  As Pete has said at length, one of his major problems is with the choice of arbitrary assumptions of standard state, as he laid out in this comment.  Of course, the real issue is the intercept and what that means.

What are the key problems the authors point out?
  • Correlation Inflation
  • Defining assumptions are arbitrary
  • Scaling (dividing measured activity by physicochemical property) and offsetting (subtracting physicochemical property from measured activity) are used but no one has ever said why you do one or the other for a given LEM
  • Scaling assumes a linear relationship between activity and property (zero intercept)
  • Offsetting implies the LEM has a unit slope
  • LEMs are not quantitative, but are presented as such.
  • Units matter!  ΔG = RTln(Kd/C) where C= concentration of standard state.  Substitution of IC50 in this equation is easy to do, but not at all correct.  A more subtle example of the problem is provided by the definition of LLEAT as the sum of a (dimensionless) number and a quantity with units of molar energy per heavy atom.[Click to embiggen.]
 So, this is a fun paper to read.  I highly recommend it.  The authors go into exquisite detail of what each and EVERY factor that goes into a LEM means and how it is used, and more importantly how it SHOULD be used.  The first conclusion is that there is no basis in science for any of the assumptions related to scaling or offsetting.  They recommend that data be modeled to determine a trend.  They argue that only in this way can a given compound be determined to have beaten the trend.  The King is dead; long live the King?

They are right, of course, and IMNSHO it doesn't matter.  I still aver that ligand efficiency metrics are useful.  I can measure accurately with a meter stick that is only 95 cm, as long as I know it is 95cm.  The same thing with any LEM; understand its limitations and use it appropriately.  And remember, its a guide, not a hard and fast rule. 

Pete and colleagues set up the obvious acronym, LEMONS (Ligand Efficiency Metrics of No Substance).  And when life gives you LEMONS, you make LEMONADE (Ligand Efficiency Metrics with No Additional Determinate Evaluation). 

09 June 2014

Fluorinated Fragments vs G-quadruplexes

Recently we highlighted an example of fragment-based ligand discovery against a riboswitch. Of course, RNA can form all kinds of interesting structures, and in a new paper in ACS Chem. Biol. Ramón Campos-Olivas (Spanish National Cancer Research Centre) and Carlos González (CSIC, Madrid) and their collaborators describe finding fragments that bind G-quadruplexes.

G-quadruplexes, as their name suggests, consist of groups of four guanine residues hydrogen bonding to one another in a planar arrangement. These individual tetrads then stack on top of one another. They can form in guanine-rich regions of RNA or DNA. Most famously, G-quadruplexes are found in telomeres at the ends of chromosomes. However, they are also found in telomeric repeat-containing RNA (TERRA), and are required for cancer cells to proliferate indefinitely.

The researchers used 19F-NMR screening to identify fragments that bound to an RNA containing 16 (UUAGGG) repeats (TERRA16). 19F-NMR is a technique about which Teddy waxes rhapsodic, and in this incarnation involves examining the NMR spectra of fragments in the presence or absence of TERRA16. Fragments that bind to the RNA show changes in 19F spin relaxation, resulting in broader, lower intensity signals. The library consisted of 355 compounds from a variety of sources, and although most of them were fragment-sized, a couple dozen had molecular weights above 350 Da.

The initial screen produced a fairly high hit rate (20 fragments), of which seven were studied in detail. Standard proton-based STD NMR confirmed the 19F-NMR results. The researchers then turned to a shorter RNA containing only two repeats (TERRA2); this RNA sequence dimerizes to form a G-quadruplex. All seven fragments stabilized this complex against thermal denaturation, consistent with binding. Six of the fragments also induced changes to the 1H NMR spectrum of TERRA2, though one also caused general line broadening that could indicate aggregation. For the well-behaved fragments, dissociation constants (KD) were determined by measuring changes in chemical shifts with increasing concentrations of ligand. KD values ranged from 120 to 1900 micromolar, with modest ligand efficiencies ranging from 0.17-0.28 kcal/mol/atom.

Of course, selectivity against other nucleic acid structures is a major concern, so the researchers used 1H and 19F NMR to assess compound binding to a tRNA, a DNA duplex, and a DNA analog of TERRA2 also able to form a G-quadruplex. Aside from the putative aggregator, none of the seven compounds bound tRNA, and only two (including the aggregator) bound duplex DNA. However, all the compounds bound to the DNA G-quadruplex. Interestingly though, the DNA sequence used can form two types of G-quadruplexes in solution (parallel or antiparallel), whereas the equivalent RNA can only form a parallel dimer. In all cases the small molecules appeared to shift the equilibrium of the DNA to the parallel conformation, consistent with their initial identification as RNA binders.

Last year we highlighted another paper in which fragments were identified that may bind to a different DNA G-quadruplex. It would be interesting to functionally compare these two sets of hits. For example, do the hits identified initially against the DNA G-quadruplex also bind RNA G-quadruplexes? Of course, as with the riboswitch effort, there is a long way to go. It should be an interesting journey.