18 December 2023

Review of 2023 reviews

The annual Practical Fragments look-back on the preceding year may not be the most highly anticipated year-end tradition, but I hope you find something of interest in this twelfth edition.
I was fortunate to attend several conferences and wrote about CHI’s Discovery on Target in Boston and Drug Discovery Chemistry in San Diego. As for reviews, Louise Walsh and collaborators at Astex, Vrije Universiteit Amsterdam, Novartis, and Frontier Medicines (me!) published our annual analysis of fragment-to-lead success stories in J. Med. Chem., this one covering the year 2021. Some twenty other reviews of interest to this readership were also published. I’ll cover them thematically below.
Crystallography is the most popular fragment-finding technique, and in Expert Opin. Drug Disc. Wladek Minor and collaborators at University of Virginia and Jagiellonian University examine “the current role and evolution of X-ray crystallography in drug discovery and development.” At the start of 2023 the Protein Data Bank (PDB) contained more than 200,000 structures, which sounds impressive until you learn that the AlphaFold database contains more than 200 million predicted protein structures. But this is not experimentalist vs machine: the researchers note how machine learning approaches can be used to more rapidly refine and improve experimental data with resources such as CheckMyBlob and PDB-REDO.
For those wishing to dig deeper, two papers in Methods Enzymol. go into experimental detail. In the first, Natalie Tatum and colleagues at Newcastle University describe “crystallographic fragment screening in academic drug discovery.” May Sharpe and collaborators at the Swiss Light Source and University of Hohenheim describe their fast fragment-screening pipeline in a comprehensive (49 page) guide. The focus is on reproducibility, and there is plenty of practical advice. For example, “the authors have even been successful in flying with crystal plates,” though getting these through airport security may be easier in some countries than others.
Protein-detected NMR was the first truly practical fragment-based approach, and another paper in Methods Enzymol. by Brian Volkman, Brian Smith, and colleagues at Medical College of Wisconsin describes “fragment-screening by protein-detected NMR.” This distills eight years of effort building their internal protein-detected NMR fragment screening platform that has been applied to 16 proteins thus far. The chapter is particularly detailed on protein and library preparation and screening.
Compared with crystallography and NMR, virtual screening can be dramatically faster; we’ve highlighted multibillion-compound screens. In WIREs Comput. Mol. Sci., Artem Cherkasov, Francesco Gentile, and colleagues at University of British Columbia and University of Ottawa discuss (open access) how computational methods are “keeping pace with the explosive growth of chemical libraries.” They cover brute force methods, fragment-based virtual screening, and machine-learning based methods, all while avoiding hype, and conclude that it will take time for these methods to “have a real impact on practical drug discovery.”
Finally, Marianne Fillet and collaborators at University of Liege and University of Namur provide a general review in Trends Anal. Chem. covering multiple methods to detect non-covalent fragments. These include established techniques such as biochemical assays, ligand-observed NMR, crystallography, thermal shifts, and SPR, as well as less common ones such as WAC, microscale thermophoresis, ACE, and DEL. The paper includes several nice tables and even a decision tree to help choose among the various approaches.
Covalent fragments
Many techniques to detect noncovalent interactions also apply to reversible covalent inhibitors, the subject of a review in Med. Chem. Res. by Faridoon and collaborators at Genhouse Bio and Olema Oncology. The researchers focus on various warheads including cyanoacrylamides, nitriles, ketones and aldehydes, boronic acids, and others, and provide multiple examples for each.
In contrast, an open-access review in Pharmaceuticals by Monique Multeder and collaborators at Leiden University Medical Center discusses methods to detect both reversible as well as irreversible covalent protein-drug adducts. Crystallography is the most informative, but the researchers also delve into various mass-spectrometry techniques including top-down (with intact proteins) and bottom-up (after digestion of modified proteins). Also covered are activity-based protein profiling (ABPP) methods, NMR, and fluorescence-based approaches. The nearly 300 references make a useful compendium.
One of the most exciting recent developments is “proteome-wide fragment-based ligand and target discovery,” the subject of an open-access review in Isr. J. Chem. by Ines Forrest and Christopher Parker, both at Scripps. This concise, highly readable account covers a lot of ground, from ABPP to fully functionalized fragments (FFFs) to phenotypic screening.
If you’re doing covalent FBLD you’ll need a library of covalent fragments, and if you’re building one, I’d recommend a review in Prog. Med. Chem. by David Mann and colleagues at Imperial College London. The paper nicely summarizes design principles such as choice of warhead and the fact that reactivity can vary considerably even among compounds with the same warhead. Synthetic methods and screening approaches are also well covered, along with methods to distinguish specific binding from nonspecific reactivity.
Most covalent fragments target cysteine residues, but there at least nine other potentially reactive amino acids, and these are the subject of an open-access review by György Keserű and colleagues at Budapest University of Technology and Economics in Trends Pharm. Sci. Lysine, serine, threonine, tyrosine, and histidine are the most common targets, though some of the warheads are so reactive that specificity will be challenging, let alone reasonable pharmacokinetic properties. This is especially true for aspartic and glutamic acids, methionine, and tryptophan.
