Targeting SARS-CoV-2 RNA – but not specifically

Last week we highlighted work suggesting that small molecule binding sites in RNA are most likely to be found in complex structures. A new open-access paper in Angew. Chem. Int. Ed. by Harald Schwalbe and collaborators at Goethe University Frankfurt and elsewhere provides both a case in point and an illustration of how difficult it is to target RNA.

 

The researchers had previously screened 15 RNAs from the SARS-CoV-2 virus, an effort we highlighted in 2021. In the new paper, the researchers focus on a portion of the frameshift element, which is important for directing viral replication from either of two partially overlapping open reading frames. The core of this RNA element is a roughly 69-nucleotide-long structure called a pseudoknot. Like most RNA sequences, this one can form multiple structures, including dimers, and the researchers used NMR, small-angle X-ray scattering (SAXS), and native gel electrophoresis to confirm that the construct was behaving as a homogenous monomer, consistent with three previously determined structures.

 

Based on some of the initial fragment hits, the researchers selected 50 similar molecules, of which only 14 were sufficiently soluble for screening. One of the more potent compounds, D05, initially showed promising activity in a ligand-detected NMR assay but turned out to be completely inactive when retested from a fresh stock. It turns out that D05 decomposes to compound 2, which was confirmed as active. Further modification led to compound 4, the most potent compound described. (Dissociation constants were determined by NMR, fluorescence, or both, and the two methods were in good agreement.)

Two-dimensional NMR with isotopically labeled RNA was used to try to determine the location of the binding site(s). Even with access to a 1.2 GHz magnet, the NMR peaks were severely overlapped, so the researchers used segmental isotopic labeling, in which just half of the RNA was labeled at a time. This exercise revealed potentially three different binding sites for compound 2.

 

The researchers also used two different computational approaches, Vina and RLdock, to predict binding sites, each of which could find one or two of the binding sites identified by NMR.

 

Several compounds were tested to see if they could block frameshifting in cell-lysates, and compound 2 showed 40% inhibition at 145 µM.

 

So far so good. But consistent with best practices, the researchers tested compounds 2 and 4 against phenylalanine tRNA. Unfortunately, the two ligands exhibited similar affinities to this control RNA as they did to the SARS-CoV-2 pseudoknot, despite the lack of sequence similarity. This suggests that these ligands bind to RNA nonspecifically. Perhaps this is not surprising given the three binding sites observed in a single 69-mer.

 

In the end, this is a thorough but sobering paper. Despite an impressive screening campaign with multiple biophysical methods, the best ligands seem to have modest affinity and low specificity. Drugging RNA still appears much more difficult than drugging proteins. But for either sort of target, this sort of careful work will be essential to find promising leads.

Identifying ligand-binding pockets in RNA, computationally and experimentally

Most drugs bind to proteins, but RNA provides many interesting targets. Unfortunately, finding drug-like small molecules that bind to RNA is difficult. A new paper in Proc. Nat. Acad. Sci. USA from Kevin Weeks and colleagues at University of North Carolina Chapel Hill provides tools to do so.

 

RNA presents several challenges for drug discovery. First, there are far fewer high-resolution structures than there are for proteins. This is in part due to the second challenge: RNA strands are often wriggly, able to form multiple conformations. And finally, RNA is highly charged and more polar than most proteins, so there are fewer opportunities for the hydrophobic interactions that often provide significant affinity in protein-ligand complexes.

 

These challenges have not deterred intrepid investigators: Practical Fragments first wrote about targeting RNA with fragments way back in 2009. However, examples of high-affinity ligands remain elusive, and in 2023 I wondered whether “most RNA is truly undruggable.”

 

The latest paper leaves me more optimistic. It describes a computational approach to find small-molecule binding sites in RNA. The researchers started with an open-source tool called fpocket, which was built for proteins. The fpocket program places virtual spheres all around a biomolecule, where each sphere contacts the center of four atoms. The size of each sphere depends on local curvature, and clusters of spheres define pockets.

