Review of 2025 reviews

Turning and turning in the widening gyre

The falcon cannot hear the falconer…

 

In our small, annual counterweight to Yeats’ “mere anarchy," Practical Fragments looks back on 2025.

 

This year marked the thousandth post on Practical Fragments, a milestone neither Teddy nor I imagined when the blog launched back in 2008. In terms of conferences, I wrote about CHI’s Drug Discovery Chemistry in San Diego here and Discovery on Target in Boston here.

 

For the past decade I’ve participated in annual J. Med. Chem. perspectives covering fragment-to-lead success stories, two of which published this year. The first, spearhead by Rhian Holvey at Astex, covers the year 2023, while Astex’s David Twigg took the lead on covering the year 2024. In addition to the tabular summaries for which these reviews are best known, both also include tables of “near misses,” none of which made the main tables because the starting points were (sometimes just slightly) too large. Four out of six of these heavyweights are covalent fragments, suggesting that the rule of three may need to be relaxed for these. The most recent paper also includes a table showcasing the eight approved FBLD-derived drugs.

 

Two more general publications are also of interest. In a brief (open-access) editorial in J. Med. Chem. Weijun Xu and Congbao Kang at A*STAR summarize fragment-finding methods and approved drugs and discuss future applications of FBLD in PROTACs and targeting RNA. And in Curr. Res. Pharm. Drug Discov., Geoffrey Wells, Exequiel Porta, and colleagues at University College London present a “graphical review” which covers library design, screening strategies, hit validation, fragment optimization, and a few case studies of approved drugs, along with current challenges.

 

In Drug Des. Devel. Ther., Bangjiang Fang and colleagues at Shanghai University of Traditional Chinese Medicine present an open-access bibliometrics analysis of 1301 fragment-based drug design papers published between 2015 and the end of 2024, which includes top ten lists of institutions, authors, and papers as well as keyword trends and analyses. Annual growth has averaged 1.4%, and the field is both global and collaborative, with 35% of publications involving more than one country.

 

Targets

Two reviews focus on oncology. The first, in Bioorg. Chem. by Milind Sindkhedkar and collaborators at Manipal College of Pharmaceutical Sciences and Lupin Ltd., briefly covers the history and practice of FBDD before providing short summaries of seven of the eight approved drugs to come from it. The second, published open-access in Chem. Rev. by Vanderbilt’s Steve Fesik, is a concise and highly readable introduction and account of the author’s groundbreaking work on BCL-2 family proteins, KRAS, and WDR5.

 

A much longer open-access review in Chem. Rev. by Paramjit Arora and colleagues at New York University covers protein-protein interactions (PPIs). Much of the focus is on larger molecules such as cyclic peptides, peptide mimetics, and other macrocycles, but there are summaries of fragment-based approaches against KRAS and 14-3-3 proteins.

 

The topic of 14-3-3 proteins is treated more fully in an open-access Acc. Chem. Res. paper by Michelle Arkin and colleagues at UCSF. While the focus of most efforts against PPIs is to find inhibitors, for 14-3-3 the goal is to find stabilizers, or molecular glues. The Arkin lab and others have been succeeding using various approaches, particularly disulfide tethering. We wrote about these efforts most recently in 2023, and the new review provides a nice update.

 

Fragment finding methods and libraries

Sahra St. John-Campbell and Gurdip Bhalay, both at The Institute of Cancer Research, published a massive open-access perspective on “target engagement assays in early drug discovery” in J. Med. Chem., covering a host of biochemical, biophysical, and cell-based assays. A table lists more than 50 different techniques, almost half of which are applicable to FBLD. Each row shows what characteristic(s) are measured as well as critical requirements for  protein, sample, and equipment. The paper is also beautifully illustrated with dozens of figures: one shows which techniques are most useful for different types of targets, and each method gets its own diagram.

 

A more focused open-access review is provided by Stefanie Freitag-Pohl and colleagues at Durham University in Biophys. Rev. After surveying various biophysical techniques, the researchers focus on spectral shift analysis, and in particular the Dianthus instrument from NanoTemper Instruments. This plate-based, high-throughput microfluidics-free instrument can detect changes in fluorescence caused by environment or temperature. Examples demonstrate affinity measurements across several orders of magnitude, up to double-digit millimolar, and a nice scheme shows use of the Dianthus in a fragment-screening workflow.

 

Moving to specific techniques, Jia Gao, Ke Ruan, and colleagues at University of Science and Technology of China Hefei provide an open-access survey of “the rise of NMR-integrated fragment-based drug discovery in China” in Mag. Res. Lett. After a brief overview of NMR approaches, they cover case studies from China, most of which are focused on fragment screening rather than optimization.

 

A less common biophysical method is native mass spectrometry (nMS), the subject of an open-access opinion in RSC Med. Chem. by Louise Sternicki and Sally-Ann Poulsen at Griffith University. This is a good survey of the approach; we highlighted a more fragment-focused review by the same authors last year.

 

The most common fragment-finding approach, X-ray crystallography, is covered in two open-access reviews. The first, in Acta Cryst. F by Sarah Bowman and collaborators at University of Buffalo and Brookhaven National Laboratory, focuses on critical early stages, from protein characterization to sample preparation and various crystallization approaches. The second, in Curr. Opin. Strut. Biol. by Martin Noble and colleagues at Newcastle University, starts by briefly reviewing crystallographic fragment screening before turning to fragment libraries. The paper includes a nice table summarizing publicly available libraries at major synchrotrons, with the text describing these in more detail.

 

The provider of one of these libraries, EU-OPENSCREEN, is the subject of an open-access review in SLAS Discov. by Robert Harmel and collaborators at EU-OPENSCREEN ERIC and Fraunhover ITMP. As we wrote last year, EU-OPENSCREEN is a broad consortium whose mission is to advance early drug discovery by providing access to technology and expertise. The new paper summarizes the four compound collections, including the European Fragment Screening Library (EFSL), and surveys progress to date. It also lays out ambitious plans, including expanding to >30 sites in nine countries.

 

Computational approaches and cryptic sites

Despite the hype about artificial intelligence in the broader world, AI in fragment-based drug discovery has been less common. In Curr. Opin. Struct. Biol., Woong-Hee Shin and colleagues at Korea University College of Medicine summarize applications to fragment growing, merging, and linking. The open-access paper includes a handy table of 13 programs, and includes GitHub links where available.

 

Cryptic binding sites, defined by Ehmke Pohl and collaborators at Durham University and Cambridge Crystallographic Data Centre “as binding pockets that exist in the ligand-bound state of a protein but not in its apo form,” are the focus of an open-access review in Bioinform. Adv. The researchers cover earlier computational approaches for finding these, especially molecular dynamics (MD) and machine learning (ML). They note that a key challenge for ML is the limited quantity and quality of experimental data: undiscovered cryptic sites would be misclassified as non-binding sites.

 

Yowen Dong, Ge-Fei Hao, and colleagues at Guizhou University review “computational methods for identifying cryptic pockets” in Drug Discov. Today. As with the previous review, these are divided between molecular dynamics and AI-based techniques, which are discussed individually and then compared. The researchers apply six approaches to the model bacterial protein TEM-1 β-lactamase and find that, for this highly studied single protein, the AI-based methods are much faster (seconds instead of days) and just as accurate, though MD-based methods provide more insight into formation mechanisms of cryptic pockets.

