23 May 2016

Calculating hotspots in detail

In the eight years since Practical Fragments first started, Moore’s law has held strong and computational power has increased accordingly. Last year we described how tools such as FTMap can be used to identify hot spots – regions on proteins where fragments are most likely to bind. Although FTMap is quite successful at identifying these, it is less able to point to specific interactions (such as hydrogen bond donors or acceptors) that are likely to drive binding. In other words, computational chemists have become adept at identifying where fragments might bind but lag in predicting how. A new paper in J. Med. Chem. by Chris Radoux at the Cambridge Crystallographic Data Centre and collaborators at UCB and the University of Cambridge addresses this challenge.

The approach starts with a set of three simple molecular probes: toluene, to look for hydrophobic interactions; aniline, to look for hydrogen bond acceptors; and cyclohexa-2,5-dien-1-one, to look for hydrogen bond donors. These probes are larger than those (such as ethanol) used in many other programs, the idea being that too-small molecules might find hot spots so small as to be useless. Indeed, with 7 non-hydrogen atoms, these probes are near the low end of the consensus size for fragments.

Calculations are performed on protein structures – either with no ligand bound or with a bound ligand computationally removed – to determine whether each surface atom of the protein is a hydrogen bond donor, acceptor, or hydrophobic, as well as how exposed the particular atom is. The three probes are then mapped onto the proteins to look for favorable interactions. Regions where multiple probes can bind are scored higher, with hotspots defined as those regions of the protein having the highest scores. The type of probe with the highest score also describes what type of interactions are likely to be favorable at various regions within a given hot spot. Although the researchers note that multiple software packages could be used for these calculations, they used a program called SuperStar, and calculations took just a few minutes on an ordinary laptop.

To validate the approach, the researchers used a previously published data set (discussed here) of 21 fragment-to-lead pairs against a variety of proteins for which crystal structures and binding affinities were available. In general, the method was able to identify the fragment binding site quite effectively; the one outright failure was on the fragment with the lowest affinity, which also had poorly resolved electron density in the crystal structure. Importantly, the fragments tended to have the highest scores, with added portions of the leads scoring lower. This data set was used to calibrate the scoring system for identifying hot spots, as well as specific molecular interactions within each hot spot.

Having thus validated the approach, the researchers took a more detailed look at two published fragment-to-lead programs for protein kinase B and pantothenate synthetase. In both these cases, group efficiency analyses had previously been performed to establish which portions of the ligands contributed most significantly to binding. Gratifyingly, the computations correctly predicted these.

Overall this approach appears promising. At a minimum, it is another tool for assessing the ligandability of potential targets. More significantly, by highlighting the hottest bits of hot spots, it could be useful for medicinal chemists trying to optimize and grow fragments and leads. Unfortunately, as currently described, the process will require a skilled modeler. It would be nice if the authors built a simple web-based interface for people to upload pdb files for analysis, as is the case for FTMap. Also, all the data presented are retrospective – a prospective example would be the true test. Does anyone have experience to share?

16 May 2016

Fragments vs JAK – but phototoxicity

The four members of the JAK family of kinases have received plenty of attention due to their role in inflammation. Two drugs that inhibit these targets, tofacitinib and ruxolitinib, have been approved for rheumatoid arthritis and myelofibrosis, respectively. Psoriasis is another possible indication, but for this disease a topical drug might be useful, particularly one with low systemic bioavailability. The search for such molecules is the subject of a paper recently published in ACS Med. Chem. Lett. by Andrea Ritźen and colleagues at LEO Pharma.

The researchers screened 500 fragments at 100 µM each using surface plasmon resonance (SPR) against JAK2. Hits were then tested in a biochemical assay against JAK1; in general, there was good correlation, suggesting a (desirable) lack of selectivity between the two family members. One of the more attractive hits was compound 1, which was characterized crystallographically bound to JAK2.

Compound 1 is an indazole, which is often seen in kinase inhibitors binding to the so-called hinge binding site where the adenine of ATP binds. Other indazoles have previously been reported as JAK2 inhibitors. Nonetheless, the sulfonamide moiety of compound 1 provides an interesting new vector. Moreover, a search of published structures suggested that adding a phenol moiety could make additional contacts to the proteins. This led to the design and synthesis of compound 2, which showed a dramatic boost in potency as well as measurable cell activity. Further optimization led ultimately to compound 34, with low nanomolar biochemical activity and mid-nanomolar cell activity. Compound 34 was also reasonably selective in a panel of 20 kinases.

A topical drug needs to be stable in sunlight, but unfortunately compound 34 showed phototoxicity. This led to the testing of a few other compounds, revealing that even the initial fragment 1 is unstable over a period of a few hours in simulated outdoor light. Indazole itself seems reasonably stable, suggesting that perhaps adding different substituents could fix the problem.

This is a brief but satisfying example of using published information as well as medicinal chemistry to advance a fragment hit. Although the program does not appear to have led to a drug lead, it is laudable that the researchers describe the photoinstability of the indazoles. With these moieties appearing so frequently in campaigns against kinases, this could be a valuable cautionary tale to others pursuing similar scaffolds.

09 May 2016

Fragments vs KEAP1 – crystallographically

Last week we discussed how soaking crystals in high concentrations of fragments could identify useful molecules. Indeed, last month’s Drug Discovery Chemistry conference featured a talk by Tom Davies (Astex) illustrating the power of this approach. In a recent paper in J. Med. Chem., Tom, Jeffrey Kerns (GlaxoSmithKline), and their collaborators provide the full story.

The researchers were interested in the protein KEAP1, which binds to and blocks the activity of the transcription factor NRF2. Small electrophilic molecules covalently react with KEAP1, causing NRF2 to dissociate and upregulate various cytoprotective genes, which could be useful for a variety of diseases. Indeed, this is at least partly how the approved drug dimethyl fumarate seems to work. However, dimethyl fumarate is quite reactive; a more specific molecule could have a better therapeutic profile, and would certainly be useful for probing the complex biology. With this in mind, the researchers sought a non-covalent inhibitor.

NRF2 interacts with a bowl-shaped “Kelch domain” of KEAP1 largely through electrostatic interactions. Thus, not only is this a challenging protein-protein interaction, coming up with a cell-permeable molecule is all the more difficult. The researchers soaked crystals of the Kelch domain against 330 fragments overnight at concentrations of 5-50 mM. Fragments were observed binding in three adjacent hot-spots, and although no functional activity could be detected, the binding modes suggested a path forward.