 

To benchmark fpocket on RNA, the researchers first constructed a curated database of drug-like ligands bound to RNA. Of 538 RNA-ligand structures solved at the fairly low bar of ˂ 3.5 Å resolution, only 48 ligands were deemed drug-like by the quantitative estimate of drug-likeness (QED) score. (Although the QED score may be overly restrictive, and many approved drugs have low QED scores, setting a strict threshold means that any pockets identified are likely to be particularly attractive.)

 

Using default (protein-appropriate) parameters, fpocket identified just 63% of known ligand-binding sites in RNA, vs 83% for proteins. Worse, many predicted RNA pockets probably aren’t actually ligandable because they are too exposed to solvent. By tweaking parameters, the researchers improved performance of the program for RNA to 92%, and they also identified several attractive pockets that had previously been missed.

 

When the researchers applied the reparametrized program, redubbed fpocketR, to two bacterial ribosomes, they found several dozen pockets in each, including known antibiotic-binding sites. To assess whether the new pockets could bind fragments, they used an experimental approach called Frag-MaP, which uses fully functionalized fragment (FFF) probes containing a variable fragment, a photoreactive diazirine, and an alkyne. Treating bacterial cells with these FFF probes in the presence of UV light crosslinks them to nearby RNA. Crosslinked probes can then be isolated using click chemistry with the alkyne, and RNA sequencing reveals the sites of modification. Impressively, 89% of ligand binding sites found in the Frag-MaP experiments were predicted by fpocketR.

 

In another validation experiment, fpocketR identified pockets where 7 out of 17 antibiotics bind to bacterial ribosomes. Notably, all but one of the undetected pockets bind antibiotics such as aminoglycosides that don’t appear conventionally drug-like and indeed are not orally bioavailable.

 

Continuing to apply fpocketR to more RNAs led to the identification of dozens of new pockets. Interestingly, most of these pockets occur in complex RNA structures, such as multi-helix junctions or pseudoknots, rather than simpler structures such as bulges and consecutive loops. This could explain the paucity of fragment hits in a study we highlighted in 2023, which focused on simple loops.

 

Now that we know where to find attractive ligand-binding pockets in RNA, hopefully we will be more successful finding high-affinity ligands.

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.

 

Methods

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.

 

Targets

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).

 

Other

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.

Fragment linking on the bacterial TPP riboswitch

Last week’s post focused on fragment screening against RNA, and we continue the theme this week with a paper published in Proc. Nat. Acad. USA by Kevin Weeks and collaborators at University of North Carolina Chapel Hill, New York University, and Université de Sherbrooke.

 

The researchers developed a screening technology called SHAPE-MaP (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension and Mutational Profiling). Essentially, RNA in the presence or absence of potential ligands is treated with an acylating agent that reacts with the 2’-hydroxyl group on ribose subunits. This addition requires the hydroxyl groups to be exposed, so ligands that bind in the vicinity may directly block or cause conformational changes to change the patterns of acylation. Conveniently, acylation causes mutations when the modified RNA is sequenced, making modified sites easy to detect. Moreover, by clever uses of “barcodes” in other regions of the RNA, multiple samples can be pooled and analyzed.

 

Because the approach uses sequencing to identify binding sites, long strands of RNA can be tested. In this case, the researchers built an RNA construct containing a ‘pseudoknot’ structure in the dengue virus genome as well as a thiamine pyrophosphate (TPP) riboswitch, which changes conformation when it binds to TPP. (We wrote about a different fragment screen against this riboswitch back in 2014). A set of 1500 rule-of-three compliant fragments from Maybridge was screened, resulting in 41 hits. These were then rescreened in triplicate, which winnowed the field to just eight fragments, of which seven bound TPP and one appeared to be nonspecific. All eight were assessed by isothermal titration calorimetry (ITC), which produced measurable affinities for six.

 

Compound 2 was the most potent hit, and when the researchers tested 16 analogs they found some, such as compound 17, with improved affinity. With an eye towards fragment linking, they took a conceptually similar approach to SAR by NMR by rescreening the original 1500 fragments in the presence of compound 2 to look for ligands that would bind at a second site. This yielded five hits, including compound 28. ITC characterization of a more soluble analog, compound 31, revealed that it had weak but measurably improved affinity in the presence of compound 2. A handful of linked analogs were made, and while most of these had affinity similar or worse than the best initial fragment, compound Z1 bound with sub-micromolar affinity as assessed by ITC.

