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.”

The Chemical Probes Portal turns ten. Use it!

Last week we highlighted a new tool to computationally predict whether a molecule might aggregate, thereby causing false positives. This doesn’t necessarily mean the molecules are bad (after all, some approved drugs aggregate), but it’s all too easy to screen molecules under inappropriate conditions. This brings up the topic of chemical probes, and as it happens the Chemical Probes Portal turns ten years old this year, as celebrated in a Cancer Cell Commentary by Susanne Müller, Domenico Sanfelice, and Paul Workman and a blog post by Ben Kolbington at the Institute of Cancer Research.

 

We first wrote about the Chemical Probes Portal in July 2015, when it contained just 7 compounds. When we returned in 2023 it contained more than 500 compounds, and by the end of last year the number was up to 803. As of today it lists 1174 probes for 622 targets. Nearly a third of the probes also have chemically related inactive controls. These seem like large numbers, but the the human genome conservatively encodes for some 20,000 proteins, and the ambitious Target 2035 initiative seeks chemical probes for all of them.

 

The new paper emphasizes that the standards are in some ways higher for chemical probes than for approved drugs: “whereas probes principally require a high degree of selectivity, drugs need ‘only’ to be safe and effective and may often hit several targets.” Dimethyl fumarate comes to mind as a highly promiscuous covalent modifier that is nonetheless a useful drug for multiple sclerosis and psoriasis.

 

Even when a compound hits a target of interest, that doesn’t mean any biological effects observed are due to the target, particularly when the readout is cell death. The researchers note that TH588 was originally reported as a potent inhibitor of MTH1, but it actually kills cancer cells by binding to tubulin, a fact not always mentioned by chemical suppliers. Another study found that ten clinical compounds were still active in cells even when their putative target was knocked out using CRISPR.

 

The tone of the Commentary is pragmatic, emphasizing that for new or difficult targets, it may be difficult to find good chemical probes. For example, LY294002 is mentioned as a “pathfinder tool” that was useful to explore the biology around the PI3 kinase family but has now been superseded by more selective molecules.

 

Unfortunately, not everyone seems to have gotten the message. Curcumin, which as we noted can aggregate, form nonselective covalent adducts, fluoresce, and generate reactive oxygen species, appears in >2600 PubMed publications – just in the past year. What a waste.

 

If you’re exploring the biology of a target, please check the Portal to see whether there are good probes. If you’re reading (or reviewing!) a paper that reports small molecule studies, please check to see whether the probe has been assessed - especially to see if it shows up as one of more than 250 Unsuitables. And if you’re interested in participating, please consider reviewing or even hosting a Probe Hackathon.

They may be cons, but they’re our CONS

Practical Fragments has written repeatedly about various assay artifacts (vide infra). Different technologies are susceptible to different interference mechanisms, making general rules difficult. Earlier this year we wrote about the Metal Ion Interference Set, or MIIS: a collection of a dozen salts that could be used to assess the sensitivity of assays to metal contaminants. In a recent open-access JACS Au paper, Huabin Hu (Uppsala University), Jonathan Baell (Monash University), and collaborators extend the concept to small molecules.

 

The researchers have compiled a Collection Of useful Nuisance compounds, or CONS, perhaps with a nod to “Chemical con artists foil drug discovery” published a decade ago, which we highlighted here. The 103 members of the CONS are divided into three categories.

 

The first set contains five aggregators: molecules that have been shown to form colloidal clusters that non-specifically interfere with biological assays, as discussed here.

 

The largest set, at 67 members, consists of PAINS, or pan-assay interference compounds, which we first wrote about in 2010. These are themselves divided into various subcategories: non-specific electrophiles such as curcumin and an isothiazolone, redox cyclers such as quinones, contaminants such as the decomposition products of certain fused tetrahydroquinolines, miscellaneous, metal chelators, and additional mechanisms including optical interference and singlet oxygen quenchers, which are particularly problematic in AlphaScreen assays.  

 

The last set consists of 31 compounds that can cause problems in phenotypic assays. Some of these non-specifically disrupt cell membranes. Others have well-defined but toxic effects, such as interfering with tubulin or intercalating into DNA. Such bioactivity is not always a bad thing: some of these molecules, such as topotecan and colchicine, are approved drugs, but it’s useful to be aware of whether these types of activities will affect your assay.

 

One criticism of the PAINS concept is that it lumps together multiple mechanisms. (Pete Kenny wrote about this recently.) Another criticism is that, by focusing on chemical substructures, true hits may be unfairly deprioritized based on structure alone. What’s nice about the CONS list is that the potentially interfering mechanisms of each molecule are documented and categorized so they can be considered when establishing an assay. For example, you may not care whether a compound interferes in a phenotypic assay if you are performing a screen on an isolated enzyme.

 

The entire set of compounds is available from Enamine, and additional vendors are provided in a supplementary table. If you’re doing a lot of assays, particularly on new targets and mechanisms, it may be worth testing the CONS to understand what kinds of false positives might occur.

Beware of fused tetrahydroquinolines

Practical Fragments has written frequently about pan-assay interference compounds, or PAINS. These molecules contain substructures that frequently show up in hits that tend not to be advanceable, often wasting considerable effort. One criticism of the PAINS concept is that the original definitions were based on a limited number of screens in one assay format. In a new (open-access) J. Med. Chem. paper, Alison Axtman and collaborators at University of North Carolina Chapel Hill, Emory University, and Oxford University characterize one class of PAINS in more detail.

