06 July 2020

Fragment events in 2020 and 2021

Remember conferences?

Those things to which you  might travel halfway around the world to present and hear about the latest research, catch up with old friends, meet new ones, and participate in the global scientific enterprise?

Crammed poster sessions where social distancing was measured in centimeters or inches, not meters
or feet?

Breakfast buffets where you could grab coffee and pastries before joining an early round-table discussion?

Late-night discussions where lack of sleep and perhaps an excess of ethanol made you wonder, "huh, what if...?"

How many ideas have originated, and collaborations initiated, at conferences?

As one small example, Practical Fragments was conceived at a CHI meeting way back in 2008.

Among the many casualties of COVID-19 have been in-person conferences, as memorialized in the list of cancellations here.

But some meetings are still planned, with the hope that conditions will improve by the time the events are held. And others are moving online. Here is the current list of particular interest to readers.

August 25-26: CHI’s Fifteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held VIRTUALLY. This is part of the larger Drug Discovery Chemistry meeting, running August 25-28. You can read impressions of the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting herehere, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

September 16-18: CHI’s Eighteenth Annual Discovery on Target will also be VIRTUAL. As the name implies this event is more target-focused than chemistry-focused, but there is an entire section on FBDD. You can read impressions of the 2019 event here and the 2018 event here.

Update as of 7 July: Pacifichem postponed till 2021 - see below

March 10-12, 2021:  Postponed from this year and not exclusively fragment-focused, the Eighth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and returns to Boston next year. You can read my impressions of the 2018 event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event herehere, and here.

March 29 - April 2: CHI’s Sixteenth Annual Fragment-Based Drug Discovery will (hopefully) be held live in San Diego. You can read impressions of the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting herehere, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

December 16-21: The second Pacifichem Symposium devoted to fragments has been rescheduled and will (hopefully!) take place in Honolulu. Nearly 40 speakers and poster presenters submitted abstracts, and a new call for abstracts will go out in January 2021 so there could be even more. Pacifichem conferences are only held every 5 years and are designed to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, Korea, New Zealand, and the US. Here are my impressions of the 2015 event.

Know of anything else? Add it to the comments or let us know.

29 June 2020

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 (MPro). 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.

22 June 2020

Fragments vs Mycobacterium tuberculosis InhA

Though this could change in a bad way, tuberculosis is currently the deadliest infectious disease worldwide, causing nearly 1.7 million deaths per year. Multidrug-resistant and extensively drug-resistant strains are widespread. Some approved drugs work by blocking the mycolic acid pathway essential for mycobacterial envelope formation. One member of the pathway, the enzyme InhA, is the target of isoniazid and ethionamide. Both of these molecules are prodrugs, and a major mechanism of resistance shuts down their bioactivation. To sidestep this problem, Mohamad Sabbah, Chris Abell, and collaborators at University of Cambridge and Comenius University in Bratislava have targeted InhA directly, as they describe in a recent open-access J. Med. Chem. paper. (See here for a previous FBLD effort against this target.)

The researchers began with a differential scanning fluorimetry (DSF) screen of 800 fragments, each at 5 mM. Forty-two fragments stabilized InhA by at least 3 °C and were tested at 1 mM in three ligand-based NMR assays: CPMG, WaterLOGSY, and STD. All 18 fragments that hit in at least two of these confirmatory assays were soaked into InhA crystals at 20 mM, yielding 5 hits.

None of the fragments inhibited enzymatic activity at 2 mM, but compound 1 was chosen for optimization based on an attractive growth vector into a hydrophobic region of the binding pocket. The carboxylic acid was replaced with an isosteric sulfonamide to yield compound 6, which has measurable activity. Various substituents were tested around the new phenyl ring, with a significant boost in activity caused by an aminomethyl moiety. Cyclizing the molecule and further medicinal chemistry ultimately led to compound 23, with high nanomolar activity. A crystal structure revealed that compound 23 bound as expected and that the primary amine was making interactions with the enzyme cofactor as well as an ordered water molecule.

Unfortunately, although compound 23 slightly inhibited the growth of M. tuberculosis, it did not inhibit synthesis of mycolic acids, suggesting that activity was through a different mechanism. The researchers suggest that the molecules may not be sufficiently cell permeable or that they are effluxed or metabolized. However, it may be that they just aren’t potent enough. Perhaps further medical chemistry will improve affinity by another couple orders of magnitude and achieve pathway inhibition. Regardless, this is a nice example of a robust biophysical assay cascade followed by fragment growing and structure-based design.

15 June 2020

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.

08 June 2020

Deconstructing an HTS hit for GyrB inhibitors

COVID-19 is deservedly engaging most of our collective mindspace when it comes to infectious diseases. Unfortunately, plenty other threats are out there, including antibiotic-resistant bacteria. A paper recently published in ACS Omega by Fumihito Ushiyama and colleagues at Taisho reports progress in this area.