Growing from fragment 1 toward a hot-spot occupied by an aromatic fragment (magenta below) led to compound 4, with detectable activity in a fluorescence-polarization assay as well as clear binding in an isothermal titration calorimetry (ITC) experiment. Growing compound 4 toward another hot-spot occupied by a sulfonamide-containing fragment (green below) led to the sub-micromolar compound 6, and further optimization resulted in the low nanomolar compound 7. As is often (but not always) the case, the fragment portion of the final molecule binds in virtually the same position as the initial fragment 1 (blue).

Comparison with the structure of the NRF2 peptide reveals that the carboxylic acid in compound 7 binds in a very similar fashion to a glutamate residue in the peptide, and some of the other peptide contacts are also mirrored, but with very different moieties. Importantly, compound 7 has only a single negative charge, balanced lipophilicity, and fills the binding pocket more effectively than the peptide. These properties translate to good biological activity in multiple different cell-based assays, where the compound causes NRF2 translocation to the nucleus, upregulates appropriate gene expression, and prevents glutathione depletion when cells are treated with an organic peroxide. Although the pharmacokinetics have yet to be optimized, it also shows encouraging activity in a rat model of ozone exposure. Finally, it is reasonably selective in a panel of 49 undesirable off-target proteins. All these properties make this at least an excellent chemical probe.

There are several important lessons in this paper. First, as we’ve noted previously, it is possible to replace a highly polar peptide with a much more drug-like molecule. Second, fragments don’t need measurable affinity to be useful starting points. Crystallography is ideally suited for finding such fragments, though Astex researchers reported a similar success story with NMR last year. Finally, progressing these fragments won’t necessarily be easy, as reflected in the 25 authors on the paper. That said, such intensive efforts can pay off, as illustrated last month by the approval of venetoclax against another difficult protein-protein interaction.

02 May 2016

A strong case for crystallography first

We noted last week that one theme of the recent CHI FBDD meeting was the increasing throughput of crystallography. Crystal structures can provide the clearest information on binding modes, and a key function of standard screening cascades is to whittle the number of fragments down to manageably small numbers for crystal soaking. Only a few groups have used crystallography as a primary screen. A team led by Gerhard Klebe at Philipps-Universität Marburg argues in ACS Chem. Biol. that crystallography should be brought to the forefront.

The researchers were interested in the model protein endothiapepsin. As discussed last year, they had previously screened this protein against a library of 361 compounds using six different methods, and the agreement among methods was – to put it charitably – poor. Nonetheless, many hits that did not confirm in orthogonal assays produced crystal structures when soaked into the protein. Thus emboldened, the researchers decided to soak all 361 fragments individually into crystals of endothiapepsin. This resulted in 71 structures, a hit rate of 20%, higher than any of the other methods (which ranged from 2-17%). Even more shocking, 31 of the fragments were not identified by any of the other methods, and another 21 were only identified by one other method. Thus, a cascade of any two assays would have found, at best, only a quarter of the crystallographically validated hits.

In agreement with other recent work, the fragments bound in multiple locations, including eight subsites within the binding cleft as well as three potentially allosteric sites. Not all of these sites were found using other methods.

But are these fragments so weak as to be uninteresting? To find out, the researchers performed isothermal titration calorimetry (ITC) to determine dissociation constants for 59 of the crystallographic hits. Three of the 21 most potent (submillimolar) binders were not detected by any of the other methods, and another seven were only found by one.

What factors led to this crystallographic bonanza? First, the researchers used the very high concentration of 90 mM for each fragment (in practice sometimes <90 mM because of precipitation). Not surprisingly, solubility was important: 97% of the hits had solubilities of at least 1 mM in aqueous buffer, and the soaking solution contained 10% DMSO as well as plenty of glycerol and PEG. Achieving such high concentrations is harder when multiple fragments are present, and the researchers argue from some of their historical data that the common use of cocktails lowers success rates.

How did different methods compare? Interestingly, functional assays such as high-concentration screening or reporter-displacement assays fared best, while electrospray ionization mass-spectrometry (ESI-MS) and microscale thermophoresis (MST) were close to random. This is in marked contrast to other reports for ESI-MS and MST, and the researchers are careful to note that “the choice and success of the individual biophysical screens likely depend on the target and expertise of the involved research groups.”

Primary crystallographic screening was an early strategy at Astex, and although this may not have been fully feasible 15 years ago, it seems they were on the right track. Of course, not all targets are amenable to crystallography, and not everyone has ready access to a synchrotron beam with lots of automation. But for those that are, it might be time to drop the pre-screens and step directly into the light.

25 April 2016

Eleventh Annual Fragment-based Drug Discovery Meeting

The first major fragment event of 2016, CHI’s Drug Discovery Chemistry, was held last week in San Diego. FBDD was the main focus of one track, and fragments played starring roles in several of the others as well, including inflammation, protein-protein interactions, and epigenetics. Also, for the first time this year the event included a one-day symposium on biophysical approaches, which also included plenty of fragments.

In agreement with our polls, surface plasmon resonance (SPR) received at least a mention in most of the talks. John Quinn (Genentech) gave an excellent overview of the technique, packed with lots of practical advice. At Genentech fragments are screened at 0.5 mM in 1% DMSO at 10°C using gradient injection, which permits calculation of affinities and ligand efficiencies directly from the primary screen. Confirmation of SPR hits in NMR is an impressive 80%. A key source of potential error in calculating affinities is rebinding, in which a fragment dissociates from one receptor and rebinds to another. That problem can be reduced by increasing the flow rate and minimizing the amount of protein immobilized to the surface. Doing so also lowers the signal and necessitates greater sensitivity, but happily the baseline noise has decreased by 10-fold in the past decade.

Some talks focused on using SPR for less conventional applications. Paul Belcher (GE) described using the Biacore S200 to measure fragments binding to wild-type GPCRs. In some cases this provided different hits than those detected against thermally stabilized GPCRs. And Phillip Schwartz (Takeda) described using SPR to characterize extremely potent covalent inhibitors for which standard enzymatic assays can produce misleading results. These screens require exotic conditions to regenerate the chip, so it helps that the SensiQ instrument has particularly durable plumbing.

