Fragment growing in three dimensions made easy

Nearly a decade ago we highlighted a paper from Astex that exhorted chemists to develop new synthetic methodologies useful for fragment-based drug discovery. Peter O’Brien has taken on the challenge, and he and his collaborators at University of York and AstraZeneca report their progress in a recent (open-access) J. Am. Chem. Soc. paper.

 

The O’Brien group has previously published synthetic routes to shapely fragments, which we wrote about here. These could be useful for expanding fragment collections, but that happens infrequently. The new paper focuses on the far more common challenge of what to do when you have a fragment hit.

 

The idea was to create a “modular synthetic platform for the elaboration of fragments in three dimensions.” The researchers designed a set of bifunctional building blocks that could be coupled to existing fragments. The two functionalities were N-methyliminodiacetic acid boronate (BMIDA) and a Boc-protected amine. The amine is a versatile handle for multiple types of chemistry, while the BMIDA moiety is particularly useful for Suzuki-Miyaura cross-coupling. (Indeed, two separate groups of researchers had previously built libraries suited for cross-coupling using halogen-containing fragments, as we discussed here.)

  

For the new building blocks, the researchers considered azetidines, pyrrolidines, and piperidines with fused or spiro-cyclopropyl groups. These are rigid “three-dimensional” units, and the relative locations of the BMIDA group and the amine could provide very different distances and vectors. After modeling 27 possibilities, the researchers chose nine building blocks based on diversity and predicted ease of synthesis. These were synthesized on gram scale, and all nine are now commercially available.

 

To demonstrate that the building blocks would be generally synthetically useful, the researchers coupled them to a variety of (hetero)aryl bromides, with yields ranging from 10-90%, and most >60%. The Boc group was then deprotected and the crude amine was used in a variety of successful reactions.

 

The building blocks were each also coupled to 5-bromopyrimidine, the Boc-group was deprotected, and the free amines were capped as methanesulfonamides. Small molecule crystallography of the resulting compounds confirmed modeling results that the two vectors had a wide range of orientations and were separated by 1.5-4.4 Å. Moreover, most compounds were rule-of-three compliant, had good measured aqueous solubility, and were even stable in human liver microsomes and rat hepatocytes.

 

As a use-case, the researchers considered the approved drug ritlecitinib, an irreversible JAK3 inhibitor. They imagined that its pyrrolopyrimidine moiety was a fragment hit, and then virtually combined it with their nine scaffolds, each functionalized with an acrylamide. These were then virtually docked, and the best two were synthesized and tested. Compound 96 was quite potent, albeit less so than ritlecitinib.

The question of whether three-dimensionality is desirable as a design feature remains unproven, as we noted recently. However, whether the high Fsp³ of the nine new scaffolds is itself a selling point, they do provide new vectors for fragment growing, and their synthetic enablement justifies including them at least in virtual campaigns.

Flatland: still a nice place to be

In 2009 we highlighted a paper reporting that approved drugs have a higher fraction of sp³-hybridized carbon atoms than discovery-phase compounds. Perhaps focusing on molecules with a high Fsp³, the ratio of sp³-hybridzed carbons to total carbons, would lead to greater success. Or not: a new analysis in Nat. Rev. Chem. by Ian Churcher (Janus Drug Discovery), Stuart Newbold, and Christopher Murray (both at Astex) finds that the relationship has not held up.

 

The 2009 “Escape from Flatland” paper was widely discussed at conferences and has been cited more than 3000 times. But according to the authors of the new study, most of these citations are from papers describing new synthetic methodologies rather than from papers discussing medicinal chemistry.

 

And not all the attention has been positive. As we noted in 2013, Pete Kenny and Carlos Montanari reanalyzed the data and found that an apparent correlation between Fsp³ and solubility disappeared when plotting all discrete data points instead of binned data.

 

More recently, we highlighted a paper that found no significant difference between the shapeliness of drugs, as assessed by their principal moment of inertia (PMI), and the shapeliness of small molecules in the ZINC database.

 

The new paper looks at Fsp³ values for drugs approved during various time periods. Among 980 drugs approved up to 2009, the average Fsp³ was 0.458. However, of the 431 drugs approved after 2009, the average Fsp³ has dropped to 0.392. The researchers speculate that this (statistically significant) decrease may be due to an increase in the number of kinase inhibitors, which are usually highly aromatic, as well as an increase in the use of metal-catalyzed cross coupling reactions.