The researchers were specifically interested in the protein DNA Gyrase B (GyrB), which is essential for bacterial replication (see here for previous work on the same target). A high-throughput screen against the E. coli protein led to a few dozen hits that were validated using a variety of biophysical methods including SPR, isothermal titration calorimetry (ITC), and crystallography. Compound 1 binds in the ATP-binding site, which is also where the natural product inhibitor novobiocin binds. The latter molecule makes an interaction with an arginine residue in the protein, but introducing a carboxylic acid moiety onto compound 1 to make a similar interaction was not successful (compound 8e).

Taking a step back, the researchers stripped compound 1 down to the core fragment 2a, which makes both polar and hydrophobic interactions with GyrB. Unfortunately, this fragment was too weak to show any affinity by ITC, as were 120 related fragments.

Looking closer at the structure of compound 1 bound to the protein revealed a small unfilled hydrophobic pocket near the 2-quinolinone fragment. Making appropriately substituted fragments was “relatively complicated,” and most of them were inactive. However, compound 2d showed binding by ITC as well as excellent ligand efficiency. Growing from this fragment ultimately led to compound 13e, with low nanomolar affinity. In addition to binding, compound 13e is a potent inhibitor of GyrB and is selective against a panel of 96 human kinases. Unfortunately though, it displays only modest antibacterial activity, likely due to efflux.

Nonetheless, this is a nice example of thoughtful structure-based design. In particular, the dramatic boost in potency gained by filling a small pocket (nearly 400-fold from compound 8e to 13e) validates the willingness to explore difficult chemistry rather than sticking with available analogs. The paper ends by noting that optimization is continuing, and I wish them well. By my count only a single fragment-derived antibacterial agent has entered clinical development, and that program is no longer active. We could use more.

01 June 2020

BETting on fast follower fragments

A common approach in drug discovery is to improve a previously reported molecule. An example of such a “fast follower” approach has just been published in J. Med. Chem. by Cheng Luo, Bing Zhou, and colleagues at Shanghai Institute of Materia Medica.

The researchers were specifically interested in bromodomains, which recognize acetylated lysine residues in proteins and play major roles in gene expression. In 2018 we described AbbVie’s fragment-based discovery of ABBV-075, which had entered phase 1 clinical trials. Although reasonably selective for BET-family bromodomains, it also strongly inhibits EP300, which could lead to toxicity. Thus, more selective molecules have been sought.

The new paper starts with a thermal shift assay of 1000 fragments against the two separate bromodomains of BRD4, BD1 and BD2. Hits were validated using an AlphaScreen assay. Compound 47 was found to be active against both BD1 and BD2, with high ligand efficiency (all IC50 values shown are for BD1; values for BD2 are similar). Modeling suggested this fragment could be merged with ABBV-075, and indeed the resulting compound 26 was quite potent. (Note: structures of compounds 26 and 38 were originally drawn incorrectly - now fixed.)

Compound 26 was metabolically unstable, but further optimization, aided by crystallography and modeling, ultimately led to compound 38. This molecule has good oral bioavailability in mice and promising pharmacokinetics in both mice and rats. It inhibits the expression of cancer-driving genes such as c-Myc and BCL-2, inhibits the growth of several cancer cell lines, and demonstrated good tumor growth inhibition in a mouse xenograft study. Compound 26 does not inhibit five cytochrome P450 enzymes or hERG. Finally, it is much more selective than ABBV-075 against EP300 and indeed most other bromodomains aside from BET family members. The researchers conclude that “compound 38 is a highly promising preclinical candidate.”

Unfortunately, selectivity for BET-family bromodomains may not be sufficient to avoid toxicity. Indeed, as we described earlier this year, AbbVie has dropped clinical development of ABBV-075 in favor of ABBV-744, which is selective for BD2 over BD1. Whether or not the same could be done for this series, the paper is still another nice example of appending a fragment onto a previously discovered molecule.

25 May 2020

Machine learning for two-dimensional NMR

Among the many methods to find fragments, only two – X-ray crystallography and protein-observed NMR – can routinely provide detailed structural information. Indeed, the first SAR by NMR paper arguably launched the field of fragment-based lead discovery nearly a quarter century ago. However, whereas crystallography has steadily increased in popularity, protein-observed NMR has lagged. A new paper in Comp. Struct. Biotech. J. by Grzegorz Popowicz and collaborators at Helmholtz Zentrum München, Technical University of Munich, and the ETH seeks to change this.

Two-dimensional NMR techniques, such as 1H-15N HSQC, produce two-dimensional plots with the chemical shift of the proton on one axis and the chemical shift of the nitrogen on the other. Different amide groups in a protein have different chemical shifts, and these can change in position or intensity when a ligand binds. Ideally these chemical shift perturbations (CSPs) can be used to tell exactly where on the protein a ligand binds, but even unassigned perturbations can give qualitative information on whether or not the protein is interacting with a fragment.