In theory, SPR can be used to measure the thermodynamics of binding by running samples at different temperatures, but John Quinn pointed out that enthalpic interactions dominate for most fragments, so the extra effort may not be worthwhile. Several years ago many researchers felt that enthalpically driven binders might be more selective or generally superior. Today more people are realizing that thermodynamics is not quite so simple, and Ben Davis (Vernalis) may have put the nail in the coffin by showing that, for a set of 22 compounds, enthalpy and entropy of binding could vary wildly simply by changing the buffer from HEPES to PBS! (Free energy of binding remained the same with either buffer.)

Thermal shift assays (TSA or DSF) continued to be controversial, with Ben finding lack of agreement between the magnitude of the shift and affinity, though there was a correlation with success in crystal trials. In contrast, Mary Harner (BMS) reported good agreement between thermal shift and affinity. She also found that it seemed to work better when the fragments bound in deep pockets than when they bound closer to the surface. However, Rumin Zhang (Merck), who has tested more than 200 proteins using TSA, mentioned that some HCV protease inhibitors could be detected despite the shallow active site. Rumin also pointed out that a low response could indicate poor quality protein – if most of the protein is unfolded it might be fine for biochemical assays but not for TSA. Negative thermal shifts are common and, according to Rumin, sometimes lead to structures, though others found this to be the case less often.

What to do when assays don’t agree was the subject of lively discussion. Mary Harner noted that out of 19 targets screened in the past two years at BMS using NMR, SPR, and TSA, 45% of the BMS library hit in at least 1 assay. However, 68% of hits showed up in only a single assay. Retesting these did lead to more agreement, but even many of the hits that didn’t confirm in other assays ultimately led to leads. All techniques are subject to false negatives and false positives, so lack of agreement shouldn’t necessarily be cause for alarm. Indeed, Ben noted that multiple different soaking conditions often need to be attempted to obtain crystal structures of bound fragments.

Crystallography in general is benefiting from dramatic advances in automation. Jose Marquez described the fully automated system at the EMBL Grenoble Outstation, which is open to academic collaborators. And Radek Nowak (Structural Genomics Consortium, Oxford) discussed the automated crystal harvesting at the Diamond Light Source, which is capable of handling 200 crystals per hour. He also revealed a program called PANDAA (to be released soon) that speeds up the analysis of crystallographic data.

Crystallography was used as a primary screen against KEAP1, as discussed by Tom Davies (Astex). A subset of 330 of the most soluble fragments was tested in pools of four, which revealed several hot spots on the protein. Interestingly, an in-house computational screen had not identified all of these hot spots, though Adrian Whitty (Boston University) noted that they could be detected with FTMap. The fragments themselves bound exceptionally weakly, but intensive optimization led to a low nanomolar inhibitor.

Another case in which extremely weak fragments turned out to be useful was described by Matthias Frech (EMD Serono). A full HTS failed to find any confirmed hits against cyclophilin D, but screening by SPR produced 168 fragments, of which six were characterized crystallographically. Although these were all mM, with unimpressive ligand efficiencies, they could be linked or merged with known ligands to produce multiple leads – a process which took roughly one year from the beginning of the screen. Matthias noted that sometimes fragment efforts are started too late to make a difference, and that it is essential to not be dogmatic.

Huifen Chen discussed Genentech's MAP4K4 program. Of 2361 fragments screened by SPR, 225 had affinities better than 2 mM. Crystallography was tough, so docking was used instead, with 17 fragments pursued intensively for six months, ultimately leading to two lead series (see here and here), though one required bold changes to the core. This program is a nice reminder of why having multiple fragment hits can be useful, as the other 15 fragments didn’t pan out.

Finally, George Doherty (AbbVie) gave a good overview of the program behind recently approved venetoclax, which involved hundreds of scientists over two decades. He also described intensive medicinal chemistry which led to a second generation compound, ABT-731, with improved solubility and oral bioavailability.

We missed Teddy at this meeting, and there is plenty more to discuss, so please add your comments. If you did not attend, several excellent events are still coming up this year. And mark your calendar for 2017, when CHI returns to San Diego April 24-26.

18 April 2016

Native mass spectrometry vs SPR

Native state electrospray ionization mass spectrometry (ESI-MS) is, in theory, a fast and easy way to find fragments: just mix protein with fragment, shoot it on the MS, and look for complex. As a bonus, the exact mass tells you whether your fragment is what you think it is (or at least whether it has the right mass). However, published examples are relatively rare, and not always favorable. A new paper in J. Med. Chem. by Tom Peat, Sally-Ann Poulsen, and their colleagues at CSIRO and Griffith University seeks to change this.

The researchers chose the fragment-friendly model protein carbonic anhydrase II (CA II) as their target. They first screened a library of 720 fragments, each at 100 µM, using surface plasmon resonance (SPR). This yielded 7 hits, with affinities ranging from 1.35 to 1280 µM. These seven hits were then assessed by ESI-MS using equimolar concentrations of protein and fragment (10 or 25 µM each). Encouragingly, all seven hits confirmed. Soaking these fragments into crystals of CA II yielded structures for six of them.

This is nice, but of course the real question is how well ESI-MS works as a primary screen. To address this, the researchers chose 70 compounds structurally related to the 7 hits and independently tested these using both SPR and ESI-MS. This yielded 37 hits, of which 24 were detected both by SPR and ESI-MS. In fact, every SPR hit was confirmed by ESI-MS. Of 14 fragments subsequently soaked into crystals of CA II, 7 provided interpretable electron density.

This is impressive, and the researchers note that the level of agreement between SPR and ESI-MS might be better still, since some of the ESI-MS hits did give signals by SPR – they were just weaker than the chosen cutoff (KD ≤ 3 mM). Thus, in contrast to a paper discussed last year, ESI-MS does seem to be a sensitive detection method. In fact, given the low concentration of fragment needed, the researchers suggest that it could be useful for screening fragments with lower solubilities.

So what’s the secret to success? One difference from some previous reports is that the researchers used a 1:1 ratio of protein to fragment. Others have used excess fragment, which could lead to nonspecific binding and aggregate formation. And of course, CA II is a pretty forgiving model protein. I look forward to seeing ESI-MS used as a primary screen on more difficult targets.

12 April 2016

Second fragment-based drug approved

Yesterday the US FDA approved venetoclax (VenclextaTM) for certain patients with chronic lymphocytic leukemia (CLL). This drug, which readers may know more familiarly as ABT-199, was co-developed by AbbVie and Genentech. The drug binds to BCL-2 and blocks its interaction with other proteins.

