Fragments from DOS, advanced

DOS – diversity-oriented synthesis – intentionally generates a disparate set of compounds from a small number of starting materials in just a few synthetic steps. The idea is that if any of these turn out to be hits, it will be straightforward to make analogs. Since figuring out what to do with a fragment is a common bottleneck, DOS-derived fragments could help. An open-access paper published in Chem. Sci. by David Spring (University of Cambridge) and collaborators from several institutions demonstrates how to use DOS to move fragment hits forward.

 

The researchers had previously disclosed (also open access) a rule-of-three-compliant DOS library of 40 compounds derived from racemic α-methyl propargylglycine. In the current paper, these molecules were screened crystallographically at 500 mM on the Diamond Light Source XChem platform against three protein targets.

 

The first, penicillin binding protein 3 (PBP3) from P. aeruginosa, is a classic antibiotic target. A single hit, compound 1, was identified. Interestingly, this turned out to be a covalent modifier, with the catalytic serine opening up the lactone. The researchers made 10 analogs exploring four different vectors, each in five synthetic steps using cheap reagents (< £3 per gram). These were screened crystallographically and six bound; one example is compound 6.

 

 

The next protein screened, cleavage factor 25kDa (CFI₂₅), is a subunit of the pre-mRNA cleavage factor Im. (No, I hadn’t heard of it either.) A crystallographic screen yielded two hits, one of which – compound 19 – was elaborated into 14 analogs. This provided some preliminary SAR around the phenyl ring as well as a surprise: compound 31 bound to a different region of the protein.

 

Finally, the researchers screened activin A, a member of the transforming growth factor β superfamily. Compound 40 was elaborated into 14 analogs exploring four vectors, and compound 42 was found to bind in a similar manner.

 

There are several take-aways from this paper. First, DOS libraries can be remarkably diverse: compounds 1, 19, and 40 are all quite different from one another. Second, although I hesitate to discuss hit rates from such a small library, it is encouraging that hits were found at all even from fairly shapely fragments. These are also the first reported small molecule binders for CFI₂₅ and activin A. Laudably, all the structures have been deposited in the protein data bank, extensive details are provided in 185 pages of supplementary material, and the library itself is available for screening at XChem.

 

One downside to crystallographic screening is that affinities are not part of the package, and some hits may be so weak as to be difficult to advance. But the researchers note they are further characterizing the compounds in the hope of producing more potent analogs. Although Teddy’s deadline for demonstrating a highly ligand-efficient molecule from DOS has long passed, hopefully the Safran Zunft Challenge will soon be met.

Fragment chemistry roundup part 3

Last week’s post discussed three papers describing new chemistries for building fragment libraries. The theme continues this week with three more.

The first, in ACS Med. Chem. Lett. from Philip Garner (Washington State University Pullman), Philip Cox (AbbVie), and colleagues describes the synthesis of a library of pyrrolidine-based fragments in just three steps. A chiral auxiliary, which is subsequently removed, enables an asymmetric cycloaddition reaction to generate pyrrolidine rings containing three defined stereocenters. Using this method, the researchers made 48 fragments from simple starting materials.

As one might predict looking at the structures, the fragments have low lipophilicity (average AlogP = 0.12) and high levels of saturation (Fsp³ = 0.47), though with an average MW = 225 they are a bit portly.

The fragments are also quite shapely, as assessed both by principal moments of inertia (PMI) or plane of best fit (PBF). The researchers acknowledge that this shapeliness increases the fragments’ molecular complexity, and they also note the difficulty of quantifying this, “as current estimates do not take into consideration 3D, let alone the multidimensional descriptors of chemical space.” Thus, they may have lower hit rates. Hopefully we’ll see screening data from this set at some point in future.

Diversity oriented synthesis (DOS) has only been occasionally applied to fragments, perhaps in part due to issues Teddy raised in his Safran Zunft Challenge. In an (open access) Bioorg. Med. Chem. Lett. paper, Nicola Luise and Paul Wyatt (University of Dundee) describe a set of 22 fragments in 12 scaffolds starting from just 3 precursors; a few examples are shown.

Although the embedded pyrazine, pyridine, and pyrimidine moieties are found in many drugs, some of the bicyclic cores are novel or rarely found in commercial sets.

