Fragment flipping during optimization

Last month we highlighted a study that asked how often the binding mode of a fragment changed during optimization. A new paper in J. Med. Chem., by Swen Hoelder and collaborators at Institute of Cancer Research, University of Oxford, and Universitat de Barcelona provides an interesting case study.

The researchers were interested in the kinase ALK2, which is implicated in an aggressive and universally fatal childhood cancer called diffuse intrinsic pontine glioma. They started by screening a library of fragments designed to target kinases, which yielded compound 1. This compound actually contains two moieties that are known kinase hinge binders, a quinazolinone and a pyrazole. Unfortunately the researchers could not obtain a crystal structure of the fragment bound to ALK2, but SAR suggested that the pyrazole was not essential, and indeed replacing this with a quinoline led to compound 7, with sub-micromolar activity.

Next the researchers introduced methyl groups at various positions around the quinazolinone and found that these neither significantly improved nor decreased binding. Modeling based on similar reported molecules led them to grow the molecule towards solvent, ultimately leading to the mid-nanomolar compound 16 (blue in figure below), which they characterized crystallographically bound to ALK2. The molecule bound as expected, with the unsubstituted nitrogen of the quinazolinone forming a hydrogen bond to the hinge region of the kinase.

So far so good, but the researchers were still curious about some of their earlier SAR. In particular, the methyl groups added to some of the molecules should have been incompatible with the observed binding mode of compound 16, suggesting an alternative binding mode for these molecules. This insight proved correct, and in fact adding two methyl groups to compound 7 led to compound 21, which is more potent than compound 7 and binds such that the amide of the quinazolinone core forms hydrogen bonds with the hinge region, as confirmed by crystallography (green in figure).

Compounds 16 and 21 have similar affinities yet different binding modes, so what about selectivity? Testing them in a panel of ~110 kinases revealed both to be quite selective for ALK family kinases, though they had different off-targets. The selectivity of compound 21 is particularly impressive given its small size – it teeters on the edge of being rule of three compliant. A related molecule also showed activity in a cell-based assay.

An interesting unanswered question is the binding mode of the initial fragment. Perhaps it binds in multiple orientations, which could explain why crystallography was unsuccessful. Regardless, this is a nice study that illustrates how close attention to confusing SAR can lead to attractive new series.

From generic fragment to selective BET-family BD2 inhibitor

Fragments have been a rich source of leads against bromodomain-containing proteins, epigenetic readers that recognize acetylated lysine residues and are implicated in a variety of diseases. The four members of the BET family in particular have been heavily explored. Each of these proteins actually contains two separate bromodomains, called BD1 and BD2, and most reported inhibitors hit both of them more or less equally. To follow up on some intriguing biological hints that this may not be necessary, Robert Law and collaborators at GlaxoSmithKline and University of Strathclyde pursued selective BD2 inhibitors, which they describe in J. Med. Chem.

The researchers started with a fragment they first reported six years ago, and which has been used by Forma as the starting point for one of their own programs. Although fragment 10 is equipotent against BD1 and BD2 of BRD4, growing led to compound 12, with an encouraging 60-fold selectivity for BD2 (all values shown below are for BRD4 BD2). A crystal structure of a close analog suggested several opportunities for further growth to improve potency.

Changing the methyl group to a cyclopropyl group improved selectivity, and introducing a hydroxymethyl substituent off the phenyl ring (compound 44a) improved potency for BD2. This molecule was fairly lipophilic, so the researchers explored adding a variety of polar substituents to improve solubility, ultimately resulting in GSK340.

GSK340 was profiled against 35 bromodomains and found to be at least 40-fold selective for the BD2 domain compared to the BD1 domain of the four BET family members. It showed the highest affinity for BRD4 but also bound tightly to the BD2 domains of BRD2, BRD3, and BRDT and was selective against non-BET family bromodomains. The compound was cell permeable and inhibited the release of the inflammatory cytokine MCP-1, supporting the notion that BD2 domain inhibition alone could have useful anti-inflammatory effects. Unfortunately GSK340 shows sufficiently high clearance in rat and human hepatocytes that the researchers suggest its utility will be limited to in vitro assays. Still, this paper provides another illustration that – with the help of creative medicinal chemistry – a generic, non-specific fragment can lead to a novel and selective chemical probe.

256th American Chemical Society National Meeting

This week thousands of chemists converged on the venerable city of Boston for the Fall National ACS meeting. One brief but rich symposium was entitled “Best practices in fragment-based drug design,” organized by Amy Hart, David Marcoux, and Heidi Perez (BMS).

After an introductory presentation by me, Anil Padyana described FBDD at Agios. The company is focused on metabolic enzymes, many of which have dynamic, shallow, and polar active sites – challenging even for fragments! Indeed, a summary of 11 targets screened using a variety of methods revealed generally low hit rates, usually < 3%. The company’s first approved drug came out of an HTS screen against IDH2 of 80,000 compounds that yielded just 24 hits. The molecule that ultimately led to enasidenib was essentially a (large) fragment, with 21 atoms. Agios’ current fragment library is just over 5000 about 10,000 molecules, though they are in the process of expanding this to 20,000 – perhaps part of a general trend. Given the history of enasidenib, they are including molecules beyond the rule of 3, with an upper molecular weight limit of 350 Da, far higher than most respondents in our recent poll.

Cullen Cavallaro presented an early, though still unpublished, FBDD story from BMS: KAT II, a brain enzyme implicated in schizophrenia. Screening 3700 fragments using NMR, SPR, and TSA yielded 236 hits, only 6 of which were common to all methods. All 236 hits were soaked into crystals of KAT II, resulting in 43 structures, 13 of which bound in the active site. Strikingly, 12 of these contained carboxylic acids, which generally don’t cross the blood brain barrier. The lucky thirteenth fragment showed no activity in an enzymatic assay, no thermal stabilization, and only a marginal STD NMR signal. However, through a combination of library synthesis and structure-based design the researchers were able to obtain nanomolar inhibitors. Unfortunately the project was stopped when BMS exited neuroscience.