The first fragment-derived drug approved, vemurafenib, illustrated how quickly FBDD could move: just six years from the start of the program to approval. In contrast, venetoclax is the culmination of a program that has been running for more than two decades; Steve Fesik and his colleagues at Abbott published the X-ray and NMR structure of the protein BCL-xL back in 1996! The original SAR by NMR work was done on this protein, leading to ABT-263, which hits both BCL-xL and BCL-2. Subsequent work revealed that a selective BCL-2 inhibitor might be preferable in some cases, and further medicinal chemistry led to venetoclax.
This drug illustrates the power of fragments to tackle a difficult target by accessing unusual chemical space. It also illustrates creative, fearless, data-driven medicinal chemistry: not only does venetoclax violate the Rule of five, it even contains a nitro group, a moiety red-flagged due to its potential for forming toxic metabolites. This is a useful reminder that in our business rules are more appropriately considered guidelines, to be discarded when necessary.

Clinical results were sufficiently impressive that the drug was given breakthrough status and granted priority review, accelerated approval, and orphan drug designation. The ultimate victory is for the thousands of patients with relapsed CLL who have the 17p deletion on chromosome 17. In the registration trial, 80% of patients showed a partial or complete remission. It is rare to create something that works this well. Congratulations to all who played a role.

11 April 2016

Fragments vs histone demethylases: docking and merging

Tweaking epigenetic machinery is increasingly popular as a therapeutic strategy. Epigenetics often involves modification to proteins – such as histones – that interact with DNA. One common type of modification is methylation of lysine or arginine residues. A couple months ago we highlighted how fragment-based approaches were used to discover inhibitors of a methyltransferase, one of many classes of protein-modifying enzymes that underlie epigenetics. Just as methyltransferases put methyl groups on, demethylases take methyl groups off. In a recent paper in J. Med. Chem., Udo Oppermann, Brian Shoichet, and Danica Fujimori and their collaborators at the University of Oxford and UCSF show that demethylases too can be successfully targeted with fragments. What’s more, the work exemplifies concrete contributions of computational approaches to both identify and advance fragments.

The demethylase KDM4C has been implicated in cancer. This enzyme uses iron, the cofactor α-ketoglutarate (α-KG), and oxygen as part of its mechanism. The researchers ran a computational screen (using DOCK 3.6) of more than 600,000 compounds in the ZINC fragment library. Top-scoring hits were triaged on the basis of novelty and good interactions with the iron atom, and 14 fragments were tested in a functional assay. Remarkably, all of them were active, with 7 showing IC50 values < 200 µM!

Several of the top hits were 5-aminosalicylates such as compound 4. Testing 80 commercial analogs led to low micromolar inhibitors, but these could not be further optimized. Moreover, despite the small size and polarity of these compounds, many of them showed signs of aggregation – a reminder that this type of artifact must always be considered.

Unfortunately, crystallography was also not successful for any of the fragments or analogs. But the researchers noticed that, according to the docking results, fragments such as compound 4 could assume two different binding modes: in one, the carboxylate and phenol interacted with the iron atom, while in the other the carboxylate interacted with lysine and tyrosine residues in the protein. This inspired several ideas for fragment merging, leading to molecules such as compound 45. Additional variations led to mid-nanomolar inhibitors such as compound 35.

As expected, these molecules are competitive with the α-KG cofactor (which normally binds to the iron atom) but not with the peptide substrate. Many also showed encouraging selectivity profiles against other demethylases, though no cell data are reported. Finally, crystallography mostly confirmed the predicted binding models for several of the merged compounds, including compound 35.

This is a lovely example of using computational approaches not just for fragment-finding, but for fragment merging as well. As the authors point out, this was done not to showcase computational methods but because crystallography didn’t initially work. Even in the short lifetime of Practical Fragments, in silico methods have made remarkable progress, and this is another milestone. It will be fun to see further optimization of these molecules.

06 April 2016

Biophysics: not just for fragments

Biophysics and fragment-based drug discovery go together like Nutella and strawberries. Indeed, SAR by NMR ushered in the dawn of fragment-based methods two decades ago, and most fragment-based programs today make use of NMR, SPR, and/or ITC – not to mention X-ray crystallography. Interestingly, the same is not necessarily true for high-throughput screening (HTS) programs. In a recent paper in Drug Discovery Today, Rutger Folmer makes a strong case for engaging biophysics early and often in HTS. He bolsters his argument with more than 20 examples from internal programs at AstraZeneca.

The first descriptions of using NMR to profile HTS hits were not published until several years after SAR by NMR, but they were rather shocking, with up to 98% of hits failing to confirm. Nor is this merely a historical problem, as discussed here. Aggregators, redox cyclers, generically reactive covalent modifiers – all of these are problems not just in fragment screening but in HTS as well. Sometimes the most potent hits are artifacts, particularly for more difficult targets. The key to triaging out pathological actors is to assess binding and not rely solely on inhibition.

That means bringing biophysics into hit profiling at the earliest stages, before trying to optimize fruitless hits. As Rutger points out, it is often difficult to rally colleagues to look at less active molecules after they have wasted months pursuing more potent dead ends.

And biophysics can make an impact even before running screens. Profiling published tool compounds or in-licensing opportunities with biophysical techniques can reveal unwelcome surprises. Testing the output of early HTS pre-screens (7000-10,000 compounds) before a full HTS (2 million compounds at AstraZeneca) can reveal whether an assay is particularly susceptible to false positives. In some cases this can result in reconfiguring the assay, for example by choosing a different detection technology or modifying the protein construct.

A key element to gaining such benefits is organizational commitment. At AstraZeneca, a biophysicist is assigned to a project team immediately after target selection – well before any screens are run. This seems like prudent practice. How many other organizations are doing this?

01 April 2016

An interview with Dr. Saysno

Readers of a certain age may fondly remember the interviews with Dr. Noitall that used to enliven the pages of Science. Sadly, he died a few years back. But his cousin, Dr. Saysno, is still very much alive. Practical Fragments caught up with him at a recent conference in Shutka.

Practical Fragments (PF): Dr. Saysno, you've stated that experts should never be trusted.

Dr. Saysno (DS): Niels Bohr defined an expert as a person who has made all the mistakes that can be made in a very narrow field. If someone has made every possible mistake, how could you possibly trust them?