In both these papers, the chemistry is sufficiently straightforward that a hit could rapidly lead to numerous analogs, which is a selling point for including them in a library. But in advancing other fragments a common problem is that the analog you most want to make is synthetically difficult. A crystal structure may reveal that an otherwise useful synthetic handle is making intimate contacts with the protein, while a hard-to-functionalize aliphatic ring is situated next to an attractive subpocket. A clear example of this is the phase 2 IAP inhibitor ASTX660 from Astex, whose fragment starting point consisted of a piperidine linked to a piperazine.

Perhaps building on this experience, Rachel Grainger, Chris Johnson, and collaborators from Astex, University of Cambridge, and Novartis have published in Chem. Sci. a high-throughput experimentation method to functionalize cyclic amines. The researchers used nanomole-scale reactions run in 1536-well plates to explore and optimize photoredox-mediated cross-dehydrogenative heteroarylation.

After optimizing conditions, the researchers moved to larger (milligram) scale to couple 64 different protected amines against heteroarene 3a and 48 heteroarenes against N-Boc-morpholine, thereby obtaining a variety of interesting molecules, many of which contain polar functionalities. Finally, they used flow chemistry to generate more than a gram of product 5g, demonstrating scalability. The paper ends with a half dozen examples of fragments taken from recent reviews, noting how the cross-dehydrogenative coupling could be used to elaborate them.

Progress often comes from expanded possibilities. By facilitating new chemistries, this paper lowers the barriers for drug hunters to make the most promising molecules. And taken together, all six of these papers advance the field of fragment chemistry.

A new library of fluorinated Fsp3-rich fragments

Among fragment-finding methods, ligand-based NMR ranks near the top in terms of popularity. Of its many variations, fluorine (¹⁹F) NMR appears to be gaining in popularity. Fluorine NMR has several advantages, including high sensitivity and the fact that many fragments can be screened simultaneously because of the wide chemical shift range for fluorine. Although more commercial fluorine-enriched libraries are available now than when we first wrote about the approach a decade ago, the diversity of these libraries is still somewhat limited. This problem has been tackled by Mads Clausen at the Technical University of Denmark and an international team of collaborators in a new Angew. Chem. Int. Ed. paper.

The researchers wanted to create a fluorinated fragment library that would be not just diverse but also contain a high fraction of sp³-hybridized carbons (high Fsp³). Some of the early claims around “three dimensional” fragments have been questioned, and there seems to be little if any correlation between the shapeliness of fragments and that of derived leads, but if you’re going to make new fragments in academia it makes sense to explore interesting molecular architectures.

Starting from just six simple building blocks, each containing a trifluoromethyl group, the researchers generated nine different cores which were further derivatized at multiple positions to yield 115 diverse fragments. Consistent with diversity-oriented synthesis, no more than five synthetic steps were used for any molecule. All molecules were made as racemates in order to further increase the diversity of the library.

The resulting “3F Library” is mostly rule-of-three compliant, though given that the trifluoromethyl moiety alone adds 69 Da the fragments do tend to be larger, with an average molecular weight of 284 Da. They are, however, less lipophilic than two commercial fluorinated fragment libraries. And with an average Fsp³ = 0.7 and 3.3 chiral centers they are also quite shapely as assessed by principal moment of inertia.

Building a library is nice, but will it provide hits? To find out, the researchers screened the 102 fragments that passed quality control against four targets. They used a transverse (T₂) relaxation assay (specifically, CPMG) in which fragments bound to a protein tumble more slowly, causing a reduction in ¹⁹F signal intensity. Hit rates ranged from 3% to 11%, and about two thirds of these confirmed in STD or WaterLOGSY assays. As seen by the examples shown here, the fragments are quite diverse.

Whether these hits will lead to more potent molecules remains to be seen. Laudably the paper ends with the statement: “we hope that the 3F library will find use for other researchers and we encourage anyone interested in screening the fragments to contact us.” If you are looking for interesting new fragments that are tailored for follow-up chemistry, I encourage you to take the team up on their offer.

Review of 2018 reviews

As 2018 recedes into history, we are using this last post of the year to do what we have done since 2012 – review notable events along with reviews we didn’t previously cover.

This was a busy year for meetings, starting in January with a FragNet event in Barcelona, then moving to San Diego in April for the annual CHI FBDD meeting. Boston saw an embarrassment of riches, from the first US-based NovAliX meeting, to a symposium on FBDD at the Fall ACS meeting, followed closely by a number of relevant talks at CHI’s Discovery on Target. Finally, the tenth anniversary of the renowned FBLD meeting returned to San Diego. Look for a schedule of 2019 events later this month.