 

In my analysis of the 2009 paper, I asked whether higher Fsp³ ratios would lead to lower hit rates, and indeed this seems to be the case, as shown in a paper we discussed in 2020. Thus, if you pursue difficult targets, you may increase your chances of finding hits by screening molecules with lower Fsp³ ratios. Also, multiple studies, including one published just last month, have found no correlation between the shapeliness of a fragment (as defined by deviation from planarity, or DFP) and the shapeliness of the resulting lead, so there appears to be no penalty to starting with a flattish fragment. 

 

The researchers conclude that their “analysis of drug development trends over the last 15 years suggests that Fsp³ may not have been a useful metric to optimize.” Importantly, the supplementary information includes a list of >1400 approved drugs and >1500 investigational drugs along with associated properties, so you can do your own analyses.

 

In the end, generalizations will only get you so far, and may even lead you astray. At least for now, there are few shortcuts in the long slog of experimental studies necessary to discover a drug.

Spacial Scores: new metrics for measuring molecular complexity

Molecular complexity is one of the theoretical underpinnings for fragment-based drug discovery. Mike Hann and colleagues proposed two decades ago that very simple molecules may not have enough features to bind tightly to any proteins, whereas highly functionalized molecules may have extraneous spinach that keeps them from binding to any proteins. Fragments, being small and thus less complex, are in a sweet spot: just complex enough.

 

But what does it mean for one molecule to be more complex than another? Most chemists would agree that pyridine is more complex than methane, but is it more complex than benzene? To decide, you need a numerical metric, and there are plenty to choose from. The problem, as we discussed in 2017, is that they don’t correlate with one another, so it is not clear which one(s) to choose. In a new (open access) J. Med. Chem. paper, Adrian Krzyzanowski, Herbert Waldmann and colleagues at the Max Planck Institute Dortmund have provided another. (Derek Lowe also recently covered this paper.)

 

The researchers propose the Spacial Score, or SPS. This is calculated based on four molecular parameters for each atom in a given molecule. The term h is dependent on atom hybridization: 1 for sp-, 2 for sp²-, 3 for sp³-hybrized atoms, and 4 for all others. Stereogenic centers are assigned an s value of 2, while all other atoms are assigned a value of 1. Atoms that are part of non-aromatic rings are also assigned an r value of 2; those that are part of an aromatic ring or linear chain are set to 1. Finally, the n score is set to the number of heavy-atom neighbors.

 

For each atom in a molecule, h is multiplied by s, r, and n². The SPS is calculated by summing the individual scores for all the atoms in a molecule. Because there is no upper limit, and because it is nice to be able to compare molecules of the same size, the researchers also define the nSPS, or normalized SPS, which is simply the SPS divided by the number of non-hydrogen atoms in the molecule. Although SPS can be calculated manually, the process is tedious and the researchers have kindly provided code to automate the process. Having defined SPS, the researchers compare it to other molecular complexity metrics, including the simple fraction  of sp³ carbons in a molecule, Fsp³, which we wrote about in 2009. 

 

The researchers next calculated nSPS for four sets of molecules including drugs, a screening library from Enamine, natural products, and so-called “dark chemical matter,” library compounds that have not hit in numerous screens. The results are equivocal. For example, the nSPS for dark chemical matter is very similar to that for drugs. On the other hand, natural products tend to have higher nSPS scores than drugs, as expected. Interestingly, the average nSPS score for compounds in the GDB-17 database, consisting of theoretical molecules having up to 17 atoms, is also quite high.

 

The researchers assessed whether nSPS correlated with biological properties, and found that compounds with lower nSPS tended to have lower potencies against fewer proteins, as predicted by theory. That said, this analysis was based on binning compounds into a small number of categories, and as Pete Kenny has repeatedly warned, this can lead to spurious trends.

 

The same issue of J. Med. Chem. carries an analysis of the paper by Tudor Oprea and Cristian Bologa, both at University of New Mexico. This contextualizes the work and confirms that drugs do not seem to be getting more complex over time, as measured by nSPS. This may seem odd, though Oprea and Cristian note that by “normalizing” for size, nSPS misses the increasing molecular weight of drugs.

 

This observation also raises other questions, such as the fact that SPS explicitly excludes element identity. Coming back to benzene and pyridine, both have identical SPS and nSPS, which does not seem chemically intuitive. One could quibble more: why square the value of n in the calculation of SPS? Why allow s to be only 1 and 2, as opposed to 1 and 5?

 

In the end I did enjoy reading this paper, and I do think having some metric of molecular complexity might be valuable. I’m just not sure where SPS will fit in with all the existing and conflicting metrics, and how such metrics can lead to practical applications.