 

The natural ligand TPP binds to the riboswitch with a K_d of 110 nM, and in doing so blocks in vitro transcription of bound RNA. Despite having a similar affinity as TPP, compound Z1 was much less effective at blocking transcription. Unfortunately, although the researchers were able to obtain crystal structures of several molecules bound to the riboswitch, including compound 17, they were unable to obtain one with compound Z1.

 

This is a rare example of fragment linking on RNA, and although the linked molecule does not show fully additive affinity, it does have reasonable ligand efficiency. But like the example last week, this paper illustrates how difficult discovering RNA binders is likely to be. The confirmed hit rate is less than 0.5%, and this is for an RNA sequence that evolved specifically to bind low molecular weight ligands. As the researchers note, none of the fragments bound the dengue virus pseudoknot. Perhaps most RNA is truly undruggable, at least with small molecules.

A cell-active fragment targets the microRNA-372 hairpin precursor

Fragment-based lead discovery against RNA has been a theme on Practical Fragments for well over a decade. Unfortunately, most of the resulting hits are either weak or non-druglike. A paper published late last year in J. Am Chem. Soc. by Matthew Disney and collaborators at the Scripps Research Institute in Florida provides a nice counter example.

 

The researchers started by building a new fragment library based on known RNA-binding molecules – a venerable approach we first described back in 2009. The most common scaffolds differ somewhat from the most common scaffolds found in drugs (see here), with pyrimidines, triazoles, furans, and benzimidazoles over-represented. A set of 2500 fragments mostly conforming to the rule of three was purchased from ChemDiv, and the structures of all of them are helpfully provided in the supporting information.

 

Most fragment screening efforts start with a target of interest, but here the researchers chose a “library-versus-library” format, in which they screened all 2500 fragments against 5120 different RNAs. Each RNA molecule consisted of an identical 40-nucleotide hairpin containing randomized 3 x 2 or 3 x 3 internal nucleotide loops. Fragments were immobilized onto an agarose-coated microarray and radiolabeled RNA was added. Interestingly, only 19 fragments were found to bind the RNA. Competition experiments with other RNA sequences and DNA reduced the number of specific binders down to three. RNA sequencing experiments revealed that two of these fragments were fairly promiscuous, each binding over 100 different RNAs, but compound 3 bound just 28 RNAs with the 3 x 2 internal loop and none with the 3 x 3 internal loop.

 

Having identified specific RNA sequences bound by compound 3, the researchers searched human microRNAs (miRNAs) and found that the pre-miR-372 contains a bulge predicted to bind. When this RNA is processed it produces miR-372, which represses translation of the tumor suppressor LATS2, thereby increasing cellular proliferation. Perhaps the binding of compound 3 to the pre-miRNA would impede its processing.

 

The affinity of compound 3 for pre-miR-372 was measured to be 300 nM, giving it an impressively high ligand efficiency. In vitro experiments showed that the compound blocked processing by the enzyme Dicer. The researchers conducted a series of cellular experiments showing that compound 3 decreased levels of miR-372 and increased pre-miR-372m while having no effect on 379 other miRNAs. Encouragingly, compound 3 also increased levels of LATS2 mRNA and protein and decreased cell proliferation. Additional experiments with siRNA, mutated versions of pre-miR-372, and cell lines with lower levels of miR-372 all support the on-target mechanism.

 

This is a lovely paper, and compound 3 appears to be a good starting point for further optimization. However, the work also suggests why finding lead-like RNA binders may be so difficult. In the library-vs-library approach described, the researchers studied 12,800,000 different molecular interactions and came up with just three (somewhat) specific binders. The ligandability of RNA – or at least the small internal loops studied here – appears to be low. To prospectively find hits against specific RNAs may require much larger fragment libraries than are typically used. Perhaps this could be an application for DNA-encoded fragment libraries, which we wrote about here.

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.