PF: But don't you think they may have learned from their mistakes?

DS: Balderdash! Hegel was right: the only thing we learn from history is that we learn nothing from history.

PF: What's your opinion of ligand efficiency (LE)?

DS: Ligand efficiency is an abomination! It's mathematically invalid!

Worse, determining the free energy of binding from a dissociation constant is not even wrong: if you change your definition of standard state, you can make ΔG° any positive or negative number you want. Just how relevant do you think your definition of standard state is on the surface of Venus? Or Pluto?

For the same reason, pH is utterly meaningless. You really ought to throw out your pH paper, not to mention your pH meters, since they all assume an arbitrary reference state.

PF: But what about all the researchers who find pH and LE useful?

DS: Usefulness is the last refuge of the scoundrel!

Look, many of the best selling drugs on the market are antibodies, and when you calculate their ligand efficiencies, they are close to zero. How can you have a metric that doesn't work on some of the most important drugs out there?

I only believe in equations that are universal and apply in all situations, unsullied by the physical world. Anything that involves standard states is just mumbo-jumbo.

PF: What do you think of pan-assay interference compounds, or PAINS?

DS: Now that’s a topic that really gets my blood boiling! PAINS were defined on the basis of just six assays. Six assays I tell you!!! [DS vigorously pounds his shoe on the desk.] Just because something hits six assays – or six hundred for that matter – doesn’t mean it will hit the six hundred and first!

PF: But aren't there some chemical substructures that are so generically reactive they should never be used in probes?

DS: Nothing is universal! All molecules are unique, like little snowflakes. If a compound comes up as a hit in your assay, by all means publish it as a chemical probe in the best possible journal, and try to encourage suppliers to start selling it so other people can use and cite your brilliant discovery.

No one has a right to criticize your molecule unless they test it against every single protein in the human body and show that it hits all of them.

When the revolution comes, the imperialist PAINS stooges will be swept into the dustbin of history along with the lackeys of ligand efficiency!

PF: So if you don't trust experts, you don't like metrics, and you can't make generalizations, how can we move forward in science short of deriving every result ourselves from first principles?

DS: That's simple: just ask me!

21 March 2016

Fragments vs bacterial GyrB

Anti-bacterial targets are not common among fragment-based lead discovery efforts. We’ve written previously about AstraZeneca’s work on DNA gyrase, which led to a clinical candidate. In a recent paper in Bioorg. Med. Chem. Lett., Michael Mesleh and collaborators at Cubist and Evotec describe their efforts on this protein.

Bacterial DNA gyrase has two subunits, GyrA and GyrB, and is essential during DNA replication. It is also well-validated, being the target of decades-old antibiotics such as the fluoroquinolones. The researchers started by screening a library of 5643 fragments against Staphylococcus aureus GyrB using STD NMR, yielding 304 hits. These were winnowed down to 46 based on intensity of the STD signal, novelty, and ease of follow-up chemistry. These were then tested using chemical shift perturbations and crystallography. Although several crystal structures of fragments bound to GyrB were obtained, these did not suggest clear ways to advance the hits.

On the other hand, compound 5, which showed only weak binding by NMR and did not result in a crystal structure, was appealing because of its novelty and polarity. The researchers knew that most ligands that interact with GyrB make a pair of hydrogen-bond donor-acceptor interactions, and they used that knowledge to surmise a binding model. This suggested that growing the fragment towards a pair of arginine residues could improve affinity and led to the synthesis of compound 8, with low micromolar activity.

A crystal structure of a related molecule confirmed the model and also suggested that removing the methyl group would stabilize a more planar conformation better matched to the binding site. Doing so (compound 9) yielded a ten-fold boost in potency. (This methyl is also a nice example of Teddy’s “Sauron Atom”). In a separate paper published last year, the researchers further optimized this molecule to compound 2, with low nanomolar potency and activity in animal models.

Several things stand out about this paper. First, the researchers were willing to pursue a fragment with an affinity lower than other hits. Second, careful modeling and conformational analyses were critical in advancing the molecules. Finally, crystallography was not used in the initial fragment growing. Of course, it helped that the researchers were working with a well-characterized protein amenable to modeling. Still, it is nice to see another example of advancing fragments in the absence of experimentally-determined structures.

14 March 2016

EthR revisited again: fragment merging this time

Fragment linking, growing, and merging: these are the main methods for enhancing affinity. Two years ago we highlighted a fragment screening effort against the tuberculosis target EthR, which involved fragment linking. A few months later we discussed fragment growing against the same target by a different group. Now the first group, led by Chris Abell at the University of Cambridge, has published a new paper in Org. Biomol. Chem. describing fragment merging.

In the original paper, a thermal shift assay had led to the discovery of a few dozen fragments, several of which were characterized crystallographically bound to EthR. In some cases, two molecules of the same fragment could bind in the large lipophilic cavity of the protein and block binding to DNA, as assessed by SPR. Capitalizing on this, two copies of compound 1 were linked together to generate a micromolar binder.

In the new paper, the researchers tried merging compound 1 with another fragment, compound 2, which also binds at two positions within the protein. Several merging strategies were attempted, and although they all stabilized the protein against thermal denaturation and could be characterized crystallographically bound to the protein, most were no better at blocking DNA binding than the original fragments. Compound 5, however, did show enhanced activity, and was the subject of additional SAR. This led to compound 15, which showed low micromolar binding by isothermal titration calorimetry (ITC) and functional activity. (Oddly, compound 1 appeared to bind considerably more tightly by ITC than suggested by its functional activity, perhaps a result of having two binding sites.) The crystal structure of the optimized, merged compound bound to EthR revealed that compound 15 binds as expected (gray), overlaying with one copy each of compound 2 (magenta) and compound 1 (cyan). 

Unfortunately, aside from compound 1, none of the molecules showed activity in a cell-based assay. The researchers propose that this is due to poor permeability across the notoriously impenetrable envelope of the mycobacterial envelope. All in all this is a nice story, as well as a sobering reminder that while potency is important, it is just one of many properties that need to be optimized.

As to the question of whether one should apply growing. linking, or merging, a single case study does not really permit generalization. However, it is satisfying that all three techniques can lead to early leads.

