12 March 2018

Fragments vs PDE10A: Astellas’ turn

The 11 members of the phosphodiesterase (PDE) family cleave cyclic nucleotides such as cAMP and cGMP to regulate cell signaling. These enzymes are established drug targets – sildenefil inhibits PDE5, for example. PDE10A inhibitors have been heavily investigated for a variety of neurological disorders, and fragments have played a role in several efforts: we’ve highlighted work from Merck, AstraZeneca, and Zenobia/PARC on this target. A new paper in Chem. Pharm. Bull. by Ayaka Chino and colleagues describes work from Astellas.

A previous HTS screen at the company had led to a series of low nanomolar inhibitors, but these had metabolic liabilities and also inhibited CYP3A4. Thus, the researchers turned to fragments. No details are given as to library size, screening method, or hit rate, though it is worth noting that Astellas has previously reported fragment screening by crystallography. Compound 2 turned out to be a hit, and examination of the crystallographically determined binding mode proved quite useful. (Astute readers will also note the similarity of compound 2 to one of the Merck fragments.)

Because the chlorophenyl moiety was pointing towards solvent, the researchers decided to lop this off  to lower both lipophilicity and molecular weight. Previous publications had also revealed the presence of a “selectivity pocket”, and the researchers therefore grew towards this pocket, yielding molecules such as compound 7. Further tweaking led to compound 13, with low nanomolar potency. In contrast to the HTS-derived lead, this molecule was metabolically stable in vitro and showed negligible inhibition against a panel of 13 CYP enzymes.

This is a nice – albeit brief – example of how fragments can generate new chemical matter even against an extensively explored class of enzymes. Plenty of questions remain around pharmacokinetics, selectivity, and brain penetration, but the paper does end by promising that more will be revealed.

05 March 2018

Fragments deliver (another) inhibitor for CBP and EP300

In 2016 we highlighted a chemical probe that binds two closely related bromodomains, CBP (cyclic-AMP response element binding protein) and EP300 (adenoviral E1A binding protein of 300 kDa). These proteins bind to acetylated lysine residues in various nuclear receptors and are implicated in several types of cancer. Multiple chemical probes are always nice to have, and in a new paper in Eur. J. Med. Chem., Yong Xu and collaborators at Guangzhou Medical University, the University of Chinese Academy of Sciences, Jilin University, the University of Hong Kong, and the University of Auckland go some way towards this goal.

The researchers started with a virtual screen of 272,741 fragments (MW < 300 Da) docked against CBP. The top 5000 were clustered into related subsets and analyzed manually. Of thirteen fragments purchased and tested in an AlphaScreen assay, two had IC50 values better than 40 µM. Compound 6 was slightly less potent, but showed good selectivity against three other bromodomains.

The docking model of compound 6 suggested that more bulk between the indole and the carboxylic acid could be beneficial. Several molecules were made and tested, with compound 25e being the most potent. A related molecule was characterized crystallographically bound to CBP; this suppored the predicted binding mode.

Next, various small lipophilic elements were added to try to pick up additional interactions, ultimately leading to compound 32h, with low nanomolar affinity. This compound, which is equally active against EP300, also showed promising selectivity: it had no activity in a panel of six other bromodomains, including BRD9, which is inhibited by the chemical probe (CPI-637) mentioned above. Unfortunately compound 32h has no activity in cells, which the researchers speculate is due to the carboxylic acid. Masking this moiety with a tert-butyl ester causes a modest reduction in the biochemical activity but does lead to low micromolar activity in several cell assays.

Although much remains to be done, this is a nice example of advancing a computationally-derived fragment with limited structural information. I suspect we’ll see more of these, particularly for well-understood target families.

26 February 2018

Computationally-enabled fragment growing without a structure

Advancing fragments without high-resolution structural information remains a challenge scientists often choose not to take on, according to our poll last year. But for many appealing targets, such as membrane proteins, structural information is difficult to obtain. In a new paper in J. Med. Chem., Peter Kolb and collaborators at Philipps-University Marburg and Vrije Universiteit Brussel describe a computational strategy.

The approach, called “growing via merging”, starts with a core fragment that binds to a target, in this case the β2-adrenergic receptor (β2AR). Ideally this interaction is structurally characterized, but if not a model can suffice. Here, the researchers started with five fragments they had previously discovered. All of these had in common a lipophilic core with a primary or secondary amine appendage; this is a known pharmacophore for β2AR, so modeling could be used to orient the fragments.

Next, this core fragment is derivatized in silico with other fragments using a selection of 58 common reactions. Since all five fragments contained an amine, reductive amination was used here. A set of nearly 19,000 fragment-sized aldehydes and ketones was extracted from the ZINC database and computationally transformed into amines – as if they were reacted with one of the core fragments. These were then docked into the receptor, and those that did not overlap with the core fragments and also placed the amine near the amine of the core fragment were kept for further analysis.

The top 500-scoring fragments were then “reacted” – again in silico – with the core fragments and again docked. Eight of these were actually synthesized and tested for binding, of which four had higher affinity than the initial fragments. The best, compound 11, showed a 40-fold boost in affinity over its starting fragment.

This is an appealing approach, and it will be interesting to see how generalizable it proves. The β2AR is a somewhat forgiving test case due to prior work on the target and the fact that the ligand’s amine interaction with a critical asparate residue helps to orient the core fragment. Laudably though, the computational toolbox (called PINGUI, for Pyton in silico de novo growing utilities) is open access. Please leave a comment and share your experiences if you’ve tried it.

19 February 2018

More hits from a complex library?

One of the cornerstones underpinning fragment-based lead discovery is molecular complexity: fragments are less complex than larger molecules, and are thus likely to bind to more sites on more proteins. In theory, then, you want relatively simple fragments, and in fact Astex has actually formalized this with the concept of the “minimal pharmacophore”, in which each fragment contains a single pharmacophore (such as a hydrogen bond donor next to a hydrogen bond acceptor). But this is not the only way to build a fragment library; in 2016 we noted a paper out of the University of Dundee describing fragment libraries built with “caps” for easy derivatization. In a new paper in ChemMedChem, Paul Wyatt, Peter Ray, and collaborators at the University of Dundee and GlaxoSmithKline describe a screen with this “functional group complexity” (FGC) library.

