Halo Library says hallo to crystals

Last week we wrote about the magical properties chlorine can impart to molecules. More generally, halogen atoms can be helpful for a variety of reasons beyond new types of interactions with proteins and improved metabolic stability. For example, fluorine NMR can be used to rapidly identify ligands, and we’ve written about a custom fluorinated library. Heavier halogens can be particularly useful for screening by crystallography, and we’ve written about two libraries (HEFLib and FragLites) containing chlorine-, bromine,- or iodine-bearing fragments. Now Francesc Ruiz, Eddy Arnold, and colleagues at Rutgers bring us the “Halo Library,” described in a new (open access) J. Med. Chem. paper.

 

The researchers assembled a library of 46 halogenated fragments. In contrast to the libraries above, which focused on either fluorine or heavier halogens, this one is multi-purpose, with about half the compounds containing fluorine, half containing bromine, and a handful of molecules containing chlorine or iodine. Most of the fragments came from their internal collection and had been screened against several targets, and the rest were commercial compounds that had been reported to bind to at least one target.

 

The Halo Library is similar in terms of molecular properties to HEFLib and FragLites, though with a slightly lower average molecular weight (172.5 Da). Like the two earlier libraries, the Halo Library is relatively “flat,” with Fsp3 = 0.2.

 

Ten years ago we highlighted work from the Arnold lab in which a library of 775 fragments was screened crystallographically against HIV reverse transcriptase (HIV-1 RT), resulting in a 4% hit rate. The researchers returned to this protein with their Halo Library, soaking crystals with individual fragments at 20 mM. This resulted in 12 hits, an impressive hit rate of 26%. Admittedly some of these halogenated fragments had been identified as binders previously, so it will be interesting to see how the library behaves on other targets.

 

In addition to the high hit rate, fragments bound to six sites not occupied by ligands in the 2013 study, and two of these sites had never been reported to be ligand binding sites, despite extensive work on this protein. (Roughly half of anti-retroviral drugs for HIV target RT, and ART regimens typically include two or three separate inhibitors.)

 

Eight of the fragments inhibited the biochemical activity of the enzyme by at least 50% at 5 mM, and three of them gave IC₅₀ values in the low mM range with ligand efficiencies as high as 0.47 kcal/mol/atom. Among these three, two bound to a single site, while the third (4-amino-3-bromopyridine) bound at four separate sites. Another library member, the “universal fragment” 4-bromopyrazole previously identified by the researchers, bound to eight sites but showed only 22% inhibition at 5 mM.

 

The binding modes of all twelve fragments are described in some detail and show the standard range of hydrogen bonds and van der Waals interactions. Halogen bonds were surprisingly rare, unlike the case of FragLites against different proteins. It would be interesting to see a summary of the types of interactions, and how many involved the halogen atoms. The identities of all the library members are provided in the Supporting Information, so you can build your own library. 

 

And on that note, this is the last week to take our survey on fragment libraries, so please make sure to vote!

Chlorine: more magic than methyl

More than a decade ago we highlighted a paper that discussed “magic methyl” groups, which can boost the affinity of a ligand for a protein by more than 100-fold. Since then we’ve noted examples where these have been used to optimize fragments. But methyl groups are just one option for fragment growing. In a recent J. Med. Chem. paper, Debora Chiodi (Scripps) and Yoshihiro Ishihara (Vividion) take a close look at chlorine – and suggest that the halogen is even more magic than methyl. (See here for Derek Lowe’s summary.)

 

Chlorine is the sixth most common element found in drugs, after carbon, hydrogen, oxygen, nitrogen, and sulfur. In terms of size it is comparable to a methyl group, but more lipophilic. It is also more electronegative, and can significantly change the electronics of a molecule. Finally, unlike methyl groups, chlorine atoms often stabilize molecules against metabolism. But what about potency?

 

The researchers examined all 50,000 papers containing matched-pair SAR published in eight medicinal chemistry journals between 2010 and 2022, a process they characterize as “painstakingly manual.” All papers in which a hydrogen to chlorine swap increased the activity by at least ten-fold were then selected for further analysis. This cutoff was used based on tradeoffs of lipophilic ligand efficiency (LLE or LipE): you want a sizable increase in potency to compensate for the fact that adding a chlorine to a molecule increases logP by nearly 1.

 

In total, the researchers found 633 articles in which the potency increased by at least 10-fold, 131 where the potency increased by at least 100-fold, and 21 where the potency increased by a whopping 1000-fold or more, far better than any methyl.

 

Case studies in the paper attribute potency improvements to multiple factors, including better van der Waals interactions, decreasing the basicity of a molecule, direct hydrogen bonds to the chlorine, and halogen bonding, in which the chlorine makes favorable interactions with a carbonyl oxygen. Moreover, chlorine can also improve membrane permeability (via increased lipophilicity) and pharmacokinetics. Indeed, many of the most dramatic improvements in activity are measured not against isolated enzymes but in whole cells.

 

Thus, unlike a methyl group which merely increases lipophilicity or changes the conformation of a molecule, chlorine provides several opportunities for enhanced interactions. As the researchers summarize, “the chlorine atom is able to combine the beneficial effects of a fluorine atom (e.g., electronegativity/electron-withdrawing ability, metabolic stability, increased acidity), a methyl group (e.g., lipophilicity, van der Waals interactions, steric effect), and even a bromine atom (e.g., halogen bonding), and is arguably the most versatile among these substituents.”

 

Of course, the researchers were looking for beneficial effects: chlorine is not a universal panacea. Increased lipophilicity is usually something you want to avoid in the later stages of lead optimization, and adding chlorine atoms often reduces solubility. The researchers mention examples in which adding a chlorine atom to a molecule decreased potency by more than 100-fold.

 

As for lessons, adding chlorine atoms to fragment hits is probably a good early step, as in this 2017 example. The researchers also highlight halogen-enriched fragment libraries (which we wrote about here). A ligand with an affinity of 100 µM will be easier to find than a millimolar binder, but systematically adding halogens to different positions on a molecule increases the number of fragments to include in a library. On that topic, please make sure to take our survey on libraries, which closes at the end of May.