Anna Vulpetti provided an overview of work done by her and her Novartis colleagues to discover inhibitors of Factor D. A high-throughput screen of the serine protease didn’t yield anything useful, but a combination of fragment screening and structure-based design led to multiple series of inhibitors. Anna is a proponent of fluorine NMR, and Novartis has recently expanded its fluorinated fragment library to 4000 members. Like Agios, they have chosen to include some larger fragments, up to 350 Da.

Finally, David Norton described the initial work done at Astex to discover an orally available ERK1/2 inhibitor, which entered phase 1/2 clinical trials in May of this year. We highlighted some of this work a couple months ago so I won’t cover it in detail, but among other lessons David emphasized the importance of initial fragment optimization before starting to grow.

There were plenty of fragment talks outside the symposium too. Last year we highlighted Ben Cravatt’s strategy for performing fragment screening in cells. Chris Parker, the first author on that paper, has just launched his independent academic career at Scripps Florida, and provided an update. Chris noted that, for phenotypic screening, the approach is essentially a target-finding method, and indeed more than 4000 proteins have been identified, varying over five orders of magnitude in abundance. Having proper controls is critical, and recent efforts include screening pairs of enantiomeric fragments and looking for differences.

Taekyu Lee provided an update of the Vanderbilt MCL-1 program, most recently described in this paper. Some of the molecules shown have low picomolar affinity, mid nanomolar cell activity, and are more than 10,000-fold selective for MCL-1 over BCL-2 and BCL-x_L. The program was partnered with Boehringer Ingelheim earlier this year, and they’ve got competition: four other molecules have entered the clinic.

Covalent fragments were also a theme. Peter Wipf (University of Pittsburgh) described the construction of a 300 compound “mercaptophilic” library. In contrast to other academic reactive fragment libraries we’ve covered (see here, here, and here), this one contains a wide variety of different warheads with varying reactivities.

Finally, Jeff Neitz (UCSF) described efforts against Taspase-1, which is involved in cancer cell proliferation. A high-throughput screen of 242,000 molecules yielded seven chemical series – all of which ultimately proved to be artifacts. The enzyme is a threonine protease but contains a cysteine residue near the active site, so the researchers conducted a Tethering screen with 1280 disulfide-containing molecules, which led to 64 hits in five classes. Converting the disulfide to more drug-like warheads ultimately led to nanomolar molecules with cell-based activity, and the researchers even had some success removing the warhead entirely.

If you missed the meeting, you still have time to catch what should be an epic conference: FBLD 2018 returns to San Diego where it originated ten years ago. People still talk fondly about that meeting, so don’t miss this one!

Poll results: the modern fragment library

Our most recent poll has just closed, and the results provide a snapshot of fragment libraries in 2018.

The first two questions asked about the smallest and largest fragments (in terms of non-hydrogen or heavy atoms) you would include in your library. As shown below, the median lower bound is 7-8 heavy atoms, while the median upper bound is 17-18 heavy atoms. This is comparable to what we saw when we last asked these questions five or six years ago.

(Methods note: of the 98 respondents to the question “What is the largest number of heavy atoms you would allow in a fragment,” 10 answered ≤ 10 atoms, but no one answered 11-12. I suspect that the 10 answers were erroneous and that they actually meant to answer the second question, “What is the smallest number of heavy atoms you would allow in a fragment,” which was answered by 89 respondents. Therefore I have excluded these answers from the figure. Please leave a comment if I’ve mischaracterized your vote!)

The third question asked how many fragments people have in their libraries, and more than 40% of the 99 respondents answered 1001-2000.

The distribution is similar to the results from four years ago when we last asked this question. Notably though, the number of respondents with very large libraries has more than doubled, admittedly from a small base.

Overall then this poll could probably be summarized as, “plus ça change.” Or, as the growing number of clinical success stories attests, if it ain’t broke, don’t fix it!

Fragment growing and merging: an inverse agonist of RORγt2

The nuclear receptor transcription factor RORγt2 is involved in the differentiation of Th17 cells, and is thus a target for inflammatory diseases. The protein contains a large, hydrophobic ligand binding site, and as a result most known inverse agonists have less than ideal physicochemical properties. In a paper recently published in J. Med. Chem., Samuel Hintermann and colleagues at Novartis have taken a fragment-based approach.

The researchers screened a library of 1408 fragments using a “differential static light scattering (DSLS)” assay, which is a type of thermal shift assay that measures denaturation and aggregation of the protein. A few dozen molecules that stabilized RORγt2 were tested in dose-response curves prior to crystallization trials, ultimately yielding 13 structures. Compound 1 was particularly interesting because it binds in the center of the cavity, providing growth vectors in two directions. It also makes a couple hydrogen bonds with the protein, as opposed to purely hydrophobic interactions.

Growing from the ethoxy position quickly led to improvements in affinity. To avoid the possibility of toxic iminoquinone metabolites, the researchers replaced the central phenyl ring with a pyridine, resulting in the low micromolar inverse agonist compound 8.

To further improve affinity, the researchers merged an element from a previously reported GlaxoSmithKline molecule (compound 2) onto compound 8, resulting in the potent compound 9, which was characterized in a battery of assays.

The crystal structure of compound 9 bound to the protein revealed that the core fragment moiety binds in the same manner as the original compound 1, though the added benzyl ether binds in a subpocket that had not previously been observed to bind ligands.