07 March 2016

Fragments vs choline kinase alpha

Fragments and kinases have a long and successful association, as demonstrated by nearly half of FBLD-derived clinical candidates. Most of the attention has been on protein kinases, which transfer phosphate groups to other proteins. But there are more kinases out there, and in a recent paper in J. Med. Chem., Stephan Zech and colleagues at Ariad describe how they developed inhibitors against choline kinase α (ChoKα), a potential anticancer target involved in phospholipid synthesis.

A screen of known kinase inhibitors came up largely empty, so the researchers used STD NMR to screen a library of 1152 diverse, rule-of-three compliant fragments in pools of five, each at 3 mM. This yielded 55 hits, which were then tested in a fluorine-detected NMR (FAXS) assay to see whether they could displace molecules that bind in either the ATP or substrate binding sites. These experiments suggested that 13 fragments bind in the choline binding site while 21 bind in the ATP site; the remaining 21 either bind elsewhere or are artifacts.

Most of the fragments showed minimal activity in functional assays, but compound 11 was an exception. SPR confirmed binding, though it also seemed to bind to an unrelated protein and displayed super-stoichiometric behavior at high concentrations. Nonetheless, it could successfully be soaked into crystals of ChoKα, and the resulting structure revealed that it binds deep in the choline-binding pocket, with the terminal methyl group in a small pocket at the bottom. This is also consistent with STD epitope mapping of a related fragment, which showed that the azepane ring was closely associated with the protein.

An initial search for commercial analogs followed by several rounds of medicinal chemistry led to compound 43, with low micromolar potency, and several more rounds of optimization led to compound 65, with nanomolar binding (by SPR) and inhibition. A crystal structure of this molecule bound to the enzyme revealed that the fragment binding mode is roughly conserved, while the added basic moiety binds close to several acidic residues near the surface of the binding pocket. A very closely related molecule showed low micromolar activity in several cell-based assays (data for compound 65 is not reported). 
This is an interesting paper for several reasons. First, it reports the successful use of fragment screening against an unusual target. Second, although multiple fragments were found to bind in the ATP-binding site (a productive starting point for many conventional kinases), these fragments could not be optimized. On the other hand, a fragment that binds in the choline-binding site could rapidly be improved to nanomolar inhibitors. Third, although fragment 11 did show some red flags, it was ultimately optimizable – a reminder that some misbehavior in a fragment should not necessarily disqualify it. Finally, the iterative and structure-based nature of the medicinal chemistry – which is well beyond what this brief blog post can cover – makes a nice case study in fragment growing. Of course, the final molecule still has high hurdles to surmount, and it will be fun to see the story progress.

29 February 2016

Fragment events in 2016 (updated)

The first FBDD-related meeting of 2016 has already come and gone, but there are still plenty of events ahead. Below are several updates as well as a new listing.

April 20-21: CHI’s Eleventh 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 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. Also as part of this event, Ben Davis and I will be teaching a short course on FBDD over dinner on April 20.

May 24: Development of Novel Therapies through Fragment Based Drug Discovery will be held in Houston, Texas. Despite being only one day, it looks like a great lineup of speakers, so check it out!

June 7-10: Although not strictly fragment-focused, the third NovAliX Conference on Biophysics in Drug Discovery is likely to have lots of relevant talks, and is a good excuse to get to Strasbourg, France. You can read Teddy's impressions of the 2013 event herehere, and here.

July 12-15: FBDD Downunder 2016 will be held at Monash University in Melbourne. This is only the second such event; the first was lots of fun and even resulted in a special issue of the Aust. J. Chem., so definitely check this out if you can.

October 9-12FBLD 2016 will be held in Boston, MA. This marks the sixth in an illustrious series of conferences organized by scientists for scientists, the last of which was in Basel in 2014. Surprisingly, this also seems to be the first dedicated fragment conference in Boston. You can read impressions of FBLD 2012FBLD 2010, and FBLD 2009. Early-bird registration is now open!

November 7-9: Finally, the OMICS Group is holding their second Drug Discovery & Designing in Istanbul, Turkey, with a track on FBDD.

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

22 February 2016

Fragments vs PRMT6

Epigenetics involves turning genes on or off without changing their sequence. This often relies on modifications to proteins or DNA that are recognized by other proteins. As Teddy pithily observed, this is a big field. However, in the realm of fragments, most of the attention has been on bromodomains; other classes of proteins, such as methyltransferases, have been largely neglected. A new paper in J. Med. Chem. by Masoud Vedadi and Matthieu Schapira and collaborators at the University of Toronto and Bayer suggests fragments are promising for these targets as well.

The researchers were specifically interested in protein arginine methyltransferases (PRMTs), which transfer a methyl group to one of the terminal side chain nitrogen atoms on specific arginine residues. PRMT6 in particular targets histone proteins to modulate transcription and has been implicated in cancer as well as neurodegenerative diseases. A few potent inhibitors have previously been reported for PRMTs, and the team started by deconstructing them to hunt for active fragments.

Ligand deconstruction involves chopping a known ligand into fragments to see whether any of these pieces will still bind. In this particular case, EPZ020411 had previously been characterized crystallographically bound to PRMT6 with the basic amine-containing “tail” in the substrate arginine-binding groove. Testing this fragment 6 by itself revealed a low micromolar inhibitor with a ridiculously high ligand efficiency.
Thus encouraged, the researchers ran a functional screen of 2040 diverse fragments (about half from Maybridge) at 1 mM concentration and retested hits at 0.5 mM. About half the resulting hits were false positives or other uglies, leaving the researchers with 14 fragments with IC50 values from 0.3 – 400 µM. As might be expected given the cationic nature of the substrate, 12 of these have basic nitrogens.

Compound 7 was particularly interesting: at 300 nM this is one potent fragment! ITC revealed a dissociation constant of 970 nM, with a favorable enthalpy and unfavorable entropy of binding. It did hit other PRMTs, but was remarkably selective against a panel of 25 other human methyltransferases.

The researchers also determined the crystal structure of compound 7 bound to PRMT6, which revealed it binding, as expected, in the arginine site, making hydrogen bonds with a conserved catalytic glutamic acid. Weirdly though, it seems to be a noncompetitive inhibitor: increasing concentrations of substrate peptide or cofactor had no effect on inhibition. The team speculates that the noncompetitive behavior could be because the substrate makes strong interactions with the protein outside the arginine-binding site. Nonetheless, the fragment did inhibit PRMT6 activity in a cell assay with IC50 = 21 µM.