From reversible to irreversible fragments vs caspase-6

The caspase family of cysteine proteases is critical for initiating and executing programmed cell death, and some members also play a role in inflammation. Humans have a dozen caspases, all of which cleave substrate peptides immediately after an aspartic acid residue. Developing chemical probes for specific family members has been challenging for several reasons, not least because of similar active sites. In a newly published open-access J. Am. Chem. Soc. paper, Michelle Arkin, Adam Renslo, and colleagues at UCSF have done just that. (Michelle spoke about this work at the CHI DDC meeting last month.)

 

The researchers were particularly interested in caspase-6, which has been implicated in neuroinflammation. They recognized that, in addition to the active site cysteine, caspase-6 has another cysteine not found in other caspases. C264 sits at the far end of the active site, on the smaller of the two separate protein subunits that come together at the active site. They used Tethering (previously described here) to screen a library of 1500 disulfide-containing fragments. Since C264 is on the small subunit and the active site cysteine is on the large subunit, it was easy to determine specificity using mass spectrometry.

 

There were quite a few hits against the small subunit of caspase-6, including compound 1. The researchers replaced the disulfide moiety with an irreversible vinylsulfonamide warhead and made a few other modifications to the core to obtain compound 2a. This showed sub-micromolar inhibition of the enzyme after just 15 minutes, and crystallography revealed that it bound covalently to C264, as expected. The crystal structure also suggested where to grow the molecule, leading to compound 3a, which was a low nM inhibitor at 15 minutes. The researchers determined the k_inact and K_i values, which are important parameters for irreversible inhibitors. The molecule was also quite selective, showing little to no inhibition of nine other human caspases at 10 µM in biochemical assays.

 

Compound 3a effectively blocked caspase-6 mediated cleavage of the substrate lamin A in a cellular assay. To assess cellular specificity, the researchers made an alkyne-containing version of compound 3a which could be used in pull-down experiments using click chemistry. This revealed a couple dozen protein targets, of which only eight could be competed by the parent compound 3a. Interestingly, all except caspase-6 were membrane-bound proteins, and none were proteases.

 

This is a nice, concise paper reminiscent of the early work that ultimately led to sotorasib. A key liability of the series is the vinylsulfonamide warhead, which is probably too intrinsically reactive for in vivo studies. The researchers did try replacing this with the clinically validated acrylamide warhead, but unfortunately the resulting molecule was not active. Nonetheless, compound 3a is worth considering as a chemical probe, and the researchers note that further optimization efforts are ongoing. I look forward to seeing the evolution of these molecules.

Fragments vs metallo-beta-lactamases

With all the attention on COVID-19 over the past few years, it’s easy to forget about bacteria. But they haven’t forgotten about us. Indeed, antibiotic resistance genes are continuing to spread throughout the microbial world. One class of genes encode the metallo-beta-lactamases (MBLs), which hydrolyze beta-lactam antibiotics. In a recent J. Med. Chem. paper, Mihirbaran Mandal, Li Xiao, and colleagues at Merck describe their efforts against these enzymes. (Mihirbaran spoke about this work at the CHI DDC meeting a few weeks ago.)

 

The researchers started by turning to the literature and previous internal efforts. Tetrazole-containing molecules had been identified as MBL inhibitors, and a virtual screen led to the selection of 76 compounds for testing, of which 29 inhibited the enzyme NDM-1, one of several known MBLs. These included fragment-sized compound 13, and a subsequent screen also identified compound 14.

 

Crystal structures of both these molecules revealed that they bind in the active site. The tetrazole makes interactions with one of the catalytic zinc ions, while the proximal sulfonamide moiety in compound 14 makes interactions with both zinc ions by displacing a bridging hydroxide ion. Merging compounds 13 and 14 led to compound 18, with nanomolar activity against NDM-1 as well as two other MBLs.

 

Despite its metal-chelating sulfonamide moiety, compound 18 was inactive against 34 mammalian metalloenzymes tested. It also didn’t inhibit any of 114 potential off-targets in a Eurofins screen. As expected, the molecule alone had no bactericidal activity, but it did enhance the activity of the beta-lactam antibiotic imipenem in MBL-expressing bacteria. Increasing the polarity of the molecule by adding a hydroxyl moiety to a solvent-exposed region and converting a phenyl to a pyridyl ring resulted in compound 23. While less active against isolated MBL enzymes, this molecule was more effective at inhibiting bacterial growth in the presence of imipenem, possibly due to accumulation in the periplasmic space.

 

Compound 23 is clean against off-targets such as hERG, ion channels, and various CYP enzymes. It has reasonable pharmacokinetic properties in mice when dosed intravenously. In a preliminary mouse efficacy study, the compound reduced levels of bacteria in spleen and kidney when dosed with imipenem.

 

This is a nice example of structure-based design starting from fragment-sized molecules. With an abundance of nitrogen atoms and a ClogP < 0, compound 23 looks unusual. Nonetheless, the researchers write that “further evolution of this class of molecules… eventually led to the discovery of several clinical candidates.” I look forward to seeing these described.

RSC Medicinal Chemistry special FBDD issue

The Royal Society of Chemistry puts out RSC Med. Chem., and last year they asked David Rees (Astex), Anna Hirsch (Helmholtz Institute for Pharmaceutical Research Saarland), and me whether a special themed issue on FBDD would be useful for the community. Naturally we said yes, and the results have now been published. You can read our introduction here.

 

Unlike olden days, when special issues were bound between covers, this is a virtual special issue, with papers published over a period of several months. Indeed, we already wrote about two of them last year: one on combining DNA-encoded libraries (DEL) with FBLD and one on inhibitors of PRMT5/MTA. (Both of these were also topics at the CHI FBDD meeting earlier this month.) In the next few paragraphs we highlight the rest.