Finally, another article in Trends Pharm. Sci. by Carlo Ballatore and colleagues at University of California San Diego describes using covalent strategies to develop stabilizers and inhibitors of protein-protein interactions (PPIs). Site-directed fragment tethering with disulfide and imine chemistry is a focus, particularly in the context of 14-3-3 proteins. Proximity-enabled covalent strategies, in which warheads are grafted onto non-covalent molecules, are also covered. There is also a short section on covalent PROTACs – more on that topic below.
Keeping with the theme of protein-protein interactions, Ge-Fei Hao, Guang-Fu Yang, and collaborators at Central China Normal University and Guizhou University discuss fragment-based approaches against “undruggable” PPIs in Trends Biochem. Sci. After describing why protein-protein interactions can be difficult, the paper presents several successful case studies, including venetoclax, sotorasib, and targeting 14-3-3 proteins.
Targeted protein degradation continues to be a major focus for drug discovery, and this is commonly achieved by hijacking E3 ligases to cause them to ubiquitinate a target of interest. Iacovos Michaelides and Gavin Collie (AstraZeneca) describe how FBLD has been used to find ligands against E3s in an open-access J. Med. Chem. paper. There are more than 600 E3s, and because their biology relies on protein-protein interactions they are often tough targets. Fragment hits can be weak and difficult to advance, though the researchers do describe several success stories including against KEAP1 and XIAP/cIAP. Covalent fragments have the potential to permanently reprogram E3 ligases, and these are covered well too.
Another difficult type of target is RNA, the topic of two reviews. In an open-access Curr. Opin. Struct. Biol. paper Kevin Weeks and colleagues at University of North Carolina Chapel Hill provide a concise and beautifully illustrated overview of the field. They note that “RNA-targeted FBLD is in its infancy,” but given that the first report dates to 2002 it is a long childhood, and the paper does a good job of describing the challenges.
A more extensive treatment of “fragment-based approaches to identify RNA binders” is provided by Matthew Disney and colleagues at UF Scripps in J. Med. Chem. The paper describes many case studies, some of which we’ve covered, and also contains a handy table comparing the pros and cons of a dozen different methods for finding RNA-binding fragments.
Tuberculosis kills more than 1.5 million people each year, and fragment-based approaches have been applied against multiple targets within the pathogen, as reviewed by Baptiste Villemagne and colleagues at University Lille in Eur. J. Med. Chem. We’ve covered many of these studies on Practical Fragments, but as the paper notes none have advanced to the clinic. This is attributed in part to cell permeability, and the researchers suggest turning to phenotypic screens (see below).
Fragment linking can be difficult but highly effective, especially for difficult targets. An overview of published linkers is provided by Isabelle Krimm and collaborators at Université Claude Bernard Lyon and Université Montpellier in Expert Opin. Drug Disc. The paper includes a table summarizing 40 fragment linking stories, noting that most linkers are short and flexible. Another table summarizes 19 examples of target-guided synthesis, including dynamic combinatorial chemistry. As the paper notes, all of these are small model studies based on known compounds. In silico approaches, the last topic covered, will probably prove more practical.
And on the subject of practical, Dean Brown (Jnana Therapeutics) provides an “analysis of successful hit-to-clinical candidate pairs” in J. Med. Chem. This is an update to his 2018 article and captures 156 clinical candidates reported in the journal between 2018 and 2021. Of these, 14 had fragments in their lineage. Most of these drugs appear in our list of fragment-derived clinical candidates (though berotralstat does not – I’ll need to look closer). The paper contains lots of interesting analyses. For example, of the 138 oral drugs, 39 had a molecular weight > 500 Da, 24 had Clog > 5, and 17 had more than 10 hydrogen bond acceptors (HBA). On the other hand, none had more than 5 HBD, emphasizing that you should be parsimonious with hydrogen bond donors.
Finally, veteran drug hunter Nicholas Meanwell provides “reflections on a 40-year career in drug design and discovery” (open access) in Med. Chem. Rev. Those of you who saw his talk earlier this year at the CHI DDC meeting will know what to expect, and those of you who didn’t will be in for a treat. A personal and entertaining romp through pharma starting in the early 1980s, the paper is full of surprises, such as the pursuit of minor impurities in a phenotypic screen that ultimately led to the hepatitis C drug daclatasvir. Nicholas notes that “you discover what you screen for, so screen design is of paramount importance.”
The paper also reveals a passion for medicinal chemistry: “In a search for inspiration for design concepts, I sat down one Saturday afternoon in early October of 1987 and perused every molecule in the United States Adopted Names (USAN) dictionary.” And, as he notes near the end, “Decision making in drug discovery and development is a delicate balancing act, inherently flawed based on absence of predictive accuracy, and knowing when to conclude a discovery program with grace is also an important trait.” That said, he provides examples of successful programs that were almost killed multiple times – and others that were killed at Bristol Myers Squibb but subsequently succeeded elsewhere. While this is frustrating on one level, Nicholas takes satisfaction in the fact that “the science that we conducted and the molecules and pharmacophores that we defined have been of benefit to mankind.”
There are still a couple weeks left in the year, but that’s it for Practical Fragments for 2023. Thanks for reading, and special thanks for commenting. And if you live in one of the 70+ countries with elections in 2024, please vote.