The researchers were interested in the protein InhA, a drug target for Mycobacterium tuberculosis, the organism causing the eponymous disease. A relatively small library of 1360 fragments was assembled from six different sources, loosely defined by the authors:
  • 573 commercial fragments
  • 170 “3D” fragments from the 3DFrag consortium
  • 326 of the designed FGC fragments
  • 46 commercial fragments chosen based on known InhA inhibitors
  • 124 “inventory” fragments
  • 121 “project” fragments
These were screened against InhA in pools of 8, with each fragment present at 0.5 mM, using STD NMR, resulting in a fairly high hit rate of 11% (149 fragments). The commercial fragments and FGC fragments both gave a marginally higher hit rate (12.6%, 72 fragments and 13.2%, or 46 fragments respectively) while the 3D fragments gave a considerably lower hit rate (5.9%, or 10 fragments).

Previous work had suggested that more potent molecules seemed to reduce the STD signals for the NADH cofactor, so these molecules (32 fragments) were prioritized. The 13 FGC fragments represented a hit rate of 4%, nearly double the 2.4% for the library as a whole.

All 149 of the initial fragments were tested in a biochemical assay at 0.5 mM, but only 4 gave measurable inhibition – too few to draw conclusions. Five compounds were characterized crystallographically bound to InhA, including two of the FGC fragments. This information was used to merge two fragments, compound 24 (an FGC fragment) and compound 12 (a commercial fragment), yielding a mid-micromolar inhibitor. Adding a “magic methyl” gave a satisfactory ten-fold boost in potency. Fragment 24 was also merged with a previously reported molecule, compound 3a, to produce compound 42.

These results suggest that more heavily functionalized fragments don’t necessarily have a lower hit rate, albeit for a small library and a single target. And as we noted last year, molecular complexity is difficult to define; it is not immediately obvious that FGC fragment 24 is actually more complex than commercial fragment 12. The old cliché still holds: more data are needed.

12 February 2018

Fragments in the clinic: ABBV-075 / Mivebresib

Bromodomains bind to acetylated lysine residues in proteins to control gene transcription. These epigenetic regulators have received considerable attention as drug targets, particularly for oncology. Last year we highlighted work out of AbbVie in which fragments found in an NMR screen were advanced to two series of molecules that potently inhibit the four members of the BET family of bromodomains. A more recent publication in J. Med. Chem. by Keith McDaniel and his colleagues at the company describes how one of the fragments was transformed into the clinical compound ABBV-075, or mivebresib.

Compound 6 was not the most potent fragment identified, but crystallography confirmed that it binds in the acetyl lysine binding pocket. The earlier work described how the pyridazinone moiety was replaced with a pyridone and another phenyl ring was added to make molecules such as compound 9, with sub-micromolar activity.

Further modification of the pyridone led to compound 19, with a nearly 20-fold boost in affinity. Crystallography revealed that the pyrrolopyridone makes a bidentate interaction with a critical asparagine residue in BRD4, and also displaces a “high-energy” water molecule.

Next, the researchers sought to pick up additional interactions, and it turned out that introducing a nitrogen off the central ring was synthetically straightforward and would point substituents towards a pocket in the protein. This led to low nanomolar inhibitors such as compound 25, and crystallography revealed that one of the sulfonamide oxygen atoms makes a hydrogen bond with a backbone amide. Happily, the improvement in potency was also accompanied by an improvement in stability in liver microsome assays.

Unfortunately, although the pharmacokinetics in mice were reasonable, these compounds showed high clearance in rats. Analysis of the metabolites revealed that this was largely due to oxidation of the unsubstituted phenyl ring, so the researchers took the classic route of introducing halogen atoms to both deactivate the ring and block metabolism sites. This ultimately led to ABBV-075.

In addition to excellent potency in biochemical, biophysical, and cell-based assays, ABBV-075 showed excellent antitumor effects in a mouse xenograft assay when dosed orally at the low concentration of just 1 mg/kg. In addition to BRD4, the compound binds tightly to the other BET family members but is selective against most of the other bromodomains. It also demonstrates good pharmacokinetic properties in mice, rats, dogs, monkeys, and humans. ClinicalTrials.gov lists a Phase 1 study currently recruiting.

This is a lovely, textbook example of how structurally-enabled fragment growing combined with careful pharmacokinetic-based optimization can lead to a clinical candidate. Obviously there is a long and uncertain road ahead for the molecule prior to approval, but getting this far is a victory in itself.

05 February 2018

Pointless stereochemistry

Designing fragments to be more “three dimensional” than the flatter aromatic molecules that dominate most libraries is a topic often discussed in fragment library design. One way to make fragments more shapely is to introduce a stereocenter, but doing so often complicates the synthesis. In fact, new methods for efficient enantioselective synthesis constitute a major theme of organic chemistry research. In a recent paper in Angew. Chem. Int. Ed., Niklaas Buurma (Cardiff University), Andrew Leach (Liverpool John Moores University) and collaborators at Hawler Medical University Erbil and AstraZeneca demonstrate that the effort is sometimes not worthwhile.

Because proteins are chiral, different enantiomers can have profoundly different activities. The classic case is thalidomide, the racemic mixture of which was sold as a sedative in the 1950s, leading to the birth of thousands of babies with profound birth defects. Only one enantiomer appears to be responsible for the teratogenic effects, and many people are taught that had the manufacturer sold just one enantiomer, the disaster would have been averted. Unfortunately, biology is not so simple: the hydrogen atom attached to the chiral center is slightly acidic, and thalidomide rapidly racemizes at physiological pH.

Such racemization is more common than generally appreciated. The researchers experimentally measured the racemization of a couple dozen compounds using either circular dichroism (CD) spectroscopy or NMR (in the latter case, this involved dissolving the molecule in deuterated buffers and measuring the rate of deuterium incorporation, which occurs through an achiral intermediate).

The experimental results were then compared with those obtained through computational methods. Initially these were intensive quantum mechanical calculations, but the researchers also developed a rapid and effective approach by considering each of the attached substituents around the stereocenter independently. Importantly, the details for doing this are provided in the supporting information.

How much of a problem is this? The researchers provide four examples of what they call “potentially pointless stereoselective syntheses,” all published in high profile journals in 2016 (interestingly, three are fragment sized).

According to calculations, all of these molecules would undergo 19 to 70% racemization in 24 hours under physiological conditions.

So before embarking on any onerous stereoselective synthesis, it would be worth running a quick calculation. If the molecule goes forward you’ll still need experimental evidence for stability, but at least you’re less likely to be unpleasantly surprised by the answer.