 

AstraZeneca has been doing FBDD since 2002, and has gained hard-won wisdom, some of which was shared in a 2016 review we wrote about here. After years of screening, their fragment library had started to deteriorate, so they rebuilt it entirely, as described by Simon Lucas and colleagues. Some of the starting fragments came from their previous library, but they also considered molecules from their larger collection. Rather than focusing on the rule of three, they developed their own multiparameter optimization function, “FragScore,” which incorporates logD_7.4, heavy atom count, number of rotatable bonds, and number of hydrogen bond donors. All compounds were inspected to make sure they would be synthetically tractable, and quality was assessed by SPR, NMR, redox activity, and solubility. The final set consists of 2741 fragments, with a subset of 1152 maximally diverse and attractive fragments for ligandability assessments or screening hard-to-make proteins. They also gathered 16,806 near neighbors for hit follow-up. So far the effort has paid off, with all four of the targets screened thus far yielding progressible hits. If you’re building or renovating a fragment library, you should read this paper.

 

Continuing on the theme of libraries, Bradley Doak, Martin Scanlon, and colleagues at Monash University describe their “MicroFrag” library, a set of 91 tiny (5-8 non-hydrogen atom) compounds similar to MiniFrags and FragLites. A crystallographic screen (at 1 M concentration!) of the MicroFrag library against the difficult E. coli target DsbA yielded a 52% hit rate, compared with a 2% hit rate with a conventional fragment library. Importantly, the MicroFrag screen identified the two main hot spots previously discovered from the conventional fragment library, along with ten others that may be less actionable. Interestingly, a crystallographic screen of 15 organic solvents at even higher concentrations (50-80%) was less informative: the primary hot spot did not distinguish itself from others. In the case of MicroFrags, not only did this hotspot bind the largest number of fragments, but all the molecular interactions seen for larger fragments were observed.

 

Fluorine NMR takes advantage of its own specialized library, the subject of a paper by Chojiro Kojima (Osaka University), Midori Takimoto-Kamimura (CBI Research Institute) and collaborators from several institutions. The researchers describe the construction of a 220-member library divided into pools of 10-21 compounds. This library was screened against four diverse proteins, yielding between 3 and 16 hits. The three hits against FKBP were characterized in more detail, including two-dimensional NMR and isothermal titration calorimetry. The researchers also discuss using ¹⁹F STD experiments to determine the binding mode of bound fragments.

 

Fluorine is not the only halogen of interest for library design. We’ve previously described the halogen-enriched fragment library (HEFLib, here and here), which consists of chlorine, bromine, and iodine-containing molecules. Frank Boeckler and collaborators at Eberhard Karls Universität Tübingen and the Max Planck Institute describe screening this library against the Y220C mutant of p53 in an expansion of work they first described back in 2012. Of 14 hits identified by thermal shift or STD NMR, ten confirmed by two-dimensional ¹H-¹⁵N-HSQC NMR. Four of these bound in the cleft created by the Y220C oncogenic mutation. Two other fragments turned out to be covalent binders, though they reacted with more than one cysteine residue. Although all the fragments have low affinities, they could potentially serve as starting points for optimization.

 

An ongoing debate is whether there is an advantage to screening more “three dimensional” fragments as opposed to planar aromatic fragments. If your taste tends towards the former, the synthetic chemistry can get tricky. According to an analysis we highlighted last year, the piperidine ring is the third most common scaffold found in drugs. Now, Peter O’Brien (University of York) and an international group of collaborators report efficient synthetic routes to all 20 cis- and trans-piperidines substituted with a methyl group and a methyl ester. A virtual library of 80 compounds in which the secondary amine is capped with simple substituents such as methyl or acetyl groups was found to be quite shapely, particularly compared with the disubstituted pyridyl starting materials. Moreover, the fragments are still reasonably sized, with no more than 15 non-hydrogen atoms and ClogP values < 2.

 

Machine learning is gaining prominence everywhere, not least in drug discovery. In 2021 we highlighted an “autoencoder” designed for constructing fragment libraries biased towards “privileged” fragments more likely to generate hits. However, the method required considerable programming savvy. Now Angelo Pugliese (BioAscent) and collaborators at the Beatson Institute have implemented their model in the open-source KNIME platform, making it accessible to a wider range of researchers. As an example they use the method to construct a GPCR-focused fragment library, with the structures of all the members provided in the supporting information.

 

On the subject of fragment libraries, please make sure to vote in our 6-question poll on library design (right side of page; you may need to scroll up).

 

Not all the papers in this special issue involve library design. Marko Hyvönen, David Spring, and collaborators at University of Cambridge and National University of Singapore describe allosteric inhibitors of the kinase CK2α, which has been implicated in cancer cell survival. We highlighted some of their work against this target in 2017, in which they used fragment linking to find high nanomolar inhibitors of the enzyme. In the new paper, the researchers describe additional fragment binders at the so-called αD pocket, distant from the ATP-binding site. Virtual screening for analogs led to a fragment with mid-micromolar activity in biochemical and cell assays, and fragment merging led to low micromolar inhibitors.

 

This is a nice collection of papers, and for those of you without easy literature access make sure to check them out soon: for the next six months all of them are free to read after free RSC registration. Enjoy!

Eighteenth Annual Fragment-Based Drug Discovery Meeting

Last week the CHI Drug Discovery Chemistry (DDC) meeting was held in San Diego, and it was the largest ever, with more than 850 participants, 87% of whom attended in person, up from 70% last year. I won’t attempt to cover all twelve tracks, but will just touch on some of the main themes.

 

Covalent fragments

Brent Martin kicked off a session devoted to covalent modifiers by describing the in-cell proteome-wide covalent ligand discovery done at Scorpion Therapeutics. Brent emphasized the importance of measuring k_inact/K_i to characterize compounds, and he went so far as to say he would recommend rejecting papers that report only IC₅₀ values. He emphasized some of the challenges finding low-affinity fragments (with high K_i values) that are not overly reactive. But Upendra Drahal (Amgen) noted that sotorasib has only weak affinity (K_i = 86 µM) but a high k_inact (0.85 s^-1) for the G12C mutant form of KRAS, despite being quite stable against glutathione. High reactivity is fine, as long as it is highly selective reactivity.

 

Jeffrey Martin (Biogen) spoke about covalent fragments applied to neuroscience, including targets for Alzheimer’s (tau) and Huntington’s disease. And in two separate talks Dan Nomura (UC Berkeley) provided numerous examples of finding covalent fragments against a variety of targets, including cMYC and E3 ligases (more on those below).