 

The researchers focused on fused tetrahydroquinolines, or THQs. Of the 51 molecules containing this substructure in the original 2010 PAINS paper, 34 hit in at least one of the assays, and one hit all six. At the time Jonathan Baell and Georgina Holloway noted that “it is not clear for some PAINS, such as the fused tetrahydroquinolines, what the relevant mechanisms of interference may be.” 

 

 

The new paper notes that fused THQs are common in screening libraries, with more than 15,000 commercially available. They also frequently show up as hits: the researchers summarize more than two dozen examples against a wide variety of targets including phosphatases, kinases, protein-protein-interactions, and more. In most cases the hits are modestly active, with low to mid micromolar IC₅₀ values, though a few are high nanomolar. 

 

Promiscuity per se is not necessarily bad. Just last week we noted that the 7-azaindole fragment was the starting point for three approved drugs. However, despite showing up as hits in so many screens, only one peer-reviewed paper reports a crystal structure of a fused THQ bound to a protein, and the researchers note that “no optimized chemical probes or approved drugs contain the chemotype.”

 

Importantly, fused THQs hit in a variety of different assay formats, including spectrophotometric, chemiluminescent, SPR, and radiochemical. Thus, these are not merely problematic in the AlphaScreen format studied in the original PAINS paper.

 

So what’s going on? The researchers found that, while molecules containing the fused THQ core were initially colorless, they darkened when dissolved in DMSO or chloroform, turning purple within 72 hours. Interestingly, the reaction seems to be light-dependent: solutions stored in the dark remained colorless. Thin layer chromatography and NMR showed new species forming, and mass spectrometry revealed oxidation with loss of two or four hydrogen atoms. The isolated double bond in the cyclopentene ring seemed to be the culprit, as the saturated analog was stable. Indeed, all of the hits shown in the paper contain the double bond, so fused THQs that lack this feature may be fine – if they ever show up in your assay.

 

It is still not clear exactly how the decomposition products light up so many assays, but in general it’s a good idea to steer clear of molecules that fall apart in ambient light, unless you’re trying to make a photosensitizer.

 

The researchers conclude that “it is tragic to continue to watch groups invest time and resources in dead-end hit-to-lead campaigns, and the medicinal chemistry community will benefit everyone if the cycle stops.”

 

This concludes our public service announcement.

A Liability Predictor for avoiding artifacts?

False positives and artifacts are a constant source of irritation – and worse – in compound screening. We’ve written frequently about small molecule aggregation as well as generically reactive molecules that repeatedly come up as screening hits. It is possible to weed these out experimentally, but this can entail considerable effort, and for particularly difficult targets, false positives may dominate. Indeed, there may be no true hits at all, as we noted in this account of a five-year and ultimately fruitless hunt for prion protein binders.

 

A computational screen to rapidly assess small molecule hits as possible artifacts would be nice, and in fact several have been developed. Among the most popular are computational filters for pan-assay interference compounds, or PAINs. However, as Pete Kenny and others have pointed out, these were developed using data from a limited number of screens in one particular assay format. Now Alexander Tropsha and collaborators at University of North Carolina Chapel Hill and the National Center for Advancing Translational Science (NCATS) at the NIH have provided a broader resource in a new J. Med. Chem. paper.

 

The researchers experimentally screened around 5000 compounds, taken from the NCATS Pharmacologically Active Chemical Toolbox, in four different assays: a fluorescence-based thiol reactivity assay, an assay for redox activity, a firefly luciferase (FLuc) assay, and a nanoluciferase (NLux) assay. The latter two assays are commonly used in cell-based screens to measure gene transcription. The thiol reactivity assay yielded around 1000 interfering compounds, while the other three assays each produced from 97 to 142. Interestingly, there was little overlap among the problematic compounds.

 

These data were used to develop quantitative structure-interference relationship (QSIR) models. The NCATS library of nearly 64,000 compounds was virtually screened, and around 200 compounds were tested experimentally for interference in the four assays, with around half predicted to interfere and the other half predicted not to interfere. The researchers had also previously built a computational model to predict aggregation, and this – along with the four models discussed here – have been combined into a free web-based “Liability Predictor.”

 

So how well does it work? The researchers calculated the sensitivity, specificity, and balanced accuracy for each of the models and state that “they can detect around 55%-80% of interfering compounds.”

 

This sounded encouraging, so naturally I took it for a spin. Unfortunately, my mileage varied. Or, to pile on the metaphors, lots of wolves successfully passed themselves off as sheep. Iniparib was recognized correctly as a possible thiol interference compound. On the other hand, the known redox cycler toxoflavin was predicted not to be a redox cycler – with 97.12% confidence. Similarly, curcumin, which can form adducts with thiols as well as aggregate and redox cycle, was pronounced innocent. Quercetin was recognized as possibly thiol-reactive, but its known propensity to aggregate was not. Weirdly, Walrycin B, which the researchers note interferes with all the assays, got a clean bill of health. Perhaps the online tool is still being optimized.

 

At this point, perhaps the Liability Predictor is best treated as a cautionary tool: molecules that come up with a warning should be singled out for particular interrogation, but passing does not mean the molecule is innocent. Laudably, the researchers have made all the underlying data and models publicly available for others to build on, and I hope this happens. But for now, it seems that no computational tool can substitute for experimental (in)validation of hits.

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.