Review of 2021 reviews

As the year winds down SARS-CoV-2 continues its relentless drive through Greek letters and the planet. But there is hope: vaccines seem to be holding, for those who have access, and two oral drugs have been granted emergency use authorization by the US FDA, one of which (PF-07321332) is covalent and looks remarkably effective. As is our custom, Practical Fragments ends the year by highlighting conferences and reviews.

 

Conferences started the year online only (CHI’s Sixteenth Annual Fragment-based Drug Discovery), moved to hybrid (CHI’s Nineteenth Annual Discovery on Target) and sadly returned to virtual (Pacifichem 2021).

 

This year produced more than twenty FBLD-related reviews, and these are grouped thematically: NMR and crystallography, computational methods, targets, library design, and covalent fragments. The most general is the sixth installment in a series of annual reviews in J. Med. Chem. covering fragment-to-lead success stories from the previous year. Iwan de Esch (Vrije Universiteit Amsterdam) took the lead (pardon the pun) on the most recent review, which details 21 examples from 2020. In addition to the centerpiece table showing fragment, lead, and key parameters, this open-access paper also includes an analysis on the molecular complexity of fragment hits.

 

NMR and crystallography

Consistent with its central role in FBLD, several reviews covered NMR. Ben Davis (Vernalis), one of the leading practitioners, discusses fragment screening in Methods Mol. Biol. The chapter is written for a non-specialist, so you won’t see detailed pulse sequences. Instead, Ben provides a very accessible and practical guide covering everything from sample preparation through data analysis and validation.

 

A more detailed description of solution NMR in drug discovery by Li Shi and Naixia Zhang (Shanghai Institute of Materia and Medica) is published (open access) in Molecules. With 180 references, this review covers considerable ground, including various ligand-detected and protein-detected methods for screening as well as for hit-to-lead and mechanistic studies. The paper also includes a nice summary of in-cell (!) NMR.

 

The Pacifichem meeting had several talks on fluorine NMR, and speaker Will Pomerantz, together with Caroline Buchholz, has published a thorough, open-access review in RSC Chem. Biol. Will has been a leading developer of protein-observed ¹⁹F NMR, so naturally this topic is well-covered, but there is plenty on ligand-observed ¹⁹F NMR as well as a good background section and musings on the future of the field.

 

And if you’re looking for a detailed how-to guide for NMR-based fragment screening, Harald Schwalbe and colleagues describe the platform they’ve built at the Center for Biomolecular Magnetic Resonance (BMRZ) at Johann Wolfgang Goethe-University Frankfurt in J. Vis. Exp. This open-access paper also describes quality control experiments of the iNEXT library, which we’ve discussed here.

 

Switching gears to crystallography, J. Vis. Exp. carries a paper by Frank von Delft and collaborators describing the XChem platform at the Diamond Light Source. This high-throughput fragment screening platform has delivered a 95% success rate on more than 150 screens, with hit rates varying from 1-30%. In addition to technical details, this open-access article also provides tips on successfully getting your screening proposal through peer review.

 

XChem has inspired similar efforts at other synchrotrons, including the Fast Fragment and Compound Screening (FFCS) platform at the Swiss Light Source. This is concisely described by May Sharpe and Justyna Wojdyla in Nihon Kessho Gakkaishi (open-access and published in English).

 

Private companies are also moving into high-throughput crystallography. Debanu Das and collaborators describe the platform at Accelero Biostructures, which is capable of screening ~500 fragments in two days. Screens against three nucleases are described in some detail in an open-access article in Prog. Biophys. Mol. Biol.; these and other components of the DNA damage response are the focus of XPose Therapeutics, Accelero’s sister company.

 

Computational methods

In addition to the experimental methods reviewed above, a couple papers describe computational approaches. In Drug Disc. Today: Tech., FragNet alum Moira Rachman and collaborators from UCSF, Universitat de Barcelona, and elsewhere focus on “fragment-to-lead tailored in silico design.” This is a nice review of the recent literature and emphasizes the fact that much of the heavy design lifting is still done by medicinal chemists – at least for now.

 

Predicting the energies of modified fragments has long been a challenge, and one promising approach is free energy perturbation, in which one ligand is “perturbed” into another and the relative energy differences calculated. Barbara Zarzycka and colleagues at Vrije Universiteit Amsterdam provide a concise review for aficionados in Drug Disc. Today: Tech.