11 December 2023

Fragments vs IP6K1 without using structural information

The three members of the inositol hexakisphophate kinase family are potential targets for a wide range of diseases, from Alzheimer’s to cancer to metabolic disease. However, current inhibitors are not specific for individual isoforms. Also, the most potent compounds contain a carboxylic acid moiety, which is usually at odds with brain penetration. In a new ACS Med. Chem. Lett. paper, James Barrow and collaborators at Johns Hopkins School of Medicine, the Lieber Institute for Brain Development, and AstraZeneca describe neutral, selective inhibitors.
The researchers started with a high-throughput biochemical screen of 17,000 fragments, each at 100 µM, against IP6K1. The library itself is available here. After dose-response follow-up studies, 90 hits confirmed, with IC50 values as good as 2 µM. Most of the hits contained carboxylic acids, but compound 5 did not, and also had good ligand efficiency.
No crystal structures of IP6K1 have been reported, so the researchers used an AlphaFold model for docking compound 5. This suggested a fragment growing approach. A variety of replacements for the pyrrolidine were attempted, and while some of these had improved activity many also proved to be chemically unstable. Removing the nitrogen and growing led to compound 24, which was both chemically stable and had sub-micromolar activity.
The quinazolinone core itself was associated with poor solubility, and the researchers made multiple attempts to modify it, such as introducing additional nitrogen atoms or methylating to remove a hydrogen-bond donor. Unfortunately, all these modifications led to significant losses in potency.
Compound 24 is highly selective for IP6K1 over IP6K2 and somewhat selective over IP6K3. Unfortunately, it showed no cellular activity, possibly due to modest biochemical potency and solubility. Nonetheless, this brief paper illustrates that starting with a larger than normal fragment library can lead to new chemotypes. Screening the larger library gave the researchers more chances to find fragments that did not contain carboxylic acids. Indeed, the difficulty of modifying the quinazolinone moiety demonstrates the utility of screening more molecules. Had the closely related molecules been in the library, they might not have turned up as hits, but their presence would suggest that relevant chemical space had been interrogated.
The paper is also a nice example of optimizing hits in the absence of structural information. Although much needs to be done to turn compound 24 into a chemical probe, the fact that it is still so small (almost rule-of-three compliant) provides hope that this can be done.