If meetings were abundant, the same can be said for reviews.

Lead optimization

Writing in J. Med. Chem., Dean Brown and Jonas Boström (AstraZeneca) asked “where do recent small molecule clinical development candidates come from?” For three quarters of the 66 molecules published in J. Med. Chem. in 2016 and 2017 the answer is from known compounds or HTS, though fragments accounted for four examples. Although average molecular weight increased during lead optimization, lipophilicity did not, suggesting the importance of this parameter.

The importance of keeping lipophilicity in check is also emphasized by Robert Young (GlaxoSmithKline) and Paul Leeson (Paul Leeson Consulting) in a massive J. Med. Chem. treatise on lead optimization. Buttressed with dozens of examples, including several from FBLD, they show that the final molecule is usually among the most efficient (in terms of LE and LLE) in a given series, even when metrics were not explicitly used by the project team. Perhaps with pedants like Dr. Saysno in mind, they also emphasize the complexity of drug discovery, and note that “seeking optimum efficiencies and physicochemical properties are guiding principles and not rules.”

Lipophilic ligand efficiency (LLE) is also the focus of a paper in Bioorg. Med. Chem. by James Scott (AstraZeneca) and Michael Waring (Newcastle University). This is based largely on personal experiences and provides lots of helpful tips. Importantly, the researchers note that calculated lipophilicity values can differ dramatically from measured values, and go so far as to say that “this variation is sufficient to render LLEs derived from calculated values meaningless.”

Turning wholly to fragments, Chris Johnson and collaborators (including yours truly) from Astex, Carmot, Vrije Universiteit Amsterdam, and Novartis have published an analysis in J. Med. Chem. of fragment-to-lead success stories from last year. This review, the third in a series, also summarizes all 85 examples published between 2015 and 2017, confirming and expanding some of the trends we mentioned last year.

Targets

Two reviews focus on specific target classes. Bas Lamoree and Rod Hubbard (University of York) cover antibiotics in SLAS Discovery. After a nice, concise review of fragment-finding methods, the researchers discuss a number of case studies, many of which will be familiar to regular readers of this blog, including an early example of whole-cell screening.

David Bailey and collaborators from IOTA and University of Cambridge discuss cyclic nucleotide phosphodiesterases (PDEs) in J. Med. Chem. The researchers provide a good overview of the field, including mining the open database ChEMBL for fragment-sized inhibitors. As they point out, the first inhibitors discovered for these cell-signaling enzymes were fragment-sized, so it is no surprise that FBLD has been fruitful – see here for an example from earlier this year. Interestingly though, although at least six fragment-sized PDE inhibitor drugs have been approved, none of these were actually discovered using FBLD.

PDEs are an example of “ligandable” targets, for which small molecule modulators are readily discovered. In Drug Discovery Today, Sinisa Vukovic and David Huggins (University of Cambridge) discuss ligandability “in terms of the balance between effort and reward.” They use a published database of protein-ligand affinities to develop a metric, LIG_exp, for experimental ligandability, and also describe their computational metric, Solvaware, which is based on identifying clusters of water molecules binding weakly to a protein. Comparisons with experimental data and with other predictive metrics, such as FTMap, reveal that while the computational methods are useful, there is still room for improvement.

We have previously written about how target-guided synthesis methods such as dynamic combinatorial chemistry have – despite decades of research – yielded few truly novel, drug-like ligands. Is this because the targets chosen were simply not ligandable? In J. Med. Chem., Anna Hirsch and collaborators at the University of Groningen, the Helmholtz Institute for Pharmaceutical Research, and Saarland University review some (though by no means all) published examples and examine their computationally determined ligandability scores. There seems to be no difference between these targets and a set of traditional drug targets.

Finding fragments

Crystallography continues to be a key tool for FBLD: as we noted in the review of the 2017 literature, 21 of the 30 examples made use of a crystal structure of either the starting fragment or an analog, and only 3 projects didn’t use crystallography at all. That said, FBLD is possible without crystallography, as illustrated through multiple examples in a Cell Chem. Biol. review by Wolfgang Jahnke (Novartis), Ben Davis (Vernalis), and me (Carmot).

In the absence of a crystal structure, NMR is best suited for providing structural information, and this is the subject of a review in Molecules by Barak Akabayov and colleagues at Ben-Gurion University of the Negev. The researchers provide a nice summary of NMR screening methods and success stories within a broader history of FBLD. They also include an extensive list of fragment library providers as well as a discussion of virtual screening.