Unfortunately, analyzing hundreds of two-dimensional spectra is a tedious manual process; think of spending several hours playing Where’s Wally with blobs instead of people. And with only two colors. Thus, the process is subject to error and human bias. To make life easier for NMR spectroscopists, and to make analysis more objective, the researchers developed an automated software package called the CSP Analyzer.

The process started with 1611 spectra taken from fragment screens against four different proteins, of which 176 had a bound ligand. From the total, a training set was assembled of 32 actives along with 68 inactive or noisy spectra. These training spectra were fed into a machine learning algorithm similar to those used for computer image processing. Building the model required quite a bit of tweaking; because inactives outnumbered actives, a simple algorithm would do better by returning more false negatives than false positives. However, when looking for a fragment needle in a haystack of spectra, you really don’t want to miss anything useful, and the researchers used strategies to minimize this problem. In the end CSP Analyzer performed quite well, with an accuracy of 87% across the entire data set. Importantly, while it returned 10.3% spectra as false positives, it only missed 3.1% of spectra as false negatives.

Teddy would often end his posts by asking whether a new technique was practical. I’m no NMR spectroscopist, so I’ll leave it to readers to weigh in with their opinions. Happily, the software is freely available here, so you can download and try it yourself. Moreover, the researchers have ambitious future plans, such as extending CSP Analyzer to other types of NMR experiments and inputs. The rise of the machines continues, in a benevolent fashion. At least thus far.

18 May 2020

Merging two of the same fragments for FABP4

The fatty acid binding proteins (FABPs) are a family of 10 proteins that – as their name suggests – shuttle fatty acids around cells. FABP4 has been implicated in a host of diseases, from atherosclerosis to nonalcoholic steatohepatitis. A recent paper in J. Med. Chem. by Yechun Xu and collaborators mostly at Shanghai Institute of Materia Medica describes how a fragment led to a compound with in vivo efficacy. It is a lesson in both recognizing and capitalizing on the fact that fragments often have multiple binding modes.

The researchers screened just 500 fragments, each at 1 mM, looking for displacement of a fluorescent ligand. Two hits were identified, of which compound 1 was by far the most potent. The researchers characterized the binding mode using crystallography, which itself was challenging because the protein co-purified with bound fatty acids. They had to denature the protein, strip fatty acids, and then refold it to obtain the apo form. When they were finally able to determine the crystal structure, they were surprised to find that compound 1 adopted three different binding modes under two different conditions (pH 6.5 and 7.5). These experimental results were supported by molecular dynamics calculations.

It is not uncommon for fragments to assume different binding modes. Indeed, the 7-azaindole fragment that led to vemurafenib, pexidartinib, and other clinical compounds has been found to bind in multiple orientations. In this case, the researchers recognized that the three binding modes put the two phenyl rings in three positions, suggesting that grafting a third phenyl ring onto compound 1 could improve affinity. This proved successful, and the resulting compound 3 had an affinity more than two orders of magnitude better as assessed both in the displacement assay and by isothermal titration calorimetry. Crystallography revealed that the molecule bound as expected.

Further structure-based design ultimately led to compound 17, with low nanomolar affinity. This molecule is also active in a cellular assay and has surprisingly good pharmacokinetic properties in mice. Given these encouraging results, the researchers tested whether the molecule could protect mice from multiorgan damage promoted by inflammatory lipopolysaccharides. The results were positive.

Unfortunately, compound 17 does show low micromolar activity against FABP3, whose inhibition would likely cause cardiac toxicity. Still, this is a nice example of fragment “self-merging”. Although merging two different fragments is common, merging a fragment onto itself is relatively rare, and – as shown here – not necessarily easy. It is an approach worth keeping in mind the next time you encounter a fragment with multiple binding orientations.

11 May 2020

Broadening the scope of 19F NMR

Over the past decade, fluorine NMR has established itself as a powerful fragment-finding method due to the advantages Teddy laid out in his classic “fluorine fetish” post. One feature of 19F NMR is that the chemical shifts of organofluorine molecules span a very wide range, in theory allowing large mixtures to be screened. However, existing NMR methods do not work across such large spectral windows, thereby requiring multiple experiments to screen an entire library. This limitation has now been overcome as described in a paper just published in Angew. Chem. by Andreas Lingel, Andreas Frank, and collaborators at Novartis and Karlsruhe Institute of Technology.

The researchers developed an experiment based on “broadband universal rotation by optimized pulses” (BURBOP). I confess that the details evade me (though they are all there in the supporting information if you wish to try it at home), but the upshot is a type of CPMG experiment in which fluorine-containing fragments bound to a protein show decreased peak intensities. Crucially, a single experiment can cover the full frequency range of pharmacologically relevant fluorine-containing molecules, spanning about 210 ppm. Previously, this required four two separate experiments.

Such increased throughput led the researchers to revamp their library, increasing the size from 1600 to 4000 fragments in an augmented library dubbed LEF4000. The paper has a nice, broadly applicable description of their curation process. Candidate members were brought in from both commercial and in-house sources and chosen to complement existing library members in terms of diversity. A modified rule of three was applied, with trifluoromethyl-containing fragments allowed to go up to 350 Da.

