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.

Small and simple, but novel and potent

Back in 2012 we wrote about GDB-17, a database of possible small molecules having up to 17 carbon, oxygen, nitrogen, sulfur, and halogen atoms, most of which have never been synthesized. Although novelty isn’t strictly necessary for fragments, as evidenced by the fact that 7-azaindole has given rise to three approved drugs, it’s certainly nice to have. In a new (open-access) J. Med. Chem. paper, Jürg Gertsch, Jean-Louis Reymond, and colleagues at the University of Bern synthesize fragments that had not been previously made and show that they are biologically active.

 

When you start drawing all possible small molecules you get lots of weird stuff, including an explosion of compounds containing multiple three- and four-membered rings, which may be difficult to make. The researchers wisely focused on “mono- and bicyclic ring systems containing only five-, six-, or seven-membered rings.” They further limited their search to molecules containing just carbon and one or two nitrogen atoms (as well as hydrogen, of course). Systematic enumeration led to 1139 scaffolds, ignoring stereochemistry, of which 680 had not been previously reported in PubChem. Out of these, three related scaffolds were chosen for investigation.

 

Computational retrosynthesis was used to devise routes to the three bicyclic scaffolds, and these were successfully synthesized, along with mono-benzylated versions, for a total of 14 molecules (including stereoisomers), all rule-of-three compliant. The online Polypharmacology Browser 2 (PPB2) was used to predict targets, and several monoamine transporters came up as potential hits. The molecules were tested against norepinephrine transporter (NET), dopamine transporter (DAT), serotonin transporter (SERT), and the σ-R1 receptor in radioligand displacement assays. None of the free diamines were active, but several of the benzylated compounds were, in particular compound 1a.

 

Compound 1a was initially made as a racemic mixture, and when the two enantiomers were resolved (R,R)-1a was found to be a mid-nanomolar inhibitor of NET while (S,S)-1a was 26-fold weaker. Compound (R,R)-1a was also a mid- to high nanomolar inhibitor of σ-R1, DAT, and SERT. Pharmacokinetic experiments in mice revealed that the molecule had poor oral bioavailability but remarkably high brain penetration and caused sedation. The researchers conducted additional mechanistic studies beyond the scope of this blog post and conclude that (R,R)-1a could be a lead for “neuropsychiatric disorders associated with monoamine dysregulation.”

 

There are several nice lessons in this paper. First, as we noted more than a decade ago, there is plenty of novelty at the bottom of chemical space. Moreover, and in contrast to our post last week, even small fragments can have high affinities. But novelty comes at a cost: synthesis of compound 2a required eight steps from an inexpensive starting material with an overall yield of just 9%, though this could certainly be optimized. Nonetheless, particularly for CNS-targeting drugs which usually need to be small in order to cross the blood brain barrier, the price might be worth paying.

 

Of course, even within this paper there are hundreds more scaffolds to look at than the three tested, and perhaps the researchers were lucky that their choices were biologically active. As computational methods continue to advance, it will be worthwhile turning them loose on GDB-17.

Crystallography first in fragment optimization: Binding-Site Purification of Actives (B-SPA)

At FBLD 2024, Frank von Delft (Diamond Light Source) announced the ambitious goal of taking a 100 µM binder to a 10 nM lead in less than a week for less than £1000. Fragment to lead optimization usually takes longer, as dozens or even hundreds of compounds need to be synthesized and tested. One way to speed things up is through “crude reaction screening,” otherwise known as “direct to biology,” in which unpurified reaction mixtures are tested directly. In a new (open-access) Angew. Chem. Int. Ed. paper, Frank, John Spencer, and collaborators at University of Oxford, University of Sussex, and Creoptix apply this approach to crystallographic screening.

 

The researchers were interested in the second bromodomain of Pleckstrin Homology Domain-Interacting Protein, or PHIP(2), an oncology target. As we discussed in 2016, they had previously run a crystallographic screen and identified multiple hits, including F709, which, despite having no measurable affinity, had good electron density and multiple vectors for optimization. Six separate libraries based on this fragment were constructed, with between 58 and 1024 targeted small-molecule products per library and up to four steps done without purification.

 

One challenge for crude reaction screening is assessing whether or not a reaction has actually generated product. Typically this is done by analytical liquid chromatography mass spectrometry (LCMS), but analyzing results manually is tedious. Fortunately academics have graduate students and postdocs, and it was presumably these intrepid souls who spent 17 days analyzing the 1876 small-molecule products attempted.

 

I can say from personal experience that spending hours perusing LCMS chromatograms is not enjoyable, so the researchers built an automated tool called MSCheck, which appears to be freely available here. This showed 83% agreement with the manually curated data, and even identified additional true positives that had been missed. All together 1077 of the reaction mixtures had the desired product, with success rates for the various libraries ranging from 39% to 97%.