Speaking of virtual screening, three reviews cover this topic. In Methods Mol. Biol., Durai Sundar and colleagues at Indian Institute of Technology Delhi touch on a number of computational approaches for de novo ligand design, though the lack of structures sometimes makes it challenging to read. A broader, more visually appealing review is published in AAPS Journal by Yuemin Bian and Xiang-Qun Xie at University of Pittsburgh. In addition to an overview and case studies, the researchers also provide a nice table summarizing 15 different computational programs. One of these, SEED, is a main focus of a review in Eur. J. Med. Chem. by Jean-Rémy Marchand and Amedeo Caflisch (University of Zürich). The researchers describe how this docking program can be combined with X-ray crystallography (SEED2XR) to rapidly identify fragments; we highlighted an example with a bromodomain. Their ALTA protocol uses SEED to generate larger, more potent molecules, as we described for the kinase EphB4. The researchers note that together these protocols have led to about 200 protein-ligand crystal structures deposited in the PDB over the past five years.

Rounding out methods, Sten Ohlson and Minh-Dao Duong-Thi (Nanyang Technological University) provide a detailed how-to guide in Methods for performing weak affinity chromatography, and how this can be combined with mass spectrometry (WAC-MS), as we noted last year.

Chemistry

One drawback of some computational approaches for fragment optimization is that they do not consider synthetic accessibility. In Mol. Inform., Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University and Institut Paoli-Calmettes focus on hit to lead approaches that do, and provide a handy table summarizing nearly a dozen computational methods. We highlighted one from the authors, DOTS, earlier this year.

DOTS is an example of using DOS, or diversity-oriented synthesis. In Front. Chem., David Spring and colleagues at University of Cambridge review recent applications of DOS for generating new fragments, some of which we recently highlighted. Only a couple examples of successfully screening these new fragments are described, but the authors note that this is likely to increase as virtual library screening continues to advance.

Perhaps the most productive fragment of all time is 7-azaindole, the origin of three fragment-derived clinical compounds. (The moiety appears in both approved FBLD-derived drugs, vemurafenib and venetoclax.) Takayuki Irie and Masaaki Sawa of Carna Biosciences devote their attention to this little bicycle in Chem. Pharm. Bull. The researchers count six clinical kinase inhibitors that contain 7-azaindole (not all from FBLD) as well as more than 100,000 disclosed compounds containing the fragment. More than 90 kinases have been targeted by molecules containing 7-azaindole, and the paper provides a list of 70 PDB structures of 37 different kinases bound to molecules containing the moiety.

Finally, in J. Med. Chem., Brian Raymer and Samit Bhattacharya (Pfizer) survey the universe of “lead-like” drugs. Among the most highly prescribed small molecule drugs, 36% have molecular weights below 300 Da. Only 28 of 174 drugs approved between 2011 and 2017 fall into this category, consistent with the increasing size of newer drugs. The researchers discuss 16 recently approved drugs, and find that 13 have very high ligand efficiencies (at least 0.4 kcal mol^-1 per heavy atom). As noted above, optimization often entails adding molecular weight by growing or linking, and the researchers suggest that alternative strategies such as conformational restriction and truncation also be investigated.

And with that, Practical Fragments wishes you a happy new year. Thanks for reading some of our 686 posts over the past decade plus, and please keep the comments coming!

Fragments vs GSK3β via DOS

Diversity-oriented synthesis, or DOS, enables the rapid and systematic synthesis of multiple related compounds from small sets of molecules and reactants. By creatively choosing the chemistry, DOS practitioners can selectively generate all diastereomers and produce more complicated molecules than are usually found in commercial screening collections. While much of the attention has been focused on larger molecules, DOS offers clear applications for addressing the chemistry challenges of FBLD. This is illustrated nicely by a recent paper in ACS Med. Chem. Lett. by Alvin Hung, Damian Young, and collaborators at the Broad Institute, Harvard, the Albert Einstein College of Medicine, A-STAR, and Baylor College of Medicine.

The researchers started with a very small (86 fragment) library, which Damian is in the process of expanding to 3000 compounds. Differential scanning fluorimetry was used to screen the molecules against the kinase GSK3β, which is implicated in cancer and Alzheimer’s disease. Three related fragments slightly increased the melting temperature of the enzyme, of which the simplest was compound 1S.