 

Covalent ligands

Allosteric sites are an important sub-class of cryptic pockets, and in J. Med. Chem. Jianing Li and colleagues at Purdue University discuss covalent allosteric inhibitors. After briefly discussing advantages of covalent molecules, they review examples targeting protein phosphatases, kinases, and GTPases, such as KRAS.

 

Of course, covalent molecules are not limited to allosteric sites. An open-access review in Bioorg. Med. Chem. Lett. by Walaa Bedewy, John Mulawka, and Marc Adler at Toronto Metropolitan University summarizes published covalent protein ligands, grouping them by target site:  active sites, residues adjacent to an active site, protein-protein interfaces, cofactor binding sites, and allosteric sites.

 

Chem. Rev. published two massive reviews on covalent ligands, each with more than 300 references. The first, by Tomonori Tamura, Masaharu Kawano, and Itaru Hamachi at Kyoto University, covers a wide range of topics, from covalent drugs, to peptide- and protein-based covalent inhibitors, to chemical biology labeling and target engagement strategies, to covalent bifunctional molecules such as PROTACs and radionucleotide-based molecules, and even covalent modification of DNA and RNA. The paper includes 68 figures, many reproduced from the original publications.

 

The second (open-access) Chem. Rev. paper, by Ku-Lung Hsu and colleagues at University of Texas at Austin, focuses on covalent ligands targeting protein residues other than cysteine, particularly lysine and tyrosine; we highlighted some of Hsu’s work recently. The paper also discusses naturally occurring molecules that bind to lysine, such as pyridoxal phosphate and aldose sugars.

 

Methods for finding covalent ligands are the focus on an open-access review in JACS Au by Mengke You, Hong Liu, and Chunpu Li at Shanghai Institute of Materia Medica. Specifically, they review disulfide tethering, activity-based protein profiling (ABPP), covalent DEL, phage and mRNA display, and sulfur(IV) fluoride exchange (SuFEx), with examples for each.

 

The last paper on this topic, in J. Med. Chem., offers a brief but important overview of all covalent FDA-approved small molecule drugs through 2023. Samuel Dalton and collaborators at Isomorphic Laboratories and Merck counted 128 covalent drugs, about 7% of all small molecule drugs. More than half are antibiotics, and more than 85% target serine or cysteine. Only 10% are reversible, but this number is rapidly increasing, with 11 of the 13 reversible covalent drugs approved since 2010. Importantly, the names, chemical structures, indication, target and target residue, warhead, and key references for all the drugs are provided in the supporting information.

 

Miscellaneous

Deconstruction of ligands to smaller fragments that are then “reconstructed” into new leads is a venerable approach in FBLD and the subject of an open-access perspective in J. Med. Chem. by J. Henry Blackwell, Iacovos Michaelidies, and Floriane Gibault at AstraZeneca. Multiple examples dating as far back as the late 1990s are provided, along with appropriate caveats about potential changes in fragment binding modes and protein conformations.

 

Finally, an open-access perspective in J. Med. Chem. by Dean Brown (Jnana Therapeutics) examines the 104 oral drugs approved from 2020 through 2024, including structures, dosing, pharmacokinetics, and safety. Roughly a third of these drugs are dosed more than once per day, and almost a quarter have a black box warning, while 42% have at least one contraindication. Dean warns that “overly prescriptive [development candidate] criteria may inadvertently stifle the development of innovative drugs,” and that it is difficult but important “to be the champion for a compound that others perceive as ‘un-drug like.’” The growing success of covalent drugs illustrates that some organizations are taking this to heart.

 

And that’s it for 2025. Thanks for reading and special thanks for commenting. And in 2026, may the best of us be filled with passionate intensity.

GAS41 revisited: a chemical probe

The YEATS domain of the protein GAS41 is an epigenetic reader that modulates gene expression by binding to acetylated lysine residues in chromatin. Multiple lines of evidence suggest it could be a useful target for various cancers, in particular non-small cell lung cancer (NSCLC). Four years ago Practical Fragments highlighted a paper from Jolanta Grembecka, Tomasz Cierpicki, and colleagues at the University of Michigan describing a fragment screen and subsequent optimization of a hit to molecules with some cellular activity. In a new J. Med. Chem. paper, the same team now describes molecules with better cell potency, as well as a negative control.

 

Compound 1, the initial fragment hit, had weak affinity for GAS41, but replacing the t-butyl group with a proline led to compound 7, with low micromolar activity in a fluorescence polarization assay. On the other side of the molecule, modification and growth of the amide moiety led to compound 16, also with low micromolar activity. In the 2021 paper, molecules related to compound 16 were dimerized to bind to two YEATS domains in close proximity in the GAS41 dimer. This yielded mid-nanomolar inhibitors, but the molecules were also large, with limited cell permeability. In the new paper, the researchers instead combined medicinal chemistry learnings and used structure-based design to generate monomeric molecules, culminating in DLG-41.

 

The affinity of DLG-41 for GAS41 was measured as 1 µM using isothermal titration calorimetry (ITC). In accordance with best practices for chemical probes, the researchers also developed a negative control by replacing the thiophene moiety with a thiazole; this compound, DLG-41nc, shows negligible activity in two different biochemical assays.

 

DLG-41 showed high nanomolar activity in a NanoBRET assay, demonstrating that the molecule is both permeable and binds to the GAS41 protein in cells. Importantly, the IC₅₀ for the negative control DLG-41nc was > 25 µM in this assay. DLG-41 blocked proliferation in a panel of NSCLC cell lines, though DLG-41nc also showed some activity, albeit at higher concentrations. Gene expression studies in one cell line showed that DLG-41 caused changes in hundreds of genes, while DLG-41nc was inactive.

 

This is a nice example of fragment optimization in academia. With both biochemical and cell-based potency around one micromolar, DLG-41 is hovering on the edge of the 2015 suggestions for a chemical probe. But used alongside the negative control, the compound should be useful for further exploring the biology of GAS41.

Surprise – a covalent histidine-targeting PDE3B inhibitor

Earlier this year I wrote about archiving crystallographic fragment data, and indeed a meeting is planned for early next year to establish guidelines. A new paper in J. Med. Chem. by Samuel Eaton and David Christianson at University of Pennsylvania illustrates why this is important.

 

The story starts with a paper published in 2024, also in J. Med. Chem., by Ann Rowley, Gang Yao, and collaborators at GSK and 23andMe. They were interested in finding inhibitors of PDE3B, a cyclic nucleotide phosphodiesterase that has been implicated in metabolic disease. However, this enzyme has a closely related counterpart significantly expressed in cardiac tissue: PDE3A, with 95% amino acid identity near the active site. So the researchers sought an inhibitor highly selective for PDE3B over PDE3A.