Kinetic studies using a reporter displacement assay revealed that compound 9 has both a slow on-rate as well as a slow off-rate, consistent with the fact that it is fully enclosed by the protein. The researchers performed molecular dynamics simulations to try to determine how the ligand could enter or leave, which suggested large conformational changes in a flexible region of the protein. Isothermal titration calorimetry (ITC) showed that the binding of compound 9 is enthalpically driven, with an unfavorable entropy. Although interpreting thermodynamics is fraught, this result makes intuitive sense given the hydrogen bonds formed and the fact that the molecule seems to rigidify the protein.

Biophysics is interesting, but of course biology is what was driving the program. Compound 9 is potent in a variety of cell assays and is also selective for RORγt2 over other nuclear hormone receptors. However, it is also mostly insoluble, and although it did show efficacy in a rodent inflammation model, plasma concentrations of compound 9 were highly variable between individual rats, which the authors attribute to poor physicochemical properties.

This is a nice application of fragment growing and merging that demonstrates how difficult it is to find useful leads for lipophilic sites: even with favorable biochemistry and biophysics, the pharmacokinetics are a slog. That said, others have made progress against similarly hydrophobic targets, so it will be fun to watch this story progress.

Conservation of fragment binding modes revisited

A common assumption when growing or linking fragments is that the binding mode will remain the same. This is often the case, but exceptions occur frequently enough to keep life interesting. Last year we highlighted a study that tried to answer the question of when ligands changed their binding mode by analyzing the protein data bank (PDB). In a new J. Med. Chem. paper, Esther Kellenberger and collaborators at Université de Strasbourg and Eli Lilly have conducted an even more exhaustive study.

The researchers considered all protein structures deposited in the PDB between 2000 and mid-2016 solved to at least 3 Å resolution. This yielded 1079 different fragments (MW < 300 Da) and 1832 larger (“drug-like”) ligands, as well as 126 crystallization additives such as buffers and detergents. In comparing the same protein with different ligands, care was taken to remove mutant proteins that could cause a change in binding mode.

This dataset was used to address several questions.

First, how often does the same fragment bind to the same pocket in the same manner? Often a crystal structure will have several different copies of the same protein in the asymmetric unit. In nearly three-quarters of cases, the fragments bound in a similar manner to the different copies. The exceptions often involved protein conformational changes, in some cases due to different crystal contacts.

Second, how often does a fragment maintain its binding mode when incorporated into a larger molecule? The data set included 359 pairs of ligands on 51 proteins. Again, about three-quarters of fragments had similar binding modes as their larger counterparts. When binding modes changed, protein flexibility often played a role. Polar contacts such as hydrogen bonds were much more highly conserved than hydrophobic contacts. As the earlier study also found, binding modes of very small fragments (MW < 110) were most likely to change, while fragments with MW > 150 almost always retained their binding modes.

Third, do fragments and larger ligands make similar interactions? The data included 235 proteins in which at least one structure contained a fragment and another structure contained a larger ligand.  (The larger ligand didn’t necessarily contain the fragment.) Obviously larger ligands are able to make more interactions than smaller ligands, but, as Stephen Roughley and Rod Hubbard observed back in 2011, enough fragments should allow you to map out the important interactions. After systematically exploring the data, the current researchers suggest that fully mapping a pocket requires nine or more different fragments, a high bar satisfied by just 11 proteins.

Finally, do crystallization additives behave as fragments? The researchers looked at all additives with MW < 300, and separately considered those bound to otherwise free (apo) proteins and those bound to proteins containing other ligands. In general additives showed more variation in their binding modes, though those binding to apo proteins often made similar contacts as made by fragments and larger molecules. Intriguingly, small polar molecules such as DMSO and glycerol often made hydrophobic interactions with proteins.

There is plenty more in the paper than can be summarized here. Laudably, the researchers have provided all of their data in a convenient web portal that even supports chemical substructure searches. Overall the results reassuringly suggest that the binding mode of a fragment usually remains the same as it is optimized. But of course these types of analyses are subject to survivor bias: fragments that change binding mode unexpectedly may be more difficult to optimize, and thus less likely to lead to larger ligands.

The odds may be ever in your favor, but look out for the exceptions.

Dimerization: elegant but not essential

A special case of fragment linking is dimerization, in which two copies of the same fragment bind to adjacent sites in a protein and are subsequently linked together (see for example here, here, and here). A recent example was published in J. Med. Chem. by Bernard Pirotte, Julien Hanson (University of Liège), Lionel Pochet (University of Namur), Jette Kastrup (University of Copenhagen) and their collaborators.

The researchers have for some time been interested in AMPA receptors, critical components in neuronal synaptic transmission. Increasing their activity could be useful for treating diseases such as depression and schizophrenia, but increasing activity indiscriminately is known to be toxic. One approach has been to develop positive allosteric modulators (PAMs), which increase the activity only in the presence of the natural ligand glutamic acid, thus amplifying the normal biological signal.

AMPA receptors themselves are dimers of dimers. Many different PAMs have been reported for AMPA receptors, and some of these are in fact dimeric molecules that span two adjacent binding sites across the dimer interface. A crystal structure of a molecule closely related to compound 35 revealed that each molecule binds to two adjacent protein subunits, so the researchers designed compound 22, which pairs the molecules through a simple ethylene moiety. The strategy paid off with a low nanomolar activator, which crystallography confirmed binds as expected.

Interestingly, conceptually cleaving the bond connecting the two fragments generates a compound (33) which is slightly less active than the initial fragment 35; it is possible the methyl groups are too close to one another when two copies of compound 33 are bound.

As the researchers point out, compound 22 is one of the most potent AMPA receptor PAMs reported. However, it is also quite large, particularly since it needs to cross the blood-brain barrier. No animal data are reported, but a simple metric called the CNS MPO desirability score is reasonably predictive. This score is based on the molecular weight, lipophilicity, total polar surface area, number of hydrogen bond donors, and basicity; higher scores are better. By this measure, compound 22 is predicted not to have high brain penetration, though of course any metric needs to be taken with caution.