28 January 2018

FragNet: The next generation

The first fragment event of 2018 was held in Barcelona last week. This was part of FragNet, established “to train a new generation of researchers in all aspects of FBLD.” Fifteen graduate students from 13 European countries are participating over the course of three years. This meeting marked the midway point for them. I was privileged to serve as a scientific advisor, and was impressed at how much they’ve been able to accomplish in just 16 months. They’ll be on the market next year, so you’ll definitely want to prioritize them if they apply to your institution.

One interesting feature of the program is that, in addition to their primary research, each student completes two “secondments” in other labs – one in academia and one in industry. This is unusual (in the US), and gives them a much broader range of experiences than is typical in graduate school.

The projects themselves are diverse, ranging from synthetic chemistry through computational approaches and biophysics. Fragment library design is a major theme: David Hamilton is building substituted cyclobutanes, Hanna Klein is focusing on pyrrolidines and piperidines, and Aaron Keely is exploring covalent fragments. Darius Vagrys, Sebastien Keiffer, Edward FitzGerald, Pierre Boronat, Lorena Zara, Eleni Makraki, Bas Lamoree, and Lena Muenzker are applying multiple (mostly) biophysical techniques against a variety of different targets. Andrea Scarpino, Moira Rachman, and Maciej Majewski are focusing on computational approaches. Finally, Angelo Romasanta is exploring the diffusion of FBLD techniques through industry. Often multiple students work on one problem from different angles: for example, Andrea is using modeling to explain some of the experimental results produced by Aaron. Plenty of interesting data are being generated in the projects, and I look forward to seeing the eventual publications.

In addition to the student presentations, there was a one day workshop open to the public, with a strong focus on computational approaches. Chris Murray (Astex) discussed how these play a role in all aspects of FBLD, from library design to finding related compounds using the Fragment Network (discussed here). Having a good set of validated experimental data is essential for benchmarking computational methods, and Astex has contributed one of these. But not every computational approach is applicable to every problem. Free energy perturbation (FEP), a rigorous method for predicting SAR, worked well retrospectively for the target XIAP but was not useful prospectively for a target in which the researchers were trying to find a less lipophilic replacement for a phenyl ring. Chris also pointed out that computational methods have a high hurdle – not just to make predictions but to do so better than experienced scientists.

Jenny Sandmark (AstraZeneca) discussed structure-guided design, with a heavy focus on crystallography. She emphasized the importance of quality control: resolution better than ~2.4 Å, with good electron density and low B factors. (Computation can help: Maciej gave an example where dynamic undocking was able to clarify an ambiguous crystal structure.) Jenny also highlighted a set of 52 crystal structures of fragments bound to the capacious binding site of soluble epoxide hydrolase that has been made publicly available for the benefit of modelers.

Chun-wa Chung (GlaxoSmithKline) discussed the importance of understanding your screening technologies and all their limitations. How to establish a cascade assay depends on the needs: if crystallography is challenging, you may want to limit the hits to those that confirm in multiple methods, as these are more likely to confirm crystallographically. If, on the other hand, you have the capacity to do lots of structures you should examine hits from all screens, as those that don’t repeat may be false negatives. Chun-wa also discussed the importance of biophysics for HTS (though this may require different protein constructs for different methods). An HTS screen of 1.7 million molecules against ATAD2 produced a 1% hit rate, of which 444 were studied using a variety of methods including fluorescence polarization, SPR, and NMR. Ultimately only 16 compounds turned out to be useful – all in a single series. (See here for their fragment efforts.)

John Overington (Medicines Discovery Catapult) gave an overview of the open-access database ChEMBL, which holds data from publications and patents on more than 11,000 targets and 14.5 million molecules, including 13,000 clinical candidates and 1500 drugs. Of course, the entries are only as good as the underlying publications: biochemical assays can vary by about 10-fold, cell-based assays can differ by about 100-fold, and in vivo results can vary by 1000-fold. Still, studying these data can produce interesting insights. For example, the observation that antibacterial compounds tend to be larger and more polar appears to be due to the fact that many antibiotics bind to bacterial RNA – those that just bind to bacterial proteins have more standard properties.

Finally, Anthony Bradley described the computational resources at XChem. We’ve recently discussed some of these, including their open-access version of Fragment Network for analog searching. XChem uses extremely high concentrations of fragments for soaking – DMSO stocks are 500 mM and are soaked at 30-50%, so the final concentration can be as high as 250 mM! This often results in multiple fragments binding to a crystal, many of which are of uncertain functional relevance; Anthony used the term “putosteric” for putative allosteric site. Achieving functional activity can be challenging, but it is encouraging that of 16 targets initiated in the past 12 months, 7 have produced compounds with IC50 values better than 100 µM.

All in all a great start to the year – and lots of good events ahead – hope to see you at some!

21 January 2018

Linking fragments on DNA

DNA-encoded chemical libraries are one of the sexier new approaches for lead discovery. Typically, small molecules are synthesized while covalently linked to DNA and then screened for binding to a target. The structure of the molecule is encoded in the sequence of the DNA, and since incredibly tiny amounts of DNA can be sequenced (wooly mammoth genome, anyone?) you can fit massive libraries into a single Eppendorf tube. Indeed, some companies boast 100-billion compound libraries, nearly three orders of magnitude more than the number of molecules indexed by Chemical Abstract Service.

One might think this has no relevance for fragments. Indeed, the only mention of DNA-encoded libraries I recall on Practical Fragments was a comment by Teddy back in 2012 that the approach is “as opposite from FBDD as you can go”. A recent paper by Dario Neri, Filippo Sladojevich, and their collaborators at the ETH Zürich and Philochem in ChemMedChem suggests otherwise.

The researchers have developed an approach called DNA-encoded self-assembling chemical (ESAC) libraries (see also their earlier paper in Nat. Chem.). Rather than synthesizing a single molecule on each strand of DNA, this approach involves assembling two separate sub-libraries of DNA-linked molecules, one attached to the 5’-end and the other attached to the 3’-end. These are then mixed together, allowed to hybridize in a combinatorial mixture, and screened against the target; if a specific combination of fragments is identified (through elegant PCR experiments), this indicates that the two fragments bind to the target in close proximity.

The researchers have focused on the protein alpha-1-acid glycoprotein (AGP), a prominent plasma protein whose function is poorly understood. In their Nat. Chem. paper, a library of 111,100 members (550 x 202 fragments) identified fragments A-117 and B-113. Neither of these fragments showed any binding themselves, but when linked together the resulting compound 1 bound with low micromolar affinity as assessed by isothermal titration calorimetry (ITC).