 

A DNA-encoded library (DEL) screen of 1.9 trillion(!) molecules was screened against both PDE3B and PDE3A. Hits were resynthesized without the DNA and tested in activity assays, leading to several chemical series, only one of which was selective for PDE3B. A key feature of this series was a boronic acid moiety, which was essential for activity. Optimization led to compounds such as GSK4394835A, with high nanomolar activity against PDE3B and >20-fold selectivity against PDE3A. The GSK researchers deposited a crystal structure of this molecule in the protein data bank (PDB), along with the structure factor amplitudes. It showed the boronic acid making non-covalent interactions with side-chain residues as well as the catalytic magnesium atoms and water molecules.

 

Further optimization at GSK led to compounds with as much as 300-fold selectivity for PDE3B, but like GSK4394835A, these were only high nanomolar inhibitors. The researchers could further improve potency, but this came at the expense of selectivity. Cell activity was modest at best, and the researchers noted that “the boronic acid is, in general, a challenge for development of an orally bioavailable drug.”

 

This is where the University of Pennsylvania researchers take up the story. As their paper points out, several drugs do contain boron, most notably bortezomib, which forms a covalent adduct with a threonine in the proteasome. When Eaton and Christianson took a closer look at the PDB entry showing GSK4394835A bound to PDE3B, they “noticed unusual features such as extra density around the boron atom of GSK4394835A, steric clashes between the boronic acid moiety and H737, and aberrant refinement statistics… from ideal bond lengths.” Upon re-refinement, they found that the boronic acid in fact makes a covalent bond with histidine 737. The structure explains why the boronic acid moiety was essential for activity, and the new paper suggest that other covalent warheads could potentially be used in place of the boronic acid. (Eaton and Christianson write that they contacted the GSK researchers in February of 2024, but it is not clear whether they heard back.)

 

This is a nice correction of the literature and a reminder not to take crystal structures at face value. The beauty of the PDB is that, with the experimental data deposited, the new researchers were enabled to re-refine the data even without input from the original authors.

 

As we’ve previously discussed, this example is not the only misleading crystal structure in the PDB. Many fragment structures have lower occupancy and more ambiguous electron density and would be even more prone to misinterpretation. As the community moves to establish guidelines for depositing fragment structures, it will be important to provide access to the raw data to facilitate this type of reanalysis.

A sharp NMR trick for rapidly measuring affinities

As noted in our poll last year, ligand-detected NMR ranks among the most popular fragment-finding approaches. The various methods are able to detect even weak binders, so determining affinities is important to effectively prioritize hits. This, however, can be time-consuming. In a recent J. Am. Chem. Soc. paper, Ridvan Nepravishta, Dušan Uhrín, and collaborators at CRUK Scotland Institute, University of Edinburgh, and Universidad de Sevilla present a clever way to speed up the process.

 

Normally, NMR spectra of small molecules show multiple spectral lines, with each line corresponding to a different atom or atoms (typically protons). Indeed, depending on the details, the signal from a single proton might be split into multiple peaks. All these signals are great for understanding the details of individual atoms, but the more lines there are, the lower the signal to noise ratio. For maximum sensitivity it would be nice to combine all the lines from all the atoms in a given molecule into a single, intense singlet. This is exactly what the researchers have done.

 

The approach is called Sensitive, Homogeneous And Resolved PEaks in Real time, or SHARPER. For the NMR aficionados out there, “when placed before the acquisition of the NMR signal, a train of spin-echoes in the form of the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence suppresses evolution due to chemical shifts and J couplings…. All these attributes of the CPMG pulse sequence are maintained when the spin-echo train is employed during the acquisition of the NMR signal. However, this time, the outcome is not a regular spectrum, but under certain conditions, a single spectral line formed as a sum of Lorentzian lines of contributing spins.”

 

The researchers initially applied SHARPER to two commonly used ligand-detected methods: ¹H STD, which we wrote about here, and ¹H CPMG, which we wrote about here. The first test system was human serum albumin (HSA) binding to naproxen. Keeping protein concentration constant at 9 µM and varying ligand concentration gave similar K_D values (210-280 µM) for standard STD, STD SHARPER, and CPMG SHARPER (conventional CPMG failed due to insensitivity at lower ligand concentrations). These values are an order of magnitude higher than those reported using SPR and ITC (25 and 10 µM, respectively) because of the high protein and ligand concentrations needed for conventional NMR approaches; when the SHARPER experiments were rerun at 1 µM HSA, the K_D values were 39 µM. Several other HSA ligands also gave good agreement with the literature.

 

Next, the researchers applied STD SHARPER to the anti-cancer target fascin, which we wrote about in 2019. An examination of 11 ligands from that study gave good agreement with the published dissociation constants. Importantly, SHARPER was faster than conventional approaches, with 15 K_D determinations per day instead of four.

 

Not content with this four-fold improvement in throughput, the researchers developed a new experiment based on line broadening called ¹H LB SHARPER. This allows the determination of 48 dissociation constants per day, and the results for HSA and fascin agreed with the other methods.

 

One of the most time-consuming aspects of most NMR-based affinity measurements is preparing and analyzing samples at multiple ligand concentrations, so the researchers turned to machine learning to choose which ligand concentrations would be most informative and choose just two of them rather than the six or more commonly used. This worked too, thereby potentially increasing throughput to 144 dissociation constants per day.

 

The researchers suggest that SHARPER could also be applied to some of the other recent NMR techniques we’ve discussed, such as PEARLScreeen and photo-CIDNP. Although I always emphasize that I’m no NMR spectroscopist, this strikes me as a neat, practical approach. What do you think?

FTO revisited: fragment linking this time

Four years ago we highlighted a fragment-merging approach targeting fat mass and obesity-associated protein (FTO), an RNA demethylase implicated in acute myeloid leukemia (AML). In a new J. Med. Chem. paper, Ze Dong, Cai-Guang Yang, and collaborators at University of Chinese Academy of Sciences Beijing describe a fragment linking approach that arrives at a similar outcome.

 

As we noted in 2021, previous research had revealed that meclofenamic acid (MA) binds in the substrate-binding site, near the binding site for the 2-oxoglutarate (2-OG) cofactor. In the new paper, the researchers synthesized an analog of MA which they then linked to analogs of 2-OG through a variety of linkers. Among the best of these was compound 8a, with low micromolar activity in an assay using PAGE (polyacrylamide gel electrophoresis) as well as mid-nanomolar activity in a different type of assay. This is an improvement over the MA analog itself (which was only tested in the PAGE-based assay, results shown in the figure). Compound 8a was also selective for FTO over two related proteins.

 

 

As is often seen in fragment linking, the SAR is quite sharp. The two-carbon linker was critical; lengthening the linker by one methylene or adding a methyl group abolished activity. The double bond in the 2-OG mimetic was also important; the saturated version of this molecule was inactive.

 

Surprisingly, a crystal structure of FTO bound to a molecule closely related to 8a revealed that while the MA moiety bound as expected, the 2-OG analog adopted a different conformation. But inexplicably, according to the experimental section, 2-OG was added to the crystallization solution, which would compete with compound 8a. Indeed, the structure deposited in the protein data bank shows the 2-OG analog N-oxalylglycine bound to the catalytic metal ion.

 

With two carboxylic acid moieties, it is no surprise that compound 8a showed no antiproliferative activity in AML cell lines. However, ester prodrugs did show low micromolar activity. Further characterization of one of these showed changes in protein levels consistent with FTO inhibition. This molecule also caused tumor growth inhibition after intraperitoneal dosing in a mouse xenograft model.