One nice feature of DOS is that – by design – analog synthesis is straightforward. Thus the researchers made a dozen or so derivatives to flesh out the SAR. This revealed that the enantiomer, compound 1R, stabilized the protein even more than the initial hit. STD and WaterLOGSY NMR confirmed binding, and isothermal titration calorimetry (ITC) revealed modest but measurable affinity. Synthesis of a few additional analogs led to compound 15R, with low micromolar affinity as assessed both by ITC and an enzymatic assay. Ligand efficiency was also good, though the ligand efficiency by atom number (LEAN) values of the molecules do not quite meet Teddy’s Safran Zunft Challenge – a wager due to be settled at FBLD 2016 in a few weeks.

A key selling point of DOS is that, by accelerating chemistry, it enables optimization even without structural information. In this case the researchers suspected that the fragment binds in the hinge region of the kinase, and subsequent crystallography revealed that this was indeed so. Interestingly though, the quality of the crystal structure was insufficient to unambiguously place compound 1R; perhaps it binds in multiple conformations. The crystal structure of compound 15R, on the other hand, was clear.

Of course, there is still a long way to go for this series, and it remains to be seen how broadly applicable DOS will be for FBLD. I look forward to seeing additional examples.

Fragments in Texas

The meeting Development of Novel Therapies through Fragment Based Drug Discovery was held last week in Houston, Texas, organized by the Gulf Coast Consortium for Quantitative Biomedical Sciences. Although it was only a single day, it was packed, with thirteen speakers, a couple vendor lunch talks, and some two dozen posters. Below is just a flavor – please add your own impressions if you were there.

I kicked off the first session by giving an overview of FBDD, highlighting both pitfalls and successes. Beth Knapp-Reed (GlaxoSmithKline) then discussed efforts against LDHA, a target previously tackled successfully using fragment linking (see here and here). In this case, an HTS screen of 1.9 million compounds produced only a single hit that resulted in a crystal structure, while fragment screens yielded 16 structures at three binding sites. An NMR-based functional screen (using ¹³C-labeled substrate) was key to obtaining robust SAR, and using information from both the HTS hit and the fragments ultimately led to nanomolar inhibitors. Next, Tom Davies provided an overview of Astex’s discovery platform, focusing on a success with KEAP1. We recently highlighted research suggesting that crystallography should be used as a primary screen, which Astex does for some targets. Tom noted that doing so currently takes about a month, though only after spending somewhere between 3 and 12 months establishing a robust protein construct as well as crystallization and soaking conditions.

Jane Withka opened the next session by discussing the continuing evolution and use of the Pfizer fragment library. Out of 32 targets screened, only one produced no hits, and this was a particularly flexible protein. Interestingly, despite being relatively balanced among basic, acidic, and neutral members, hits were strong enriched in neutral compounds and strongly depleted in basic fragments. Neutral fragments were exactly what was sought by Daniel Cheney (Bristol-Meyers Squibb), who discussed successful efforts to replace a basic amidine moiety in Factor VIIa inhibitors. And Brad Jordan (Amgen) discussed the successful application of ¹⁹F NMR screening to find fragments that could be linked to previously discovered inhibitors to obtain selective picomolar inhibitors of BACE1.

Alex Waterson (Vanderbilt) started the first afternoon session by discussing how fragments had been successfully applied to RPA, RAS, and MCL-1. In the last case, the best compounds now have dissociation constants in the picomolar range, are active in cells, and show activity in xenograft models. IND-enabling studies are slated to begin as early as this year, with the hope of developing a cousin of venetoclax. Inna Krieger (Texas A&M) described how fragments could be used to understand the mechanism of M. tuberculosis malate synthase, while Dawn George (AbbVie) described selective (but inexplicably toxic) PKCθ inhibitors, which are now being made available to researchers to probe the biology. Finally, Damian Young (Baylor) gave an update on his sp³-carbon enriched fragments. Jane had mentioned that following up on hits with multiple stereocenters was not always easy, but Damian’s DOS approach efficiently and systematically yields each possibility. Whether these will meet the Safran-Zunft challenge remains to be seen.

The last session was focused on success stories. Michael Mesleh (Broad Institute) discussed Cubist’s bacterial DNA gyrase inhibitors. Marion Lanier (Takeda) described how fragment screening and careful medicinal chemistry led to a low nanomolar, selective inhibitor of BTK. With a molecular weight of just 318, the molecule is scarcely larger than a Texas fragment, and has good pharmacokinetics and activity in a rat arthritis model. And Yi Liu (Kura) discussed the optimization of covalent KRAS inhibitors originally discovered using Tethering.