However, a separate J. Med. Chem. paper by many of the same researchers revealed that dimerizing the molecules is not essential: simply growing compound 35 could also generate a low nanomolar AMPA receptor PAM (compound 8). Crystallography revealed that the added phenyl group binds where the second molecule of compound 35 would normally bind. Moreover, compound 8 has a higher ligand efficiency as well as a higher CNS MPO desirability score than the dimeric compound 22, suggesting that it is more likely to be able to cross the blood-brain barrier.

In the absence of pharmacological or pharmacokinetic data, if forced to choose I would probably focus on compound 8 rather than compound 22. All of which is to say that although there is a certain elegance to dimerizing molecules, you might be able to replace one of them with a smaller, simpler moiety.

Fragments score a win against WDR5-WIN

Protein-protein interactions (PPIs) can be difficult targets for multiple reasons. First, the contacts often cover large, flattish areas with few “ligandable” pockets. Second, they can involve multiple proteins; imagine trying to disrupt a huge multicomponent machine with a little widget. The protein WDR5 falls into the second category. It serves as a scaffold around which other proteins assemble to regulate epigenetics. One of these proteins, MLL1, is implicated in certain leukemias and binds to WDR5 through the WDR5 INteraction (WIN) motif, making this protein-protein interaction an intriguing anti-cancer target. In a recent paper in J. Med. Chem., Stephen Fesik and colleagues at Vanderbilt University describe their efforts towards this target.

Unlike some PPIs, the WIN motif does contain a nice little pocket which normally recognizes arginine residues. However, since the highly basic guanidine moiety of arginine is undesirable in drugs, the researchers conducted a fragment screen to find new WIN-site binders. A two-dimensional (¹H-¹⁵N HMQC) NMR screen of a large fragment library (>13,800 fragments, more than the majority of respondents in the poll to the right) identified 47 hits that produced similar spectral changes as a peptide that binds in the WIN site. Compound F-1 was the most potent.

A crystal structure of compound F-1 bound to WDR5 revealed that the imidazole moiety binds in the same deep pocket normally occupied by the arginine side chain, with the phenyl ring pointing up out of the pocket. Initial growing off the phenyl ring into nearby hydrophobic pockets produced more potent compounds, but at best these were still micromolar binders. The researchers had more success by targeting a slightly more distant pocket with compounds such as 4a and subsequently compound 4i. A crystal structure of compound 4a bound to WDR5 suggested that the biologically active conformation might not be the lowest energy conformation of the free molecule. Introducing a ring to restrict the conformation led to more potent molecules such as 6e, with sub-nanomolar affinity.

Unfortunately, though potent in biochemical assays, compound 6e and related molecules were about 2800-fold less potent in cell-based assays. The compound is cell permeable and not effluxed, so the disconnect must be due to something else – perhaps the multiple other proteins in the cellular environment. Anyone who has spent much time doing medicinal chemistry will have encountered frustrating situations like this. Perhaps a new chemotype is needed, or perhaps the compounds need to be made even more potent. Indeed, several years ago the Fesik group reported nanomolar binders of MCL-1, but it was not until they improved affinity to picomolar that they saw good cell potency. Stay tuned!

Rise of the machines for fragment optimization

Our latest poll (please vote on the right-hand side of the page!) is about fragment libraries. Once you have your library, you can screen it using a variety of approaches. But what do you do once you get hits? Computational methods are increasingly being adopted; just this year we’ve discussed two approaches: growing via merging and AutoCouple. A new paper in J. Med. Chem. by Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University and several other institutions describes a method that combines virtual screening with automated real-world synthesis in a platform called diversity-oriented target-focused synthesis (DOTS).

The process is best described with an example, and the test case presented is the first bromodomain of BRD4, BRD4(BD1). The researchers, who had previously identified a xanthine-containing series of inhibitors, pared this back to fragment-sized compound F1. Crystallography revealed a nearby pocket, which the researchers attempted to target with DOTS.

The researchers built a virtual library of 576 sulfonamides extending off the para position of the phenyl ring of compound F1. These were then virtually screened against BRD4(BD1) using the S4MPLE molecular modeling tool in which the F1 portion was constrained in the crystallographically observed conformation while the variable bits were allowed to move. The 100 top-scoring molecules were examined more closely, and 17 representatives were chosen to be synthesized on an automated robotic platform. This was actually a fairly modest set, as the Chemspeed system they used can run up to 96 parallel reactions. The crude products were then tested in a fluorescence assay, and all of them showed improved activities compared to the initial fragment. The majority, such as compound 17, showed high nanomolar inhibition.

The 13 submicromolar compounds were then resynthesized, purified, and validated in thermal shift and isothermal titration calorimetry (ITC) assays; these orthogonal methods confirmed their activities. The crystal structure of compound 17 bound to BRD4(BD1) was also solved, and this revealed that – as designed – the initial fragment retained its binding mode while the added portion makes new interactions with the protein.

The fact that 14 of the 17 molecules synthesized were at least an order of magnitude more potent than the initial fragment is satisfying, though it is worth noting that bromodomains are not the most difficult targets. Also, all of the new molecules have lower ligand efficiencies than the initial fragment. Still, advances and combinations of computational and robotic approaches will certainly increase the throughput of synthesis and testing, and I expect to see more of these examples.

Practical Fragments turns ten, and celebrates with a poll on the modern fragment library

Ten years ago today Teddy launched Practical Fragments with a simple question about screening methodologies. More than 660 posts later we've returned to that topic several times, most recently in 2016. But before you can start screening you need a fragment library, which is the subject of our new poll.