 

Targets

Three reviews cover applications of FBLD to various target classes. Kinases have been particularly successful, with four of the six approved fragment-derived drugs targeting these enzymes. In Trends Pharm. Sci., Ge-Fei Hao and collaborators, mostly at Central China Normal University, review the state of the art. In addition to background and several case studies, the paper includes a nice table showing structures and summaries of clinical-stage kinase inhibitors.

 

Epigenetics has been another fruitful area, and in J. Med. Chem. Miguel Vaidergorn, Flavio da Silva Emery (both University of São Paulo) and Ganesan (University of East Anglia) detail the “successful union of epigenetic and fragment based drug discovery (EPIDD + FBDD).” This thorough summary (with 165 structures!) of the literature is particularly detailed when it comes to bromodomains, four inhibitors of which have entered the clinic with the help of fragments. The researchers point out that EPIDD and FBDD both began around the same time, and in fact the oncology drug vorinostat could be described as “a unique case of solvent-based drug discovery.”

 

RNA has long been a target of FBLD, and in ChemMedChem Mads Clausen (Technical University of Denmark) and collaborators review the state of the art. The various established and emerging methods to find fragment hits are covered in depth, and there is also a nice discussion as to whether RNA-focused fragment libraries will be useful.

 

Library design and molecular properties

In Expert Opin. Drug Discov. Zenon Konteatis (Agios) asks “what makes a good fragment in fragment-based drug discovery?” His answers provide a concise summary touching on the rule of three, molecular complexity, “three-dimensionality”, and other topics.

 

The topic of three-dimensional fragments is covered in several other reviews. In Drug Disc. Today: Tech., Iwan de Esch and collaborators at Vrije Universiteit Amsterdam and University of York assess 25 so-called 3D libraries reported in the literature, mostly since 2015. The researchers manually drew all 897 fragments so they could calculate various properties. While most of the molecules are rule-of-three compliant, just under half could be called 3D by both plane of best fit (PBF) and principal moment of inertia (PMI). PBF and PMI measurements correlated with one another, while Fsp³ correlated with neither measurement, leading to the conclusion that “Fsp³ is a poor measure of 3D shape.”

 

Shapely or not, sp³-rich fragments are interesting from a diversity point of view, and in Chem. Sci. Max Caplin and Dan Foley (University of Canterbury) discuss synthetic methods for advancing these. This is an excellent open-access review of the recent literature around C-H bond functionalization and well worth reading for the chemists in the audience.

 

3D fragments are often chiral, and the importance of chirality in drug discovery is the focus of a paper in ACS Med. Chem. Lett. by Ilaria Silvestri and Paul Colbon (University of Liverpool). The researchers note an opportunity for chemical suppliers: only 245 of 9751 heterocyclic building blocks offered by Sigma-Aldrich are chirally pure.

 

“Library design strategies to accelerate fragment-based drug discovery” is the topic of a Chem. Eur. J. review by Nikolaj Troelsen and Mads Clausen (Technical University of Denmark). The researchers provide a highly accessible overview of different libraries appropriate for different fragment-finding methods, including covalent approaches.

 

Covalent fragments

This year saw the approval of sotorasib, the first covalent fragment-derived drug, so it is no surprise that several papers focus on this topic. Sara Buhrlage, Jarrod Marto, and colleagues at Dana-Farber Cancer Institute provide a thorough introduction to “chemoproteomic methods for covalent drug discovery” in Chem. Soc. Rev. The review covers both isolated protein screening as well as proteome-wide methods and includes multiple case studies.

 

Nir London and colleagues at The Weizmann Institute of Science focus on “covalent fragment screening” in Ann. Rep. Med. Chem. This is an excellent review of the recent literature and also includes an analysis of six commercial covalent fragment libraries.

 

And finally, in RSC Chem. Biol. (open access), Nathanael Gray and collaborators mostly at Dana-Farber Cancer Institute discuss strategies for “fragment-based covalent ligand discovery”, including computer-aided approaches, as well as target classes and new modalities such as PROTACs. They end by asking whether sotorasib was “a lucky, one-off case” or “a preview of continued and increased impacts that these approaches will have on drug discovery as the improved methods, larger libraries, and increased focus start to bear fruit.”

 

I’m betting on the latter.

 

And that’s it for 2021. Thanks for reading, special thanks for commenting, and here’s hoping we’ll be able to meet in person in 2022.