This was my first visit to Houston, and I was struck by the number of researchers who had relocated from around the world, particularly from (previously) large(r) pharma companies. Whenever scientists meet the talk often turns to funding shortages, but not here: everyone seemed to have plenty of money and resources, and one of the organizers announced that he was trying to fill several positions. This was the first major fragment meeting in Texas but likely not the last – there is talk of turning it into a recurring event. And there are still several good upcoming events this year; early registration for FBLD 2016 closes in just a few weeks.

Safran Zunft Challenge

Dan has already hit the highlights of FBLD 2014.  I won't do the lowlights.  They were few and far between.  I will try to give some flavor of the conference.  If you missed it, they t-shirts given out had this as a design

 

This was designed by Lukasz Skora of Novartis.  It is keywords used here at the blog, sized by frequency.  That is pretty cool.  It also gives us an idea of what we are really talking about here.  I have some other "flavor of Basel pictures" posted here.  

The conference was excellent, just the right size to allow people to interact at a high level.  The dinner was especially good for this (and the unending wine/beer didn't hurt!).   I have been lucky this year to be at many conferences with many different people.  Damian Young at the Baylor College of Medicine Center for Drug Discovery (and recently of the Broad) has been speaking at all of them about his Diversity Oriented Synthesis (DOS) approach to generating fragments.  Well, this has bothered me, DOS is not Fragments.  Am I some sort of Luddite?  Am I being too purist?  Could be.  

Well, an eminent group of FBLD-ers was gathered around a table during the conference dinner, including Justin Bower and Martin Drysdale of the Beatson, Chris Smith of Takeda, the aforementioned Dr. Young, myself, Terry Crawford of Genentech, and Beth Thomas of the CCDC.  So, out of this discussion, comes the Safran Zunft Challenge, administered by Dr. Bower.  I bet Damian that his molecules are too complex to be "fragments".  What will this mean?  I am betting that a "bad" interaction is worse than a good one, that is going all the way back to the Hann Model.  

So, this one molecule from Damian's presentation.  I have nothing against it per se, but for illustrative purposes.  I bet that his molecules will not have a LEAN (pIC50/HAC) >0.3.  [This is the metric I like, Pete.  I understand the limitations.]  By FBLD2016, Damian expects to have data on his molecules (and he is looking for partners).  If I lose, I owe the undersigned a beer.  Below we have preserved for posterity the discussion and those who were there (no hopping on the beer bandwagon late people!).  

I also think this is a good way for us to discuss the ontology of a "fragment"?  To me, its not just size, it is more of its "nature".  Fragments rely on simple molecules, adding complexity even with small molecules, strips away the "fragment-ness", IMNSHO. 

Ninth Annual Fragment-based Drug Discovery Meeting, Part 2

The first major fragment event of 2014 drew around 500 people to San Diego last week. This is part of CHI’s three-day Drug Discovery Chemistry conference, and although the official FBDD track was only one of six, it is a testimony to the vitality of the field that fragments made appearances in most of the other sessions. With 17 talks in the FBDD track alone this post will not attempt to be comprehensive; Teddy has already shared some impressions here.

Jim Wells (UCSF) gave a magisterial keynote address that emphasized how useful fragments can be for tackling difficult targets such as protein-protein interactions (PPIs). In fact, many of the talks in the protein-protein interaction track relied on fragments. That’s not to say it’s easy. Rod Hubbard (University of York and Vernalis) emphasized that advancing fragments to leads against such targets can take a long time and often requires patience that strains the management of many organizations. Fragment hits against PPIs usually have lower ligand efficiencies (0.23-0.25 kcal/mol/HA if you’re lucky), and improving potency can be a bear. Rhian Holvey (University of Cambridge) presented a nice example of how she was able to find millimolar fragments that bind to the anti-mitotic target TPX2, potentially blocking its interaction with importin-alpha, but even structural information was not enough to get to potent inhibitors.

G-protein coupled receptors (GPCRs) were thought to be unsuitable for fragments until recently, but both Iwan de Esch (whose work has been profiled several times, including here and here) and Jan Steyaert (Vrije University) presented success stories. In fact, Jan has only been working with the Maybridge fragment library for a few months, but has found agonists, antagonists, and inverse agonists for several GPCRs.