Overall then it seems that the PRMTs are amenable to FBLD. They are interesting drug targets, and at the very least having more probes will help to unravel the biology.

15 February 2016

Selectivity in STD

Among NMR-based fragment screening methods, saturation transfer difference (STD) came in as most popular in a recent poll. The technique is very sensitive and thus able to identify weak fragments. Unfortunately, it’s a bit too sensitive; hit rates of >30% are not uncommon. Many of these hits interact non-specifically with the protein. These can be weeded out using orthogonal screening methods or competition assays, but it would be nice to make STD itself more discriminating. In a paper published late last year in J. Med. Chem., Olivier Cala and Isabelle Krimm describe how to do just this.

In an STD experiment, the protein target is irradiated and transfers some of its magnetization to bound ligands. When these dissociate they retain some of the magnetization, and so the NMR signals of the fragments decrease. The problem is that lots of fragments interact non-specifically with proteins. For example, if a lipophilic fragment dances across greasy patches on a protein surface to escape from water without making any specific interactions, it will still get magnetized. Can such signals be distinguished from fragments that bind in a single, well-defined manner?

Within a ligand that binds to a protein, a proton that binds closer to the protein will show a stronger STD effect than one that is exposed to solvent. This is in fact the basis for STD “epitope mapping”, which allows one to roughly model how a ligand binds to a protein – or at least which parts of a ligand are closest to the protein. In the new paper, the researchers argue that simply observing differences in the STD effect between different protons in a ligand can distinguish whether or not that ligand is binding in a single binding mode.

Several examples support this assertion. For one protein, all the fragments that showed significant epitope mapping could be competed with a known reference molecule, suggesting binding to a specific site; this was less often the case for fragments that did not show epitope mapping. In another example, the privileged fragment 7-azaindole was found to bind to two different proteins with different epitope maps, suggesting different (but specific) binding modes for each protein. The technique also seems fairly robust to the affinity of the fragments (KD 50 µM to > 1 mM), the details of the NMR experiment (saturation time from 0.5 to 4 seconds), and ligand/protein ratios between 66 to 1 and 400 to 1.

As the researchers note, there are caveats. For example, if a fragment can bind in two different but nonetheless specific binding modes, it may show uniform STD effects and will be a false negative. Nonetheless, comparing STD effects across a ligand does seem a worthwhile exercise. Not only could it help prioritize fragments, it could also reveal which protons are further from the protein, and so suggest growth vectors.

08 February 2016

Dihydroisoquinolones as fragments

It’s a common problem: you find a fragment that binds to your target and want to grow it to improve affinity. A search for commercial analogs comes up empty, so you look into modifying the hit, only to discover that you’ve got a six-step synthesis on your hands. Or worse; perhaps there is no precedent at all. The chemical literature is replete with total syntheses of complicated natural products, but seemingly simple fragments are often not well-represented. Last year, researchers from Astex exhorted chemists to develop synthetic routes for attractive fragments, and in a recent paper in Org. Biomol. Chem. David Rees and colleagues take up their own challenge in the case of dihydroisoquinolones.

Dihydroisoquinolone itself is a nifty little fragment. It has just 11 atoms, cLogP = 1.0, and its solubility is > 5 mM in aqueous buffer. Its cis-amide moiety can serve as a hydrogen bond donor and acceptor, and the adjacent phenyl ring provides a bit of grease for interacting with hydrophobic protein residues.

The researchers built on existing methodology using a rhodium catalyst to introduce polar groups (such as hydroxymethyl and dimethylamino) at the R position. Depending on the nature of the R group, regioisomers in which the substituent ends up at the 4-position could sometimes also be isolated.

The methodology is robust and tolerates air, moisture, and various substituents. The alkene starting material is easy to come by, and the aromatic starting material is easy to make. By varying this, the researchers could generate 6- or 7- substituted dihydroisoquinolones, though 5- and 8- substituted versions seem harder to access. The team was also able to use other aromatics as starting materials, including thiophene, thiazole, and pyridine.

Thus, if dihydroisoquinolone comes up as a hit, this paper will allow you to quickly explore most of the vectors. So how often does this fragment show up? It is not clear why some fragments, such as 7-azaindole and 4-bromopyrazole, show up again and again, while others languish so lazily in the library that they might as well not even be there. We’ve highlighted at least one case where a dihydroisoquinolone was a useful hit.

Practical Fragments would love to know your experience. Do you have dihydroisoquinolones in your library? How often do they show up as hits? And what other fragments do you find that are in need of better synthetic routes for further exploration?

01 February 2016

Fragment-Based Drug Discovery: Lessons and Outlook

In 2006, Wolfgang Jahnke and I co-edited the very first book on fragment-based drug discovery. Half a dozen books have followed, most of which have been reviewed at Practical Fragments (see right-hand column). These are now joined by a new book edited by Wolfgang and me in Wiley’s Methods and Principles in Medicinal Chemistry series.

At 500 pages and 19 chapters, this is the most extensive treatment since the Methods in Enzymology volume five years ago. In the interest of space I can’t write more than a sentence or two about each chapter, but I would like to thank all the contributors. Although I’m undoubtedly biased, I believe this work will set the standard for years to come.

The book is divided into three sections, starting with The Concept of FBDD. Rod Hubbard (Vernalis and University of York) opens with a chapter on the role of FBDD in lead-finding, which provides an introduction, historical overview, and summary of current thinking and future challenges. One particularly interesting section compares the contents of the 2006 book with the state of the art today, highlighting the fact that many of the basic techniques were already in place a decade ago, but the number of success stories has increased dramatically.

Chapter 2, by Glyn Williams and colleagues at Astex, discusses how to choose targets for FBDD, including concepts such as ligandability. Key principles are nicely illustrated with several important targets including the IAPs and HCV-NS3.

The last two chapters in this section focus more on numbers. Chapter 3, by Jean-Louis Reymond and colleagues at the University of Berne, covers the computational enumeration of chemical space, with a special emphasis on the contents and uses of their GDB-17 set of the 166 billion possible molecules with up to 17 non-hydrogen atoms. And chapter 4, by György Ferenczy and György Keseű at the Hungarian National Academy of Sciences, provides an overview of various metrics (such as ligand efficiency and LELP) and how these can be useful for fragment optimization.