 

Superficially, compound 8a resembles compound 11b from the 2021 paper. Like that molecule it is probably too weak to serve as an ideal chemical probe. That said, with one fewer aromatic ring, compound 8a may be better suited for further optimization.

xLE: solving problems or missing the point?

Ligand efficiency (LE) has been discussed repeatedly and extensively on Practical Fragments, most recently in September. Two criticisms are its dependence on standard state and the observation that larger molecules frequently have lower ligand efficiencies than smaller molecules. In a just-published open-access ACS Med. Chem. Lett. paper, Hongtao Zhao proposes a new metric, xLE, to address these concerns.

 

LE is defined as the negative Gibbs free energy of binding (ΔG) divided by the number of non-hydrogen (or heavy) atoms, and of course ΔG is state-dependent. Standard state assumptions are 298K and 1M concentrations, choices that some people see as arbitrary since few biologically relevant molecules ever achieve concentrations near 1M. To remove the dependence on standard state, Zhao proposes to remove the translational entropy term of the unbound ligand from the free energy calculation.

 

Zhao also addresses the second criticism, that larger molecules often have lower ligand efficiencies. This phenomenon was observed in an (open-access) 1999 paper titled “the maximal affinity of ligands,” which found that, beyond a certain threshold, larger ligands do not have stronger affinities; there are very few femtomolar binders even among the largest small molecules. Thus, Zhao proposes attenuating the size dependence.

 

The new metric, xLE, is defined as follows:

 

xLE = (5.8 + 0.9*ln(M_w) – ΔG)/(a*N^α) - b

Where N is the number of non-hydrogen atoms, α is chosen to reduce size dependence, and a and b are “scaling variables.” He chooses α=0.2, a=10, and b=0.5, with little explanation.

 

To assess performance, Zhao examined nearly 14,000 measured affinities from PDBbind. When plotted by number of atoms, median affinity increased up to about 35 heavy atoms but then leveled off. Median LE values decreased sharply from 6 to 12 heavy atoms and then leveled off somewhere in the 20s. But median xLE values were consistent regardless of ligand size.

 

Zhao also examined LE and xLE changes for 175 successful fragment-to-lead studies from our annual series of J. Med. Chem. perspectives. LE decreased from fragment to lead for 48% of these, but xLE increased for all but a single pair.

 

And this, in my opinion, is a problem.

 

In the seminal 2004 paper, LE was proposed as "a simple ‘ready reckoner’, which could be used to assess the potential of a weak lead to be optimized into a potent, orally bio-available clinical candidate." The metric was particularly important before FBLD was widely accepted, when chemists were even less inclined to work on weak binders.

 

Here is the situation for which LE was devised. Imagine two molecules, compounds 1 and 2. The first has just 12 non-hydrogen atoms, a molecular weight of 160, and a modest 1 mM affinity for a target - similar to some fragments that have yielded clinical compounds. The second is much larger: 38 non-hydrogen atoms, a molecular weight of 500, and 10 µM affinity for the same target. Considering potency alone, compound 2 is the winner.

 

However, the LE for compound 1 is a respectable 0.34 kcal/mol/atom, while the LE for compound 2 is 0.18 kcal/mol/atom. So while a 10 µM HTS hit may initially look appealing, the LE suggests that this is an inefficient binder, and further optimization may require adding too much molecular weight to get to a desired low nanomolar affinity.

 

In contrast, the xLE values for both compounds are nearly identical, 0.38, and so this metric would not help a chemist prioritize which hit to pursue. In other words, xLE does not provide the insight for which LE was created. It might even lead to suboptimal choices. 

 

Moreover, unlike LE, xLE is non-intuitive. And finally, with three scaling or normalization factors, xLE is arguably even more arbitrary than a metric dependent on the widely-accepted definition of standard state.

 

Personally I find the practical applications of xLE limited, but I welcome your thoughts.

Searching monstrously large chemical space with FrankenROCS

Back in 2023 we highlighted a computational fragment linking/merging approach which was used to find high nanomolar inhibitors of the SARS-CoV-2 macrodomain (Mac1), a COVID-19 target. However, those molecules contained carboxylic acids, often associated with poor cell permeability. In a new open-access Sci. Adv. paper, James Fraser and collaborators at UCSF, Relay Therapeutics, Enamine, and Chemspace describe a related approach to find new, non-charged inhibitors.

 

The new approach, called FrankenROCS, “takes pairs of fragments as input to query a database using the rapid overlay of chemical structure (ROCS) method of comparing 3D shape and pharmacophore distribution;” the goal is to find larger molecules that most closely resemble the initial fragment pairs. As with the previous publication, the team started with more than 200 crystallographic fragment hits published in 2021. A set of 7,181 pairs of adjacently-bound fragments were searched against 2.1 million compounds commercially available from Enamine. The top 1000 were inspected, and 39 were purchased and soaked into crystals of Mac1. This led to 10 successful structures, of which AVI-313 did not contain a carboxylic acid. This molecule had weak but measurable activity in an HTRF competition assay.

 

Two million compounds is a lot but pales in comparison to Enamine’s “make-on-demand” REAL space, which at the time this research was done consisted of more than 22 billion molecules. The REAL space molecules are constructed from 960,398 building blocks that can be combined using 143 reactions. We previously described an approach called V-SYNTHES to screen Enamine’s REAL space. FrankenROCS takes a different active-learning approach called Thompson Sampling, which dates back nearly a century.

 

Imagine two sets of 1000 building blocks, R1 and R2, which could be coupled to generate 1,000,000 molecules. Rather than searching all possibilities, each R1 building block is linked to three random R2 building blocks, and each R2 building block is linked to three random R1 building blocks. These are virtually screened, and the R1 or R2 building blocks from those with the highest scoring compounds are used for further iterations. In theory, after tens of thousands of iterations, the best compounds will have been identified.

 

The researchers fed 97 fragment pairs from the 2021 paper into Thompson Sampling FrankenROCS to find molecules that would best overlay with the fragment pairs.  Ultimately 32 compounds were purchased, six of which were successfully crystallized with Mac1. Unfortunately, the most potent was a weaker inhibitor than AVI-313 and contained a carboxylic acid. The researchers speculate that the inability to find better molecules in larger chemical space may have stemmed from limitations of the scoring function, a problem we’ve previously discussed.

 

The researchers returned to focus on AVI 313, making substitutions at multiple positions, ultimately synthesizing 148 compounds, 121 of which could be characterized crystallographically. Importantly, several compounds had low micromolar activity, even without a carboxylic acid. The crystal structures show the binding site to be somewhat flexible, as evidenced by side chain and main chain movements to accommodate some of the binders.

This is a nice, thorough investigation, and the 137 protein-compound crystal structures deposited into the protein data bank provide useful training data for next-generation computational approaches. Moreover, the fact that immeasurably weak fragments can be advanced to low micromolar, ligand-efficient hits is yet another reason for the research community to figure out how to make crystallographic fragment screening data more widely available, as we exhorted here.