 

The successful reactions were soaked into crystals and screened, and nearly 90% of these generated usable data. A total of 29 crystals had interpretable density in the ligand binding site: 7 were starting materials and 22 were desired products. Of the products, 19 bound with the piperazine core in a similar position as the initial fragment, while three bound in an alternate manner.

 

Of course, the whole point of this exercise is to find improved binders, so the researchers tested pure versions of each of the 22 crystallographic hits in two different assays. Only compound PHIP-Am1-20 had measurable affinity, with modest ligand efficiency.

This is not the first example of crude reaction screening by crystallography; we wrote about REFiL_x and a related technique in 2020. In one of those papers, the crude reaction mixtures were assessed by SPR as well as crystallography, which revealed that the crystallographic screen missed some binders, and there is no reason to think the same did not happen here. Indeed, molecules that bind tightly in a different conformation may be more likely to shatter the crystal lattice and thus go undetected.

 

The researchers state that for non-crystallographic crude reaction screening “only strong assay readouts are informative.” But is this bug, or a feature? A 2019 publication that used crude reaction screening to identify KRAS ligands (which I wrote about here) used an assay cascade to quickly select the most potent hits. Even the fastest crystallographic screens can’t compete with plate-based assays in terms of speed.

 

Perhaps PHIP(2) is a particularly challenging test case. As we discussed in 2022, multiple computational screens performed poorly in predicting crystallographic binding modes of ligands for this protein. But as I wrote at the time, it may be that many crystallographic ligands are just too weak to be useful.

 

Although there is a strong case for using crystallography first for finding fragments, I am not yet convinced the same applies for optimizing fragments.

Fragments win in a virtual screen against the 5-HT2A receptor

Virtual screening is continuing to make impressive strides. The latest example, in Nature, comes from William Wetsel (Duke), John Irwin (UCSF), Georgios Skiniotis (Stanford), Brian Shoichet (UCSF), Bryan Roth (UNC Chapel Hill), Jonathan Ellman (Yale), and a large group of collaborators. The paper has received considerable attention (for example In the Pipeline), but in my opinion the connection to FBLD has been understated.

 

The researchers were interested in finding new agonists for the 5-HT_2A receptor (5-HT_2AR). This GPCR is the target for LSD and psilocybin, both of which have been shown to reduce depression and anxiety. Is it possible to find molecules with similar therapeutic activity but without the accompanying psychedelic properties?

 

LSD contains a tetrahydropyridine (THP) moiety, which is relatively rare in screening libraries. The researchers developed convergent routes to THPs in which they could independently and efficiently vary multiple substituents. Using this chemistry, they constructed a virtual library of 4.3 billion compounds, all with molecular weights ≤ 400 Da and cLogP ≤ 3.5.

 

At the time the research began, there were no structures of 5-HT_2AR, so the researchers built a homology model based on the closely related 5-HT_2BR, which differs by only four amino acid residues in the orthosteric pocket where LSD binds. This model was then screened against a subset of the THP library, those ≤ 350 Da. Despite screening some 7.45 trillion complexes (sampling an average of 92 conformations and 23,000 orientations per molecule), the process took only nine hours on a 1000-core CPU cluster. The result was 300,000 hits in nearly 15,000 families. To ensure novelty, only compounds quite different from known ligands were further considered, and 17 “richly functionalized” THPs were synthesized and tested in radioligand assays. Four were active, including racemic compound 28. Searching the 4.3 billion compound library for analogs ultimately led to compound 70 and a related, slightly more potent molecule lacking the methyl substituent on the amine. A cryo-EM structure subsequently validated the predicted binding mode.

 

The paper spends considerable time characterizing these two compounds. Both are agonists and somewhat selective for 5-HT_2AR over 5-HT_2BR and 5-HT_2CR. They are highly selective over 318 other GPCRs and 45 off-targets. GPCRs can signal through arrestin and/or G-protein, and while LSD works (mainly) through the arrestin pathway, the new molecules work (mainly) through the G-protein route. Importantly, the compounds showed anti-depressive and anti-anxiety effects in mouse models. Although you can’t ask mice if they are tripping, the molecules did not cause “head-twitch responses” and other behavioral effects seen with LSD, suggesting that they may not have hallucinogenic properties.

 

This is a lovely piece of work, and a few observations relevant to FBLD stand out. First, the best molecules are actually rule-of-three compliant, despite the fact that larger molecules were included in the virtual screen. Indeed, the top two molecules are actually smaller than the initial hits. This suggests that choosing more richly functionalized molecules may not have been the most efficient approach. We’ve written previously about V-SYNTHES, which entails stepwise selection and growing of fragments; it would be interesting to retroactively test whether this type of approach would have more quickly gotten to compound 70.