Fragments vs SARS-CoV-2 RNA

The first mention of SARS-CoV-2 on Practical Fragments in early March of last year highlighted a crystallographic fragment screen against the main viral protease. As discussed last week this effort has now led to compounds with nanomolar activity in cells. We’ve also highlighted a separate crystallographic screen against this target as well as a screen against the Nsp3 macrodomain. But proteins are not the only potential viral targets.

 

A recent (open access) paper in Angew. Chem. Int. Ed. by Harald Schwalbe and a large group of collaborators mostly at Johann Wolfgang Goethe-University focuses not on proteins but on RNA. Harald also presented this work at Discovery on Target last week, where he noted that the effort is part of the COVID19-NMR project, a collaboration of 240 people in 18 countries.

 

The researchers investigated 15 RNA regulatory elements that are conserved between SARS-CoV-2 and SARS-CoV, ranging from 29-90 nucleotides, as well as 5 larger multielement RNAs (118-472 nucleotides). These were screened against the DSI-poised library (discussed here): 768 fragments designed for rapid follow-up chemistry.

 

Three different ligand-detected NMR methods were used for screening: chemical shift perturbation (CSP) or line-broadening, waterLOGSY, and T2-relaxation. Fragments were screened at 200 µM in pools of 12 against 10 µM RNA. Compounds that hit in at least two assays were investigated individually.

 

In total 40 fragments bound to one or more of the 15 shorter RNAs, and an additional 29 fragments bound to the five longer RNAs. Between 5 and 49 hits were found for all but two of the RNAs. Selectivity varied: some fragments bound to just one RNA while one fragment bound to 18 of 20.

 

Given the negatively-charged phosphate backbone of RNA, it is not surprising that many of the fragment hits are positively charged at physiological pH. Nearly one-third of the 40 hits against the shorter RNAs contain a basic amine; pyrimidine and benzimidazole moieties are enriched, and not one of the hits contain a carboxylic acid. All the hits have at least one aromatic ring and most have two or three, perhaps suggesting intercalation. Moreover, as seen in a previous ambitious RNA screen from the same group, hits tend to have fewer sp³-carbons than non-hits.

 

The highest affinity fragment had a dissociation constant of just 64 µM but an impressive ligand efficiency of 0.38 kcal/mol/atom. A search of commercial analogs yielded a compound with low micromolar affinity against two RNA targets. In his presentation Harald noted that this series has since been optimized to a 200 nM binder.

 

This paper is a tour de force, but as I have noted, there remains a dearth of high-affinity specific RNA binders. The researchers also note another potential problem: viral RNA accounts for roughly two-thirds of total RNA in cells infected with SARS-CoV-2. Would this necessitate high concentrations of drug for effective antiviral activity?

 

Whether or not the work leads to drugs, it should further basic research. Laudably, structures of all the hits and non-hits are provided in the paper, and the extensive supporting information provides more details. Hopefully we will soon see whether fragments poised for ready elaboration really will enable rapid progress against RNA.

Fragments vs (lots of) RNA

RNA is hot. Hundreds of millions of dollars have gone to startups focused on finding small molecules that bind RNA, and plenty of large pharmaceutical companies are pursuing this class. As with all difficult targets, fragments have a role to play, as demonstrated by a paper just published (open access!) in ChemBioChem by Harald Schwalbe and collaborators at Johann Wolfgang Goethe-University Frankfurt and Saverna Therapeutics.

 

The researchers assembled a collection of 101 fluorine-containing fragments and pooled these into five sets of 20-21 compounds each. These were then screened (at 50 µM) against 14 different RNA targets, ranging from 14-nucleotide hairpins to a 127-nucleotide riboswitch. The primary screen was ligand-detected ¹⁹F NMR using CPMG, in which ligand binding causes a change in relaxation which is detected as a decreased signal. All hits from the mixtures were confirmed as single molecules. To assess selectivity, all the compounds were also screened against 5 DNA targets and 5 proteins.

 

The results are not entirely unexpected. Some of the fragments did not hit any targets, while others were rather promiscuous: a couple showed strong binding to 8 of the 14 RNA targets. That said, strong is a relative term; the highest affinity of any fragment measured was 375 µM.