Fragments vs RhoDGI2: Towards a chemical probe

Many readers of this blog will be familiar with KRAS, a mutant form of which was successfully targeted a few years ago by a covalent, fragment-derived drug, sotorasib. KRAS is just one member of a large family of molecular switches which are on when bound to GTP and off when bound to GDP. This exchange is facilitated or inhibited by other proteins, including guanine nucleotide dissociation inhibitors (GDIs). GDIs bind to the GDP-form of RAS proteins, keeping them in the off state, but they can also stabilize Ras proteins against proteasomal degradation, keeping them around longer.

 

RhoGDI2 is a GDI that regulates Rho GTPases, which are involved in multiple cell pathways. The biology is complicated though, and RhoGDI2 has been implicated as both a cancer driver and inhibitor. Clearly a chemical probe would be useful. In a new ACS Chem. Biol. paper, Wei He and collaborators at Tsinghua University and University of Science and Technology of China Hefei report the first steps.

 

The story begins with a 2017 paper in Biochim. Biophys. Acta. Gen. Subj. by Ke Ruan (one of the authors of the new paper) and colleagues. A ligand-detected NMR screen of just under 1000 fragments yielded 14 hits, three of which were confirmed by two-dimensional protein-observed NMR. Further experiments suggested these bound in the hydrophobic pocket that binds gerarnylgeranylated Rho GTPases. Compounds 1 and 2, though weak, became the starting points for fragment growing.

 

Borrowing from compounds 1 and 2 and adding a phenyl moiety led to compound 2102, which was crystallographically confirmed to bind in the substrate binding pocket. Further fragment-growing, guided by structure-based design, ultimately led to HR3119, with low micromolar affinity for RhoGDI2 as assessed by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). HR3119 has four diastereomers, and those with an R-configuration at the benzylic position (6R) were almost 100-more potent than 6S.

 

HR3119 blocked the interaction of RhoGDI2 with the Rho GTPase Rac1 in cell lysates. (6R)-HR3119 stabilized RhoGDI2 in a cellular thermal shift assay, while (6S)-HR3119 did not. (6R)-HR3119 also decreased migratory activity of a cancer cell line, consistent with the role of RhoGDI2 in actin dynamics. However, (6S)-HR3119 also showed activity in this assay, albeit at a higher concentration, suggesting off-target effects.

 

The biochemical and cell activity are still too weak to nominate (6R)-HR3119 as a chemical probe against RhoGDI2; ideally biochemical activity should be better than 100 nM and cell activity should be better than 1 µM. Nonetheless, this is a good starting point for further optimization, and a nice example of fragment-based lead discovery in academia.

Fragments vs FEN1: A chemical probe

Synthetic lethality is a relatively new approach to treating cancer by targeting proteins whose inhibition is lethal to cancer cells that have specific mutations. Disruption of flap endonuclease 1 (FEN1), an enzyme important for DNA replication and repair, becomes synthetic lethal combined with BRCA1 and BRCA2 loss of function mutations. However, although a few inhibitors have previously been reported, these had poor cell activity and physicochemical properties. In a new J. Med. Chem. paper, Sam Mann and collaborators at Artios Pharma, Merck KGaA, and several other organizations describe a chemical probe.

 

FEN1 is a member of the RAD2 nuclease family, all of which contain two magnesium atoms in the active site. Thus, the researchers set out to build a metal-chelating library, a strategy we’ve written about previously. Noting that a bivalent metal chelator requires at least three hydrogen bond acceptors, the researchers included fragments that were not strictly rule-of-three compliant. More than 300 fragments were screened in a biochemical assay against FEN1 and three related proteins, EXO1, GEN1, and XPG. Hits were selected based on potency, ligand efficiency, and selectivity. Two related fragments came out on top, one of which was characterized crystallographically bound to FEN1, confirming engagement with the catalytic magnesium ions

 

 

Compound 6 was trimmed back to the chelating core compound 7 before attempts were made to grow the molecule in several directions, leading eventually to compound 21, the first molecule to show target engagement in cells. Modeling suggesting the presence of a high-energy water that might be displaced, which was attempted by expanding the core to include a morpholine moiety. Further modulation of properties ultimately led to MSC778, with modest oral bioavailability in mice, rats, and dogs. The paper describes some nice medicinal chemistry that goes beyond the scope of this post. For example, there was a correlation between cellular target engagement and off-rates as determined by surface plasmon resonance (SPR). One wonders if a covalent inhibitor, with essentially no off-rate, could be even more effective.

 

MSC778 is at least 65-fold selective for FEN2 over related RAD2 family members. It is also clean in a panel of off-target safety assays. The molecule is cytotoxic to a cancer cell line in which the BRCA2 gene had been knocked out but less so to the same cell line carrying wild-type BRCA2. Surprisingly though, no tumor growth inhibition was seen in a mouse xenograft model using the mutant BRCA2 cell line at the highest tolerable dose. However, tumor stasis was seen when the compound was dosed in combination with niraparib, a PARP inhibitor, consistent with earlier cell experiments suggesting that PARP inhibitors could synergize with FEN1 inhibitors.

 

The lack of single agent activity seen with MSC778 was undoubtedly disappointing, though the researchers note that it is unclear whether this is “due to insufficient target coverage, or an unexpected disconnect between the phenotypic consequences of FEN1 inhibition in vitro and in vivo.” Nonetheless, MSC778 looks to be a useful chemical probe for further understanding the biology of FEN1. This paper is also a nice application of building and screening a metal-chelating fragment library, which could be useful for targeting additional metalloproteins.

Checking halogen bonds

Halogen bonds (or X-bonds) are one of the less appreciated protein-ligand interactions. As we discussed in 2022, the polarized nature of a carbon-halogen bond creates a partially-positively charged “σ-hold” at the bit of the halogen furthest from the carbon, and this can make favorable interactions with lone pairs on oxygen or sulfur atoms (or nitrogen, but in most proteins this is limited to histidine residues and is rare.) Halogens can also interact with aromatic π-systems such as the side chains of phenylalanine, tryptophan, histidine, and tryptophan. Since many fragments contain halogen atoms by design, halogen bonds may occur frequently. But how do you decide whether “a halogen in proximity of a possible acceptor” actually contributes to binding? In a new (open-access) paper in Protein Science, Ida de Vries, Robbie Joosten, and colleagues at Oncode Institute and The Netherlands Cancer Institute provide a new metric.

 

The researchers examined structures of halogen-containing ligands bound to proteins in PDB-REDO, a database of carefully vetted and refined structures from the Protein Data Bank. They only included structures solved to better than 2.5 Å resolution and omitted structures where halogens had high B-factors, which may be the result of radiation damage. This led to 8423 structures in which a halogen possibly interacted with an oxygen or sulfur atom and 8096 potential halogen-π interactions, which were analyzed in detail.

 

A halogen bond to an oxygen or sulfur atom can be described by the interatom distance and two angles: θ1 (carbon-halogen-oxygen/sulfur) and θ2 (halogen-oxygen/sulfur-carbon). Halogen-π-system bonds can be defined by distance to the centroid of the π-system and θ1, the carbon-halogen-centroid angle. (The paper has a nice diagram.) These parameters were calculated and annotated for all the structures.