An in-house analysis of 25,000 fragments revealed that only about half of those with a clogD7.4 greater than 3 were soluble above 0.5 mM, so this was applied as an upper limit. Fragment solubilities were experimentally measured, and only compounds with solubilities above 0.2 mM were kept. (Although fluorine NMR is often done at low concentrations, complementary biophysical experiments are not.) Additional quality control measures included NMR and LC-MS purity assessments and removal of compounds that formed soluble aggregates as assessed by CPMG. Ultimately, 3969 of 5600 candidate molecules passed the gauntlet, and were combined in 131 mixtures of about 30 compounds each.

Having built their library, the researchers screened it against the antibacterial target CoaD, which is involved in coenzyme A synthesis. The screen took just two days, and automated hit identification took only a few hours on a standard laptop. The overall hit rate was ~6%, and some of the hits were confirmed using two-dimensional protein-observed NMR methods, revealing that they bind in the enzyme active site with affinities in the mid micromolar to low millimolar range.

Pushing the technique further, the researchers built a “Supermixture” of 152 compounds, including five of the hits spanning a wide range of chemical shifts, from -50 to -220 ppm. Even under these conditions the binders were readily identifiable, and the paper states that libraries exceeding 20,000 fragments could in principle be screened in a few days.

In 2009 I wondered why 19F NMR was not used more widely. How things change! At Novartis the LEF4000 library has been screened against “a wide variety of disease-related targets” and identified “tractable hits for each of the screened targets, among them many considered undruggable by small molecules such as transcription factors, a cytokine, a nuclear receptor, and a repeat RNA.” Practical Fragments looks forward to seeing some of these appear in the growing list of FBDD-derived clinical candidates.

04 May 2020

Fragment merging on the WBM site of scaffold protein WDR5

Two years ago we highlighted work out of Stephen Fesik’s lab at Vanderbilt University describing potent binders of WDR5, a molecular scaffold that interacts with dozens of other proteins. Those molecules bind at the so-called WIN site, disrupting interactions with proteins such as MLL1. Other proteins, such as the famous anticancer target MYC, bind at a completely different location – the WBM site. This is the focus of a new paper from the same group in J. Med. Chem.

The researchers had previously completed a traditional high-throughput screen and identified molecules such as compound 1. These were further optimized, but, as one might expect looking at the chemical structure, the best molecules had “challenging physicochemical profiles.” The researchers turned to fragments for help.

A two-dimensional (1H-15N HMQC) NMR screen of ~14,000 fragments yielded 43 hits, all of them quite weak, with dissociation constants in the millimolar range. The tetrapeptide portion of MYC that binds to the WBM site, Ile-Asp-Val-Val, contains a carboxylic acid flanked by lipophilic residues, and as one would expect many hits were hydrophobic acids. Crystal structures were determined for five, and these suggested a fragment merging opportunity.

The carboxylic acid moiety of fragment F2 makes similar interactions with an asparagine residue in WBM as the sulfonamide moiety of compound 1. The resulting merged compound 2a showed improved potency. More than a dozen replacements for the cyclohexyl ring were attempted but none improved potency significantly. Similarly, moving the cycloalkyl group around the 5-membered heterocycle was not productive. However, introducing a methyl sulfone moiety to engage a lysine residue led to a ten-fold boost in potency for compound 12. The molecule disrupted WDR5-MYC complex formation in cell lysates and also reduced MYC binding to target genes in cells.

This is another nice example of using fragment merging to fix problems across early lead series. Of course, compound 12 still has a long way to go; as the researchers note, the phenol is a likely site of glucuronidation. Still, this and the 2018 paper demonstrate the power of fragments to target two separate protein-protein interfaces on the same protein.

27 April 2020

PhABits: photoaffinity-based fragment screening

Three years ago we highlighted work out of Ben Cravatt’s lab describing “fully-functionalized fragments” that – in addition to a variable portion – contain a photoreactive diazirine moiety and an alkyne moiety. These were incubated with cells and irradiated with UV light to crosslink the fragments to bound proteins. The alkyne was then used in click chemistry to isolate and identify the bound proteins. Cell-based screening is not for the faint of heart, but as demonstrated in a paper recently posted on ChemRxiv by Jacob Bush and collaborators at GlaxoSmithKline and University of Strathclyde, the functionalized fragments can also be used in biophysical screening. (Emma Grant presented a nice poster on some of this work at FBLD 2018.)

A small library of 556 fragments, rebranded as PhotoAffinity Bits (or PhABits), was synthesized by coupling the alkyne- and diazirine-containing carboxylic acid with a diverse set of amines (each with < 16 heavy atoms). These were then screened at 200 µM against six pure recombinant proteins, irradiated with UV light, and analyzed using intact protein mass spectrometry as in Tethering and other forms of covalent FBLD. Hit rates varied tremendously, from less than 3% for myoglobin to 47% for lysozyme. It would be interesting to see whether this approach, like other fragment finding methods, is able to assess protein ligandability.