Quality control of the iNEXT poised fragment library

Two weeks ago we described fragment libraries built for crystallographic screening, and this week we continue the library theme. A primary challenge after validating a fragment hit is what to do next. You can look for similar compounds, either in-house or from vendors, but ultimately you’ll need to do chemistry. And this might not be trivial: you don’t want to embark on a multistep synthesis if you can avoid it. One solution is to build “poised fragments,” which contain at least one functional group amenable to easy chemistry. Characterization of such a library has just been published (open access) in J. Biomol. NMR. by Harald Schwalbe (Goethe University) and a large multinational group of collaborators.

The researchers are part of the European iNEXT (Infrastructure for NMR, EM, and X-rays for Translational research) consortium. A set of 11,677 commercial fragments was computationally analyzed, and 782 were purchased from several vendors. To efficiently cover chemical space, the researchers took a “minimum fragments and maximum diversity” approach: similarity analysis revealed 391 clusters, most with just 1-3 members. The fragments are mostly rule of three compliant, though a bit on the large side, with 80% of fragments in the 200-250 Da range and an average molecular weight of 220 Da.

Fragments were characterized by NMR and LC-MS. NMR experiments were done in both d₆-DMSO (at 50 mM) and phosphate buffer (at 1 mM). The DMSO solutions were spiked with 10% D₂O; this mixture remains liquid at 4 °C, thus avoiding freeze-thaw problems. Fragment concentrations were established by NMR using either external or internal standards.

Some 30% of fragments did not pass quality control. This probably will not come as a shock to long-time readers, though it is a bit worse than some previous studies. Just like unhappy families, fragments failed QC for a variety of reasons, including impurities, degradation, solubility, and even inconsistency with the expected structures. There were also a couple cases where two fragments were mixed together, suggesting operator error while assembling the library. NMR spectra for various types of QC failures are provided in the paper.

The researchers are honest about deficiencies in the library, noting that it contains PAINful molecules such as catechols and hydroquinones, though one wonders why these were not removed in the first place. Laudably, they provide SMILES for all 782 compounds along with an extensive set of physicochemical calculations (see here – opens as an Excel spreadsheet). Weirdly though, the researchers do not specify which molecules failed QC or which vendors they came from. At first I thought these had been weeded out of the final set, but five examples shown as failures appear in the spreadsheet. The library is sold commercially by Enamine as the DSI-poised Library, and since this library contains only 768 compounds perhaps the most egregious bad actors have been removed. Enamine was rated highly in a reader poll, so presumably the compounds have all passed QC.

So how does the fragment library perform? That – unfortunately – is not addressed in this paper, though the DSI-poised library was among those screened against the SARS-CoV-2 main protease (M^Pro). Have you used it? And if so, has the “poised” nature of the library allowed you to efficiently grow fragment hits? Hopefully these questions will be answered in the literature, if not in the comments.

Crystallographic fragment screening roundup

Last year’s poll revealed that crystallography has become the most popular technique for FBLD. Three recent papers discuss some of the whys and hows.

The first publication, published in Structure by Manfred Weiss and collaborators at Helmholtz-Zentrum Berlin, Philipps-Universität Marburg, Freie Universität Berlin, and Lund University, describes the construction of the F2X-Universal Library for crystallographic screening. Starting from 1.4 million commercially available fragments, the researchers filtered out undesirable molecules such as PAINS and then clustered the remaining compounds by similarity. Among clusters with at least 1000 members they chose one fragment from each. The resulting library, sourced from roughly a dozen vendors, contains 1103 fragments individually dissolved in DMSO at 0.5 M concentration. Outside of the Diamond Light Source, screening 1000 crystals is still a sizable effort, so the researchers have also made a 96-membered subset library called F2X-Entry.

F2X-Entry was screened against two targets, the model protein endothiapepsin (EP) and the spliceosomal protein-protein complex Aar2/RNaseH-like domain of Prp8 (AR). Crystals of each were screened with 100 mM fragments and processed with the assistance of PanDDA (described here).

The results were quite impressive: 29 hits for EP, several of which bound in more than one site, and 20 hits for AR. The overall hit rates of 30% and 21% compare favorably with a previous crystallographic screen against EP that yielded a 20% hit rate. The solvent DMSO sometimes doesn’t play well with crystals, but the researchers were able to obtain 72-75% of the original hits when soaked in the absence of solubilizing DMSO.

One bit of data I would have liked to have seen is how the physicochemical properties of the fragment hits compare to the overall library, similar to what was reported here. Both libraries can be accessed by collaborators at the BESSY II synchrotron, and the smaller library can also be accessed through an MTA, so hopefully as these are screened against more targets this information will be published.

A second paper, in ChemMedChem by Gerhard Klebe and collaborators at Philipps-Universität Marburg and AstraZeneca, also discusses crystallographic screening of a 96-compound library, one sold commercially by Jena Biosciences. The target chosen was tRNA guanine transglycosylase (TGT), an enzyme important for the pathogenicity of the sometimes lethal bacteria Shigella. Soaking each of the compounds at 100 mM yielded 8 structures, 5 of which bound in the active site.

The researchers also screened the library using orthogonal methods, SPR and ligand-detected NMR. As in a previous study from the same group, the results were “puzzling”: none of the hits were detected in all three assays. (The first author, Engi Hassaan, presented some of this work at a CHI meeting last year.) Indeed, only one of the crystallographic hits was found among the 10 hits from SPR, and a different crystallographic hit was found among the 22 NMR hits.