The linker connecting the two fragments is long, flexible, and not particularly drug-like; its improvement is the focus of the ChemMedChem paper. The researchers increased the size of their second fragment library from 202 to 428 elements, and an ESAC screen revealed that the pair of fragments A-117 and B-217 – both still attached to DNA – had a dissociation constant of 110 nM; B-217 itself (attached to DNA) was around 9900 nM.

To find out how these fragments could be productively linked, the researchers coupled them to 11 different scaffolds, each of which was attached to DNA. All of these bound to AGP, with dissociation constants ranging from 9.9 to 1300 nM. The moment of truth came when the researchers resynthesized some of the molecules no longer attached to DNA. Compound A117-L1-B217 bound with a Kd of 76 nM as assessed by SPR, while the weakest on-DNA binder (Kd = 1300 nM) showed no binding by itself. Although no explanation is provided for this discrepancy, it could be due to low solubility.

This is an interesting approach, though the molecules reported do tend towards molecular obesity (A117-L1-B217 weighs 765 Da and has a ClogP approaching 8). Indeed, this may be an inherent liability – the minimum allowable distance between two fragments that are each attached to DNA may be larger than desirable for most targets. Still, it will be fun to watch this develop.

15 January 2018

Fragments vs USP7, two ways, both allosteric

Proteins in cells are constantly synthesized and degraded in a complex, highly regulated manner managed in part by the ubiquitin proteasome system. Simplistically, a ubiquitous small protein called ubiquitin is conjugated to other proteins, targeting them for destruction, and some of the proteins thus targeted control the stability of still other proteins. But ubiquitination is not destiny: ubiquitin can be removed by more than 100 deubiquitinating enzymes, or DUBs.

As I said, this is complex. But complexity has never stopped folks from pursuing drug targets, and multiple groups are interested in a particular DUB called USP7, which is implicated in cancer and other indications. USP7 is one of more than 50 members of a subfamily of DUBs that use cysteine as a catalytic residue. Selectivity is an obvious challenge, and since cysteine is chemically reactive, any screening result carries a high risk of being an artifact. Two recent papers describe how fragment-based approaches led to potent, selective inhibitors.

The first, published in J. Med. Chem. by Paola Di Lello, Vicki Tsui, and coworkers at Genentech, started with an NMR fragment screen. This identified molecules such as compound 1, which NMR data suggested bound near the active-site cysteine. This and other fragments were used to conduct virtual screens of the much larger Genentech library, and 21 of these were then tested experimentally. Most of these either didn’t bind, bound to multiple sites, or caused protein aggregation, but four of them, including compound 2, showed clear binding to a specific site on USP7 and also inhibited the enzyme in a biochemical assay.

Surprisingly, protein-detected NMR suggested that these four molecules did not bind in the active site as expected but rather in an adjacent “palm site”, a hypothesis that was confirmed by a crystal structure of compound 2 bound to USP7. This led the researchers to reexamine other hits from the original NMR screen, where they identified several aminopyridinephenols, such as compound 13.

Meanwhile, a biochemical HTS against USP7 had identified 76 hits, but most of these turned out to be artifacts, and none of them yielded co-crystal structures with the enzyme. The fragment findings led the researchers to revisit some of the weaker hits that had been overlooked, such as compound 15. This led to a crystal structure showing binding in the palm site, and further medicinal chemistry ultimately led to molecules such as compound 28 (GNE-6640), with nanomolar activity in both biochemical and cell-based assays. A separate paper in Nature characterizes the biology in more detail, revealing that molecules in this series interfere with ubiquitin binding and are highly selective for USP7.

Another fragment effort on this target was reported by Timothy Harrison and collaborators at Almac and Queen’s University, Belfast in Nat. Chem. Biol. An SPR screen of 1946 fragments against the catalytic domain of USP7 led to compounds such as fragment B. This was combined with molecules from other groups that had been reported in the literature, leading to compound 1. Subsequent medicinal chemistry, informed by crystallography, led to compound 4, with low nanomolar biochemical and cell-based activity and excellent selectivity. The enantiomer is much less active, and compound 4 should be a useful chemical probe to further understand the biology of USP7.

Remarkably, not only do the two series of molecules bind some distance away from the active site cysteine (yellow, upper right), they bind in completely different, non-overlapping sites!

These papers illustrate the importance of allosteric sites for tackling specific members of large protein families. They are also both cases of “fragment-assisted drug discovery.” Unlike many success stories we’ve highlighted, it is difficult or impossible to find the initial fragment in the final molecules. Heck, Genentech’s best molecules bind in a completely different site from where the first fragment hits bound. Being open to such possibilities, and using all available data from every possible source, are keys to success.

08 January 2018

Fragments vs Lp-PLA2 – third time’s the charm?

The enzyme lipoprotein-associated phospholipase A2 (Lp-PLA2) cleaves phospholipids into inflammatory molecules. As such, it has been pursued as a target for several indications, from atherosclerosis to Alzheimer’s disease. In 2016 we highlighted two fragment success stories against this target (here and here). A recent paper in J. Med. Chem. provides a third, this one by Jianhua Shen, Yechun Xu, and colleagues at the Shanghai Institute of Materia Medica, ShanghaiTech University, and the University of Chinese Academy of Sciences. The fact that all the scientists are from China illustrates the growth of FBLD in that country, as we reported last November.

The researchers started by screening a 500 fragment library in an enzymatic assay. Compound 10 was a weak hit but had good ligand efficiency and was unlike known Lp-PLA2 binders. Moreover, crystallography revealed multiple interactions between the sulfonamide and the protein. This information was used to perform a similarity search followed by docking of 200,000 compounds. The top 500 were manually inspected and 100 were purchased and tested, with compound 11 showing low micromolar activity.

A crystal structure of compound 11 bound to the protein revealed a similar binding mode as the initial fragment, and also suggested further improvements, such as adding substituents to fill a small pocket (as in compound 14a). Further optimization for both affinity and stability ultimately led to compound 37, which inhibited Lp-PLA2 in human and rat plasma. It also exhibited good oral bioavailablilty in rats and promising pharmacokinetics. The researchers state that further optimization is ongoing.