Most of the PhABits did not react with the proteins tested, though 58 crosslinked to at least four, and 10 crosslinked to all six. For one of the proteins screened, the bromodomain BRD4-BD1, a known high-affinity ligand could compete 68 of the 89 fragment hits, suggesting a specific interaction at the acetyl lysine pocket. Of the 21 fragments that were not competed, 19 bound to at least three other proteins. Interestingly, the physicochemical properties and solubilities of these fragments were not notably different from the rest, and the researchers speculate that their non-specificity may be due to a longer-lived reactive intermediate generated after UV irradiation.

Several of the BRD4-BD1 fragments were confirmed as binders using a TR-FRET assay, some with low micromolar affinities, though the tighter ones tended to contain known bromodomain binding motifs such as isoxazoles. A couple of these were successfully used to generate PROTACs, as suggested here. Protein digestion and LC-MS/MS sequencing revealed that the fragments crosslinked residues near the acetyl lysine binding site, and this binding mode was confirmed using X-ray crystallography for one of the fragments.

In addition to BRD4-BD1, another target the researchers highlight is KRAS4BG12D. Of the 11 unique hits, some resembled previously reported molecules, and LC-MS/MS studies suggested that they do in fact bind in the same pocket. Competition studies confirmed this, and the resulting IC50 values were similar to those previously determined using HSQC NMR.

As the researchers point out, this photoaffinity-based screening approach is limited to homogenous proteins that are suitable for mass spectrometry. Also, the crosslinking efficiency is not necessarily related to the affinity of the fragment. Still, this is an interesting approach to both find fragments and identify their binding sites. It will be fun to see how it develops.

19 April 2020

Back to the Future: HIV protease offers lessons for SARS-CoV-2

Today’s guest post is by Glyn Williams (University of Cambridge). Fragment aficionados will recognize Glyn as the former VP of Biophysics at Astex, but before that he worked at Roche. His experiences there in the 1990s have lessons for today. -Dan Erlanson

In two recent Practical Fragments posts (here and here), Dan Erlanson noted efforts which will allow the scientific community to contribute to drug design efforts against the SARS-CoV-2 main protease (Mpro). Leading the charge at the moment is the COVID Moonshot consortium who have already received design proposals for covalent inhibitors, based on the structures of fragments bound to Mpro that have been generated by researchers at the Diamond Light Source. At the same time, more information about Mpro, including its substrate preferences, is being published. Soon there will be an urgent need to define a selection procedure which will allow valuable drug candidates to be progressed.

A similar situation was faced in 1985 when HIV protease was being considered as a drug target for AIDS. An excellent description of a pragmatic, and ultimately successful, procedure was published in 1993 by Noel Roberts and Sally Redshaw of Roche in The Search for Antiviral Drugs:Case Histories from Concept to Clinic.

When the project began there was no definitive proof that this aspartyl protease was essential for viral replication in human cells and that it could not be substituted by a cellular protease. However, its in vitro ability to cleave a Phe-Pro or Tyr-Pro peptide bond (amongst others) marked it out as unusual, and that was sufficient encouragement for Roche to initiate a discovery programme. Inhibitor design then took advantage of this feature to build in selectivity over human aspartyl proteases, ultimately giving a high therapeutic index while also improving inhibitor absorption after oral administration. 
Critical issues, such as the decision to target the HIV-1 viral strain, access to suitable protease constructs and clear criteria for project progression, were defined early on. Novel protease and anti-viral assays were then developed in parallel with transition-state mimetic leads. From the start, it was recognised that the low aqueous solubility of the optimal peptide substrates could imply that peptidomimetic inhibitors were also likely to have poor physico-chemical properties. At the time there was no structural information on the enzyme or its complexes, so there was little opportunity to avoid these shortcomings.

As with COVID-19, the worldwide health implications of HIV were obvious and scientific interactions between different research groups were driven by a spirit of cooperation. Public laboratories contributed clinical data and provided access to assays for viral activity. In 2020 the ability to share data has improved beyond recognition but the ability to interpret and act on it is still subject to political and commercial pressures. At Roche, a series of hydroxyethylene inhibitors was not pursued due to its prior inclusion in multiple patents for renin inhibitors. In addition, sensitivity to criticism from AIDS activist groups during the project discouraged Roche from developing follow-up candidates later.

Many current predictions and public expectations about COVID-19 now depend on the availability of vaccines in 2021. After more than three decades of research, no preventative vaccine is yet available for HIV. However, the ability to treat a viral infection, even with a drug that contains and controls the infection rather than eliminates it, should not be undervalued. In 1993 the Roche HIV protease clinical candidate, Ro 31-8959, was in Phase 2 evaluation. Roberts and Redshaw pointed out then that lowering a patient’s viral load would reduce the risk of further infections amongst health-care workers and social contacts, while the persistence of immature and non-infectious viral material in cells could stimulate the patient’s own immune system to eliminate the virus.