Several plausible reasons for the low overlap are provided, including different buffer compositions and concentrations of fragments. Solubility likely played a role: a dozen fragments could not be screened by NMR at 0.2 mM, and 15 fragments interfered with the SPR assay and thus could not be screened. Indeed, some of the fragments are PAINS, including an eyebrow-raising dinitrocatechol, so the fact that they were not observed by crystallography is perhaps unsurprising.

Finally, in a Biomolecules paper, Gerhard Klebe and collaborators screen the same library against human carbonic anhydrase II (hCAII). This resulted in 9 hits – 8 from the library and 1 from a cryoprotectant used at 2.6 M in some experiments. Among the library hits, four bound at the active site, including a couple hydrazides which make interactions with the catalytic zinc ion. Surprisingly, two fragments bind covalently to the N-terminus of  hCAII. One seems to form a formaldehyde-mediated linkage, while the other – an aminomethylheterocycle – has likely oxidized to an aromatic aldehyde that can form an imine linkage.

All of the fragment hits in this case were identified through visual inspection of the electron density maps, and in this system PanDDA was actually not helpful, revealing only three of the fragment hits. The researchers note that they used different soaking times for the different crystals, and suggest that shorter soaking times in particular may not allow time for crystals to equilibrate, as assumed in PanDDA.

So in summary, fragment screening by crystallography is becoming easier and is likely to give high hit rates, particularly when conducted at high concentrations. Also, libraries matter: it is interesting that the F2X-Entry library gave considerably higher hit rates than the second 96-compound library, albeit on different targets. Of course, a fragment hit is only the beginning of a long journey, but at least a structure provides guidance to begin optimization.

Review of 2017 reviews

The year is done, and the darkness

Falls from the wings of Night.

As we've done since 2012, Practical Fragments is using the last post of the year to highlight conferences as well as reviews not previously discussed.

Significant events included the venerable CHI FBDD meeting in San Diego, the NovAliX Biophysics conference in Strasbourg, and the first-ever fragment conference in Shanghai. We discussed a special issue of Essays in Biochemistry devoted to structure-based drug design, and Teddy came out of retirement to provide an entertaining summary of his experience putting together a book on biophysics in drug discovery - well worth reading if you're ever tempted to edit one yourself.

As in years past, several reviews were devoted to the broad topic of FBDD. Below, I’ll outline the general reviews, followed by those focusing on particular targets, techniques, and other topics.

György Keserű (Hungarian Academy of Sciences) and Mike Hann (GlaxoSmithKline) ask “what is the future for fragment-based drug discovery?” in Fut. Med. Chem. After a concise summary of the topic, they answer that it “includes target discovery and validation, the development of chemical biology probes, pharmacological tools and more importantly drug-like compounds.” In other words, the future looks bright.

FBDD is more comprehensively covered by Ben Davis and Stephen Roughley (Vernalis) in Ann. Reports Med. Chem. This is a complete, self-contained guide to the field, covering everything from history, theory, fragment library design, and fragment-to-lead approaches. It is ideal for a newcomer, but there are enough insights throughout that it makes a rewarding read for experts too.

Of the thirty-plus fragment-derived drugs that have made it to the clinic, none are directed against neglected diseases. Gustavo Henrique Goulart Trossini and colleagues at Universidade de São Paulo review some of the work that has been done in this area in Chem. Biol. Drug Des.

And rounding out general reviews, Christopher Johnson (Astex) and collaborators examined all 28 successful fragment-to-lead programs published in 2016, defined as at least a 100-fold improvement in affinity to a 2 µM or better compound. This is a sequel to our analysis of the 2015 literature, also published in J. Med. Chem., and many of the trends are similar. Interestingly, many leads maintained high ligand efficiencies, and there was no correlation between the “shapeliness” (deviation from planarity) of fragments and that of the resulting leads. Consistent with our recent poll on the importance of structural information, 25 of the 28 examples used crystallography at some point.

Targets

Three of the success stories from 2016 involved bromodomains, the subject of an entire month of Practical Fragments’ posts last year. In Arch. Pharm., Mostafa Radwan and Rabah Serya (Ain Shams University, Cairo) review this target class, with a particular emphasis on the four BET family proteins.

More than 30% of enzymes are metalloenzymes, yet these are targeted by fewer than 70 FDA-approved drugs. One of the first published examples of FBDD involved a metalloenzyme, but most efforts have been focused on a limited set of metal-binding pharmacophores, such as hydroxamic acids. Seth Cohen (University of California, San Diego) has been steadily building libraries of metallophilic fragments, and in Acc. Chem. Res. he describes how this approach can lead to new classes of inhibitors.

Protein-protein interaction inhibitors are another underrepresented class of drugs, though one approved FBDD-derived molecule falls into this category. In Methods, Daisuke Kihara and collaborators at Purdue University look at in silico methods to discover PPI inhibitors, including fragment-based approaches.

Unlike PPIs, kinases have been highly successful drug targets. We recently highlighted one review of cyclin-dependent kinases (CDKs), and in Eur. J. Med. Chem. Marco Tutone and Anna Maria Almerico (Università di Palermo) provide another. Although the main focus is on in silico methods, there is a section on FBDD.

Techniques

As noted above, X-ray crystallography has played a role in most successful fragment to lead programs. In the open-access journal IUCrJ, Sir Tom Blundell (University of Cambridge) provides an engaging and personal view of protein crystallography, a field in which he has played a starring role, starting with his early involvement in determining the crystal structure of insulin. He also notes that the interchange of ideas and techniques between academia and industry has long been a crucial driver of advances.