 

Keynote speaker Michelle Arkin (UCSF) described using disulfide Tethering to find reversible covalent binders of caspase 6 that were subsequently optimized to cell-active irreversible inhibitors. She provided an evocative visual metaphor of proteins participating in an English country dance, moving from partner to partner in a dynamic yet choreographed fashion. The most popular dancers are the 14-3-3 proteins, which act as hubs mediating binding to hundreds of other proteins. Michelle has found stabilizers of some of these interactions, which could shut down aberrant disease signaling.

 

Covalent ligand discovery is best done with mass spectrometry, and two talks revealed useful new methods. Jim Nonomiya described an approach he and his Genentech colleagues developed called CoMPAS, covalent mapping by peptide attenuation screening. This method uses an isotopically labeled peptide as an internal standard to assess disappearance of covalently modified peptides from enzymatically digested proteins. Sensitivity can be much better than for intact protein mass spectrometry, allowing lower consumption of scarce recombinant protein. Depending on the type of setup, throughput can also be higher.

 

Throughput is the name of the game in a method presented by Nate Elsen (AbbVie) called IR-MALDESI-MS. The home-built system uses a laser to gently ionize aqueous solutions in 384-well plates before running them through an electrospray mass spectrometer. The system can analyze up to 20 samples per second, including intact proteins.

 

Non-covalent fragments

Turning to non-covalent methods, Rod Hubbard (Vernalis) provided an update of the PAC-FragmentDEL approach which we highlighted last year. DNA-encoded libraries can be mind-bogglingly large, with more than a trillion molecules at Hitgen. The fragment set is much smaller, at just 130,000 members, but this is still two orders of magnitude larger than a typical fragment library. (Speaking of which, please make sure to fill out our library survey on the right-hand side of the screen if you haven’t already done so.) This increased chemical diversity increases the odds of finding rare molecules, such as molecular glues that bind to protein complexes rather than individual components.

 

Success stories are always plentiful at conferences. Timo Heinrich (EMD Serono) described using SPR to identify fragments that were optimized to orally bioavailable inhibitors of the anti-cancer target TEAD1, which we wrote about here. Mihir Mandal (Merck) described the use of fragment concepts in the development of clinical candidates targeting metallo-β-lactamases, an important cause of antibiotic resistance. Chris Smith described Mirati’s discovery of inhibitors against the anti-cancer target PRMT5/MTA, one of which has gone into the clinic (see here and here). And Tanweer Khan traced the origins of renin and plasmepsin inhibitors to work on the aspartyl protease BACE1 at Merck, which we wrote about here.

 

Sometimes we learn just as much from projects that don’t move forward, as illustrated in a nice talk by Haihong Wu. He and his AbbVie colleagues were interested in the tau protein, which is intrinsically disordered, making structure-based design difficult. Although a two-dimensional NMR screen identified several dozen fragment hits, these were hard to optimize, with sharp, non-linear SAR. Perhaps the covalent binders we mentioned above will be more advanceable.

 

Targeted protein degradation

Targeted protein degradation and induced proximity were major themes of the conference. We speculated several years ago that FBLD could be useful here, and this has turned out to be abundantly true.

 

E3 ligases attach ubiquitin to other proteins, marking them for degradation. More than 600 exist in the human genome, but only a handful have been co-opted for targeted protein degradation, and everyone is racing to find new and better ligands for the unexplored E3 ligases. Mary Matyskiela described how she and her colleagues at Neomorph are using fragment screening to identify such ligands. She said they have been able to use cryo-electron microscopy to guide structure-based design.

 

Dan Nomura’s second talk focused on finding ligands against E3 ligases, including UBE2D and RNF126. For both of these proteins small covalent ligands seem to be generally useful for causing degradation of target proteins, and because the chemical structures have been disclosed it will be fun to see them explored in more systems.

 

A significant focus for targeted protein degradation is to find ligands for E3s that are restricted to specific tissues or tumors. Steve Fesik (Vanderbilt) described using SAR by NMR to find ligands against three E3 ligases, including one not expressed in the heart, which could avoid cardiotoxicity. Steve also described using SAR by NMR to find nanomolar binders for β-catenin. These are being used to make degraders for this anti-cancer target.

 

Many of the molecules used for targeted protein degradation are well beyond conventional rule-of-five space as they contain binding moieties for the target of interest as well as an E3 ligase. Reflecting on his forty-year career as a medicinal chemist at Bristol Myers Squibb, keynote speaker Nicholas Meanwell noted that a beautiful drug is one that helps patients, not one that fits a set of metrics. For small molecule therapeutics, he observed, “the opportunities have never looked better.”

 

We’ll end on this note, but please feel free to leave comments about your highlights. And mark your calendar for April 1-4, when DDC returns to San Diego.

New poll: fragment libraries in 2023

The quality of a fragment hit depends on the quality of the library. Virtual screens can be done with billions of compounds, but if you’re going to build a physical library you need to be pickier. Researchers today are increasingly avoiding pathological molecules such as PAINs, but even sticking to “reasonable” molecules leads to choice overload.

 

So for those building or refurbishing their libraries, Practical Fragments launches a new poll. It’s been almost five years since we’ve asked readers about their libraries, and the current poll is our longest yet, with six questions.

 

The first three questions focus on size: how large is your fragment library and what are the minimum and maximum number of heavy atoms in each fragment.

 

Next, we ask about whether you include chiral molecules in your library, and whether these are present as enantiomerically pure compounds or racemates.

 

Chiral compounds can introduce synthetic challenges which may impede hit follow-up, so our next question asks whether you consider synthetic tractability before adding fragments to your library.

 

Finally, building a proper fragment library requires considerable time and resources, so you want to take good care of it. Our last question probes how you store your fragment library. Note that this question asks about your working library, the one that you screen and access on a regular basis, not the master stocks which may be squirreled away as solids in the deep freeze.

 

Please vote on the right-hand side of the page. If you have multiple fragment libraries (perhaps one for crystallographic screening and another for biochemical screening) feel free to vote for each; you need to press "Finish Survey" at the end.

 

There are, of course, more complex elements to library design that can’t be captured in simple multiple-choice polls. For example, we’ve written previously about the importance of function rather than functional groups, and the types of rings found in approved drugs. If you have opinions about these or other subjects, please share.