Roberts and Redshaw concluded their 1993 analysis with the statement that "although there is still much work to be done, we remain very hopeful that Ro 31-8959 will make a positive contribution to the therapy of AIDS". Two years later Ro-31-8959, as Saquinavir, was approved by the FDA and, with Ritonavir, a second protease inhibitor from Abbott Labs, led to a 64% reduction in deaths from AIDS in the US over the next 2 years. Let us now hope for the same degree of success from new COVID-19 treatments.

13 April 2020

Fragment chemistry roundup part 3

Last week’s post discussed three papers describing new chemistries for building fragment libraries. The theme continues this week with three more.

The first, in ACS Med. Chem. Lett. from Philip Garner (Washington State University Pullman), Philip Cox (AbbVie), and colleagues describes the synthesis of a library of pyrrolidine-based fragments in just three steps. A chiral auxiliary, which is subsequently removed, enables an asymmetric cycloaddition reaction to generate pyrrolidine rings containing three defined stereocenters. Using this method, the researchers made 48 fragments from simple starting materials.

As one might predict looking at the structures, the fragments have low lipophilicity (average AlogP = 0.12) and high levels of saturation (Fsp3 = 0.47), though with an average MW = 225 they are a bit portly.

The fragments are also quite shapely, as assessed both by principal moments of inertia (PMI) or plane of best fit (PBF). The researchers acknowledge that this shapeliness increases the fragments’ molecular complexity, and they also note the difficulty of quantifying this, “as current estimates do not take into consideration 3D, let alone the multidimensional descriptors of chemical space.” Thus, they may have lower hit rates. Hopefully we’ll see screening data from this set at some point in future.

Diversity oriented synthesis (DOS) has only been occasionally applied to fragments, perhaps in part due to issues Teddy raised in his Safran Zunft Challenge. In an (open access) Bioorg. Med. Chem. Lett. paper, Nicola Luise and Paul Wyatt (University of Dundee) describe a set of 22 fragments in 12 scaffolds starting from just 3 precursors; a few examples are shown.

Although the embedded pyrazine, pyridine, and pyrimidine moieties are found in many drugs, some of the bicyclic cores are novel or rarely found in commercial sets.

In both these papers, the chemistry is sufficiently straightforward that a hit could rapidly lead to numerous analogs, which is a selling point for including them in a library. But in advancing other fragments a common problem is that the analog you most want to make is synthetically difficult. A crystal structure may reveal that an otherwise useful synthetic handle is making intimate contacts with the protein, while a hard-to-functionalize aliphatic ring is situated next to an attractive subpocket. A clear example of this is the phase 2 IAP inhibitor ASTX660 from Astex, whose fragment starting point consisted of a piperidine linked to a piperazine.

Perhaps building on this experience, Rachel Grainger, Chris Johnson, and collaborators from Astex, University of Cambridge, and Novartis have published in Chem. Sci. a high-throughput experimentation method to functionalize cyclic amines. The researchers used nanomole-scale reactions run in 1536-well plates to explore and optimize photoredox-mediated cross-dehydrogenative heteroarylation.

After optimizing conditions, the researchers moved to larger (milligram) scale to couple 64 different protected amines against heteroarene 3a and 48 heteroarenes against N-Boc-morpholine, thereby obtaining a variety of interesting molecules, many of which contain polar functionalities. Finally, they used flow chemistry to generate more than a gram of product 5g, demonstrating scalability. The paper ends with a half dozen examples of fragments taken from recent reviews, noting how the cross-dehydrogenative coupling could be used to elaborate them.

Progress often comes from expanded possibilities. By facilitating new chemistries, this paper lowers the barriers for drug hunters to make the most promising molecules. And taken together, all six of these papers advance the field of fragment chemistry.

06 April 2020

Fragment chemistry roundup part 2

It has been more than a year since we devoted a post solely to fragment library synthesis (though see here for an example describing library synthesis and screening). Since you can’t screen fragments without a library, Practical Fragments will spend the next two posts focusing on recent library design papers.

The first, from David Spring (University of Cambridge) and collaborators at the Technical University of Denmark, California State Polytechnic University Pomona, and University of Leeds, was published earlier this year in Chem. Commun. David Spring has long been interested in fragments that resemble natural products (NPs), such as those with multiple sp3 stereocenters.

The researchers focus on 3-hydroxy-2,2-disubstituted-cyclopentan-1-ones, which are found in natural products and derived drugs. The two building blocks syn-1 and anti-1 were elaborated in fewer than six synthetic steps into a total of 38 small molecules in 20 scaffolds, a few of which are shown.
Close attention was paid to physicochemical properties, and consequently the library is rule-of-three compliant, with a mean molecular weight of just 208 Da. The library is also quite shapely, as judged either by a high (0.70) mean Fsp3 or by individual members' principal moments of inertia (PMI).

Another paper from David Spring’s lab was published last year in Eur. J. Org. Chem. In it, the researchers describe the synthesis of nine heterocyclic spirocycles, a couple of which are shown here.