NMR was the first practical method used for FBDD, so it is not surprising that there are several reviews on the topic. In Arch. Biochem. Biophys., Michael Reily and colleagues at Bristol-Myers Squibb provide a detailed overview of NMR in drug design. This covers not just the ligand- and protein-detection methods often used in fragment screening, but also more intensive techniques to characterize protein-ligand interactions.

A briefer look at many of these topics is provided by Yan Li and Congbao Kang (A*STAR) in Molecules. This review also highlights more unusual approaches such as NMR experiments on living cells.

Artifacts are a fact of life in both FBDD and HTS, and it is always important to recognize these early. In J. Med. Chem. Anamarija Zega (University of Ljubljana) discusses how NMR can help. This includes methods to detect aggregators and covalent modifiers. Of course, NMR methods can introduce their own artifacts, and these are also covered.

Other topics

Speaking of artifacts, PAINS are responsible for quite a few. The term “PAINS” has also been somewhat controversial, and in a new paper in ACS Chem. Biol. Jonathan Baell (Monash University) and J. Willem Nissink (AstraZeneca) examine the “utility and limitations” of the term Jonathan coined seven years ago. As they acknowledge, the PAINS filters were derived from just 100,000 compounds run in a limited set of assays. This means that not every bad actor will be recognized by PAINS filters, and some compounds that are may only be PAINful in certain assay formats. Like Lipinski’s rule of 5, it is important to recognize the limits of applicability. As the authors note, “the key is to remain evidence-based.”

Another sometimes controversial topic is ligand efficiency and associated metrics, the subject of an analysis in Expert Opin. Drug Disc. by Giovanni Lentini and collaborators at the University of Bari Aldo Moro. This includes extensive tables of rules and metrics, both common and obscure. The authors note that, while metrics can be useful, it is important not to use them as a “magic box.” As they quote William Blake, “to generalize is to be an idiot.”

Shawn Johnstone and Jeffrey Albert (IntelliSyn Pharma) discuss pharmacological property optimization for allosteric ligands in a review in Bioorg. Med. Chem. Lett. As we recently noted, fragments are particularly suited for discovering allosteric sites, and this paper discusses how to characterize these.

Finally, Jörg Rademann and collaborators at Freie Universität Berlin discuss protein-templated fragment ligations in Angew. Chem. Int. Ed. Earlier this year we highlighted some of his work, and this review provides a thorough analysis of both reversible and irreversible approaches, with good discussions of detection methods, chemistries, and case studies.

That’s it for the year. Thanks for reading, and especially for commenting.

And may 2018 be filled with music, and light.

Twelfth Annual Fragment-based Drug Discovery Meeting

CHI’s Drug Discovery Chemistry meeting took place over four days last week in San Diego. This was easily the largest one yet, with eight tracks, two one-day symposia, and nearly 700 attendees; the fragment track alone had around 140 registrants. On the plus side, there was always at least one talk of interest at any time. On the minus side, there were often two or more going simultaneously, necessitating tough choices. As in previous years I won’t attempt to be comprehensive but will instead cover some broad themes in the order they might be encountered in a drug discovery program.

You need good chemical matter to start a fragment screen, and there were several nice talks on library design. Jonathan Baell (Monash University) gave a plenary keynote on the always entertaining topic of PAINS. Although there are some 480 PAINS subtypes, 16 of these accounted for 58% of the hits in the original paper, suggesting that these are the ones to particularly avoid. But it is always important to be evidenced-based: some of the rarer PAINS filters may tag innocent compounds, while other bad actors won’t be picked up. As Jonathan wrote at the top of several slides, “don’t turn your brain off.”

Ashley Adams described the reconstruction of AbbVie's fragment libraries. AbbVie was early to the field, and Ashley described how they incorporated lessons learned over the past two decades. This included adding more compounds with mid-range Fsp³ values, which, perhaps surprisingly, seemed to give more potent compounds. A 1000-member library of very small (MW < 200) compounds was also constructed for more sensitive but lower throughput biophysical screens. One interesting design factor was to consider whether fragments had potential sites for selective C-H activation to facilitate fragment-to-lead chemistry.

Tim Schuhmann (Novartis) described an even more “three-dimensional” library based on natural products and fragments. Thus far the library is just 330 compounds and has produced a very low hit rate – just 12 hits across 9 targets – but even a single good hit can be enough to start a program.

Many talks focused on fragment-finding methods, old and new. We’ve written previously about the increasingly popular technique of microscale thermophoresis (MST), and Tom Mander (Domainex) described a success story on the lysine methyltransferase G9a. When pressed, however, he said it did not work as well on other targets, and several attendees said they had success in only a quarter to a third of targets. MST appears to be very sensitive to protein quality and post-translational modifications, but it can rapidly weed out aggregators. (On the subject of aggregators, Jon Blevitt (Janssen) described a molecule that formed aggregates even in the presence of 0.01% Triton X-100.)

Another controversial fragment-finding technique is the thermal shift assay, but Mary Harner gave a robust defense of the method and said that it is routinely used at BMS. She has seen a good correlation between thermal shift and biochemical assays, and indeed sometimes outliers were traced to problems with the biochemical assay. The method was even used in a mechanistic study to characterize a compound that could bind to a protein in the presence of substrate but not in the presence of a substrate analog found in a disease state. Compounds that stabilized a protein could often be crystallized, while destabilizers usually could not, and in one project several strongly destabilizing compounds turned out to be contaminated with zinc.