Fragment mixtures vs protein mixtures

In FBLD – as in most areas of research – speed and efficiency are prized. The faster you can find quality fragments, the faster you can advance them. NMR-based screening remains one of the most popular fragment-finding methods, and in a recent Molecules paper William Pomerantz and collaborators at the University of Minnesota and Gustavus Adolphus College provide an accelerated workflow.

 

The Pomerantz lab is well known for protein-observed ¹⁹F (PrOF) NMR, in which fluorine-labeled residues are incorporated into proteins. This is easily accomplished by supplementing the media with fluorine-containing amino acids during protein expression. To date more than 15 fluorinated amino acids have been tested in more than 70 proteins, ranging from 7 to 180 kDa in size. Because the chemical shift of fluorine is so sensitive to its environment, a fragment binding nearby can be readily detected by PrOF NMR.

 

When a single type of amino acid is fluorinated, the resulting protein spectrum is considerably simpler than in traditional protein-observed NMR methods. Taking advantage of this, the researchers mixed two different bromodomain proteins: the human oncology target BPTF and PfGCN5 from the malarial parasite Plasmodium falciparum. Both of these bromodomains contain a tryptophan in their N-acetyl-lysine binding sites, so each protein was labeled with 5-flurotryptophan. The proteins were then screened (at 50 µM each) against 467 fragments from Life Chemicals in pools of 4-5 (at 400 µM each). Chemical shift perturbations of the binding-site tryptophan were seen for half of the 98 pools. To determine which fragments were responsible for these shifts, the researchers tested their fragment mixtures against the relevant proteins using (ligand-detected) CPMG NMR. Since they had previously determined the ¹H NMR spectra of all their fragments, it was easy to pick out the binders.

 

Hit rates were similar for both BPTF (9.8%) and PfGCN5 (9.2%), and 4.1% of fragments hit both bromodomains. The researchers had previously screened this library, which is enriched for shapely fragments, against the bromodomain BRD4 D1 (see here) and obtained a similar hit rate. Statistical analyses revealed that the 3D-character for PfGCN5 hits is similar to the library as a whole, as had also been seen for BRD4 D1, while the BPTF hits tended to be flatter.

 

The researchers also followed up on several  fragments individually. One in particular had low micromolar affinity for PfGCN5 as assessed with both PrOF NMR and ¹H-¹⁵N HSQC NMR titrations. Interestingly, this fragment also caused a chemical shift in a different 5-fluorotryptophan residue some 22 Å away from the canonical binding site. Binding at this site could not be competed by a known high-affinity ligand, and a computational screen using FTMap suggested that this does appear to be a secondary binding site.

 

Overall this approach appears to be an appealing workflow as judged by comparing required time, protein, and ligand amounts to other NMR-based screening cascades. As the researchers note, it is advantageous to assess both protein and ligand behavior, as done here. Have you tried using PrOF, and if so how has it performed for you?

Flatland: a nice place to be

The ideal shape of compounds used for biological screens is a subject of vigorous debate, with some arguing that shapely molecules may be superior in various ways to the “flatter” aromatic compounds that tend to dominate libraries. This view was expressed more than a decade ago in the paper, “Escape from Flatland: Increasing Saturation as an Approach to Improving Clinical Success.” However, those conclusions have been challenged. Since many of us are trying to discover drugs, it is worth asking what actual drugs look like. This is the subject of a new ACS Med. Chem. Lett. paper by Seth Cohen and colleagues at University of California, San Diego.

Assessing shapeliness is itself contentious. Here the researchers chose the intuitive metric, principal moment of inertia (PMI), which uses a simple triangle plot to assess whether a molecule is more rod-like, disk-like, or sphere-like. The degree of shapeliness (3D Score) can be calculated by summing the x- and y-coordinates to give values between 1 (rod- or disk-like) and 2 (sphere-like).

The researchers first extracted more than 8500 drugs and nutraceuticals from DrugBank, all of which had associated three-dimensional structures and MW >100. PMI calculations revealed that nearly 80% were linear or planar, with 3D Scores < 1.2. Another 17.5% had 3D Scores up to 1.4, while only 0.5% were greater than 1.6. Interestingly, this distribution is similar to that of the ZINC database of small molecules. You might expect a correlation between size and shapeliness, with larger molecules being more three-dimensional, but this was not the case. Perhaps related, a separate analysis found no correlation between shapeliness of fragments and resulting leads.