How far will this go? The most advanced Lp-PLA2 inhibitor to make it to the clinic, darapladib, failed two phase 3 clinical trials (with nearly 30,000 patients!) for coronary diseases, casting a pall over the target. Darapladib, which was not fragment derived, can fairly be described as molecularly obese. Molecules such as compound 37 and the other fragment-derived series we previously mentioned do appear more attractive, but whether anyone will invest the massive resources needed to move them forward remains the billion yuan question.

03 January 2018

Fragment events in 2018

The new year has finally arrived, and brings quite a few interesting events.


January 24: There will be a one-day FBLD workshop in Barcelona. This is part of FRAGNET, a European Commission training program for the next generation of fragment scientists. Registration is free but you need to email fragnetworkshop@gmail.com. The subject should be Surname, Name – Institution (e.g. Potter, Harry – Hogwarts) and the body of the email should contain the word "register."

January 28 - February 1: The First Alpine Winter Conference on Medicinal and Synthetic Chemistry will take place in St. Anton am Alberg, Austria. This looks like a fun event, and includes a section on FBDD.

April 2-6: CHI’s Thirteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of last year's meeting here, the 2016 meeting here; the 2015 meeting herehere, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here. Mary Harner and I will be presenting a FBDD short course on the afternoon of April 2.

June 13-15: Although not exclusively fragment-focused, the Fifth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and will be held for the first time in Boston. You can read my impressions of last year's Strasbourg event here and Teddy's impressions of the 2013 event herehere, and here.

August 19-23: The 256th National Meeting of the American Chemical Society, which will also be in Boston, includes a session on "Best practices in fragment-based drug design", currently scheduled for August 20.

October 7-10: Finally, FBLD 2018 returns to San Diego, where it was born way back in 2008. This will mark the seventh in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2016FBLD 2014,  FBLD 2012FBLD 2010, and FBLD 2009.

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

26 December 2017

Review of 2017 reviews

The year is done, and the darkness
Falls from the wings of Night.

As we've done since 2012, Practical Fragments is using the last post of the year to highlight conferences as well as reviews not previously discussed.

Significant events included the venerable CHI FBDD meeting in San Diego, the NovAliX Biophysics conference in Strasbourg, and the first-ever fragment conference in Shanghai. We discussed a special issue of Essays in Biochemistry devoted to structure-based drug design, and Teddy came out of retirement to provide an entertaining summary of his experience putting together a book on biophysics in drug discovery - well worth reading if you're ever tempted to edit one yourself.

As in years past, several reviews were devoted to the broad topic of FBDD. Below, I’ll outline the general reviews, followed by those focusing on particular targets, techniques, and other topics.

György Keserű (Hungarian Academy of Sciences) and Mike Hann (GlaxoSmithKline) ask “what is the future for fragment-based drug discovery?” in Fut. Med. Chem. After a concise summary of the topic, they answer that it “includes target discovery and validation, the development of chemical biology probes, pharmacological tools and more importantly drug-like compounds.” In other words, the future looks bright.

FBDD is more comprehensively covered by Ben Davis and Stephen Roughley (Vernalis) in Ann. Reports Med. Chem. This is a complete, self-contained guide to the field, covering everything from history, theory, fragment library design, and fragment-to-lead approaches. It is ideal for a newcomer, but there are enough insights throughout that it makes a rewarding read for experts too.

Of the thirty-plus fragment-derived drugs that have made it to the clinic, none are directed against neglected diseases. Gustavo Henrique Goulart Trossini and colleagues at Universidade de São Paulo review some of the work that has been done in this area in Chem. Biol. Drug Des.

And rounding out general reviews, Christopher Johnson (Astex) and collaborators examined all 28 successful fragment-to-lead programs published in 2016, defined as at least a 100-fold improvement in affinity to a 2 µM or better compound. This is a sequel to our analysis of the 2015 literature, also published in J. Med. Chem., and many of the trends are similar. Interestingly, many leads maintained high ligand efficiencies, and there was no correlation between the “shapeliness” (deviation from planarity) of fragments and that of the resulting leads. Consistent with our recent poll on the importance of structural information, 25 of the 28 examples used crystallography at some point.

Three of the success stories from 2016 involved bromodomains, the subject of an entire month of Practical Fragments’ posts last year. In Arch. Pharm., Mostafa Radwan and Rabah Serya (Ain Shams University, Cairo) review this target class, with a particular emphasis on the four BET family proteins.

More than 30% of enzymes are metalloenzymes, yet these are targeted by fewer than 70 FDA-approved drugs. One of the first published examples of FBDD involved a metalloenzyme, but most efforts have been focused on a limited set of metal-binding pharmacophores, such as hydroxamic acids. Seth Cohen (University of California, San Diego) has been steadily building libraries of metallophilic fragments, and in Acc. Chem. Res. he describes how this approach can lead to new classes of inhibitors.

Protein-protein interaction inhibitors are another underrepresented class of drugs, though one approved FBDD-derived molecule falls into this category. In Methods, Daisuke Kihara and collaborators at Purdue University look at in silico methods to discover PPI inhibitors, including fragment-based approaches.

Unlike PPIs, kinases have been highly successful drug targets. We recently highlighted one review of cyclin-dependent kinases (CDKs), and in Eur. J. Med. Chem. Marco Tutone and Anna Maria Almerico (Università di Palermo) provide another. Although the main focus is on in silico methods, there is a section on FBDD.

As noted above, X-ray crystallography has played a role in most successful fragment to lead programs. In the open-access journal IUCrJ, Sir Tom Blundell (University of Cambridge) provides an engaging and personal view of protein crystallography, a field in which he has played a starring role, starting with his early involvement in determining the crystal structure of insulin. He also notes that the interchange of ideas and techniques between academia and industry has long been a crucial driver of advances.

NMR was the first practical method used for FBDD, so it is not surprising that there are several reviews on the topic. In Arch. Biochem. Biophys., Michael Reily and colleagues at Bristol-Myers Squibb provide a detailed overview of NMR in drug design. This covers not just the ligand- and protein-detection methods often used in fragment screening, but also more intensive techniques to characterize protein-ligand interactions.

A briefer look at many of these topics is provided by Yan Li and Congbao Kang (A*STAR) in Molecules. This review also highlights more unusual approaches such as NMR experiments on living cells.

Artifacts are a fact of life in both FBDD and HTS, and it is always important to recognize these early. In J. Med. Chem. Anamarija Zega (University of Ljubljana) discusses how NMR can help. This includes methods to detect aggregators and covalent modifiers. Of course, NMR methods can introduce their own artifacts, and these are also covered.