Crystallography continues to advance, due in part to improvements in automation described by Anthony Bradley (Diamond Light Source and the University of Oxford): their high-throughput crystallography platform has generated about 1000 fragment hits on more than 30 targets. Very high concentrations of fragments are useful; Diamond routinely uses 500 mM with up to 50% DMSO, though this obviously requires robust crystals.

Among newer methods, Chris Parker (Scripps) discussed fragment screening in cells, while Joshua Wand (U. Penn) described nanoscale encapsulated proteins, in which single protein molecules could be captured in reverse micelles, thereby increasing the sensitivity in NMR assays and allowing normally aggregation-prone proteins to be studied. And Jaime Arenas (Nanotech Biomachines) described a graphene-based electronic sensor to detect ligand interactions with unlabeled GPCRs in native cell membranes. Unlike SPR the technique is mass-independent, and although current throughput is low, it will be fun to watch this develop.

We recently discussed the impracticality of using enthalpy measurements in drug discovery, and this was driven home by Ying Wang (AbbVie). Isothermal titration calorimetry (ITC) measurements suggested low micromolar binding affinity for a mixture of four diastereomers that, when tested in a displacement (TR-FRET) assay, showed low nanomolar activity. Once the mixture was resolved into pure compounds the values agreed, highlighting how sensitive ITC is to sample purity.

If thermodynamics is proving to be less useful for lead optimization, kinetics appears to be more so. Pelin Ayaz (D.E. Shaw) described two Bayer CDK kinase inhibitors having either a bromine or trifluoromethyl substitution. They had similar biochemical affinities and the bromine-containing molecule had better pharmacokinetics, yet the trifluoromethyl-containing molecule performed better in xenograft studies. This was ultimately traced to a slower off-rate for the triflouromethyl-substituted compound.

The conference was not lacking for success stories, including MetAP2 and MKK3 (both described by Derek Cole, Takeda), LigA (Dominic Tisi, Astex), RNA-dependent RNA polymerase from influenza (Seth Cohen, UCSD), and KDM4C (Magdalena Korczynska, UCSF). Several new disclosures will be covered at Practical Fragments once they are published.

But these successes should not breed complacency: at a round table chaired by Rod Hubbard (Vernalis and University of York) the topic turned to remaining challenges (or opportunities). Chief among these was advancing fragments in the absence of structure. Multiprotein complexes came up, as did costs in terms of time and resources that can be required even for conventional targets. Results from different screening methods often conflict, and choosing the best fragments both in a library and among hits is not always obvious. Finally, chemically modifying fragments can be surprisingly difficult, despite their small size.

I could go on much longer but in the interest of space I’ll stop here. Please add your thoughts, and mark your calendars for next year, when DDC returns to San Diego from April 2-6!

Cussed curcumin

Teddy’s retirement from the blog has cut down on the number of PAINS-shaming posts, and truth be told there are so many candidate papers that they could easily swamp fragments, which I suspect would drive away most of the readership. That said, I did want to highlight an exhaustive Perspective about a particularly diabolical natural product just published today in J. Med. Chem. by Mike Walters and collaborators at the University of Minnesota, Brigham and Women’s Hospital, and the University of Illinois (and also covered in a news story in Nature.)

We’ve previously discussed some of the types of artifacts that can plague small molecule screens: aggregation, covalent adducts, redox cycling, fluorescence, photoreactivity, and more. Curcumin is a jack of all trades in that it is capable of all of the above. It’s also unstable even at neutral pH, and can decompose into other reactive species. It is the quintessential chemical con artist: if you have an assay, curcumin will probably be active in it.

The new paper is a thorough investigation (18 pages, with 164 references) of the chemistry and biology of curcumin, covering in gruesome detail all the many ways it can deceive. After discussing the history and physicochemical properties (and liabilities), several literature case studies where curcumin is proposed as having biological activity are explored and thoroughly demolished; one of these has been retracted but continues to be cited uncritically years later.

One might expect that something which hits so many assays would be toxic. This turns out not be the case: curcumin is present at 1-6% in tasty turmeric and only seems to show any adverse events at very high doses – several grams per day. The reason, the researchers show, is that curcumin’s pharmacokinetics are lousy, with oral bioavailability of less than 1%. This is a very literal example of the cliché “garbage in, garbage out.”

Sadly, these properties have not dampened interest in testing curcumin in people. The researchers identify 135 registered clinical trials, only eight of which have reported study results, with 49 either recruiting or not yet recruiting. The few examples where results have been reported are not particularly encouraging.

Typing curcumin into PubMed pulls up close to 10,000 papers, with more than 150 published in J. Med. Chem. alone. Will this devastating exposé help? For honest and diligent researchers, it should serve as a flashing warning to be extremely careful with any data gathered using curcumin. Unfortunately, some in the scientific community may not care as long as they are able to pump out papers. Indeed, according to Wikipedia, at least one prominent curcumin researcher had to retract several papers because of questionable “data integrity”. And there may be still darker motives: type curcumin into Google and the top results are ads touting the stuff. There’s money to be made, and even more if you slap on some scientific lipstick.

And despite specific J. Med. Chem. author guidelines to be cautious about “interference compounds” and “provide firm experimental evidence in at least two different assays that reported compounds with potential PAINS liability are specifically active and their apparent activity is not an artifact”, the journal recently published a paper fully devoted to the synthesis and SIR of rhodamine derivatives, with no consideration of mechanism nor mention that they can be problematic. (Indeed, the researchers do not even bother to include detergent in their enzymatic assay!)