As with the newer paper, the physicochemical properties conform to the rule of three, and the molecules are quite shapely as assessed by their Fsp3 values.

Wrapping up this week’s installment is a paper in Chem. Eur. J. from Richard Bayliss, Stuart Warriner, Adam Nelson (all at University of Leeds) along with collaborators at University of Leicester, Diamond Light Source, University of Oxford, and University of Johannesburg. The researchers set out to assemble a diverse set of 80 shapely fragments for general use. Several rounds of computational pruning arrived at 60 commercial compounds and 20 that were synthesized de novo. Both approaches ran into problems: some “commercially available” compounds proved “difficult to obtain in practice,” while several synthetic approaches that looked good on paper turned out to be anything but. The final library is quite shapely though: all the synthesized compounds have at least one stereocenter, and only two fragments in the entire set are “close to the rod-disk axis” of a PMI plot.

Usefully, this paper presents screening data, in this case a high-concentration (80-200 mM) crystallographic screen against Aurora A kinase. This yielded just four hits, a 5% hit rate much lower than some other crystallographic screens. Interestingly none of these bound at the kinase hinge region where fragments often bind but instead were found at an allosteric site. The authors do not speculate on the low hit rate, which could be due either to the shapeliness of the fragments or their portliness, with 18-22 heavy atoms, considerably above the optimum suggested by Astex. The fragments are available for screening at Diamond’s XChem, though they don’t seem to have been used in the recent SARS-CoV-2 main protease screen.

We’ll cover three more papers next week. In the meantime, stay safe and please leave comments!

01 April 2020

Fragment screening in cells with cryo-EM

Of all the biophysical advances so far this century, cryogenic electron microscopy (cryo-EM) has probably made the most impressive strides. Frequently dismissed as “blobology” just a few years ago, the technique now regularly produces three-dimensional structural models that rival those from X-ray crystallography. Indeed, it is rare to pick up an issue of Science or Nature that doesn’t contain a cryo-EM structure. Earlier this year, researchers from Astex described the structures of fragment hits against two proteins determined using cryo-EM. Now, the boffins from DREADCO (who previously brought us universal crystallography) have begun fragment screening in cells using cryo-EM.

Fragment screening in cells is not new: we previously highlighted work using either covalent or non-covalent fragments. However, figuring out which proteins the fragments bind can be challenging, which is one of the reasons structural information is so useful.

The researchers from DREADCO incubate their fragment library against cells – human or otherwise – for varying lengths of time. They then flash-freeze the cells in liquid ethane, collect, and process the data, using standard cryo-EM workflows. Of course, given the complexity of cells, the computational processing power needed is enormous – but nothing their SkyFragNet platform can’t handle.

One of the advantages of cryo-EM is that larger structures are more easily solved, so the researchers are focusing on organelles such as mitochondria, as well as ribosomes. Already they’ve found dozens of hits that resolve to high resolution, and they are in active fragment-to-lead optimization. Surely it is only a matter of time before our list of fragment-derived drugs includes one discovered with the aid of cryo-EM.

29 March 2020

A crowdsourcing call to action: FBLD vs SARS-CoV-2 Protease

In less than a week the number of cases of COVID-19 worldwide has more than doubled, beyond 720,000, as have the number of deaths, to more than 34,000. For those of us in drug discovery but not on the front lines of clinical care, it is frustrating to watch these numbers climb relentlessly while doing nothing to help other than physical distancing. The temporary closure of so many labs accentuates this feeling.

In early March we highlighted an effort by Dave Stuart, Martin Walsh, Frank von Delft, and others at the Diamond Light Source to screen fragments against crystals of the main protease (MPro) of SARS-CoV-2. The enzyme is a cysteine protease, ideal for covalent fragment screening, and indeed Nir London and coworkers at the Weizmann Institute used intact protein mass-spectrometry to pre-screen 993 fragments. In total, these combined efforts yielded crystal structures of 44 hits bound covalently to the active-site cysteine, 22 non-covalent hits in the active site, and 2 non-covalent hits at the protein dimer interface. Full details and structures can be found here.

In our previous post we showed an overlay of the seven fragments that had been released at the time showing multiple high-quality interactions with the protein. You can look at them all interactively here, and some of the chemical structures are shown below.

This is where crowdsourcing comes in. A group called PostEra (corrected: part of a consortium called COVID MoonShot), consisting of academic and industrial researchers around the world, is trying to use these data and more to develop drugs against SARS-CoV-2. Everyone is invited to contribute, from first year graduate students through industry veterans and emeritus professors.

Do you have ideas how you might grow or merge some of the fragments? If so, you can propose structures, and those that pass a series of filters including synthetic accessibility and toxicity predictions will be synthesized at Enamine and tested at various laboratories (including yours, if you’re interested). We’ve previously highlighted Enamine’s “make on demand” model, which has turnaround times of just a few weeks. At least a couple computational companies, including BioSolveIT and Nanome, are offering free access to their platforms to help you design molecules. Already more than 350 molecule ideas have been submitted.