Back in 2012 we asked readers the maximum size (in terms of "heavy", or non-hydrogen atoms) they would consider for fragments in their library. The results were mostly consistent with the Rule of 3, so beloved by Teddy that he compared it to a powerful wizard.

There has since been a trend toward smaller fragments, driven in part by empirical findings that smaller fragments have better hit rates, in agreement with molecular complexity theory.

At some point, though, ever smaller fragments will mean lower hit rates: fragments that are too small will bind so weakly they will be difficult to detect. And practical issues arise: organic molecules with just a few non-hydrogen atoms are often volatile.

Therefore, we’re revisiting this question: What is the smallest fragment you would put in your library?

As long as we're on the subject of libraries, how many fragments do you have in your primary screening library, or how many do you screen on a regular basis?

Please vote on the right-hand side of the page. If you have multiple fragment libraries (for example one for crystallographic screening and one for biochemical screening) you can respond for each library; you will need to press "vote" after each answer. Please feel free to leave comments too.

Thanks to all of you for making Practical Fragments a success and for your comments over the years – looking forward to the next decade!

Fragment events in 2018 and 2019

Hard to believe we're already halfway through the year, but there are still some exciting events ahead, and 2019 is already starting to take shape.

2018

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

September 25-28: CHI's Discovery on Target will also be held in Boston, and there will be lots of presentations of interest to readers of this blog, particularly in the Lead Generation Strategies track. Mary Harner and I will be presenting a FBDD short course over dinner on September 27.

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

2019

March 20-22: Although not exclusively fragment-focused, the Sixth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and will be held in lovely Nice. You can read my impressions of the 2018 event here, last year's Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here.

March 24-26: The Royal Society of Chemistry's Fragments 2019 will be held in the original Cambridge. This is the seventh in an esteemed conference series that alternates years with the FBLD meetings. You can read impressions of Fragments 2013 and Fragments 2009.

April 8-12: CHI’s Fourteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of the 2018 meeting here, last year's meeting here, the 2016 meeting here; the 2015 meeting here, here, 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.

November 20-22: If you can't make it to Nice, NovAliX will also be holding a biophysics meeting for the first time in the lovely city of Kyoto.

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

Fragments towards the clinic: ERK1/2

The first fragment-based drug to reach the market, vemurafenib, targets a mutant form of the kinase BRAF. Initial responses can be miraculous, but metastatic melanoma is an implacable foe, and patients often relapse. One mechanism of resistance involves upregulation of the kinases ERK1 and EKR2, which are downstream of BRAF. These are the subject of a paper just published in J. Med. Chem. by Tom Heightman and collaborators at Astex and Sygnature.

Most kinase drugs bind to the so-called hinge region of the protein, where the adenine moiety of ATP binds. Previously reported ERK1/2 inhibitors do indeed bind here, but one molecule from Merck (Schering) also binds in a second pocket some distance away. This molecule both inhibits the kinase and also blocks it from becoming phosphorylated itself, thereby preventing it from becoming activated.

Unfortunately this molecule did not have good pharmacokinetic properties, so the researchers sought a new series. They began with virtual, crystallographic, and thermal-shift fragment screens against ERK2. Compound 5 was active in a biochemical assay with impressive ligand efficiency. The bound structure showed multiple interactions with the protein as well as good vectors for further growth. Recognizing that spanning from the hinge region to the second pocket would require a large molecule, the researchers first sought to increase the sp³ character of the fragment to maximize solubility by replacing the pyrazole with a tetrahydropyran (compound 7), which also provided a nice boost in potency. 

Next, the researchers started growing towards the second pocket, guided by docking. This led to another large jump in potency, to the low nanomolar compound 11. Further growth to compound 16 led to marginal improvements in biochemical potency but did show antiproliferative activity in the Colo205 cell line, which contains the oncogenic BRAF mutation. Building into the second pocket, again guided by both modeling and crystallography, significantly improved the cell activity, ultimately leading to compound 27. Consistent with the design, the molecule blocks ERK activity as well as phosphorylation of ERK. It is also orally bioavailable, has good pharmacokinetics, and causes tumor regression in mouse xenograft models. Moreover, Compound 27 is quite selective for ERK1/2 in a panel of 429 kinases.

This is a lovely example of fragment-based and structure-based design. Although the final molecule is on the large side, careful attention to molecular properties maintained acceptable pharmacokinetics. The paper ends by noting that “further pharmacological characterization of 27 will be published elsewhere.” Indeed, Astex has taken an ERK1/2 inhibitor called ASTX029 into the clinic. Practical Fragments wishes everyone involved the best of luck.

Fifth NovAliX Biophysics in Drug Discovery Conference

Last week NovAliX held its biophysics meeting outside of Strasbourg for the first time. Naturally they chose Boston, one of the most European of US cities and a major hub of drug discovery. The event brought together 118 participants from 15 countries, roughly 80% from industry. Although the food and drink could not compare to France, the science and discussion were every bit as satisfying. With 30 talks and 22 posters I won’t attempt to be comprehensive, but as with last year just try to capture a few themes. 

One particularly noteworthy session was devoted to single particle cryo-electron microscopy (cryo-EM), which was recently reviewed in Nat. Rev. Drug Discov. by conference chairman Jean-Paul Renaud and a multinational team of experts. The approach involves flash-freezing a thin film of sample and using transmission electron microscopy to capture two-dimensional “projection” images of your target. If the protein is randomly oriented you can computationally combine thousands of individual images into a three dimensional structure. Although the technique has been around for decades, until recently the resolution was too low to be useful for structure-based drug design. Recent advances in hardware and computation have led to what’s come to be known as the “resolution revolution,” explained Gabe Lander (Scripps).