All of which is to say that it’s easy to publish crap. But hopefully now, more people will recognize it as such.

An interview with Dr. Saysno

Readers of a certain age may fondly remember the interviews with Dr. Noitall that used to enliven the pages of Science. Sadly, he died a few years back. But his cousin, Dr. Saysno, is still very much alive. Practical Fragments caught up with him at a recent conference in Shutka.

Practical Fragments (PF): Dr. Saysno, you've stated that experts should never be trusted.

Dr. Saysno (DS): Niels Bohr defined an expert as a person who has made all the mistakes that can be made in a very narrow field. If someone has made every possible mistake, how could you possibly trust them?

PF: But don't you think they may have learned from their mistakes?

DS: Balderdash! Hegel was right: the only thing we learn from history is that we learn nothing from history.

PF: What's your opinion of ligand efficiency (LE)?

DS: Ligand efficiency is an abomination! It's mathematically invalid!

Worse, determining the free energy of binding from a dissociation constant is not even wrong: if you change your definition of standard state, you can make ΔG° any positive or negative number you want. Just how relevant do you think your definition of standard state is on the surface of Venus? Or Pluto?

For the same reason, pH is utterly meaningless. You really ought to throw out your pH paper, not to mention your pH meters, since they all assume an arbitrary reference state.

PF: But what about all the researchers who find pH and LE useful?

DS: Usefulness is the last refuge of the scoundrel!

Look, many of the best selling drugs on the market are antibodies, and when you calculate their ligand efficiencies, they are close to zero. How can you have a metric that doesn't work on some of the most important drugs out there?

I only believe in equations that are universal and apply in all situations, unsullied by the physical world. Anything that involves standard states is just mumbo-jumbo.

PF: What do you think of pan-assay interference compounds, or PAINS?

DS: Now that’s a topic that really gets my blood boiling! PAINS were defined on the basis of just six assays. Six assays I tell you!!! [DS vigorously pounds his shoe on the desk.] Just because something hits six assays – or six hundred for that matter – doesn’t mean it will hit the six hundred and first!

PF: But aren't there some chemical substructures that are so generically reactive they should never be used in probes?

DS: Nothing is universal! All molecules are unique, like little snowflakes. If a compound comes up as a hit in your assay, by all means publish it as a chemical probe in the best possible journal, and try to encourage suppliers to start selling it so other people can use and cite your brilliant discovery.

No one has a right to criticize your molecule unless they test it against every single protein in the human body and show that it hits all of them.

When the revolution comes, the imperialist PAINS stooges will be swept into the dustbin of history along with the lackeys of ligand efficiency!

PF: So if you don't trust experts, you don't like metrics, and you can't make generalizations, how can we move forward in science short of deriving every result ourselves from first principles?

DS: That's simple: just ask me!

Fragments vs Trypanosoma cruzi spermidine synthase, allosterically

Chagas disease, caused by Trypanosoma cruzi, is spread throughout Central and South America by a nasty blood-sucking insect. A couple drugs are approved to treat it, but they can cause severe nausea and peripheral neuropathy, so there is room for improvement. In a recent paper in Acta Cryst D., Yasushi Amano and colleagues at Astellas Pharma describe their efforts against the T. cruzi spermidine synthase (TcSpdSyn).

TcSpdSyn transfers an aminopropyl moiety from the cofactor decarboxylated S-adenosylmethionine (dcSAM) to the evocatively-named putrescine (1,4-diaminobutane) as one step in the synthesis of an essential antioxidant. Small amines can bind in the putrescine-binding pocket and inhibit the enzyme with low micomolar activity, so the researchers decided to find other fragments that could bind in this pocket. They screened in the presence of dcSAM, using surface-plasmon resonance (SPR), with each fragment present at 0.25 mM, as well as in thermal shift assays, with each fragment present at 2 mM. Although nothing is reported about library size or hit rate, hits from either assay were taken into crystallography, resulting in six structures described in detail and deposited in the Protein Data Bank (pdb).

Two fragments were found that bind in the putrescine-binding pocket, and in both cases the enzyme shows some conformational changes to accommodate the fragments. Although these two fragments have only modest potency (IC₅₀ = 0.18-0.48 mM), they do have satisfying ligand efficiencies, and are good starting points for structure-based design.

Unexpectedly, the other four fragments bound not in the putrescine-binding pocket but at an interface between two proteins of TcSpdSyn, which forms a homodimer. One of these fragments, an isothiazolinone, showed mid-nanomolar activity in a functional assay. Readers may recall a paper we pilloried earlier this year which also reported an isothiazolinone as a screening hit. In that case, the researchers failed to recognize that this PAINS compound has ample precedent for reacting with thiols. Happily, in the current paper the researchers are not only aware of this, they actually see covalent bond formation between the fragment and a cysteine residue in the crystal structure. Interestingly though, the fragment reacts with only a single cysteine residue at the dimer interface, despite the presence of six other cysteine residues in the protein.

The researchers carefully analyzed this structure and found that binding of the fragment disrupts the putrescine-binding pocket; in other words, the fragment is an allosteric inhibitor. Moreover, the other three fragments that bind at the dimer interface also appear to act allosterically, and one of them is a single digit micromolar inhibitor.

