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.

Review of 2024 reviews

Long winter is here in the global north, with its dim days and gaping nights. As is tradition, Practical Fragments looks back on the year almost done. 2024 was the best year for conferences since the arrival of COVID. I wrote about CHI’s Drug Discovery Chemistry in San Diego, FBDD-DU in Brisbane, FBLD 2024, and CHI’s Discovery on Target, both in Boston. 

Another tradition is the annual J. Med. Chem. fragment-to-lead success story review; the latest covers the year 2022 and was written by Andrew Woodhead (Astex) and collaborators, including yours truly.

 

For a timely and accessible overview of “how to find a fragment,” look no further than a review of that title in ChemMedChem by Marcio Vinicius Bertacine Dias and collaborators at University of São Paulo and University of Warwick. This covers crystallography, cryo-EM, NMR, SPR, thermal shift, virtual screening, functional screening, ITC, mass spectrometry (including HDX-MS), MST, and BLI, and concludes with a nice comparison table. Some twenty other reviews were also published throughout the year, and these are discussed thematically.

 

Structure-based methods

Three reviews cover NMR. In J. Med. Chem., Janet Caceres-Cortes and colleagues at Bristol-Myers Squibb provide “perspectives on nuclear magnetic resonance spectroscopy in drug discovery.” Ligand- and protein-detected screening are covered thoroughly, with examples such as the discoveries of BI-2852 and venetoclax. Applications beyond hit finding are also discussed, such as the characterization of atropisomers in sotorasib, the identification and characterization of impurities and metabolites, in-cell NMR, and much more.

 

“Perspectives on applications of ¹⁹F-NMR in fragment-based drug discovery” is the title of an open-access review in Molecules by Qingxin Li and CongBao Kang at Guangdong Academy of Sciences and A*STAR, respectively. As we discussed in 2020, fluorine NMR is becoming increasingly common in FBLD, and this paper covers the various methods, including using ¹⁹F-NMR to measure ligand affinity. The authors also include a table summarizing 17 fragment screens that used fluorine NMR.

 

The rise of powerful permanent magnets has enabled low-maintenance benchtop NMR instruments that can be yours for as little as $50,000, compared to upwards of $1 million for a 600 MHz superconducting machine. Although sensitivity is at least 150-fold lower, hyperpolarization techniques such as photo-CIDNP, which we wrote about here, can close the gap. The latest developments are described (open access) in Chemistry–Methods by Felix Torres and collaborators at NexMR and the ETHZ.

 

X-ray crystallography has retained the top position among fragment-finding methods according to our most recent poll. In an open-access Applied Research paper, Daren Fearon, Frank von Delft, and collaborators describe high-throughput crystallographic fragment screening at the Diamond Light Source. As of August 2024 they have collected more than 240,000 academic data sets on hundreds of targets, and the paper distills some of the key lessons, some of which were applied to the COVID Moonshot, which we last wrote about here. The paper also describes future developments and needs, such as how and where to house such massive quantities of data.

 

Another center for high-throughput crystallographic screening is the Helmhotz-Zentrum Berlin (HZB) F2X-Facility at the BESSY II synchrotron, and in an open-access Applied Research paper Manfred Weiss and collaborators provide an overview of workflows and capabilities. One unique offering is the F2X-GO kit, in which F2X fragment libraries (which we wrote about here) as well other supplies are shipped to users to do soaking experiments in their own laboratories prior to shipment to the synchrotron.

 

“Structure-based virtual screening of vast chemical space” is the topic of an open-access review in Curr. Opin. Struct. Biol. by Jens Carlsson (Uppsala University) and Andreas Luttens (MIT). The Enamine REAL collection currently contains 40 billion compounds, and the researchers predict that “make-on-demand chemical libraries will likely reach more than one trillion compounds in the next few years,” which presents both opportunities and challenges, particularly given the existence of the “virtual cheaters” we recently discussed. Machine learning and fragment-based methods such as V-SYNTHES could help.