04 December 2023

Screening tough proteins by SPR

Surface-plasmon resonance (SPR) is among the most popular methods for finding fragments. However, as we have noted, SPR can be very prone to operator error and misinterpretation. In a recent (open access) SLAS Discovery paper, U. Helena Danielson (Uppsala University) and a who’s-who team of biophysicists from across Europe provide experimental strategies for screening difficult proteins.
The researchers chose five different proteins, some of which were screened in two or three different forms for a total of nine protein constructs. Six of these were screened against their FL1056 library, a custom-built 1056-member library which include molecules from the FragNet program. The library includes a number of “three dimensional” molecules as assessed by principal moment of inertia (PMI). The other library, FL90, is a small set of commercially available fragments we highlighted here.
Before screening compounds against proteins, the researchers conducted a “clean screen.” This involved injecting fragments (at 500 µM each) over the sensor surface using the same buffer that would be used in the actual screen to pre-identify fragments that stick to the surface. This typically disqualified about 1% of fragments, though for one set of conditions the number was closer to 3%.
That work done, the researchers turned to the actual screens. After proteins were immobilized on the sensor chips, the fragments were typically screened at a single concentration of 250 µM each. The threshold for the initial hit cutoff was set low, often around 10% of the library, to minimize false negatives. Subsequent follow-up studies at varying concentrations were used for confirmation. This led to a significant winnowing, with the final number of confirmed hits between 0.5 and 7% of the library.
The proteins themselves were intentionally chosen to present various difficulties. Acetylcholine binding protein (AChBP, which we wrote about here) forms a large (125 kDa) pentameric complex with multiple binding sites. Lysine demethylase 1 (LSD1) is a multidomain, cofactor-dependent protein that requires a partner protein, CoREST, for activity. LSD1 was screened in the presence or absence of CoREST. Farnesyl pyrophosphate synthase (FPPS, which we wrote about here) is a target for cancer and osteoporosis, and the microbial forms are targets for trypanosomiasis drugs. Human as well as Trypanosoma cruzi and Trypanosoma brucei proteins were screened. Protein tyrosine phosphatase 1B (PTP1B, which we recently wrote about here) is a difficult enzyme with a couple allosteric sites. The C-terminal region is intrinsically disordered, and the protein was screened with or without this region. Finally, human tau is both intrinsically disordered and prone to aggregation. As we noted earlier this year it is of interest due to its potential role in Alzheimer’s disease.
Happily, hits were identified against all the proteins, some with ligand efficiency values above 0.5 kcal/mol per heavy atom. The chemical structures for selected hits are shown, and the researchers do appropriately caution that validating them using orthogonal (non-SPR) methods is essential before further studies.
I do wish the researchers had noted whether shapely hits were enriched or depleted among the confirmed hits. To my eye most seemed fairly flat, and some seemed dubiously PAINS-like, including an eyebrow-raising dinitro-catechol. Nonetheless, the paper is a nice summary of multiple SPR campaigns. If you’re about to embark on one yourself, it is worth carefully perusing.