 

The poll was already long so we decided not to ask about library vendors, which we addressed in 2018. On this topic too, please feel free to share your thoughts.

 

Happy voting!

Profitable Fragment$

Practical Fragments has always been free and worth every penny. But over the past 15 years and 900+ posts we believe we've generated a lot of value, so today we're launching our for-profit sister site, Profitable Fragment$. Here you'll be able to buy all your favorite branded items, such as Sauron Atoms jerseys, Voldemort Rule mugs, and Dr. Saysno baseball caps. But the real moneymaker, we believe, is our new line of NFTs.

 

Non-fungible tokens, or NFTs, are one-of-a-kind digital thingamajigs that represent ownership of something else, such as works of art. And what are molecules if not works of art? Every drug that has ever been invented will have an NFT. But Profitable Fragment$ has no intention of stopping there. No, we intend to create NFTs for every fragment ever published. Fragments that have led to drugs, or even those with associated crystal structures, will go for a premium.

 

We'll also have a special line of PAINS NFTs, akin to Garbage Pail Kids. Will toxoflavin sell for more than vemurafenib?

 

Even if you can't afford something fancy like sotorasib or 7-azaindole, don't despair: there are nearly 170 billion cheaper options drawn from GDB-17. Heck, if these sell for just 10 cents per fragment it should generate more than enough cash for us to buy back Twitter!

Crystallography heats up, it seems for the better

Of the roughly 150,000 crystal structures in the Protein Data Bank (PDB), about 94% were collected at cryogenic temperatures (≤ 200 K), typically after being frozen in liquid nitrogen. Frozen crystals are easier to store and transport and can better survive bombardment by intense X-rays, but are the resulting structures still physiologically relevant? A newly published open-access paper by Daniel Keedy (CUNY) and a multinational team of collaborators suggests caution.

 

The researchers were interested in the protein tyrosine phosphatase PTP1B, a diabetes target that has implacably thwarted generations of drug hunters. A previous screen had identified dozens of fragments that bound at cryogenic temperatures to multiple sites on the protein, most prominently the active site and three secondary sites. In the new work, 143 fragments were chosen for reanalysis at room temperature, of which more than half were hits and the rest were non-hits that had been included in the previous screen. The goal was to assess whether the same fragments would bind in the same manner, and whether any of the non-hits would bind at room temperature.

 

Two methods were used for room-temperature crystallography. In both cases, fragments were soaked into crystals of PTP1B. In the first, crystals were harvested from drops as normal, but rather than being flash frozen they were enclosed in plastic capillaries to keep them hydrated. The second “in situ” method was performed by mounting the crystallization trays directly onto the goniometer at the synchrotron.

 

Both methods gave similar results, though the average resolution for the in situ method (1.99 Å) was better than the capillary method (2.30 Å) and even surpassed the previous cryo screen (2.10 Å). Fragment hits were initially identified using the automated PanDDA method (see here), followed by manual analysis and careful data processing to ensure that even the weakest binders were not overlooked.

 

Surprisingly, the hit rate was quite low: only about a third of the fragment hits that had been identified under cryogenic conditions were found in the screen at ambient temperature. Moreover, the room-temperature fragments tended to have lower occupancy factors, meaning that a higher fraction of a given binding site was empty. The researchers took special care to ensure that the results were robust, for example by checking to make sure that fragments had been correctly added to the droplets.

 

Of the fragments that confirmed, the binding modes often differed, for example by flipping 180°. In some cases water molecules around the fragment varied depending on the temperature; as we’ve written, water can be essential for binding interactions, so these differences could be significant. And in some cases the room-temperature fragments bound to different sites entirely, including one site that had not been observed to bind any fragments under cryogenic conditions.

 

Only one of the fragments that had previously not produced hits under cryogenic conditions showed up as hit at room temperature, but interestingly this one formed a covalent bond to two different lysine residues.

 

So what are we to make of all this? The researchers speculate that the overwhelming focus on cryogenic structures “may favor protein-ligand interactions that overweight enthalpic considerations and underweight entropic ones, feature inaccurate solvation environments, or suggest artificially rigid proteins.” The fact that most machine-learning models of protein-ligand interactions are trained on cryogenic structures may systematically bias their results.

 

In 2016 we highlighted an argument for moving crystallography to the front of a screening cascade, but as we noted last year doing so may lead to myriad hits too weak to advance. Perhaps room temperature crystallography can bridge this gap.

Versatile fragments from the Protein Data Bank

Four years ago we highlighted an analysis of fragments taken from the Protein Data Bank (PDB). Of 462 unique fragments, just 21 bound in more than one pocket. With the assumption that such “versatile” fragments may be particularly valuable starting points, Esther Kellenberger and colleagues at CNRS Univeristé de Strasbourg have done their own exploration of the PDB, as reported (open access) in Front. Chem.

 

Structures deposited in the PDB starting in 2000 with resolution better than 3 Å were examined to find those containing fragment-sized molecules (MW < 300 Da). Crystallization additives, phosphate and sulfate ions, and other unlovable molecules such as PAINS were excluded. Further triaging for fragments that bound in more than one pocket and in more than one binding mode (ie, different types of interactions) ultimately yielded a set of 203 versatile fragments. (One reason why so many more fragments were found in this study is the fact that the previous analysis required the word “fragment” to be present in the PDB entry.)

 

The versatile fragments are mostly compliant with the rule of three, with violations mostly related to the number of hydrogen bond donors or acceptors. Only a single molecule had ClogP > 3, though 50 were quite hydrophilic, with ClogP < 0. Interestingly, 45 of the molecules are listed as small molecule drugs, and 98 are substructures of approved drugs. Perhaps this is not surprising; drugs themselves are studied particularly intensively and frequently included in screening libraries.

 

The researchers had previously analyzed commercial libraries, and in the new paper they compared versatile fragments with the SpotXplorer library we wrote about here and the functionally diverse fragments used at XChem. Surprisingly there was very little overlap, even though most of the versatile fragments or analogs are commercially available. That said, some of the versatile fragments are molecules one may not want in a fragment library, such as the cofactor lipoic acid and the metal chelator 1,10-phenanthroline.