Other topics
Speaking of artifacts, PAINS are responsible for quite a few. The term “PAINS” has also been somewhat controversial, and in a new paper in ACS Chem. Biol. Jonathan Baell (Monash University) and J. Willem Nissink (AstraZeneca) examine the “utility and limitations” of the term Jonathan coined seven years ago. As they acknowledge, the PAINS filters were derived from just 100,000 compounds run in a limited set of assays. This means that not every bad actor will be recognized by PAINS filters, and some compounds that are may only be PAINful in certain assay formats. Like Lipinski’s rule of 5, it is important to recognize the limits of applicability. As the authors note, “the key is to remain evidence-based.”

Another sometimes controversial topic is ligand efficiency and associated metrics, the subject of an analysis in Expert Opin. Drug Disc. by Giovanni Lentini and collaborators at the University of Bari Aldo Moro. This includes extensive tables of rules and metrics, both common and obscure. The authors note that, while metrics can be useful, it is important not to use them as a “magic box.” As they quote William Blake, “to generalize is to be an idiot.”

Shawn Johnstone and Jeffrey Albert (IntelliSyn Pharma) discuss pharmacological property optimization for allosteric ligands in a review in Bioorg. Med. Chem. Lett. As we recently noted, fragments are particularly suited for discovering allosteric sites, and this paper discusses how to characterize these.

Finally, Jörg Rademann and collaborators at Freie Universität Berlin discuss protein-templated fragment ligations in Angew. Chem. Int. Ed. Earlier this year we highlighted some of his work, and this review provides a thorough analysis of both reversible and irreversible approaches, with good discussions of detection methods, chemistries, and case studies.

That’s it for the year. Thanks for reading, and especially for commenting.

And may 2018 be filled with music, and light.

18 December 2017

New tools for NMR: 2017 edition

NMR was the first practical fragment-finding method, and continues to be popular. Just over the past year we’ve discussed several new techniques, (here, here, and here), and this post highlights three more.

In Angew. Chem. Int. Ed., Jesus Angulo and colleagues at the University of East Anglia describe differential epitope mapping by STD NMR (DEEP-STD NMR). STD NMR, the most popular of ligand-detected methods according to our poll, can provide some information as to which portions of a ligand are close to a protein, but doesn’t show where on a protein the ligand binds. In DEEP-STD NMR, two separate NMR experiments are conducted and the results compared to provide this information.

The researchers provide two implementation of the technique. In the first, the protein is “irradiated” at two different frequencies; for example, the aliphatic and aromatic regions. Protein residues that are directly irradiated will show a stronger STD to ligand protons than those that are indirectly irradiated, thus revealing whether one region of the ligand is closer to an aromatic or an aliphatic amino acid side chain. If the structure of the protein is known, this can then reveal the orientation of the ligand within the binding site. A similar experiment can be done using H2O vs D2O to determine whether a portion of a ligand is in close proximity to polar residues in the protein.

Water is the subject of the second paper, in J. Med. Chem., by Robert Konrat and colleagues at the University of Vienna and Boehringer Ingelheim. As we’ve previously noted, water often plays a critical role in protein-ligand interactions. The new method, called LOGSY titration, involves doing a series of WaterLOGSY experiments at different protein concentrations and plotting the signals for each proton in the ligand as a function of protein concentration; ligand protons close to the protein show steeper slopes. The researchers examine pairs of bromodomain ligands and demonstrate that LOGSY titration can confirm changes in binding mode previously seen by crystallography. The technique could also reveal what portions of the ligands make interactions with disordered water molecules, which are more difficult to detect in crystal structures.

Both of these techniques provide useful but incomplete information about ligand binding modes. A paper in J. Am. Chem. Soc. by Andreas Lingel and his Novartis colleagues describes how to generate more detailed models. The researchers used a deuterated protein in which all methyl groups (in methionine, isoleucine, leucine, valine, alanine, and threonine) were 13C-labeled. Multiple intermolecular NOEs between the protein and several previously characterized ligands were collected and the resulting distances fed into modeling software to produce good agreement with the known structures. More significantly, the researchers were able to use the method prospectively with two weak (0.9 and 2.8 mM) fragments. The binding models were sufficiently accurate to guide chemical optimization, resulting in molecules with 30-50 µM affinities. Subsequent crystal structures revealed that these bound as predicted. Impressively, this was done on a protein that forms 115 kD hexamers – larger than those typically tackled by NMR.

Teddy would normally close his NMR posts by stating – usually quite forcefully – whether he felt the technique was practical or not. I’m no NMR spectroscopist, so I’ll throw this question out to readers – do you plan to try any of these approaches?

11 December 2017

Flipping fragments in PDE2

A common assumption in fragment growing is that the binding mode of the fragment remains the same throughout optimization (for example here, here, and here). However, this is not always the case (as described here, here, and here). A recent paper in Bioorg. Med. Chem. Lett. by Ashley Forster and colleagues from Merck falls into this latter category.

The researchers were interested in phosphodiesterase 2 (PDE2), which hydrolyzes the cyclic nucleotides cAMP and cGMP. PDE2 is highly expressed in the frontal cortex and hippocampus and has been implicated in cognition and proposed as a target for Alzheimer’s Disease. But because PDE2 is just one member of a large class of enzymes, selectivity is important. Indeed, Merck researchers previously used fragment-based methods to discover selective inhibitors of another member of the family, PDE10A.

In this case the researchers used both high-concentration biochemical screens as well as an SPR screen of a library of 1940 fragments, all with molecular weights < 250 Da. This resulted in 54 competitive inhibitors of PDE2 with affinities better than 200 µM. (No details were provided on numbers of hits from each screen.) Compound 1 was progressed into lead optimization due to its high ligand efficiency and attractive physicochemical properties.

A crystal structure of compound 1 bound to PDE2 revealed the potential to grow into a hydrophobic pocket exploited by previously reported molecules, leading to compound 5. Modeling suggested that bulking up the benzylic linker could improve the binding mode, and indeed compound 8 had submicromolar affinity. Surprisingly however, a crystal structure of a related molecule (having a single methyl group off the linker instead of two) revealed that the initial fragment had flipped orientation.

Further modeling suggested replacing the two methyl groups with a cyclopropyl group, as in compound 12. This simple change gave a 100-fold boost in potency, which was attributed to the free form of the compound more closely matching the bound form. Finally, the remaining methyl group was removed to reduce lipophilicity and remove a potential metabolic liability, leading to compound 16. Crystallography revealed that this binds as expected (gray), with the fragment moiety in the “flipped” conformation.