One advance is the 300 keV Titan Krios – a massive (and massively expensive) instrument that is so widely coveted that Gabe showed pictures of happy scientists hugging newly delivered crates. Indeed, of the ~1000 structures solved to < 4 Å resolution, the vast majority of them were solved on one of more than 130 Krios instruments throughout the world. But Gabe showed that high resolution structures can be obtained with more common 200 keV instruments, including a 2.6 Å resolution structure of aldolase (150 kD), a 2.9 Å structure of hemoglobin (64 kD), and a 2.9 Å resolution structure of alcohol dehydrogenase (81 kD) with bound NAD⁺ cofactor. Although only a handful of sub-2 Å structures have been reported, he thought these would become routine in the next few years.

Bridget Carragher (New York Structural Biology Center) described challenges and how to overcome them. Currently it takes at best eight hours to go from data to structure, but she thought getting this to under one hour would be achievable. Moreover, cryo-EM can be used to characterize different conformational or oligomeric states present in a single sample, as Giovanna Scapin (Merck) demonstrated with insulin binding to its receptor. Indeed, even simple visualization – without fancy computational processing – can provide useful information about protein aggregation, as demonstrated by Wen-ti Liu (NovAliX).

Although primary fragment screening still looks a long way off for cryo-EM, it should start to provide useful structural information for fragments bound to targets less amenable to conventional biophysical techniques, such as membrane proteins – the topic of another session.

Miles Congreve (Heptares) discussed how their stabilized “StaR” GPCRs can provide high-resolution crystal structures suitable for FBDD (see for example here). This has allowed them to discover less lipophilic, more ligand-efficient drug candidates against a variety of targets.

According to Anass Jawhari, it isn’t even necessary to make mutant GPCRs: Calixar has developed proprietary detergents that can stabilize full length adenosine A_2A receptor for a week – more than enough time to perform STD NMR screens of 100 fragments and identify 19 hits, some of which turned out to be functional antagonists. Matthew Eddy (University of Southern California) used two-dimensional NMR on this same protein to reveal dramatic differences in conformational dynamics when bound to agonists vs antagonists.

Indeed, conformational changes and dynamics were a running theme throughout the conference. Keynote speaker and Nobel-laureate Martin Karplus (Harvard) quoted fellow Nobelist Richard Feynman: “everything that living things do can be understood in terms of the jiggling and wiggling of atoms.” (As an aside, Martin’s MCSS method pioneered computational FBDD approaches, predating SAR by NMR.) Göran Dahl (AstraZeneca) described how large scale conformation changes well outside of the active site of PI3Kgamma were responsible for freakishly high selectivity of a class of inhibitors.

But how do you detect conformational changes? We’ve previously mentioned Biodesy’s SHG approach, and Parag Sahasrabudhe (Pfizer) described how this proved useful for classifying ligands for IL-17A. Gerrit Sitters (Lumicks) described a completely different “dynamic single-molecule” (DSM) approach, which involves trapping a single fluorescently labeled protein between DNA strands tethered to two microspheres. Changes in protein conformation caused by ligand binding change the distance between microspheres, and these can be detected to within 1 Å.

Kinetics is intimately linked to dynamics, but the factors responsible for slow binding and dissociation are still poorly understood. Chaohong Sun (AbbVie) examined an archive of 8000 data points and found that on-rates and off-rates each varied by more than five orders of magnitude. There was no correlation with ClogP of the ligands, though larger ligands were more likely to have slower kinetics. There were also significant target effects; on-rates were consistently slow for one target.

As we’ve previously discussed, off-rate screening (ORS) can be used to identify hits in crude reaction mixtures, and Menachem Gunzburg (Monash University) described how this technique is being used in hit-to-lead efforts. Lowering the temperature to 4 °C and adding 5% glycerol further slows dissociation, allowing weaker hits to be discovered.

At the extreme, irreversible inhibitors have an off-rate of 0, and Gregory Craven (Imperial College London) described quantitative irreversible tethering of electrophilic fragments to cysteine residues in proteins using a fluorimetric plate-based assay. As we’ve noted, one challenge with irreversible tethering is deconvoluting intrinsic reactivity from proximity-directed reactivity, which Gregory addresses using a reference thiol such as glutathione.

There is much more to say but in the interest of time I’ll stop here. If you missed the conference you have two chances next year: June 4-7 when it returns to Strasbourg, and November 20-22 when it will be held in Kyoto. And there are still excellent events coming up this year – hope to see you at one!

The origins and development of FBDD

Most of the papers Practical Fragments cover are limited in scope: a new chemical probe, say, or an NMR method. Even in our annual review of reviews, most of the publications have a focus, such as a particular technique. But a paper just published in Drug Discovery Today, by Iwan de Esch (VU University Amsterdam) and an international group of collaborators (including FragNet scholar Angelo Romasanta), is a rather different beast.

The (open access) paper is a bibliometric analysis of FBDD. The researchers first assembled all papers in Thomson-Reuter’s Web of Science which had “fragment” and one of several other terms as a keyword. If you try this at home you will find all sorts of irrelevant topics (such as antibody fragments), so these were manually removed, leaving 2781 publications. But many early papers did not refer to fragments, so all references that had been cited at least ten times were added, resulting in a total of 3642 papers published between 1953 and 2016. What can be learned with such a data set?

For one thing, the term “fragment-based drug discovery” didn’t appear until 2002. In the early 2000s “fragment-based lead discovery” was more common, though for roughly the past decade the former term and “fragment-based drug design” have co-dominated.

The researchers also examined the number of citations each paper has received to reveal interesting trends. For example, in the early years (1996-2001), industry dominated. Indeed, 9 of the 10 most cited papers of all time come from industry, and the sole outlier describes the protein data bank (PDB). In the past decade academics have become significant contributors, which is not surprising given their stronger incentive to publish.