The 3D structures of compounds in DrugBank are calculated for energy-minimized conformations, which are not necessarily the biologically relevant conformations. So the researchers next went to the protein data bank (PDB) and its crystal structures of 502 unique DrugBank molecules bound to various proteins. Some molecules were represented multiple times (1036 structures of sapropterin!), and for these the PMIs were averaged. The results of this analysis were similar, with 83.5% of molecules having a 3D Score < 1.2 and just three molecules with a 3D Score > 1.6. As with the DrugBank data, there was no correlation between 3D Score and molecular weight.

Further analyses of compounds with multiple crystallographic structures was interesting. For diclofenac, with 51 PDB entries, 3D Scores ranged from 1.03 to 1.52, with the minimized score being 1.22. However, some of these structures are likely low affinity with questionable biological relevance. In contrast, for five approved HIV drugs, the PMIs remained very similar for molecules bound in the active sites.

Getting out of flatland is surprisingly difficult: the researchers examined the PMIs for several fragments from libraries designed to have shapely members and found that none had 3D Scores > 1.4. They suggest clever ways of increasing three dimensionality, such as building organometallic molecules. While this is likely to increase novelty and patentability, it also introduces unknown biological risks. One analysis that would be interesting is whether natural-product-derived drugs are significantly shapelier than their purely synthetic counterparts.

The researchers conclude:

The true need for topological diversity in feedstocks and final drug molecules remains unclear given the overwhelming number of linear and planar drugs. The question remains as to whether more 3D compounds represent attractive and untapped therapeutic space, or if more linear/planar molecules are indeed the best topologies for bioactive molecules.

This is indeed an interesting question, and I hope that chemists – particularly those in academia – continue to make and test ever more exotic molecules. But since the first word of this blog is “Practical,” I would not discount the more planar molecules that make up most of our pharmacopoeia.

Fragments vs RIP2: from flat fragment to shapely selectivity

Last week we highlighted the utility of shapely fragments. However, as the latest review of fragment-to-lead success stories again shows, starting with a “flat” fragment does not condemn a lead to flatland. This is illustrated in a recent J. Med. Chem. publication by Adam Charnley and colleagues at GlaxoSmithKline.

The researchers were interested in receptor interacting protein 2 kinase (RIP2), which is implicated in various inflammatory diseases. A fluorescence polarization screen of 1000 fragments at 400 µM yielded 49 hits with inhibition constants ranging from 5-500 µM. Thirty of these confirmed in a thermal shift assay, and 20 were characterized crystallographically bound to the enzyme. Hit-to-lead chemistry was pursued for five series; the most successful started with compound 1a.

The crystal structure revealed that the carboxamide of compound 1a makes interactions with the hinge region of the kinase, with the phenyl group in the back pocket. A search of related molecules available in-house led to compound 2a, with a satisfying boost in potency. Interestingly, the crystal structure of this molecule bound to RIP2 revealed that the binding mode of the pyrazole moiety had flipped to keep the phenyl ring in the back pocket (compound 1a in cyan, 2a in gray). Enlarging the phenyl group to better fill the pocket led to compound 2k.

This molecule had relatively poor selectivity against several other kinases, but introducing a ring as in compound 8 improved the situation. Crystallography suggested that installing a bridged ring would pick up further interactions with the protein, and although the resulting molecule did not have better affinity, selectivity improved. Finally, a hydroxyl group was introduced (compound 11) to try to pick up interactions with a non-conserved serine residue. This addition did not improve biochemical activity, and in fact a crystal structure revealed that the hydroxyl group was pointing towards solvent, but the activity in human whole blood improved. Importantly, compound 11 was remarkably selective for RIP2: just 1 of 366 other kinases tested at 1 µM showed >70% inhibition.

This is a lovely fragment-to-lead success story that reiterates several important lessons. First, a generic (in this case commercial) and nonselective fragment can lead to novel, selective series. Second, as has been seen multiple times, fragment binding modes can flip unexpectedly, especially during early optimization. Finally, despite the relative flatness of fragment 1a (Fsp³ = 0, though the two aromatic rings are slightly twisted), it could be optimized to a more shapely lead, and the increased complexity is likely responsible for the impressive selectivity. Left unreported is the stability and pharmacokinetics of compound 11: the hydroxyl and all those sp³-hybridized carbons are likely metabolic hotspots. As is so often the case in lead discovery, what solves one problem can too often create another.

Three dimensional fragments revisited

A long-running debate in the fragment world centers on the utility of “three dimensional” fragments. Proponents argue that these (often aliphatic) fragments may be more novel, have better physicochemical properties, and have more vectors for elaboration than “flatter” (mostly aromatic) molecules. Skeptics retort that hit rates are likely to be lower for these more complex molecules, and good luck making analogs. Two papers published late last year add more data to the debate.