Compound 16 is at least 100-fold selective for PDE2 against a panel of other PDEs. The attention to physicochemical properties paid off in the form of good oral bioavailability, low clearance, and a satisfactory half life in rats. Although the paper does not mention how long the program took, it does state that only 25 analogs were made to get from the initial fragment to compound 16, and also mentions further optimization. This is another nice example of how the union of crystallography, modeling, and medicinal chemistry can rapidly lead to useful molecules.

04 December 2017

Fragment activators of AMPK

Kinase inhibitors are common. Some 40% of the fragment-derived clinical compounds in our latest list target kinases. Kinase activators, on the other hand, are rare. It is easier to interfere with something than to enhance it, and of the 630+ posts on Practical Fragments, I believe only two discuss enzyme activators. A new paper (here) by Ping Lan, Iyassu Sebhat, and colleagues at Merck and Metabasis provides a third example.

The researchers were interested in adenosine monophosphate-activated protein kinase (AMPK), which plays a critical role in metabolism. As its name suggests, this kinase has naturally occurring activators, though these are nucleotides and thus not particularly useful as chemical probes. It also has fiendishly complex biology: the active enzyme is a heterotrimer of three distinct proteins, each of which comes in two or three flavors, leading to 12 different isoforms with their own unique tissue distributions – which vary among different animals.

After multiple HTS screens failed to produce anything of value, the researchers turned to fragments. Realizing that AMPK was a tough target, they assembled a library of 25,000 highly diverse fragments tending towards super-sized (all of them were greater than 200 Da, and they had up to 22 non-hydrogen atoms). A biochemical screen yielded just three hits, including compound 4.

Despite the generally larger size of fragments in the library, it is interesting that compound 4 follows the rule of three, with just 16 non-hydrogen atoms. Although only modestly active, the small size gave an impressive ligand efficiency, and the activation was nearly 80% that of the natural ligand AMP.

Rigidification of the linker between the benzimidazole and the acid moiety led to compounds such as 27, with low micromolar potency, while growing from the benzimidazole itself led to high nanomolar compounds such as compound 36. Combining these two modifications and further optimization for pharmacokinetic properties led to MK-3903, which was chosen as a development candidate.

MK-3903 activates 10 of the 12 AMPK isoforms and is fairly selective against a panel of off-targets. As predicted mechanistically, administering the compound to mice increases the phosphorylation of downstream substrates of AMPK. It also causes decreased fatty acid synthesis and increased insulin sensitivity. However, a related molecule causes cardiac hypertrophy in rats and monkeys.

In addition to the fact that MK-3903 is an enzyme activator, there are several other notable features about this story. First, despite the difficulty of the target, the team made rapid progress, moving from the initial screen to useful tool compounds in less than a year. Second, as near as I can tell, this optimization was done in the absence of direct structural information on how the compounds bind. (A publication by a separate team, who was closely monitoring the patent literature, describes the crystal structure and mechanistic analysis of a related molecule.) Third, all of this work stemmed from a single fragment: although more ligandable targets may produce lots of hits, in the end you only need one.

Finally, this paper illustrates the lag time that can occur between research and publication: several of the authors are from Metabasis, which was acquired by Ligand Pharmaceuticals way back in 2010. That was also when the patent publication describing these molecules was filed, suggesting the work could have been done a decade ago. That’s something to keep in mind when using the literature to guess who is working on fragments.

27 November 2017

Fragments in China

The 2017 International Symposium on Fragment Based Lead Discovery (pdf here) was held in Shanghai, China last week. I was fortunate to be able to attend what I believe was the first significant FBLD meeting in Asia. Antimicrobials were a major theme, particularly against drug-resistant pathogens. The two days were filled with nearly 20 talks, so I’ll just try to capture a few impressions.

Ian Gilbert discussed the fragment-based efforts underway at the University of Dundee, focusing especially on library design. Among initially purchased commercial compounds, only 56% passed quality control, with 26% insufficiently soluble (at least 2 mM in water) and most of the rest either unstable or impure, similar to what has been seen by others. Ian has also enlisted undergraduate students to make “capped” fragments ready for optimization, as well as novel heterocycles.

Biophysics was a major theme of the conference, and Ian made a strong case for biolayer interferometry (BLI), one of the lesser-used fragment finding techniques. A screen can be completed in just a few days with less than a milligram of protein. In particular, BLI may be useful for assessing ligandability: Ian tested 31 targets, 13 known to be ligandable and 5 known to be not ligandable, and found good agreement with previous research. Ligandable targets generally gave primary hit rates >4.5%.

Ismail Moarefi (Crelux, now part of WuXi AppTec) highlighted microscale thermophoresis (MST) and differential scanning fluorimetry (DSF). NMR had identified ten hits against Pim1, but only six had yielded crystal structures, despite considerable effort. Of the four that didn’t, three had no activity by MST, while the fourth was very weak. Ismail also discussed the Prometheus nanoDSF instrument, which is sufficiently sensitive that it can resolve two-stage melting curves for a two-domain protein.

Another lesser used fragment-finding technique, affinity mass spectrometry, was described by Wenqing Shui (ShanghaiTech University). This uses ultrafiltration to separate protein-bound ligands from unbound molecules and mass spectrometry to identify hits; up to 1000 molecules can be screened in a single assay! Wenqing provided several success stories, including fragment hits with very weak (millimolar) affinity. She also demonstrated that the technique works against a membrane preparation of a GPCR.

Among more common biophysical methods, NMR was represented by Ke Ruan (University of Science and Technology of China). The challenge was characterizing a low-solubility ligand which caused extensive line-broadening of the protein due to intermediate exchange rates. This was solved by examining the distance between a fluorinated ligand and a paramagnetic label on the protein and using this to model the binding mode.

But by far the star of the show was crystallography. We’ve previously mentioned the high-throughput capabilities developed at the Diamond Light Source, and part of the impetus for this conference was to bring these technologies to China. Frank von Delft (Diamond and University of Oxford) noted that since the XChem platform launched in late 2015 more than 50,000 crystals have been screened against more than 40 targets, resulting in more than 1000 fragment structures. The group is committed to removing barriers and bottlenecks and today can process 1000 crystals per week through compound soaking, harvesting, data collection, and processing (using specially developed programs such as PanDDA). More than 30 external groups have used the facility, and every target has yielded at least one hit.