Moving beyond raw citations, the researchers manually classified papers into scientific disciplines (methods, molecular basis, applications, and crystallography) to explore the diffusion of knowledge. This reveals the centrality of the 1996 “SAR by NMR” article, which was the first to cite theoretical and computational approaches and also bring in biophysics. Deservedly, this is the most highly-cited paper (454 citations within the set of articles, and currently >2100 total according to Google Scholar).

Our most recent poll of fragment-finding methods revealed a spike in crystallography, driven both by higher hit rates as well as technical advances, and this is also seen in the paper, where the 2011-2016 period shows a significant increase in crystallography over earlier five-year periods. As we’ve also noted, there has been a shift in content: while many earlier publications focused on techniques, medicinal chemistry has become a much more common subject in recent years.

There is plenty more here, and the paper is fun reading for anyone in FBDD, whether you have lived through the history or are new to the field. My one quibble is that the list of 3642 papers is not provided as supplementary material. Indeed, it is the open nature of the PDB that has made it such a valuable resource. Hopefully the authors will release their underlying data so others can build upon it.

Fragments in the clinic: ETC-206

A few weeks ago we highlighted the story of eFT508, a clinical MNK1/2 inhibitor derived from a previously published fragment. One of the comments to that post mentioned another example describing a clinical compound against the same targets – also derived from a previously published fragment! This work was recently published in J. Med. Chem. by Kassoum Nacro and a large, multinational group of collaborators from A*STAR and other institutes.

The kinases MNK1 and MNK2 are responsible for phosphorylating and thereby activating eIF4E, a protein that regulates messenger RNA translation. All three proteins are overexpressed in various cancers, particularly blast crisis chronic myeloid leukemia (CML), in which patients stop responding to drugs such as dasatinib. An inhibitor of MNK1/2 could thus potentially resensitize the cancer cells. Moreover, MNK knockout mice are healthy, suggesting that the therapy might be minimally toxic.

The researchers started with a 2010 paper which reported a virtual screen against MNK1; nearly three quarters of the hits were fragments. The A*STAR researchers were particularly attracted to molecules such as ETP-38766, and they used modeling along with a previously reported structure of MNK2 to scaffold-hop to compound 4, with sub-micromolar activity. (MNK1 and MNK2 are closely related, and most reported compounds show similar activity against both; values for MNK1 are given here.)

Building out the molecule further did not do much for biochemical potency but did yield molecules with improved solubility, permeability, and cell-based activity – such as compound 27. Further tweaking of the core and replacement of the metabolically labile methyl piperazine ultimately led to ETC-206, with nanomolar potency in biochemical and cell-based assays. It also shows good pharmacokinetics, is orally bioavailable, and is remarkably selective for MNK1/2: in a panel of 104 kinases screened at 1 µM compound, only one other kinase showed significant inhibition. As expected, the molecule showed little antitumor activity in a xenograft assay when dosed by itself, but significantly improved the activity of dasatinib. Indeed, the molecule has recently entered a phase 1 clinical study in combination with dasatinib.

Several lessons can be drawn from this paper. First, it appears that ETC-206 was derived solely with the aid of modeling, without recourse to experimental structural data for any molecules in the series. Second, both ETC-206 and eFT508 had their origins in fragments previously discovered by others – a reminder that, with the increasing number of publications, you don’t necessarily have to do your own fragment screen in order to do FBLD. (An important corollary is that a fragment does not itself need to be novel to generate patentable chemical matter.) Finally, ETC-206 and eFT508 are both selective MNK1/2 inhibitors but look very different from one another – a reminder that many roads can lead to different clinical candidates for the same target.

Fragments vs the common cold (via NMT)

Anyone who has spent much time in drug discovery will have been asked what they've done to cure the common cold. In a paper just published in Nature Chemistry, Robert Solari, Edward Tate and collaborators from Imperial College London and institutions throughout the UK have taken a stab at this challenge.

One of the problems with rhinovirus, which causes the common cold, is that there are more than 100 different serotypes, thwarting vaccine development. To make matters worse, the virus replicates rapidly and sloppily, thereby increasing the odds of resistance mutations. To sidestep both problems, the researchers decided to target a host protein rather than a viral protein.

After the rhinovirus genome is translated in cells as a single polyprotein, it is cleaved and processed into component proteins which self-assemble to form the virion. One of the proteins, VP0, has a fatty acid attached to its N-terminus by host proteins called N-myristoyltransferases (NMT1 and NMT2 in humans). Mutagenesis studies had previously suggested that this modification is important for infectivity, so the researchers sought inhibitors against the NMTs.

High-throughput screens had previously identified two unrelated series of compounds, and crystallography revealed that they bind at adjacent but overlapping regions within the enzyme active site. Fragment-sized compound IMP-72 makes multiple interactions with the protein; an inhibitor from the other series makes a key interaction with an active-site serine. This molecule was trimmed back to a fragment (IMP-358) which showed minimal enzyme inhibition on its own but which dramatically increased the potency of IMP-72. Crystallography confirmed that the two fragments could bind NMT1 simultaneously.

A sort of fragment linking was conducted in which the key hydrogen bond acceptor of IMP-358 was attached to the more potent fragment, leading to a low nanomolar inhibitor. Further structure-guided optimization led to IMP-1088, which inhibits both human NMT1 and NMT2 with IC₅₀ < 1 nM and shows picomolar binding by surface plasmon resonance (SPR).

So does it work? IMP-1088 is able to block myristoylation of VP0 in human cells. More importantly, the molecule shows antiviral activity against a range of rhinovirus serotypes and is able to rescue cells from viral cytotoxicity. Further mechanistic work suggests that inhibiting NMT activity blocks virus assembly.