The first paper, published in J. Med. Chem. by William Pomerantz and collaborators at the University of Minnesota and Eli Lilly, describes the results of a fragment screen against the bromodomain BRD4(D1), a popular member of the BET family. The 467 fragment library was enriched for shapely fragments as assessed by plane of best fit (PBF), which is the “average distance of a non-hydrogen atom from a plane drawn through the compound such as to minimize the average.” For example, "flat" benzene has a PBF of 0 while the cofactor NADPH has a PBF of 1.53.

The library was screened using ligand-observed (CPMG) NMR, and 34 hits were confirmed using protein-observed fluorine (PrOF) NMR. All of these were competitive with the known ligand (+)-JQ1, consistent with binding at the acetylated lysine recognition site. The average PBF of the hits was 0.44, essentially the same as the library itself (0.46). This is higher than the average PBF (0.36) of all fragments crystallized with BRD4 in the protein data bank.

Structures of all the hits are provided, and some of them are indeed quite unusual. The researchers characterized a substituted thiazepane crystallographically and were able to optimize this to a 32 µM binder with good ligand efficiency. This fragment was also selective against a handful of other bromodomains.

The researchers had previously screened BRD4(D1) under identical conditions with a more traditional, “flatter” library with an average PBF of 0.26. Interestingly, in that case the hits were less shapely than the library as a whole, with an average PBF of 0.17. The confirmed hit rate was also higher: 20% vs 7%. That said, the fragments in the traditional library tended to be smaller (averaging 180 Da vs 241 Da), so the molecular complexity of this library was likely to be lower, which could account for the higher hit rate.

The second paper, published in Bioorg. Med. Chem. Lett. by Ulrich Grädler and collaborators at Merck KGaA, EMD Serono, Edelris, and Proteros, focuses on cyclophilin D (CypD), which has been implicated in cardiovascular disease and multiple sclerosis. Unlike BRD4, this is a tough target: an HTS screen of 650,000 compounds in a biochemical assay yielded just 178 hits, none of which confirmed. Undeterred, the researchers screened 2688 fragments by SPR at 2 mM, resulting in 58 confirmed hits, all quite weak (millimolar). Crystallography was attempted on most of them, yielding six structures, including such shapely specimens as compounds 3 and 7.

Compound 3 binds in the lipophilic S2 pocket of CypD, overlapping with the aniline moiety of previously reported compound 2. Fragment merging led to compound 14, with nearly 40-fold improved affinity over compound 2. A similar strategy merging compound 3 with fragment 8 led to low micromolar compound 27, two orders of magnitude more potent than the starting fragments. Perhaps most impressively, fragment linking compound 3 with compound 7, a shapely fragment which binds in the S1’ pocket, led to submicromolar compound 39, with affinity more than 10,000-fold higher than either fragment.

So in the end, fanciers of shapely fragments and detractors alike can feel vindicated by these papers. Hit rates might be lower for three dimensional fragments, but the resulting hits are likely to be less precedented. In the case of CypD, a shapely fragment led to three different series for a target that had resisted HTS. Of course, there is still some way to go: no cell, permeability, or stability data are provided for any of the molecules, and medicinal chemists may blanch at the seven stereocenters in compound 39. But these are interesting starting points, and it will be fun to see where they end up.

Pointless stereochemistry

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

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

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

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

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

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

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

Twelfth Annual Fragment-based Drug Discovery Meeting

CHI’s Drug Discovery Chemistry meeting took place over four days last week in San Diego. This was easily the largest one yet, with eight tracks, two one-day symposia, and nearly 700 attendees; the fragment track alone had around 140 registrants. On the plus side, there was always at least one talk of interest at any time. On the minus side, there were often two or more going simultaneously, necessitating tough choices. As in previous years I won’t attempt to be comprehensive but will instead cover some broad themes in the order they might be encountered in a drug discovery program.

You need good chemical matter to start a fragment screen, and there were several nice talks on library design. Jonathan Baell (Monash University) gave a plenary keynote on the always entertaining topic of PAINS. Although there are some 480 PAINS subtypes, 16 of these accounted for 58% of the hits in the original paper, suggesting that these are the ones to particularly avoid. But it is always important to be evidenced-based: some of the rarer PAINS filters may tag innocent compounds, while other bad actors won’t be picked up. As Jonathan wrote at the top of several slides, “don’t turn your brain off.”