 

The RNA targets spanned a variety of structures, but the most hit-rich were aptamers and riboswitches, which bind specific small molecules or ions. These had 7 to 26 hits each. In contrast, the other RNA targets tested all had six or fewer fragment hits. For the aptamers, competition experiments with the natural ligands suggested binding at the orthosteric sites in some cases but not others.

 

Hit rates against four of the five proteins were also high, with 16 to 55 hits each. The fifth protein, a challenging phosphatase, had only four hits. This was still better than the 24-nucleotide DNA duplex, with a single hit. The four G-quadruplex DNA targets had between 12 and 20 hits, consistent with prior research. Taken together, the results suggest that RNA riboswitches and aptamers may be reasonably ligandable, while RNA targets that do not normally bind small molecules may be more challenging.

 

The researchers also conducted cheminformatic analyses. Not surprisingly given the relatively small library, there were no strong correlations between molecular features and targets bound, though fragments that hit had a slight tendency towards more aromatic atoms and fewer sp³-hybridized carbons. This is consistent with a vigorously debated paper we highlighted in July.

 

Finding weak hits is one thing, but advancing them has been challenging for RNA targets. The researchers provide an example in which they link a fragment to an intercalator to generate a low micromolar binder, but intercalators are often nonspecific, and affinity would still need to be further improved. Practical Fragments first highlighted a fragment screen against RNA more than a decade ago, and earlier this year we noted a high nanomolar binder, but I have yet to see an attractive low nanomolar lead emerge.

 

Nonetheless, this paper provides a solid launching point for such an effort to succeed. In particular, the researchers laudably include structures of all the library members as well as the raw screening data of all the hits on all the targets in the Supporting Information. If you are feeling adventurous, you now have plenty of starting points to choose from.

In situ click chemistry on RNA

In templated or in situ reactions, bonds form between two fragments that are brought together in the context of a larger molecule such as a protein. We have written previously about dynamic combinatorial chemistry (DCC), which depends on reversible bond formation where the larger molecule shifts the equilibrium toward the linked fragments. For irreversible bond formation, the larger molecule effectively catalyzes the formation of an inhibitor (or at least a binder). In a recent Angew. Chem. Int. Ed. paper Jyotirmayee Dash and colleagues at the Indian Association for the Cultivation of Science describe an application of the latter that uses RNA as the template.

The target of interest was TAR RNA, a short region of viral RNA essential for HIV replication. We have previously highlighted a few examples of fragment screening against RNA (here, here, and here), including TAR, but most of the hits were weak.

The researchers used azide-alkyne cycloaddition, the quintessential click chemistry reaction. They built a small library of four alkynes (only one of which was fragment-sized) and 11 azides. All of these were incubated together (4 µM of each alkyne and 1 µM of each azide) in the presence of 5 µM biotin-labeled TAR RNA for 72 hours. (The reaction is typically slow at room temperatures unless catalyzed by metal ions.) Magnetic streptavidin-coated beads were then used to capture the RNA and any bound ligands, which were identified by HPLC-MS. Control experiments were run with TAR DNA or a mutant form of TAR RNA lacking an essential bulge. The result was one fairly potent compound (below) that was specific for TAR RNA, as well as a couple other molecules that were both weaker and less specific.

The affinity of compound 3ba for TAR RNA was measured by isothermal titration calorimetry (ITC) as well as by a fluorescence assay, which were in good agreement. Importantly, the ITC data suggested 1:1 binding, which is particularly important given that the ligand contains two 2-aminothiazoles, a moiety that has been called a PrAT for its promiscuous behavior. Finally, the ligand could displace the Tat peptide at low micromolar concentrations, suggesting that it is binding at the biologically relevant site of TAR.

I do have a few quibbles. It would have been interesting if the researchers had reported the affinity of the azide and alkyne themselves to see how much of a boost they got by linking. And since the most potent molecule is not always selected from target-guided synthesis, it would have been interesting to make and test other possible cycloaddition products to see if they missed anything useful.

Still, it is nice to see a submicromolar RNA binder come out of an in situ screen. Targeting RNA with small molecules has recently become trendy, and it will be fun to see how far approaches like these can go.