 

Continuing the virtual theme, Li Wang and collaborators at Nantong University discuss “molecular fragmentation as a crucial step in the AI-based drug development pathway” in an open-access Commun. Chem. paper. This summarizes 15 different computational methods for dissecting larger molecules into fragments, and also includes a list of 11 library vendors.

 

Other methods

Among experimental methods, few can match the throughput of fluorescence techniques, the subject of an open-access Heliyon review by Neelagandan Kamariah and colleagues at inSTEM & NCBS in Bangalore. It has short sections on “fluorescence polarization (FP) and anisotropy (FA), Förster resonance energy transfer (FRET), time-resolved Förster resonance energy transfer (TR-FRET), fluorescence lifetime (FLT), protein-induced fluorescence enhancement (PIFE), fluorescence thermal shift assay (FTSA) and microscale thermophoresis.” It also describes applications to GPCRs, protein-protein interactions, and other biological systems.

 

Native mass spectrometry (nMS), which we wrote about most recently in 2022, is the subject of an RSC Med. Chem. review by Louise Sternicki and Sally-Ann Poulsen at Griffith University. Sally-Ann is a leading expert in nMS, and the paper describes the technique and how it compares to other fragment-finding methods. It also includes a nice table summarizing 17 studies published between 2013-2023 that used nMS for FBLD; a 2013 review of nMS covered earlier examples.

 

Covalent Fragments

Mass spectrometry plays a prominent role in finding covalent fragments, as discussed in an open-access SLAS Discovery review by Simon Lucas and colleagues at AstraZeneca. “Covalent hits and where to find them” also describes other biophysical, biochemical, cellular approaches, and even DEL screening. It also discusses covalent libraries (which we wrote about earlier this month) and successful examples such as the discovery of sotorasib. In my opinion the researchers succeed in their “hope that this review will help serve as a useful roadmap to those seeking to drug the undruggable.”

 

A concise open-access review in Curr. Opin. Struct. Biol. by Katrin Rittinger and collaborators at The Francis Crick Institute and GSK focuses on using covalent fragments to assess target tractability, specifically ligandability and functionality. Target-based, proteome-wide and function-first approaches are summarized, and the researchers also discuss the importance of negative control compounds such as inactive enantiomers.

 

Continuing the theme of “assayability,” Micah Niphakis (Lundbeck) and Ben Cravatt (Scripps) review “ligand discovery by activity-based protein profiling” (ABPP) in a Cell Chem. Biol. paper. Because ABPP is usually conducted in cells or cell lysates, full-length proteins are assayed in their native environment, facilitating the discovery of allosteric ligands as in the case of WRN, which we wrote about earlier this year. The paper summarizes multiple examples of finding covalent ligands for challenging targets, and also highlights future challenges such as increasing throughput and targeting residues beyond cysteine.

 

Reversible covalent inhibitors are the topic of an open-access review by Dustin Duncan and colleagues at Brock University in ACS Chem. Biol. The researchers argue that reversible covalent inhibitors may cause less accumulation of the off-target adducts that could form with irreversible inhibitors. The paper includes a figure showing reversible covalent warheads, details on how to characterize them, a nice summary of general considerations, and success stories for JAK3, BTK, and proteases.

 

Targets

Covalent ligands have been particularly important for cancer targets, as reviewed by Xiaoyu Zhang (Northwestern University) and Ben Cravatt (Scripps) in an open-access Annual Review of Cancer Biology paper. The focus is on use of chemical proteomics “to expand the druggability of cancer proteomes.” The paper presents examples of finding and characterizing covalent ligands for a variety of oncology targets including KRAS^G12C.

 

E3 ligases are briefly mentioned, and this target class is the focus of an Expert Opin. Drug Discov. review by Jongmin Park and colleagues at Kangwon National University. As we’ve discussed recently, the 600 or so human E3 ligases are potentially valuable for targeted protein degradation applications such as PROTACs. The review focuses on “fragment-based approaches to discover ligands for tumor-specific E3 ligases.” In addition to summarizing successes against targets such as BCL6 and XIAP, it includes a list of 113 tumor-specific E3 ligases and another list of 52 E3 ligases that are overexpressed in certain tumors.