 

Finally, this approach can easily be extended to other scaffolds for which syntheses are readily available. Six years ago we wrote about the synthetic accessibility of dihydroisoquinolines, and last year Practical Fragments published our fifth “fragment library roundup.” The marriage of clever chemistry with virtual screening seems to have a bright future.

How fragments become leads

Our most recent poll asked how often synthetic challenges had kept researchers from pursuing a particular fragment or had impeded a fragment-to-lead project. Around two-thirds replied sometimes or often. A new open-access paper in Chem. Sci. by Rachel Grainger, Rhian Holvey, and colleagues at Astex does a deep dive into fragment-to-lead chemistry, provides a powerful visual tool, and ends with something of a call to action.

 

The researchers take as their starting points 131 fragment-to-lead success stories published from 2015 to 2019 and collated in a series of five J. Med. Chem. Perspectives. All of these started with fragments (< 300 Da) for which affinity increased by at least 100-fold, with the resulting leads having affinities of 2 µM or better. As the new paper points out, this could introduce “survivorship bias,” in that less successful projects are not included. However, as the point of the paper is to figure out what works, this likely strengthens the conclusions.

 

The targets themselves are fairly diverse: 24% kinases, 9% proteases, 36% other enzymes, 11% bromodomains, 14% other protein-protein interactions, and 6% other types of targets. The researchers closely examined how the leads related to the initial fragments. Full details are provided in the Supplementary Material (pdf). The researchers have also constructed a handy interactive viewer you can use to do your own analyses. Here is an overlay of an initial fragment (taupe space-filling) with the final lead (yellow surface).

 

What are the results? The first observation is that 93% of leads have at least one polar interaction (such as a hydrogen bond) that is conserved from the initial fragment. The most common functional groups making direct contacts to proteins are N-H hydrogen-bond donors (35%) followed by aromatic nitrogen hydrogen-bond acceptors (23%) and carbonyl oxygen hydrogen-bond acceptors (22%).

 

The second observation is that over 80% of fragments are grown from one or two vectors (examples of one and two, with the second the subject of the figure above). This is perhaps not surprising; growing from three or more vectors would likely result in portly molecules that may be more difficult to advance, venetoclax notwithstanding.

 

But the really interesting observation is that the majority of growth vectors (~80%) originate from carbon atoms. Moreover, more than half of the bonds formed are carbon-carbon bonds. For the non-chemists in the audience, this is significant because carbon-carbon bond forming reactions are not always straightforward, particularly in the presence of polar moieties.

 

In the early days of FBLD, one hope was that including functional groups such as amides in a fragment collection would facilitate fragment growing. The new paper suggests that this is naïve: a functional group in a fragment is likely to interact with the protein and so block potential growth vectors. Indeed, only 18% of growth vectors come from N-H groups, despite the fact that these are among the most synthetically accessible.

 

These findings thus explain why fragment-to-lead efforts can be so challenging. The researchers provide an example of a chemical series they ultimately abandoned due to poor synthetic tractability.

 

The paper also builds on earlier papers from Astex exhorting chemists to further advance chemical methodology. As they conclude:

An “ideal synthesis” of a lead would allow: (1) site-selective formation of bonds at all growing points of a fragment, (2) whilst being mild enough to be compatible with essential polar functionality, and (3) proceeding with minimal or no need for protecting groups….

 

We believe that further development of C-H functionalisation that is tolerant to polar fragments has the potential to transform FBDD.

If you’re in academia, this looks like a good opening for a grant proposal!

Poll results: synthetic challenges are pervasive in FBLD

Last month we wrote about “sociable” vs “unsociable” fragments: the former are amenable to straightforward chemistry for fragment growing, merging, or linking, while the latter are not. This categorization prompted a poll on how often synthetic challenges have kept you from pursuing a particular fragment or impeded a fragment-to-lead project. The results are now in.

 

(Methods note: the poll was run on Crowdsignal from 14 June through 16 July. There were 42 responses to the first question and 44 to the second. Respondents came from a dozen countries with the most from the US, UK, Germany, Denmark, Netherlands, Australia, France, and Italy.)

 

 

Responses were similar for both questions: a fifth to a quarter of respondents often found that synthetic challenges kept them from pursuing a particular fragment or impeded work on a fragment-to-lead project, nearly half sometimes found this to be the case, while a fifth to a third rarely experienced this. Only a handful of people never found synthetic challenges limiting.

 

It is heartening that around a third of respondents are rarely if ever impeded by chemistry, perhaps because they have intentionally constructed their libraries with sociable fragments. Still, the fact that nearly two thirds of researchers sometimes or often run into problems suggests a continuing need for synthetically enabling fragment chemistry.

 

For academic chemists looking to make a practical impact, this could be a fertile area. And fragment library vendors looking to differentiate themselves may want to consider providing robust synthetic routes and methodologies for their available compounds.