 

Binding modes for the same fragment in different pockets could vary considerably. The “universal fragment” 4-bromopyrazole, which we wrote about here, bound in two different binding modes, while the nucleoside thymidine showed a whopping 26 different binding modes. Conformations of the fragments could vary too, with only 43% of fragments showing a conserved conformation in all binding sites (defined as < 0.5 Å RMSD). Conformational changes, along with different protonation states, could be among the reasons why predicting fragment binding continues to be challenging.

 

This is a nice analysis, and it may be worth adding some of these versatile fragments to your own library. Laudably, SMILES strings for of all of them are provided in the supplementary material.

A very useful list: common linkers and bioisosteric replacements

Last week’s post highlighted an example of fragment linking, which despite being less common than fragment growing can still be effective. But how do you choose the linker? We’ve previously written about the most common rings found in drugs. In a new Bioorg. Med. Chem. paper Peter Ertl and colleagues at Novartis tabulate the most common linkers found in bioactive molecules.

 

The researchers start by defining linkers “as moieties connecting 2 ring systems.” To focus on druglike molecules, linkers could contain no more than eight non-hydrogen atoms total and no more than five consecutive bonds between the two ring systems. This means that para-disubstituted phenyl or 1,4-disubstiuted butyl would both be considered in the analysis, but longer linkers such as this recent example would not.

 

Molecules were extracted from the databases ChEMBL and ZINC, yielding a total of 1686 unique linkers. Various descriptors were calculated for all, which in addition to size and length included the number of heteroatoms and electronic properties. Bioactivity data for molecules in ChEMBL was used to assess which replacements were most frequently tolerated. If one linker could be replaced by another without causing a drop in affinity (or inhibition, etc.), the two linkers were considered to be bioisosteres.

 

So, what are the most common linkers? A single methylene is the most common, followed by an amide bond. I was surprised that, of the 40 most common linkers, only five are rings: para-disubstituted phenyl, 1,4-piperzine, 1,4-piperidine, 1,2,4-oxadiazole, and meta-phenyl, in that order. Not coincidentally, phenyl rings, piperidines, and piperazines are also the most common rings found in drugs, according to an analysis last year.

 

Last year we highlighted a paper from the Ertl group that included a link to a “Ring Replacement Recommender,” which suggests bioisosteric replacements for any ring. Alas, there is no “Linker Replacement Recommender,” but the new paper does provide a “bioisosteric replacement network,” which is a full-page 10 x 15 grid with the 150 most common linkers arranged such that nearby linkers are likely to be bioisosteric. For example, para-phenyl is adjacent to 2,5-thiophene and quite some distance from sulfone. These make sense, but there are also less obvious examples: the table suggests that a 1,4-pyrazole makes a good replacement for a carbamate.

 

The next time you’re doing SAR, it may be worth consulting the bioisosteric replacement network for ideas.

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.

FragLites and PepLites meet bromodomains

The last two Practical Fragments posts focused on bromodomains, epigenetic readers that recognize acetylated lysine residues. Today’s post could thus be considered part of a trilogy, though the focus is less on bromodomains themselves than a specific type of fragment library.

 

In 2019 we highlighted FragLites, small fragments containing pairs of hydrogen bond acceptors and/or donors along with a bromine or iodine atom. FragLites were designed to assess ligandability as well as identify what types of interactions would be favorable at various sites. The original test protein was the kinase CDK2. In an open-access paper published late last year in J. Med. Chem. by Martin Noble, Michael Waring, and colleagues at Newcastle University, FragLites are screened against two members of the bromodomain family.

 

The first bromodomain (BD1) of BRD4 is considered highly ligandable, with multiple inhibitors disclosed (see for example here). In contrast, ATAD2, a bromodomain in another subfamily, is more challenging, in part because it lacks a hydrophobic region useful for increasing affinity for small molecules. Thirty-three FragLites were individually soaked at 50 mM into crystals of either bromodomain. The halogen atom on each FragLite facilitates analysis by anomalous dispersion, allowing more sensitive detection of low-occupancy binders. This, along with Pan-Dataset Density Analysis (PanDDA), was used to identify specific protein-ligand “binding events.”

 

In total, 26 binding events at five sites were identified for BRD4; four ligands bound at more than one site. Of these, 17 FragLites bound at the orthosteric site of BRD4 (which recognizes N-acetyl lysine). In contrast, ATAD2 displayed 16 binding events total over seven sites; only three bound at the orthosteric site, consistent with its lower ligandability. ATAD2 had previously been screened crystallographically against the 776-membered DSI-poised fragment library, and this effort also identified seven ligand-binding sites, six of which were common to those discovered here, suggesting that the small FragLite set is able to identify most pockets.

 

As far as specific types of interactions, the average FragLite made 1.1 hydrogen bond, suggesting that the second donor or acceptor is often not engaged. In contrast, the bromine or iodine atom makes protein contacts in 33 of 42 binding events. In half a dozen cases no hydrogen bond to the protein was observed, with the primary interaction being a halogen bond.

 

The FragLites are small, relatively “flat” aromatic molecules, but of course most proteins interact with other proteins. To try to explore such interactions, the researchers developed a library of “PepLites:” N-terminally acetylated amino acid residues with a C-terminal bromopropargyl group. These were also screened crystallographically against the two bromodomains and produced considerably lower hit rates, with six bound to BRD4 (all at the orthosteric site) and nine bound to ATAD2 (of which five bound to the orthosteric site). Reassuringly, the N-acetylated lysine PepLite bound to both proteins in a similar manner as seen in larger peptides.

 

The researchers conclude that FragLites and PepLites “represent highly valuable components of a larger crystallographic screen, and we anticipate that this is where they will fit into most drug discovery programs.” Indeed, this is already happening; last year we wrote about how FragLites were screened against the bromodomain PHIP2 as part of a larger screen, and I was surprised this paper was not mentioned here. Laudably, all the atomic coordinates have been deposited in the Protein Data Bank, so folks are able to do their own analyses.

 

As FragLites and PepLites are screened against ever more targets, it will be fun to see what they can teach us about intermolecular interactions and starting points for new leads.