Another example of a difficult target is lactate dehydrogenase A (LDHA). We’ve previously highlighted cases where fragment linking was used to get to nanomolar binders (here and here); Mark Elban (GlaxoSmithKline) presented an example of fragment growing and using information from a high-throughput screen (HTS) to get to nanomolar binders. Mark also discussed a particularly disturbing false positive: HTS had generated dozens of confirmed hits spanning 7 chemotypes, but upon closer inspection it turned out that all of them came from a single vendor, and that – unreported by the vendor – they were all oxalate salts. Oxalate is a low micromolar inhibitor of LDHA, and is invisible in proton NMR, so I’m sure this was not fun to track down.

Ben Davis (Vernalis) also presented great examples of false positives and false negatives, and how to avoid them. In particular, the WaterLOGSY NMR technique is great for weeding out aggregators when run in the absence of protein.

A common theme throughout the conference was the integration of fragments with other methods, such as HTS. Nick Skelton (Genentech) actually titled his presentation “Fragment vs. HTS hits: does it have to be a competition?” Kate Ashton (Amgen) discussed how using information from a fragment screen helped solve pharmacokinetic issues with an HTS-derived hit. And Steven Taylor (Boehringer Ingelheim) presented a similar example (also covered here) of using fragments to fix a more advanced lead. Steven noted that fragment-based methods are now fully integrated into the organization, which marks a significant change from Sandy Farmer’s presentation at this meeting four years ago.

The roundtables are great opportunities to swap ideas and get feedback; Teddy already mentioned the excellent roundtable he chaired, but I wanted to also give a shout-out to one organized by Derek Cole (Takeda) focused on "practical aspects of fragment screening." We recently discussed discussed fragments that destabilize proteins in thermal shift assays, and it turns out that folks from both the Broad Institute and Takeda have also crystallographically characterized such fragments. There was the sense that either stabilizers or destabilizers should be considered hits, though the latter were less likely to lead to crystal structures than the former.

Finally, on the subject of library design, Damian Young (Baylor College of Medicine) described using diversity-oriented synthesis (DOS) to generate more “three-dimensional” fragments. He is planning to build a library of roughly 3000 fragments which he hopes to make widely available to the community; these should help answer the question of whether the third dimension is really an advantage.

The importance of library design was also emphasized by Valerio Berdini (Astex); they are currently on their seventh generation library, about 40% of which is non-commercial, and half of whose members have been solved in one or more of 6000+ crystal structures. Relevant to the rule of three, Astex is moving to ever smaller fragments, with an average of 12.6 non-hydrogen atoms, ClogP = 0.6, and MW = 179. Indeed, despite assertions that PPIs may require larger fragments, Rod noted that at Vernalis the average fragments hits against PPIs are only slightly larger (MW = 202 vs 189 against all targets) and more lipophilic (ClogP 1.2 vs 0.8).

CHI has already announced that next year’s meeting will be held in San Diego from April 21-23. As it will be the ten year anniversary, they’re planning something big, so put it on your calendar now!

Fragmenting natural products – sometimes PAINfully

Many drugs have their origins in natural products. But as any synthetic organic chemist will tell you, natural products often have complex architectures that can take years of effort and dozens of chemists to make in the lab. Thus, many of the compounds made in industry look quite different from natural products, particularly in the past few decades. High failure rates in drug discovery have led folks to return to natural products or similar compounds, such as those from diversity oriented synthesis (DOS). In a recent issue of Nature Chemistry, Herbert Waldmann and colleagues at the Max-Planck Institute in Dortmund examine whether natural products can serve as starting points for new fragments.

The researchers started by computationally deconstructing 183,769 natural products into 751,577 component fragments. After various filters (size, lipophilicity, reactivity, etc.) they arrived at 110,485 fragments sorted by similarity into 2000 clusters. The resulting fragments differ in their overall calculated properties from commercial fragments. This is all highly reminiscent of the Emerald (nee deCODE) “fragments of life”, though surprisingly that work is not referenced.

One challenge of designing new fragments is that you may not be able to buy them. In this case, nearly half of the clusters did have a compound that could be purchased – though perhaps this somewhat defeats the purpose of trying to explore novel chemical space. At any rate, 193 fragments were either bought or synthesized. These were tested in functional assays against p38a MAP kinase and several protein phosphatases. A number of hits were identified, and in the case of p38a, nine kinase-fragment co-crystal structures were solved. Some of these were similar to previously reported fragments, but others were more unusual. Together with the crystal structures, these fragments provide new ideas for a well-studied target.