The next nine chapters comprise the longest sub-section of the book, Methods and approaches for FBDD. To start screening fragments, you need a library, and designing one is the subject of chapter 5, by Martin Drysdale and colleagues at the Beatson Institute. This chapter also touches on concepts such as molecular complexity and “three-dimensional” fragments.

Screening techniques are best used in combination, and in chapter 6 Ben Davis (Vernalis) and Tony Giannetti (Google[x]) describe the synthesis of results from SPR, NMR, X-ray, ITC, functional screens, and other techniques to overcome challenges in several discovery programs. They emphasize that universal agreement among different methods is not always necessary, but carefully analyzing discrepancies can reveal unexpected problems with the screening conditions, target, or hits.

Differential scanning fluorimetry (DSF) – or thermal shift (TS) – is perhaps the most controversial screening method, and in chapter 7 Chris Abell and colleagues at the University of Cambridge cover this approach in depth. The chapter starts with a thermodynamically detailed yet nonetheless lucid discussion of the theory behind DSF, including the interpretation of negative thermal shifts. The chapter also includes plenty of practical advice and case studies, some of which we’ve covered briefly (for example here and here).

Chapter 8, by Sten Ohlson and Minh-Dao Duong-Thi at Nanyang Technological University, covers three emerging fragment screening technologies: WAC, native MS, and MST. And Chapter 9, by Sandor Vajda (Boston University) and collaborators, does an excellent job of summarizing computational approaches.

As others have noted, some of the biggest challenges are not technical but organizational, and in chapter 10 Michelle Arkin and colleagues at UCSF describe how to make FBDD work in academia. The chapter also includes some interesting polling data, concise but cogent summaries of fragment-finding techniques, and case studies on p97 and caspase-6. And in chapter 11, Jim Wells and colleagues – also at UCSF – describe using Tethering to find allosteric sites in proteins.

One area that has grown dramatically since 2006 is the use of FBDD in complex systems (such as membrane proteins), the subject of a chapter by Miles Congreve and John Christopher at Heptares. Chapter 12 also includes successful case studies, some of which we’ve covered. But finding fragments against these targets is still not easy, as illustrated in the final figure: of 18 fragment hits on 15 targets, almost all have ligand efficiency values > 0.3 kcal/mol per atom, and most of them are relatively potent, with affinities in the mid-micromolar range or better. While everyone wants to find strong binders from the start, such numbers suggest many weak-binding hits are overlooked.

Chapter 13, by Jörg Rademann and colleagues at Freie Universität Berlin, covers protein-templated fragment ligation methods, both reversible and irreversible. The chapter is wide-ranging and includes methods such as dynamic libraries and various types of “Click” chemistries.

The last section of the book, which was mostly absent a decade ago, is entitled Successes from FBDD. This starts with a chapter by Daniel Wyss, Andrew Stamford, and colleagues from Merck on BACE inhibitors. As we’ve noted, fragments have had a major role in most of the BACE inhibitors to enter the clinic, with phase III results from Merck’s verubecestat expected next year.

Epigenetics has also been strongly influenced by fragments, and in chapter 15 Aman Iqbal (Proteorex) and Peter Brown (Structural Genomics Consortium) survey the field, with case studies on several proteins that modulate epigenetic marks. These include BRD4, ATAD2, BAZ2B, SIRT2, and others.

One of the original selling points of fragment-based methods is the ability to go after difficult targets such as protein-protein interactions, and this is the subject of chapter 16, by Feng Wang and Stephen Fesik (Vanderbilt University). In addition to general guidelines, the researchers describe a number of case studies, including RPA, MCL-1, and K-Ras.

Some enzymes can be just as difficult as protein-protein interactions, and in chapter 17 Alexander Breeze (University of Leeds) and former AstraZeneca colleagues describe programs to find inhibitors of LDHA (see here and here). They also discuss how some previously reported inhibitors turned out to be artifacts.

More than two dozen kinase inhibitors have been approved by the US FDA, including the first drug derived from FBDD. In chapter 18, Gordon Saxty (Fidelta) surveys a number of kinase programs, including most of the fragment-derived inhibitors in clinical trials.

And finally, in chapter 19 Simon Rüdisser and colleagues from Novartis present an extensive discussion of renin, with special attention to their campaign, which involved a combination of HTS and fragment-based approaches.

While it may not be possible to judge a book by its cover, the cover of this book does illustrate some of the fruits of the field, with structures of three fragment-derived drugs that have entered the clinic. These are just a small fraction of the 30+ drugs working their way through the pipeline, and of the many more that will spring from the research described and informed by the work presented.

25 January 2016

Fragments vs plasmepsins

Plasmodium, which causes malaria, is a nasty bag of tricks. These include the plasmepsins, aspartic proteases that – among other duties – digest the hemoglobin in red blood cells. In a recent paper in J. Med. Chem., an international group of collaborators led by Kristaps Jaudzems and Aigars Jirgensons at the Latvian Institute of Organic Synthesis describe how they discovered inhibitors.

The team started by performing a fragment screen against plasmepsin II (Plm II), one of ten plasmepsins encoded in Plasmodium falciparum. A library of 976 rule-of-three compliant fragments (from ChemBridge) were screened in pools of six using three different NMR methods: STD, WaterLOGSY, and T1ρ. A total of 49 fragments hit at least two assays and were competitive with a known aspartic protease inhibitor, and ten of these showed functional inhibition in an enzymatic assay. Fragment 1 was the most potent.

Crystallography was unsuccessful, but the researchers were able to use ILOE NMR to show that another aromatic fragment could bind near fragment 1. Based on this information, the researchers appended a phenyl moiety to produce compound 3a and obtained a 10-fold boost in potency.

Crystallography still didn’t work, but modeling based on similar compounds on a different aspartic protease suggested that adding another hydrophobic substituent could fill another pocket, leading to compound 3b. At this stage the researchers were finally able to obtain a crystal structure of compound 3b bound to Plm II, which confirmed the predicted binding mode and also revealed another pocket that could be grown into as in in the case of compound 4b. This and several related compounds inhibited the growth of Plasmodium falciparum at low micromolar concentrations and were minimally cytotoxic to mammalian cells.

There is still much to do. Selectivity for the one human aspartic protease tested was generally modest. Also, as the researchers acknowledge, the most active compounds are seriously lipophilic. Still, this is another example of fragment-based lead discovery in academia. More importantly, it provides more ideas on how to tackle a pernicious parasite.