Fragments vs PBRM1 bromodomains revisited, more selectively

Last week we highlighted the discovery of a selective inhibitor for the BET family of bromodomains. The 61 human bromodomains fall into eight subfamilies, of which the BET family has probably been most heavily studied. In contrast, family VIII has received less attention, in part due to the lack of selective inhibitors. This deficit is beginning to be addressed by Shifali Shishodia, Brian Smith, and collaborators at Medical College of Wisconsin and Purdue University in J. Med. Chem.

 

The researchers were particularly interested in the aptly-named protein Polybromo-1 (PBRM1), which contains six of the 10 family VIII bromodomains. The protein has normally been considered a tumor suppressor, but it has also been implicated as a tumor promoter in prostate cancer. Chemical probes would be very useful to unravel the complicated biology. A few pan-inhibitors of family VIII have been developed, one of which we wrote about back in 2016, but none of these are selective for the PBRM1 protein.

 

The researchers started with an NMR screen of the second bromodomain of PBRM1, BD2, the structure of which had previously been solved by NMR. A ¹H-¹⁵N SOFAST-HMQC screen of 1968 fragments (all rule of three compliant, from Maybridge and Zenobia) in pools of 12 ultimately yielded a dozen hits, all of which are shown in the paper. Of these, compound 5 was the most potent, with dissociation constants of 45 µM by NMR titration and 18 µM by isothermal titration calorimetry (ITC). 

 

 

One of the previous pan-family VIII inhibitors described in the literature was structurally similar to compound 5, and borrowing a chlorine atom from this led to compound 11, with improved affinity. Further exploration around both phenyl rings ultimately led to compound 16, which displayed low micromolar affinity by ITC and high nanomolar activity in an inhibition assay.

 

Differential scanning fluorimetry (DSF) is commonly used to measure binding of small molecules to bromodomains, and the researchers tested some of their best compounds in a panel of bromodomains that included 9 of the 10 family VIII members. Encouragingly, compound 16 only showed a strong thermal shift (ΔT_m = 5.4 °C) to PBRM1-BD2 and moderate shifts (ΔT_m = 1.8 °C) to PBRM1-BD3 and PBRM1-BD5. No significant stabilization of the 18 other bromodomains was observed.

 

A series of shRNA experiments by the researchers revealed that the prostrate cancer cell line LNCaP was dependent on PBRM1, and compound 16 was active against these cells, albeit weakly (EC₅₀ ~ 9 µM). In contrast, the compound did not show activity against two other cancer cell lines that do not seem to be dependent on PBRM1.

 

This work is a nice example of academic fragment-based lead discovery. Although the cell activity of compound 16 is probably insufficient for a serviceable chemical probe, it does show that selectivity is possible. Hopefully these researchers, or others, will continue improving it.

Efficiency metrics in action for a bromodomain inhibitor

The metrics ligand efficiency (LE) and lipophilic ligand efficiency (LLE or LipE) are frequently used during fragment-to-lead optimization. A recent paper in J. Med. Chem. by Philip Humphreys and colleagues at GSK describes how they were useful in developing an “oral candidate quality” inhibitor of BET-family bromodomains.

 

Practical Fragments has written frequently about bromodomains, which bind to acetyl-lysine residues in histones to epigenetically modulate gene transcription. Some 17 bromodomain inhibitors have entered the clinic, of which at least three (pelabresib, PLX51107, and ABBV-744) came from fragments. GSK was an early pioneer in the field, and researchers there were interested in using fragments to develop a differentiated class of molecules that would inhibit all four members (BRD2, BRD3, BRD4, and BRDT) of the BET family, each of which contains two separate bromodomains designated as either BD1 or BD2.

 

GSK already had BRD4 BD1 binding data for 50,000 compounds, and these were analyzed to find molecules with LE>0.3 kcal/mol per atom that were structurally differentiated from known bromodomain binders. Compound 9 was quite potent and had high LE as well as respectable LipE. (As the researchers note, LE is “the more relevant metric” for fragments, with LipE becoming increasingly important during later optimization.) A crystal structure of this molecule superposed onto another bromodomain inhibitor suggested that adding a methyl group to fill a small pocket could boost affinity, and this was confirmed by compound (R)-10. This molecule showed cell activity and good permeability, although hepatocyte stability was poor, likely due to the two methoxy groups. Removing these led to compound 12, the most ligand-efficient compound that had been seen. (All values in the figure below are for binding to BRD4 BD1.)

 

Compound 12 mimics the N-acetyl lysine residue of the natural ligand, and previous research had revealed two additional regions of the bromodomain that could be targeted for enhanced affinity, the so-called “WPF shelf” and the “ZA channel.” Structure-based design was used to independently explore both areas, leading to compounds such as 24 and 31. In addition to assessing LE and LipE, the researchers paid close attention to other factors such as permeability. Virtually combining the best moieties that bind at the WPF shelf and ZA channel led to 770 potential molecules to make, which were winnowed down to just 40 on the basis of predicted lipophilicity (specifically chromLogD_pH7.4), molecular weight, and TPSA. The best of these were more extensively profiled, including in pharmacokinetic studies. I-BET432 emerged as the winner.

 

I-BET432 binds tightly to both bromodomains of the four BET family proteins and is at least 80-fold selective against two dozen other bromodomains. It shows excellent oral bioavailability in rats and dogs, does not inhibit hERG, is not mutagenic in an Ames test, and does not inhibit CYP3A4. The molecule is also clean in a panel of four dozen off-target proteins. Human oral dose predictions come in at 5-18 mg per day. A crystal structure of the molecule bound to BRD2 BD2 showed the expected binding pose, and that the two alcohol substituents may be forming an intra-molecular hydrogen bond, which could explain the high permeability.

 

This is a nice case study in metric-driven optimization. As the researchers note, I-BET432 “has the highest LipE (6.2) and LE (0.43) of the candidate quality GSK pan-BET inhibitors disclosed to date.” Although the molecule does not seem to have gone forward into development, the story is nonetheless worth reading to see how metrics can yield quality molecules.