 

Median distances were 3.5 Å between halogen and oxygen/sulfur, regardless of the halogen. Median θ1 angles were smaller than the 150º-180º expected, particularly for fluorine atoms, while median θ2 angles were more consistent with theory, at 90º-120º.

 

For halogen-π-systems, median distances were 4.8 Å for all halogens except iodine, which came in slightly higher. But θ1 angles were still smaller than expected, mostly between 110º-140º.

 

Armed with this tranche of high-quality data, the researchers established a Halogen Bond Score, or HalBS. For any potential halogen bond in a new crystal structure or other structural model, the distance, θ1, and, if applicable, the θ2 values are calculated, and if any of these diverge too far from the median values, HalBS flags them. Importantly, the researchers acknowledge that “the current HalBS cannot be used as a direct validation metric but can provide an indication of genuine halogen bonds and ‘not so proper’ halogen bonds.”

 

With this caveat HalBS could be useful, and the researchers have made the source code available at https://github.com/PDB-REDO/HalBS (though the link doesn’t seem to work for me). As they note, more data, such as might be provided by widespread deposition of large crystallographic fragment screens, could further refine HalBS. Of course, the existence of a halogen bond exists says little about how much binding energy it contributes, but it’s a start.

Ivermectin postmortem: PAINful experiences with a good drug

Ivermectin is a miracle drug. It cures infections caused by several types of parasitic roundworms, including those that cause river blindness and elephantiasis, two highly unpleasant diseases. Half of the 2015 Nobel Price for Physiology or Medicine was awarded to researchers who discovered the drug.

 

More recently, ivermectin was touted as a treatment for SARS-CoV-2. Unfortunately, after more than 90 human clinical trials, the preponderance of evidence shows that it is ineffective against COVID-19. A new (open-access) Perspective in J. Med. Chem. by Olaf Andersen, Jayme Dahlin, and collaborators at Weill Cornell Medicine, the National Institutes of Health, University of North Carolina Chapel Hill, and University of California San Francisco explores why it proved so misleading, with lessons for other drug repurposing efforts.

 

In 2011, ivermectin was one of 480 compounds tested in a biochemical assay at 50 µM and appeared to disrupt the interaction between HIV integrase and a mammalian protein involved in viral trafficking. In April 2020, low micromolar concentrations of ivermectin were reported to have anti-SARS-CoV-2 activity in a cellular assay. Given a terrifying new disease with no treatments, an approved drug that showed even tenuous activity looked like a lifeline. Researchers around the world began studying ivermectin. PubMed citations jumped from 459 in 2019 to 734 in 2021.

 

The new paper examines some of this past work and dives deeply into the biological effects of ivermectin. The molecule is poorly soluble in water (around 1-2 µM) and partitions into cellular membranes, where it activates a chloride channel receptor in worms, paralyzing them. At this it is quite potent: when taken as directed, human plasma concentrations of ivermectin are around 60 nM, but because the drug is highly protein bound the free concentration is only around 6 nM.

 

The astute reader may notice that the apparent antiviral activity was observed at low micromolar concentrations of ivermectin, three orders of magnitude higher than physiologically relevant, and the initial biochemical assay was conducted at an even higher concentration. In fact, that work was done using an AlphaScreen, which is notoriously sensitive to artifacts. The new paper demonstrates that ivermectin forms aggregates at low micromolar concentrations, and that these aggregates interfere with the AlphaScreen. (We previously wrote about how many of the early reports of compounds active against SARS-CoV-2 proteins were in fact aggregators.)

 

If ivermectin perturbs membrane proteins in worms, what does it do in human cells? The researchers tested the drug against panels of G protein-coupled receptors (GPCRs) and found that in one assay format it inhibited more than a quarter of 168 GPCRs at 10 µM, much higher than physiologically relevant but comparable to the in vitro experiments with SARS-CoV-2. Further studies revealed that ivermectin changes the properties of membranes at low micromolar concentrations, as assessed by multiple methods including electrophysiological assays. Several ivermectin analogs were also tested and found to have similar activity, consistent with nonspecific effects. Impressively, these experiments were done blinded to the experimenter.

 

You might think that messing with membranes would not be good for cells, and you would be right. The researchers found that ivermectin decreased cell viability at low micromolar concentrations in a variety of assay formats through multiple mechanisms, both cytotoxic and apoptotic. Importantly, the concentrations at which ivermectin was active against cells were similar to the concentrations where it showed activity against SARS-CoV-2. The researchers also analyzed 766 PubChem assays and found that ivermectin is active in nearly a third of those assessing cellular toxicity. Killing a host cell is certainly one way of killing a virus, but likely not a useful one.

 

In summary, the original data suggesting that ivermectin is a developable antiviral agent was flawed. The researchers describe this as “a saga of the damage that can be done by assay interference compounds” and a “cautionary tale for the dangers of ‘pandemic exceptionalism.’” They continue:

 

The fact that a repurposed drug is well-characterized clinically, or that there is an ongoing pandemic, may justify performing clinical and mechanistic experiments in parallel, but not skipping mechanistic studies, where the key experiments could have been done in a matter of a few weeks/months.

 

This J. Med. Chem. paper is a meticulous, comprehensive study; with 71 pages of supporting information there is far more to cover than I can do justice to in a blog post. The paper also includes a useful flowchart for derisking nonspecific membrane perturbation. It is well worth reading, particularly for those new to drug discovery. As Richard Feynman warned, “the first principle is that you must not fool yourself -- and you are the easiest person to fool.”

Exploiting avidity for finding fragments

As our poll last year demonstrated, there is no shortage of methods to find fragments. But that doesn’t mean new approaches aren’t welcome, particularly when they also apply to fragment growing. This is the promise of a recent paper in J. Med. Chem by Thomas Kodadek and collaborators at University of Florida Scripps and Deluge Biotechnologies. (Tom and first author Isuru Jayalath also presented this at the DDC meeting earlier this year.)

 

The researchers were inspired by the concept of avidity, the observation that multiple copies of a ligand bound to a multiprotein assembly can form a more stable complex than monomeric ligands bound to monomeric proteins. Could this phenomenon be exploited to find weak fragments?

 

A previous DNA-encoded library screen on streptavidin had identified 28 macrocycles, all of which contained one of two closely related fragments. The affinity of the more potent fragment came in at 706 µM using SPR. The researchers coupled this fragment to TentaGel beads, 10 µm wide polystyrene spheres covered in polyethylene glycol (PEG) chains terminated by amine groups. The PEG makes the beads water soluble. The beads were soaked in a solution of fluorescently labeled streptavidin, washed, and analyzed. Importantly, streptavidin exists as a tetramer, so each tetramer could bind up to four bead-bound fragments.

 

Streptavidin bound avidly to the beads, even when incubated at low (50 nM) concentrations. A control protein did not bind, nor did streptavidin bind to beads modified with a negative control fragment. Moreover, a monomeric version of streptavidin did not bind to the beads, illustrating the importance of avidity. Finally, adding the natural ligand biotin kept streptavidin from binding to the beads.

 

TentaGel beads have long been used in combinatorial synthesis, so the researchers built a small library in which the initial fragment was coupled to 48 carboxylic acids. These were then incubated with labeled streptavidin, and some of the beads showed more intense fluorescence, suggesting more protein binding. SPR analysis revealed that these new molecules had improved affinity, with the best coming in at 90 µM as a monomer. Thus, the primary screen can rank order affinities.