This is a nice example of how even PAINS compounds can be useful if they are well-characterized and not hyped. Moreover, the structures suggest new approaches for tackling a target for a neglected tropical disease, either covalently or more conventionally.

Introducing the Chemical Probes Portal

Chemical probes can be incredibly powerful reagents for understanding biology. A potent, selective, and cell-active modulator of a specific protein can be invaluable for figuring out what that protein actually does. Fragment-based methods can be effective at identifying these tool compounds, as we've described here and here.

Unfortunately, good chemical probes are difficult to discover, and scientists are left struggling with suboptimal reagents that hit multiple targets, often through pathological mechanisms. This leads to "pollution of the scientific literature," in Jonathan Baell's memorable phrasing. Despite our occasional PAINS Shaming, high-profile articles in C&EN and Nature, and even a dedicated blog, the problem continues. What is to be done?

Yesterday, a team of 53 authors from 46 academic and industrial organizations published a Commentary in Nature Chemical Biology entitled "The promise and peril of chemical probes" (see here for excellent coverage in Nature, here for Science's take, and here for In the Pipeline). This provides a good working definition for a chemical probe. According to the Structural Genomics Consortium, a chemical probe for epigenetics targets must have:

• Potency < 100 nM against the desired target
• >30-fold selectivity vs related targets
• On-target cell activity < 1 µM

It should also be profiled against a larger panel of potential off-targets, and a related inactive compound (such as a stereoisomer) should be available as a control.

After discussing examples of high-quality probes, the researchers turn their attention to what they term – rather charitably – "probes of lesser value:"

The continued use of these probes poses a major problem: tens of thousands of publications each year use them to generate research of suspect conclusions, at great cost to the taxpayer and other funders, to scientific careers and to the reliability of the scientific literature.

The authors then go on to describe best-practices. For example, even high-quality probes can give spurious results when used at high concentrations. As Paracelsus recognized five centuries ago, the dose makes the poison.

All of this is important, but as the authors acknowledge, it's been said before. What really differentiates the Commentary is the simultaneous launch of a companion web site, the Chemical Probes Portal. Its creators hope that this will lead to vigorous community discussion around questions such as:

Is there a probe for my target protein?

Which ones should I use?

How should I use this probe properly?

Is this probe suitable for use in animal models?

Currently the Portal lists just seven probes with links to references and descriptions of selectivity, solubility, and the like. All of these are “good probes,” but hopefully this will expand: the paper itself discusses the shortcomings of molecules such as staurosporine, chaetocin, obatoclax, and gossypol, and including them in the portal with detailed warnings would be valuable for the scientific community.

I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments. Practical Fragments will check back in a year or so to see how the site is doing. In the meantime, probe cautiously!

Sloppy science

As regular readers may have discerned, I’m favorably disposed to most of the papers I highlight. They may have flaws or inconsistencies, but, with rare exceptions, I generally just ignore particularly problematic publications. Last year Teddy introduced the term PAINS-shaming to draw attention to – how shall we phrase it? – less salubrious specimens. Building on this alliterative theme, today’s post is about sloppy science. A fundamental tenant of sound science is to consider alternate explanations for results. Ignore this at your peril.

An example was published in J. Cancer Prev. The researchers were interested in a mutant of isocitrate dehydrogenase 1 (IDH1), a hot cancer metabolism target. They screened 500 fragments in a functional spectrophotometric assay, with each fragment present at the very low concentration of 5-10 µM. One of these inhibited the mutant protein by 80% – pretty impressive for a fragment. Until you look at the structure: 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one (shown below).

Fifty years ago, researchers showed that this chemical class (isothiazolinones, also called isothiazolones) could react with thiols, like this:

Isothiazolinones have been categorized as PAINS, though they do not show up in the original computational filters. However, Pete Kenny has (repeatedly) stated that having a dubious structure should not automatically disqualify a compound from further investigation, so what else do we know about isothiazolinones?

Well, there’s this paper, which concludes a discussion of isothiazolinones by stating:

We could not develop these into useful compounds and ultimately the structure–activity relationship (SAR) was uninterpretable. Most insidiously, there were encouraging aspects of sharp SAR as there always are with these PAINS, but this is eventually overwhelmed by flat and nonsensical SAR. Unpredictable nonspecific cytotoxicity was manifest. We found our compounds to be rapidly reactive with thiols under assay conditions.

Of course, one could argue that this is anecdotal. But then there’s this paper, with the unambigious title “Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCA”. The first line of the abstract states:

Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides.

The researchers go on to demonstrate this using both small molecules and proteins, and some of the compounds they investigate are structurally quite similar to the hit here.

So in all likelihood the fragment described in the most recent paper reacts with one or more cysteine residues in IDH1, of which there are several. It is notable that the researchers conducted their assay in the absence of added thiol reducing agents, so modification of the cysteines would effectively be irreversible under their assay conditions.

What we have here is the re-identification of a known thiol-reactive molecule without any acknowledgement or apparent awareness that the molecule is reactive. I have no problem with covalent inhibitors, but I do have a problem with a generically reactive molecule being touted “for a future lead development”, as the researchers state in the abstract. It took me just minutes to track down the references above, and the fact that neither the researchers nor the reviewers did so is inexcusable.

Granted, this paper is not published in a high profile journal, and the easiest response would be to ignore it. It is certainly not the only one of its kind. Doing so, however, implicitly endorses sloppy science. This paper will undoubtedly pad the resumes of the authors. Highlighting its problems will hopefully make others wary of wasting time with this new "selective inhibitor."