Fragments in the clinic: MK-8189

Just over seven years ago Practical Fragments highlighted work out of Merck describing the discovery and optimization of potent, selective inhibitors of phosphodiesterase 10A (PDE10A), a potential target for schizophrenia (see here and here). An open-access paper in the latest issue of J. Med. Chem. by Mark Layton and colleagues tells how these were ultimately advanced to a clinical compound.

 

To recap, a biochemical fragment screen identified the highly ligand-efficient compound 1, which was optimized to the potent compound 2. However, this molecule had poor pharmacokinetics and multiple other liabilities. Further optimization led to Pyp-1, which we noted at the time would make a good chemical probe.

 

The new paper continues SAR around the central ring, in particular to try to reduce lipophilicity. Also, the methyl-pyrazole in Pyp-1 was associated with high clearance in rats, so this substituent was replaced with a methyl-1,3,4-thiadiazole moiety. To cut a long story short, this ultimately led to MK-8189.

 

Not only is MK-8189 a picomolar biochemical inhibitor of PDE10A, it is a low nanomolar inhibitor in cells. Moreover, it shows excellent pharmacokinetics in rats and rhesus monkeys as well as selectivity against various off-targets such as CYPs and hERG. Importantly for a drug intended to reach the brain, the molecule is permeable, not effluxed, and achieves relevant concentrations in the rat striatum after oral dosing. Finally, it decreased psychomotor activity and improved episodic memory in rat models of schizophrenia. With all these positives, MK-8189 has been taken into the clinic.

 

Several lessons emerge from this story. First, as experienced drug hunters will recognize, systematic exploration of multiple positions is important to generate a molecule with the right balance of properties to become an investigational drug. Second, as the researchers note, ligand efficiency was roughly maintained throughout the optimization process. Finally, this publication is a reminder of the long lag that can occur between research and publication. We already mentioned that the first papers describing this series appeared in 2015, but according to ClinicalTrials.gov MK-8189 first entered the clinic a year earlier, in 2014. Our list of fragment-derived clinical compounds will forever be incomplete and out of date. But on a positive note, this means that fragments may be having even more of an impact than the list shows.

The Chemical Probes Portal at Eight

Back in 2015, Practical Fragments highlighted a new resource calling itself “The Chemical Probes Portal.” At the time it included just seven probes, and my post concluded, “I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments.”

 

Well, take off it has, as illustrated by a new (open access) paper in Nucleic Acids Res. by Susanne Müller (Goethe University Frankfurt), Bissan Al-Lazikani (MD Anderson Cancer Center), Paul Workman (Institute of Cancer Research), and collaborators.

 

The paper notes that “the widespread use of small molecule compounds that are claimed as chemical probes but are lacking sufficient quality, especially being inadequately selective for the desired target or even broadly promiscuous in behavior, has resulted in many erroneous conclusions in the biomedical literature.” As an antidote, the Portal is an “expert review-based public resource to empower chemical probe assessment, selection, and use.”

 

Any scientist can suggest a potential probe, and these are then internally reviewed and curated. Assuming enough public information is available about the molecule, probes are then sent to three members of a Scientific Expert Review Panel for further vetting. Reviewers rate probes from one to four stars for use in cellular and/or animal models and recommend relevant concentration ranges. Importantly, reviewers can also include comments to highlight off-targets, lack of certain data, oral bioavailability, or anything else.

 

From a mere seven probes in 2015 the Portal has grown to include more than 500 molecules covering more than 400 protein targets in about 100 protein families. About two thirds of the probes have three or more stars, meaning they are recommended. The Portal is very easy to use and can be searched by probe or protein. Laudably, all the data can also be easily downloaded in bulk.

 

In addition to the chemical probes, the Portal also contains around 250 “Historical Compounds” that have been described in the literature but “are not recommended to be used to study the function of specific proteins as they are seriously flawed.” These include molecules such as gossypol, a known aggregator that has been reported as an inhibitor of multiple proteins, and curcumin. If you see a molecule used as a probe in the literature, it’s worth checking to see whether it shows up in the Portal.

 

The Chemical Probes Portal features heavily in a Conversation between Cheryl Arrowsmith (Structural Genomics Consortium) and Paul Workman published (open access) last year in Nat. Commun. The researchers concisely define chemical probes as “small-molecule modulators to interrogate the functions of their target proteins, as opposed to protein location, or other physical properties.” Importantly, they differentiate chemical probes from drugs. “Drugs don’t necessarily need to be as selective as high-quality chemical probes. They just need to get the job done on the disease and be safe to use. In fact, many drugs act on multiple targets as part of their therapeutic mechanism.” I have frequently heard people make comments such as, “this is just a probe, not a drug,” but a good probe should actually be more selective than many drugs.

 

That said, you do want a drug to actually hit the target of interest. The researchers highlight iniparib, a putative PARP inhibitor that made it all the way to phase 3 clinical trials for breast cancer and was tested in >2500 cancer patients. It failed. Moreover, that failure cast a pall over the field which likely delayed the development of actual PARP inhibitor drugs.

 

The researchers also discuss aggregators, which are still being reported uncritically in the literature, along with PAINS. “Such compounds should never be considered further or used as chemical probes. They should be excluded from compound libraries. Yet many are sold by commercial vendors as chemical probes and widely used.”

 

This statement raised the hackles of Pete Kenny. In a recently published critique, he states: “it is asserted in the conversation that commercial vendors are selling compounds as chemical probes that are unfit for purpose and I strongly recommend that anybody making such assertions should carefully examine the supporting evidence.”

 

Dear reader, please try the following experiment. Enter “iniparib supplier” in your favorite search engine and see what comes up. For me, the first 10 results include several that describe it as a PARP inhibitor. I won’t link to them here because I don’t want to encourage traffic to their sites. (This is also part of the reason Practical Fragments has discontinued PAINS shaming, as it only increases the profile of sloppy or harmful papers.)

 

Pete goes on to write: “I would strongly advise against making statements that a compound is unfit for use as a chemical probe unless the assertion is supported by measured data in the public domain for the compound in question.”

 

Frankly, I don’t understand Pete’s position, which I parodied here. Life is short and biology is complicated, so why waste time with dirty or inadequately characterized reagents? For me, everything is an artifact until proven otherwise. And the Chemical Probes Portal goes a long way towards demonstrating whether a particular probe is fit for purpose.