A cynic could say that these efforts are misguided given the slow pace of drug discovery. Vemurafenib, the first fragment-based drug approved, took six years from the start of the program to approval, and this is lightening speed. However, as Derek Lowe observed, all of the drugs currently being clinically tested against COVID-19 were originally developed for other indications. Stephen Burley suggested recently in Nature that we probably would already have drugs against COVID-19 had we spent more effort fighting SARS.

Hopefully we will have a vaccine long before any drugs coming out of this effort enter the clinic. But there will be a SARS-CoV-3, and a SARS-CoV-4. Having more drugs in our pipeline may prevent those from killing so many people.

23 March 2020

Fragments in the clinic: 2020 edition

As I write this, more than 350,000 people worldwide have tested positive for SARS-CoV-2. More than 15,000 of them have died.

It is important to stay aware of what's going on and take appropriate measures to stop the spread of COVID-19. But to paraphrase Nietzsche, one can spend too much time staring into the abyss. In the spirit of hope, Practical Fragments offers an updated list of FBLD-derived drugs.

The current list contains 47 molecules, 7 more than the last compilation, with 4 approved. As always, this table includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). Because of this, the Phase 1 list contains a higher proportion of compounds that are no longer progressing. Drugs reported as still active in clinicaltrials.gov, company websites, or other sources are in bold, and those that have been discussed on Practical Fragments are hyperlinked to the most relevant post. The list is almost certainly incomplete, particularly for Phase 1 compounds. If you know of any others (and can mention them) please leave a comment.


PexidartinibPlexxikonCSF1R, KIT
VenetoclaxAbbVie/GenentechSelective BCL-2
Phase 3

Phase 2

AMG 510Amgen KRASG12C
AT9283 AstexAurora, JAK2
IndeglitazarPlexxikonpan-PPAR agonist
MAK683NovartisPRC2 EED
Navitoclax (ABT-263)AbbottBCL-2/BCLxL
Phase 1

ABBV-744AbbottBD2-selective BET
ABT-518AbbottMMP-2 & 9
AT13148AstexAKT, p70S6K, ROCK
AZD5099AstraZenecaBacterial topoisomerase II
BI 691751Boehringer IngelheimLTA4H
HTL0014242Sosei HeptaresmGlu5 NAM
NavoximodNew Link/GenentechIDO1

We live in scary times. But, as this list demonstrates, by working together we can still achieve marvels.

16 March 2020

Fragments vs a Pseudomonas aeruginosa virulence factor

The world is understandably focused on SARS-CoV-2; see for example last week’s post. But there are many other threats out there, including infectious Pseudomonas aeruginosa, which is particularly problematic for immunocompromised people. A recent (open access!) ChemMedChem paper by Martin Empting and collaborators at the Helmholtz Centre for Infection Research and elsewhere describes a clever approach to tackle this pathogen.

An age-old problem for antibiotics is that they provoke resistance: nothing like death to kick evolution into high gear. One way to sidestep this is to develop drugs that target virulence rather than essential microbial pathways. The protein PqsR is part of the Pseudomonas Quinolone Signal Quorum Sensing system, and is important for pathogenicity.

A previously published screen of 720 fragments by SPR yielded about 40 hits, including compound 3. Not only does this compound have impressive ligand efficiency, it also has high enthalpic efficiency; the binding is largely enthalpy-driven. Although the utility of thermodynamics for lead optimization is questionable, the researchers were cognizant of the hydrophobic nature of the ligand binding site for PqsR, and sought molecules that would make polar interactions from the start rather than having to engineer them; a similar strategy proved successful for Astex.

Crystallography with compound 3 was unsuccessful, but SAR by catalog led to compound 7, which has higher affinity for PqsR as assessed by isothermal titration calorimetry (ITC) and also shows activity in a reporter gene assay. Fragment growing led to compound 11, which the researchers were able to characterize crystallographically. The two aromatic rings are at a sharp angle to one another, and attempts at rigidifying the linker proved unsuccessful. But further growing led to compound 20, with submicromolar activity in the reporter assay. This molecule also reduced release of a toxic virulence factor from a clinical isolate of P. aeruginosa.

Interestingly, despite the increased activity of compound 20 over compound 11 in the reporter assay, it seems to have lower affinity for PqsR by ITC. The researchers suggest that the full protein in cells likely behaves differently than the truncated version studied in the biophysical assays.

The researchers also emphasize that flexible linkers were more successful than rigid linkers in improving potency – a phenomenon we’ve previously highlighted here and here. Intuitively a more flexible linker is likely to be more forgiving, as a fraction of an ångström can make the difference between binding or not.

There is still much to do: in particular, activity will need to be improved further, and no pharmacokinetic or other animal data are provided. Moreover, a clinical trial with an anti-virulence strategy would be difficult to design. Still, this is an interesting approach, and I hope the authors or others will follow up on it.