Ashley Adams described the reconstruction of AbbVie's fragment libraries. AbbVie was early to the field, and Ashley described how they incorporated lessons learned over the past two decades. This included adding more compounds with mid-range Fsp³ values, which, perhaps surprisingly, seemed to give more potent compounds. A 1000-member library of very small (MW < 200) compounds was also constructed for more sensitive but lower throughput biophysical screens. One interesting design factor was to consider whether fragments had potential sites for selective C-H activation to facilitate fragment-to-lead chemistry.

Tim Schuhmann (Novartis) described an even more “three-dimensional” library based on natural products and fragments. Thus far the library is just 330 compounds and has produced a very low hit rate – just 12 hits across 9 targets – but even a single good hit can be enough to start a program.

Many talks focused on fragment-finding methods, old and new. We’ve written previously about the increasingly popular technique of microscale thermophoresis (MST), and Tom Mander (Domainex) described a success story on the lysine methyltransferase G9a. When pressed, however, he said it did not work as well on other targets, and several attendees said they had success in only a quarter to a third of targets. MST appears to be very sensitive to protein quality and post-translational modifications, but it can rapidly weed out aggregators. (On the subject of aggregators, Jon Blevitt (Janssen) described a molecule that formed aggregates even in the presence of 0.01% Triton X-100.)

Another controversial fragment-finding technique is the thermal shift assay, but Mary Harner gave a robust defense of the method and said that it is routinely used at BMS. She has seen a good correlation between thermal shift and biochemical assays, and indeed sometimes outliers were traced to problems with the biochemical assay. The method was even used in a mechanistic study to characterize a compound that could bind to a protein in the presence of substrate but not in the presence of a substrate analog found in a disease state. Compounds that stabilized a protein could often be crystallized, while destabilizers usually could not, and in one project several strongly destabilizing compounds turned out to be contaminated with zinc.

Crystallography continues to advance, due in part to improvements in automation described by Anthony Bradley (Diamond Light Source and the University of Oxford): their high-throughput crystallography platform has generated about 1000 fragment hits on more than 30 targets. Very high concentrations of fragments are useful; Diamond routinely uses 500 mM with up to 50% DMSO, though this obviously requires robust crystals.

Among newer methods, Chris Parker (Scripps) discussed fragment screening in cells, while Joshua Wand (U. Penn) described nanoscale encapsulated proteins, in which single protein molecules could be captured in reverse micelles, thereby increasing the sensitivity in NMR assays and allowing normally aggregation-prone proteins to be studied. And Jaime Arenas (Nanotech Biomachines) described a graphene-based electronic sensor to detect ligand interactions with unlabeled GPCRs in native cell membranes. Unlike SPR the technique is mass-independent, and although current throughput is low, it will be fun to watch this develop.

We recently discussed the impracticality of using enthalpy measurements in drug discovery, and this was driven home by Ying Wang (AbbVie). Isothermal titration calorimetry (ITC) measurements suggested low micromolar binding affinity for a mixture of four diastereomers that, when tested in a displacement (TR-FRET) assay, showed low nanomolar activity. Once the mixture was resolved into pure compounds the values agreed, highlighting how sensitive ITC is to sample purity.

If thermodynamics is proving to be less useful for lead optimization, kinetics appears to be more so. Pelin Ayaz (D.E. Shaw) described two Bayer CDK kinase inhibitors having either a bromine or trifluoromethyl substitution. They had similar biochemical affinities and the bromine-containing molecule had better pharmacokinetics, yet the trifluoromethyl-containing molecule performed better in xenograft studies. This was ultimately traced to a slower off-rate for the triflouromethyl-substituted compound.

The conference was not lacking for success stories, including MetAP2 and MKK3 (both described by Derek Cole, Takeda), LigA (Dominic Tisi, Astex), RNA-dependent RNA polymerase from influenza (Seth Cohen, UCSD), and KDM4C (Magdalena Korczynska, UCSF). Several new disclosures will be covered at Practical Fragments once they are published.

But these successes should not breed complacency: at a round table chaired by Rod Hubbard (Vernalis and University of York) the topic turned to remaining challenges (or opportunities). Chief among these was advancing fragments in the absence of structure. Multiprotein complexes came up, as did costs in terms of time and resources that can be required even for conventional targets. Results from different screening methods often conflict, and choosing the best fragments both in a library and among hits is not always obvious. Finally, chemically modifying fragments can be surprisingly difficult, despite their small size.

I could go on much longer but in the interest of space I’ll stop here. Please add your thoughts, and mark your calendars for next year, when DDC returns to San Diego from April 2-6!