 

The “impact of fragment-based drug design on PROTAC degrader discovery” is also the subject of a review in Trends in Analytical Chemistry by Xiaoguang Lei and colleagues at Shenzen Bay Laboratory. Here, the focus is more on using FBLD to discover ligands for target proteins rather than for E3s. For example, the researchers describe how the fragment-derived drug navitoclax was used as a starting point for developing DT2216, a clinical-stage BCL-x_L degrader.

 

As Vicki Nienaber noted more than a decade ago, fragment-based drug discovery is ideally suited for targeting the central nervous system, particularly when combined with a ruthless focus on molecular properties. This is the topic of an open-access review in Front. Chem. by Michael Kassiou and collaborators at University of Sydney, CSIRO, and Vast Bioscience. After a brief summary of FBLD the researchers present case studies published since 2015, the last year this topic was reviewed. We’ve covered quite a few on Practical Fragments, including apoE4, Notum, and PDE10A.

 

Other

We mentioned allostery above, and in an open-access FEBS Open Bio. article Andrea Bellelli and collaborators at Sapienza University of Rome and elsewhere ask “is allostery a fuzzy concept?” Digging into half-century old publications from Jacques Monod, the researchers conclude that the concept was “born with an original sin: two definitions.” Indeed, the first mathematical model did not even apply to monomeric proteins. Most readers of this blog will probably be satisfied with the notion that an allosteric ligand is one that binds outside of an active site, but it is worth remembering that “allostery is an umbrella that covers more than a single reaction mechanism and cannot be defined by a single mathematical expression.”

 

Structure comes up frequently on Practical Fragments, but James Fraser (UCSF) and Mark Murcko (Disruptive Biomedical) remind us in Cell that “structure is beauty, but not always truth.” We’ve written multiple posts about getting misled by crystal structures, and in this brief commentary the authors provide “four harsh truths: a structure is a model, not experimental reality; representing wiggling and jiggling is hard; in vitro can be deceiving; drugs mingle with many different receptors.” They conclude that “truth is a molecule that transforms the practice of medicine.”

 

Stumbling towards truth is a little easier with the help of a good chemical probe, and in Nucleic Acids Res. Paul Workman (Institute of Cancer Research) and collaborators provide an updated description of The Chemical Probes Portal. This free community resource now contains 803 probes against 570 targets, including 28 covalent ligands and 51 degraders. Moreover, 332 of the probes have structurally related negative controls. Importantly, the Portal also includes 258 “Unsuitable” compounds that are insufficiently potent or selective to serve as chemical probes. Checking this list can save you valuable time when reading papers about unfamiliar targets.

 

Finally, a brief open-access interview with Nobel Laureate Katalin Karikó in Issues in Science and Technology is an inspiring reminder that “you learn more from failure,” and that the pleasure of doing science can be its own reward.

 

Thanks for reading. Good luck in 2025, and remember that the sun is always out there, even when you can neither feel nor see it.

Current Topics in Medicinal Chemistry Special FBDD Issue

This year ends with an entire issue of Current Topics in Medicinal Chemistry devoted to FBDD. Rob van Montfort and Ian Collins provide a brief editorial overview of the six papers. Collins and colleagues also describe their application of fragment-based methods to develop inhibitors of the anti-cancer target protein kinase B (AKT).

Half the papers cover various forms of fragment screening: the Medical Structural Genomics of Pathogenic Protozoa Consortium describes their crystallographic approach, Helena Danielson discusses the use of SPR, and Gregg Siegal and Johan Hollander present their Target Immobilized NMR Screening (TINS) methodology.

Charles Reynolds and colleagues review metrics, such as ligand efficiency and fit quality, for evaluating fragment hits.

Finally, in the longest article in the collection, Vicki Nienaber discusses how fragment-based methods may be particularly useful for discovering compounds that will cross the blood-brain-barrier to target the central nervous system.

As 2009 comes to a close, Practical Fragments would like to thank our readers, old and new. May you all have a happy and productive 2010!