 

This is great for oligomeric proteins, but what about the large number of targets that are monomeric? Many recombinant proteins are expressed as fusions with glutathione S-transferase (GST), which facilitates purification. Importantly, GST exists as a homodimer in solution. The researchers screened a GST fusion of the oncology target Rpn13 against a small library of 94 fragment-coupled beads and found five hits. SPR studies confirmed weak (K_D > 2 mM) binding for two hits to pure Rpn13 (ie, without the GST fusion), and this binding could be competed with a known peptide ligand of Rpn13.

 

Screening beads in individual wells is one thing, but to really increase throughput it would be nice to be able to screen mixtures of different beads. To do so, the researchers developed a photocleavable linker between bead and fragment. The linker also contained an alkyne group that could be modified with a brominated imidazopyridinium moiety. This tag is UV active, ionizes well, and the bromine’s unique isotopic signature helps distinguish true hits from noise. Beads containing more than 50 different compounds, including the two fragment hits we mentioned above, were incubated with labeled streptavidin. Beads to which protein bound were separated by fluorescence-activated cell sorting (FACS), clicked with the tag, cleaved from the beads, and analyzed by mass spectrometry. Only the two known binders were identified, demonstrating the specificity of the approach.

 

This is a neat paper well worth reading. I particularly like the fact that the method can be done with minimal equipment. I look forward to seeing how it works against more targets.

Twentieth-Third Annual Discovery on Target Meeting

The CHI Discovery on Target (DoT) meeting was held last week in Boston. More than 850 people from 24 countries attended, 75% from industry. As usual I’ll just touch on some broad themes.

 

Covalent approaches

Covalent approaches were prominent throughout the conference. One of the very first talks was by Stefan Harry (Harvard/MGH), who described screening 416 cancer cell lines with three reactive “scout probes,” identifying some 6000 cysteine residues that could be covalently liganded. There are some interesting cell and context-dependent differences, and all the data are publicly available and easily searchable through a free online portal called DrugMap. He is now profiling a library of dual-electrophile-containing compounds to identify molecular glues.

 

Knowing which cysteines can be targeted is the first step for covalent drug discovery, and Sherry Ke Li described how she and colleagues at Genentech go about finding ligands. They’ve experimentally determined the reactivity of more than 6400 compounds against free cysteine and used this to train a machine-learning model to predict chemical (as opposed to specific) reactivity. Mass spectrometry (MS) using isolated proteins is the workhorse screening approach, but Sherry also described using variable temperature surface plasmon resonance (SPR) to dissect the individual components of k_inact/K_I.

 

AstraZeneca has also been doing considerable covalent screening, and Hua Xu briefly described the BFL1 story we wrote about here. In addition to pure proteins, they are now also starting to screen their covalent library in cells. Hua also presented earlier work from Pfizer on the discovery of the covalent kinase inhibitor ritlecitinib, which started with a noncovalent binder. Proteomic studies revealed that in addition to the intended target JAK3, it hits other TEC-family kinases too.

 

Adding a covalent warhead to a reversible binder is also the approach taken by MOMA Therapeutics in the discovery of their clinical WRN inhibitor MOMA-341, as presented by Momar Toure. They ended up targeting the same cysteine as Vividion (see here), though the binding mode is somewhat different. MOMA is also pursuing covalent fragment screening using intact protein MS, and Brian Sosa-Alvarado described how they were able to identify nanomolar inhibitors of RAD54L within six months of starting the program, aided by DNA-encoded libraries (DEL).

 

Not everyone is pursuing cysteine: Ken Hsu described how he and his team at University of Texas Austin are using sulfur-triazole exchange chemistry (SuTEx) to target tyrosine residues across the proteome. He noted that although cysteine could react with this warhead, the resulting thiosulfonate would be unstable. This is true in general, but I wonder if, just like the reversible cyanoacrylamide warheads we wrote about more than a decade ago, they could be stabilized within folded proteins.

 

Noncovalent approaches

Covalency is not the only game in town, as exemplified in a talk by Emma Rivers on “integrated hit discovery” at AstraZeneca. I was tickled that she grouped FBLD with HTS as “traditional” approaches, onto which they’ve added DEL and peptide libraries. Importantly, they’re focused on generating and capturing as much high-quality data as possible to enable machine learning – a topic we’ll touch on more below.

 

Nor are proteins the only target; there was a whole track on RNA- and DNA-targeting small molecules, where Benjamin Brigham described the plate-based equilibrium dialysis-based approach taken at Atavistik to screen metabolites and metabolite-like molecules. This led to two fragment-sized hits against RNA encoding SERPINA1, and although the affinities are modest, they do inhibit translation in a cell-free system.

 

At FBLD 2018 Astex presented the first cryo-EM structure of a fragment-protein complex, noting that throughput was an issue. The company has leaned into that challenge and now has three microscopes, including a top-of-the-line Krios, with another on the way. Miguel Zamora-Porras described how they have now solved hundreds of structures. Their Krios can collect data on two compounds per day, and the full cycle time from protein-ligand preparation to structure is about a week. Miguel described how structures of ligands bound to the ion channel protein TRPML1 helped reveal why some were agonists and others antagonists.

 

Data and its discontents

On the subject of structures, Steve Burley (Rutgers) gave an eloquent history and defense of the “RCSB Protein Data Bank: an open access research resource that benefits all humanity.” From its humble beginnings with just seven structures in 1971, the PDB now contains more than 240,000. And these are not just of scientific interest: all 88 of the new molecular entities the FDA approved for oncology between 2010 and 2023 had PDB structures that informed the biology or druggability, and 75% of the efforts involved structure-based design. Steve also mentioned that the question of where and how to store large-scale crystallographic data will be discussed in a meeting sometime in the spring of next year. Finally, Steve is hoping to retire from his position as Director of the RCSB PDB, so if you’re looking to make an impact, please apply.

 

The dramatic advances in protein structure prediction exemplified by AlphaFold would not have been possible without the PDB, but unfortunately the same high-quality information on protein-ligand binding modes and affinities is not available, as noted by Woody Sherman of Psivant. To illustrate the importance of training data, Woody asked ChatGPT to produce a picture of an analog clock set to 6:32. The result? A clock with three hands, one at 10, one at 2, and one at 6, because most images of clocks are set to 10:10.

 

Woody asked whether machine-learning-based docking can extrapolate or just interpolate. Although impressive results have been reported for some protein-ligand complexes, it turns out that there are often similar ligands in the training data. For truly novel ligands, the predictions tend to fall flat. Similarly, allosteric ligands are often (mis)placed into an orthosteric site – just because the model has been trained that that’s where ligands should go. Indeed, although Psivant is heavily invested in computational approaches, Woody mentioned that they often use “wet” approaches for finding initial chemical matter.