Of course, to collect data on 1000 crystals requires you to reproducibly grow lots of well-diffracting crystals that can handle the rigors of soaking, and Diamond has released a handy list of tips and tricks. Getting the right crystals was also the theme of two talks, one by Sheng Ye (Chinese Academy of Sciences) and the other by Carien Dekker (Novartis). Sheng emphasized the importance of optimizing the protein construct, which could include trimming flexible termini or disordered loops, mutating flexible surface residues, or considering different species. He also noted that adding heavy metal ions can actually improve the quality of the crystals as well as making the structures easier to solve. Carien also emphasized the importance of getting the construct right and discussed how seeding (crushing a hard-won crystal and using this to seed new drops) can be very useful. As we’ve noted, screening fragments at extremely high concentrations seems to be the current state of the art, with Novartis moving to 50 mM in the final soak and Diamond going beyond 200 mM! (In contrast to other types of screens at high concentrations, crystallography should not yield false positives, though hits might bind so weakly as to be undetectable by any other method.)

Such a wealth of structures can be daunting, and Anthony Bradley (Diamond) described the construction and use of a “poised library” for follow-up studies. The 768 fragments are (mostly) soluble to 500 mM in DMSO and are designed such that simple chemistry could generate 1.4 million analogs based on reagents currently in stock at Enamine. Potential analogs can be searched using the Fragment Network approach described here, and I was happy to see that Diamond has released their own open-source version (updated link as of 3 Jan 2018).

Jianhua He (Chinese Academy of Sciences) described the facilities at the Shanghai Synchrotron Radiation Facility (SSRF). This is the first third-generation synchrotron in China and has hosted more than 200 research groups since it opened in 2009. Feng Ye, who works at SSRF, gave a talk (in Mandarin) about screening a bacterial protein at XChem; the movies showing liquid handling and robotics would be impressive in any language. Renjie Zhang (Diamond), who also spoke in Mandarin, gave a talk describing (I’m told) not just XChem but how outside users can apply for access. Although there is currently a long waiting list, this should be addressed within the next year or so when SSRF gains Diamond status.

At the 2015 Pacifichem meeting there were only a few speakers from China. Given the level of interest and expertise I saw last week, I predict that the 2020 meeting will see many more.

19 November 2017

Essays in Biochemistry special issue: Structure-based drug design

Structure-based drug design is often an integral part of fragment-based drug discovery. Indeed, a majority of respondents in a recent poll would not work on a fragment without experimental structural information. Given the close relationship between SBDD and FBDD, I was pleased to learn that a recent issue of Essays in Biochemistry is completely devoted to SBDD.

The collection begins with an editorial by issue editors Rob van Montfort and Paul Workman, both at the Institute of Cancer Research. It briefly introduces SBDD and FBDD and provides an overview of the rest of the issue. It also contains a laudable call for rigor, awareness of artifacts, and making data publicly available.

The first full review, by Martin Noble and collaborators at Newcastle University, discusses the role of SBDD in discovering inhibitors of cyclin-dependent protein kinases (CDKs), with a particular focus on selectivity. Several small molecules are discussed, though I do wish the paper included the fragment-derived compound AT7519, which made it to phase 2 clinical trials.

The following paper, by Bas Lamoree and Rod Hubbard (University of York), is completely devoted to FBLD. This is a concise and self-contained review of the field, and is also sufficiently up to date that it provides a good primer on the state of the art.

Chris Abell and collaborators at the University of Cambridge discuss mass spectrometry for fragment screening in the next paper, including ultrafiltration, WAC, HDX-MS, and native mass spectrometry (though not Tethering). The review also includes a handy table summarizing the advantages and limitations of commonly used fragment-finding methods.

Next up is another review devoted to FBDD, this one from Benjamin Cons and his Astex colleagues. The focus is on challenging drug targets such as BCL-family proteins and KEAP1 where SBDD was pivotal, and the researchers particularly emphasize the utility of X-ray crystallography.

NMR was the first experimental technique used for FBDD, and this is the topic of a paper by Gregg Siegal and colleagues at ZoBio. The review includes examples where NMR revealed that crystallographically-determined binding sites were not biologically relevant. Newer techniques, such as NMR2, are also discussed.

Frank von Delft and collaborators describe the fourth funding phase of the Structural Genomics Consortium (SGC), which includes generating a couple dozen “target enabling packages” around new genetic targets. The ten year goals are certainly ambitious: “no crystal structure is complete without a careful analysis of the target’s disease linkage, a fully analysed fragment screen, and a series of follow-up compounds with demonstrated potency and rationalized SAR.” Given the tools and partnerships they have already established, I wouldn’t bet against them.

Hitting a single protein target can be difficult enough, but Scott Hughes and Alessio Ciulli (University of Dundee) focus on ternary interactions, in which a small molecule acts as a “molecular glue” to bring proteins together. PROTACS, molecules designed to target proteins for degradation, comprise one class that has garnered significant attention recently, and as we’ve noted previously FBDD could play a role in discovering and optimizing them. Targeted protein degradation is also the subject of the next paper, by Honorine Lebraud and Tom Heightman (Astex). In particular, the researchers focus on the use of click chemistry to rapidly build chemical probes that degrade specific target proteins.

Crystallographers have steadily been shrinking how big a crystal must be for analysis, in part due to brighter X-ray beams. Michael Hennig and collaborators at leadXpro discuss X-ray free electron lasers, which were experimentally realized less than a decade ago. The energy of these photons is more than a billion times higher than in the newest synchrotrons – so powerful that they destroy the crystals almost instantaneously, but not before producing a diffraction pattern. This means that tens of thousands of individual crystals need to be studied in order to obtain a full dataset. Needless to say the technical and computational demands are intense and still being optimized. The rewards include being able to use weakly-diffracting microcrystals, such as those of membrane proteins, and the ability to collect data at physiological temperatures, as opposed to the cryogenic temperatures typically used.

The last paper, by David Barford and collaborators at the MRC Laboratory, discusses the use of cryo-electron microscopy – which was recognized by a Nobel Prize this year. Single particle cryo-EM does not require a crystal at all, and recent advances have made near-atomic resolution possible. The idea is to image thousands of individual proteins and then computationally reconstruct them. The review discusses multiple protein-ligand complexes, and although none of these are from fragment programs, some of the ligands are approaching the size of fragments.

This collection of papers nicely captures where SBDD currently stands and illuminates the path ahead. For at least a while all the articles are free to download – so check them out now!