Looking at the structures of some of the phosphatase inhibitors, however, I started to worry. One strong point of the paper is that it is very complete: the chemical structures of all 193 tested fragments are provided in the supplementary information. Unfortunately, the list contains some truly dreadful members; 17 of the worst are shown here, with the nasty bits shown in red. All of these are PAINS that will nonspecifically interfere with many different assays.

Compounds 15, 44, 49, 159, 166, 173, 174, and 175 are catechols; compounds 89 and 151 (yes, they are the same molecule – guess they really liked this one), 165, 166, 167, and 168 are quinones; compounds 55, 89/151, and 166 are hydroquinones; compound 20 is a Michael acceptor; compound 76 is an epoxide; and compound 184 is a redox cycler. In other words, these fragments are a depressing example of life imitating art (or at least satire).

To be blunt: none of these molecules should appear in a screening library today.

I don’t want to pick on these researchers; it is after all laudable that they fully disclosed the structures of their molecules.

However, I am concerned that other people may build libraries containing some of these fragments, or worse, that opportunistic vendors will start selling “natural-product derived fragments.” Indeed, most of these molecules are commercially available. It is disappointing that so many nuisance compounds would find their way into research published in a Nature family journal, and I think it is important to call it out. Only by publicizing the problems that can arise will people be made aware of the dangers.

Not fragments versus DOS, fragments from DOS

A few months ago we highlighted a forum in Nature comparing fragment-based lead discovery with diversity-oriented synthesis, or DOS. This was quite a vigorous debate and was covered on our sister blog as well as In the Pipeline and second messenger. Personally I’ve never been a this or that kind of guy – more of a this and that – so it is refreshing to see a paper in this week’s issue of PNAS describing a DOS approach to building fragments.

Damian Young and colleagues at Harvard and the Broad Institute, ground zero for DOS, noted that many commercial fragments contain a sizable percentage of sp2 carbons: aromatic rings, for example. Because a larger number of aromatic rings correlates with lower solubility and higher attrition in lead development, the researchers focused on using DOS to generate fragments that would have a higher fraction of sp3 carbons at the expense of sp2 carbons. They used a “build/couple/pair” approach, in which chiral “building blocks” (in this case proline derivatives) were “coupled” to another building block and then functional groups were “paired” to generate bicyclic molecules. The result was about three dozen fragments.

So how do they look? Actually, not so bad. Superficially they all resemble one another, but because they contain up to three stereocenters they cover quite a bit of chemical space while still conforming to the rule of 3. Significantly, they are in fact more three-dimensional than commercially available fragments (from ZINC) having the same molecular formula or the same set of calculated physical properties (molecular weight, cLogP, number of hydrogen-bond donors and acceptors, etc.). The DOS fragments contain a larger fraction of methyl esters and carboxylic acids than I would want to see in a library overall, but this was intentional, and none of them are downright ugly.

Unfortunately the paper provides no screening data, so it is anyone’s guess whether any of the fragments will turn out to be active. Still, the approach is likely to probe new areas of chemical space. Hopefully some of the commercial purveyors of fragments will start making and selling these types of molecules.

Fragments in Nature

The most recent issue of Nature has a brief but trenchant summary of fragment-based screening (FBS) by Abbott’s Phil Hajduk, of SAR by NMR fame. This is the first half of a drug discovery forum comparing FBS with diversity-oriented synthesis, or DOS, covered by Warren Galloway and David Spring of the University of Cambridge.

Hajduk summarizes the advantages of FBS:

Fragment libraries are more diverse, synthetic resources are used more efficiently and the leads identified from FBS are more likely to yield drug candidates that have optimal physico-chemical properties.

He also points out that fragment-based approaches have led to a number of drugs in the clinic.

In the spirit of “vigorous debate,” Hajduk also takes aim at DOS. In comparison with fragment-based approaches, which start with small libraries of small fragments, DOS generally makes use of larger libraries of structurally diverse molecules which are usually drug-sized and are often inspired by natural products. However, Hajduk alleges that:

Most compounds in DOS libraries would be excluded from many corporate screening collections because of their poor physico-chemical properties.

I don’t know about “most”, but I will say that many DOS compounds look suspiciously like PAINS. Still, DOS does have at least one strength: FBS is generally limited to well-characterized systems with purified proteins, whereas DOS libraries can be used in complex phenotypic assays where the target may not be known. Whether these will ultimately yield new drugs remains to be seen.

What do you think?