 

On the subject of dubious data, Al Edwards (University of Toronto) noted that a third of all immunofluorescence images in the literature use antibodies that give signals in knockout cells. And as we wrote just last month, many reported small molecule “probes” are just as bad. Al is CEO of the Structural Genomics Consortium, whose ambitious Target 2035 aims to find a pharmacological probe for every target in the human genome. As a starter, they’re aiming for 2000 probes in the next five years. They’re using affinity-selected mass spectrometry (ASMS), screening pools of 500 compounds and 8 proteins at a time, and are getting micromolar hits against about 30% of targets. They’re accepting protein submissions, so if you’re looking for starting points against your favorite protein contact them.

 

I’ll end here, but please leave comments. And mark your calendar for Sep. 28 to Oct. 1 next year, when DoT returns to Boston.

Fragment merging without crystallography for CGRP receptor antagonists

Migraines are the third leading cause of disability worldwide. Although the pathology is complex, blocking the interaction of calcitonin gene-related peptide (CGRP) with its receptor, thereby decreasing vasodilation, has proven successful in the clinic. However, some of the early small molecule antagonists were discontinued due to hepatotoxicity. In a recent J. Med. Chem. paper, Naohide Morita, Isao Azumaya, and collaborators at Kissei Pharmaceutical and Toho University describe a new class of inhibitors.

 

CGRP binds at the interface of a heterodimeric receptor comprised of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). To find hits, the researchers screened a library of 2500 fragments (which could be up to 350 Da) at 500 µM against the extracellular CLR/RAMP1 domains using SPR. This yielded 565 hits, which were clustered based on similarity, and 250 were chosen for dose-response studies, leading to 38 confirmed hits. Competition studies with a known CGRP antagonist whittled this number down to just four, with compound 1 being chosen for further study due to ease of analog synthesis.

 

Compound 1 was confirmed as a binder using isothermal titration calorimetry (ITC). Unfortunately, co-crystallography with CLR/RAMP1 was unsuccessful, so the researchers turned to docking using information from known small molecule inhibitors. This work suggested that compound 1 binds to the CGRP receptor but does not interact with RAMP1, a conclusion further supported by mutagenesis studies.

 

To find fragments that bind RAMP1, the researchers performed a second fragment screen, again using SPR. This time the fragments were chosen from those in the first set that had not been tested in dose-response studies, supplemented with several hundred more selected based on structures of known CGRP antagonists. Of 784 fragments screened, 114 were taken into dose-response studies, leading to 8 hits. Compound 2 was the most potent, and mutagenesis studies suggested it interacted with RAMP1.

 

Crystallography of compound 2 was also unsuccessful, but docking, supported by NMR studies, suggested a possible binding mode. Compounds 1 and 2 were merged to yield compound 3, which had a satisfying 2000-fold improvement in potency compared to compound 1. Compound 3 also showed cell activity.

 

Compound 3 contains three stereocenters, so the researchers sought to simplify the molecule. They also needed to improve potency and metabolic stability. Multiparameter optimization ultimately led to compound 15, with picomolar(!) affinity for the receptor, subnanomolar activity in cells, and good pharmacokinetic properties. A standard model for migraine is inhibition of facial blood flow in marmosets, and compound 15 was active. The compound was also clean in tests for hepatotoxicity.

 

Although no further development of compound 15 is reported, this is a nice case study in fragment merging. As the researchers note, it is also one of just a handful of examples that succeeded in the absence of crystallographic data (we wrote about another one here). Hopefully this will further embolden researchers to pursue fragment merging and linking without direct structural information.

Covalent ligand efficiency

Ligand efficiency (LE) was proposed more than two decades ago as “a useful metric for lead selection.” The concept is simple: divide the binding energy of a ligand by the number of non-hydrogen (or heavy) atoms (HA). The higher the number, the higher the binding energy per atom, and thus the more “efficiently” the ligand binds to the protein. LE is particularly useful in fragment-based lead discovery when prioritizing among differently sized hits to ensure that small, weak molecules are not overlooked. While some have criticized the metric’s dependence on standard state, drug hunters have repeatedly found it to be useful, as we’ve discussed here, here, and here.

 

Irreversible covalent drugs are a horse of a different color - or perhaps a different species entirely. Because of their two-step mechanism, binding followed by bonding, time is an essential parameter, and the proper way to characterize them is with the ratio k_inact/K_I. Is it possible to develop a covalent ligand efficiency metric? This is the task that György Ferenczy and György Keserű at HUN-REN Research Centre for Natural Sciences and Budapest University of Technology and Economics set for themselves in a recent (open-access) Drug Discovery Today paper.

 

As we wrote just a couple months ago, an important distinction for covalent drugs is specific vs chemical reactivity: you want the first to be high and the second to be low. For cysteine-reactive molecules, this distinction is often assessed by measuring the rate of reaction with the abundant cellular thiol glutathione (GSH). The researchers sought to incorporate this parameter into their definition of covalent ligand efficiency (CLE) as follows:

 

CLE = LE – LE(GSH) = (-1.4*log₁₀(IC_50,t)/HA) - (1.4*log₁₀(k^2nd_sur*t)/HA)

Where IC_50,t is half maximal inhibitory concentration at time “t” and k^2nd_sur is the second-order rate constant of the ligand reacting with a surrogate nucleophile such as GSH.

 

The researchers cataloged multiple covalent modifiers from the literature. Some had reported glutathione reactivity data. For the rest, the researchers estimated these values based on analogs. They went on to calculate CLE values for the protein-ligand pairs. Laudably, all of these data are provided in the supplementary data.

 

So, how useful would CLE have been in prior lead discovery campaigns? The researchers calculated CLE values for the BFL1 covalent fragment hits we wrote about here. The potencies of the six reported fragment hits varied, reflected in k_inact/K_I values, from 0.7 to 9.5 M^-1s^-1. But their CLE values spanned a narrower range, from 0.08 to 0.12. The fragment that was successfully optimized was one of the most potent, with a k_inact/K_I of 7.5 M^-1s^-1, but had a CLE of just 0.09. If anything, CLE would have deprioritized this fragment, at odds with the stated goal that “CLE is designed to support compound priorization.”

 

As we discussed earlier this year, the researchers previously proposed that covalent fragments may need to be larger than reversible fragments. If this is true, then normalizing for size may be less important for covalent ligands than for noncovalent ones, which can be very small and weak yet still valuable. Indeed, the researchers’ analysis of covalent ligands from the literature shows a smaller range of CLE values than LE values.

 

The researchers acknowledge other oddities too: “there is no smooth transition from CLE to LE as the reactivity of ligands decreases. Moreover, CLE can take negative values for compounds with low affinity and high reactivity.”

 

But for me, the biggest liability is the fact that – unlike LE for reversible binders or k_inact/K_I – the value of CLE depends on the time the measurement was taken. (In the paper, the researchers use a 1 hour incubation, so I would propose the annotation CLE_1h.) This makes it difficult to compare CLE values taken at different time points.

 

The first word of this blog is “practical,” and I’m not convinced this adjective applies to CLE, though I applaud the effort. The popularity of LE spawned a cottage industry of other metrics, some of which we summarized in a 2011 post. I confess that I had nearly forgotten about some of them, but I think they were a useful way for the field to grapple with what characteristics mattered. As covalent drug discovery becomes increasingly popular, perhaps we will see a similar proliferation of metrics. (Indeed, we already wrote about another one here.) It will be interesting to revisit these a decade hence to see which ones have caught on.