Of course, lots of human proteins are myristoylated – NMT1 and NMT2 are human enzymes after all. Reassuringly, IMP-1088 itself did not reduce viability of uninfected cells. Although SPR had shown very slow off-rates, NMT proteins are constantly being resynthesized, and NMT activity had fully recovered after 24 hours. The researchers suggest that an early diagnosis and short treatment could be both safe and effective.

There is still much to do, notably pharmacokinetic and animal efficacy studies. And of course, the fear of toxicity will hang all the more heavily over antiviral strategies that target host proteins. So the next time someone asks whether scientists have invented a cure for the common cold, you’ll still have to tell them no. But at least we’re working on it.

Fragments vs PKC-ι: 7-azaindole strikes again

A common question in library design concerns novelty: should you populate your library with custom-made, hitherto unseen molecules, or just buy off-the shelf compounds? While the first strategy might make it easier to get patentable leads, the second approach is faster and has a long history of success. Indeed, simple 7-azaindole served as a starting fragment for three clinical compounds: vemurafenib, PLX3397, and AZD5363. A new paper in J. Med. Chem. by Alvin Hung and colleagues at A*STAR illustrates just how versatile this scaffold can be.

The researchers were interested in protein kinase C iota (PKC- ι), one of a family of 10 kinases that has been implicated in cancer. A high concentration screen of 1700 fragments yielded 15 hits with measurable IC₅₀ values, three of which were substituted 7-azaindoles. Compound 1, which has the highest ligand efficiency, was chosen to pursue.

Initial SAR quickly revealed that the bromine could be replaced with larger substituents, and a combinatorial library led to more potent molecules, such as compound 25. This was docked into a previously reported crystal structure of PKC- ι, which suggested the possibility of adding a positively charged moiety to interact with a couple aspartic acid residues. This strategy was successfully accomplished in compound 36, with low micromolar activity.

  

Adding a methoxy substituent to force a twist in the molecule led to an additional increase in potency, and rigidifying the amine led to compound 39, with mid-nanomolar activity. This was profiled against 101 kinases and found to be reasonably selective, though it did hit some other PKCs. The molecule was also not very permeable, and perhaps for this reason did now show good cellular activity.

To further optimize the series the researchers turned to group efficiency analysis, which revealed that the central benzimidazole element was the least efficient portion of the molecule. Earlier SAR and modeling had suggested that the unsubstituted nitrogen was making an important hydrogen bond to the protein, but “moving” the other nitrogen led to a more potent molecule. Further tweaking led to low nanomolar compound 49, which also had improved cellular activity.

Overall this is a nice example of advancing a generic, promiscuous fragment to a novel, potent, and selective lead – all without crystallographic support. Though further characterization of these molecules is not reported, the authors do mention optimization of a second series starting from a different fragment. Stay tuned!

Fragments vs Gram-negative bacterial PPAT

Of the 30+ fragment-derived drugs that have entered the clinic, only one is an antibiotic. In part this reflects a shift away from this therapeutic area by many companies. Novartis, though, has continued to invest, as demonstrated by two consecutive papers in J. Med. Chem.

The researchers were interested in the enzyme phosphopantetheine adenylyltransferase (PPAT, or CoaD), which catalyzes the penultimate step of coenzyme A biosynthesis from ATP and 4'-phosphopantetheine. Although the enzyme is present in all organisms, the bacterial form is highly conserved across prokaryotes and significantly different than the human form. It is also essential for bacterial growth, thus making it an attractive target.

In the first paper, Robert Moreau and colleagues start big: a high-concentration screen (at 500 µM) of 25,000 fragments as well as NMR-based screens of their core 1408 fragment library. Triaging both hit sets led to a cornucopia of 39 crystal structures of bound fragments; the chemical structures of a dozen are provided in the paper, with IC₅₀ values from 31 to >2500 µM. Perhaps surprisingly, all of these bound at the pantetheine binding site of the enzyme, suggesting that this is a “hotter” hot spot than the ATP-binding site.

Three of the fragments are described in more detail. The first was optimized from 273 µM to 4.3 µM, but subsequent advancement was unsuccessful. The second fragment, with an IC₅₀ of 230 µM against E. coli PPAT, could be optimized to mid-nanomolar inhibitors; unfortunately these were much less active against PPAT from P. aeruginosa, so this series was also abandoned. But the third fragment discussed, compound 6, proved to be more tractable.

Initial optimization based on other hits led to compound 32, and addition of a methyl to the benzylic linker provided a satisfying 30-fold improvement in potency for compound 33. This “magic” methyl appeared to help desolvate the adjacent NH as well as pre-orient the molecule in the bound conformation. Further growing from this position led to compound 53, which provided a further 7-fold improvement in potency. Crystallography revealed a hydrogen bond between the nitrile nitrogen and a protein backbone amide. Unlike the previous series, this compound was active against PPAT from both E. coli and P. aeruginosa.

The second paper, by Colin Skepper and colleagues, describes further optimization of the molecules to picomolar binders. There’s a lot of lovely medicinal chemistry in both papers, but unfortunately all the molecules displayed at best only modest antibacterial activity. One problem is that Gram-negative bacteria have two membranes: an outer one which blocks lipophilic molecules and an inner one which blocks hydrophilic molecules. Compounds that can make it past these barriers also face an array of diverse efflux pumps, and these seemed to be the downfall of this project. The core of the molecule makes multiple hydrogen bonds to PPAT; about twenty different heterocycles were tested, but most of these had significantly lower potency, and the active ones were efflux pump substrates.

These difficulties in part explain why companies have been moving away from antibiotics. This was not a minor effort: each paper listed more than twenty authors. The second ends somewhat wistfully. “Although none of the series disclosed… yielded a clinical candidate, it is our hope that these studies will help pave the way toward the discovery of new Gram-negative antibacterial agents with novel modes of action.” It is a worthy – if arduous – quest.