Flatland: a nice place to be

The ideal shape of compounds used for biological screens is a subject of vigorous debate, with some arguing that shapely molecules may be superior in various ways to the “flatter” aromatic compounds that tend to dominate libraries. This view was expressed more than a decade ago in the paper, “Escape from Flatland: Increasing Saturation as an Approach to Improving Clinical Success.” However, those conclusions have been challenged. Since many of us are trying to discover drugs, it is worth asking what actual drugs look like. This is the subject of a new ACS Med. Chem. Lett. paper by Seth Cohen and colleagues at University of California, San Diego.

Assessing shapeliness is itself contentious. Here the researchers chose the intuitive metric, principal moment of inertia (PMI), which uses a simple triangle plot to assess whether a molecule is more rod-like, disk-like, or sphere-like. The degree of shapeliness (3D Score) can be calculated by summing the x- and y-coordinates to give values between 1 (rod- or disk-like) and 2 (sphere-like).

The researchers first extracted more than 8500 drugs and nutraceuticals from DrugBank, all of which had associated three-dimensional structures and MW >100. PMI calculations revealed that nearly 80% were linear or planar, with 3D Scores < 1.2. Another 17.5% had 3D Scores up to 1.4, while only 0.5% were greater than 1.6. Interestingly, this distribution is similar to that of the ZINC database of small molecules. You might expect a correlation between size and shapeliness, with larger molecules being more three-dimensional, but this was not the case. Perhaps related, a separate analysis found no correlation between shapeliness of fragments and resulting leads.

The 3D structures of compounds in DrugBank are calculated for energy-minimized conformations, which are not necessarily the biologically relevant conformations. So the researchers next went to the protein data bank (PDB) and its crystal structures of 502 unique DrugBank molecules bound to various proteins. Some molecules were represented multiple times (1036 structures of sapropterin!), and for these the PMIs were averaged. The results of this analysis were similar, with 83.5% of molecules having a 3D Score < 1.2 and just three molecules with a 3D Score > 1.6. As with the DrugBank data, there was no correlation between 3D Score and molecular weight.

Further analyses of compounds with multiple crystallographic structures was interesting. For diclofenac, with 51 PDB entries, 3D Scores ranged from 1.03 to 1.52, with the minimized score being 1.22. However, some of these structures are likely low affinity with questionable biological relevance. In contrast, for five approved HIV drugs, the PMIs remained very similar for molecules bound in the active sites.

Getting out of flatland is surprisingly difficult: the researchers examined the PMIs for several fragments from libraries designed to have shapely members and found that none had 3D Scores > 1.4. They suggest clever ways of increasing three dimensionality, such as building organometallic molecules. While this is likely to increase novelty and patentability, it also introduces unknown biological risks. One analysis that would be interesting is whether natural-product-derived drugs are significantly shapelier than their purely synthetic counterparts.

The researchers conclude:

The true need for topological diversity in feedstocks and final drug molecules remains unclear given the overwhelming number of linear and planar drugs. The question remains as to whether more 3D compounds represent attractive and untapped therapeutic space, or if more linear/planar molecules are indeed the best topologies for bioactive molecules.

This is indeed an interesting question, and I hope that chemists – particularly those in academia – continue to make and test ever more exotic molecules. But since the first word of this blog is “Practical,” I would not discount the more planar molecules that make up most of our pharmacopoeia.

Fragments vs JAK1, a sequel from LEO

Four years ago we highlighted a paper from LEO Pharma describing inhibitors of the kinase JAK1, which is implicated in a host of inflammatory conditions. Although they developed low nanomolar inhibitors, these showed phototoxicity, which was unacceptable for the topical applications the researchers had in mind. The molecules were also not selective against closely related JAK2, whose inhibition can cause neutropenia and anemia. A recent paper in J. Med. Chem from Andrea Ritźen and collaborators at LEO and GVK Biosciences describes a more selective series.

As mentioned previously, hits came from about 500 fragments screened against JAK2 using SPR and validated in a biochemical assay against JAK1. Most fragments had similar activities against both proteins, but compound 1 was moderately selective for the latter. Initial SAR around the fragment revealed that the methyl group was essential to activity and that methylating the pyrazole nitrogen atoms also obliterated binding. The molecule looks like a hinge-binder, but because it can assume four different tautomers docking was difficult. Fortunately, replacing the difluoromethyl substituent with a phenyl ring in compound 6 improved affinity and led to a crystal structure, which showed the methyl group making lipophilic interactions with the protein.

The crystal structure also revealed that the phenyl ring didn’t quite fill the lipophilic ribose-binding pocket, so the researchers replaced this with the more three-dimensional cyclohexyl substituent in compound 7, which yielded a ten-fold improvement in biochemical potency as well as the first cellular activity. The philosophy behind further optimization is described eloquently: “in the spirit of fragment-based drug design – start small and make every added atom count – small substituents with balanced polarity were added to the cyclohexyl analogue 7.” Unfortunately, although several polar substituents introduced onto the cyclohexyl ring improved biochemical potency, they did not do much for cell activity.

Replacing the cyclohexane moiety of compound 7 with a norbornane led to compound 11, which was not only more potent against JAK1 but also less lipophilic and more soluble. The researchers then borrowed a nitrile from an approved pan-JAK inhibitor, leading to compound 40. This molecule has low nanomolar activity against JAK1 and is somewhat selective against closely related JAK2, JAK3, and TYK2. It is quite selective against a panel of 50 other kinases and does not inhibit several cytochrome P450 enzymes or bind to hERG. Oral bioavailability in rats is fairly low, but this should not be a problem for topical indications.

As noted in the paper, the researchers were able to benefit from published work from multiple other companies that has led to five approved JAK inhibitors plus several more in clinical development. While another JAK inhibitor may not be the most pressing medical need, this paper is still a nice example of structure-based design that illustrates several points. First, ligand efficiency was improved during the optimization process, in contrast to common perceptions. Second, fragment selectivity was also improved during optimization. And finally, although this sounds banal, it matters what you have in your fragment library: had the des-methyl version of compound 1 been the only representative of this core in the LEO library the researchers would not have discovered it. In other words, while simplifying your fragments will decrease molecular complexity, sometimes a single methyl group can make all the difference.

Failing honorably and openly on PrP

Many of the posts on Practical Fragments – and indeed much of what appears in the literature – describe successes. This is obvious in the list of fragment-derived clinical compounds, and discoveries of high-affinity tool molecules or even advanceable fragment hits make up a large share of the 750+ posts on this blog. But of course, most of what we do in science fails, and such failures can also be informative. Eric Minikel and 25 collaborators from the Broad Institute and multiple other organizations have just published an illustrative case study on bioRxiv. (Eric also has a detailed and eloquent blog post of his own about the work.)

The researchers describe a five-year effort to find small-molecule binders of the prion protein, PrP, which misfolds and forms aggregates that lead to neurodegeneration. The hope was that binders could be turned into PrP stabilizers or perhaps even degraders. PrP has been studied for decades and there are plenty of literature reports of small molecules that seem to interact with the target, but none of these have been convincingly validated. Moreover, the crystallographic structure of the protein does not reveal attractive binding pockets.

Fragment-based methods have succeeded for other difficult targets, so the researchers performed STD and ¹⁹F NMR screens. Of 6630 pooled fragments, 238 initial hits were retested by STD NMR, leading to 80 hits that were then assessed by two-dimensional (TROSY) NMR. This led to a single hit, a substituted benzimidazole. Unfortunately the binding site could not be determined: chemical shift perturbations were spread across the protein. Differential scanning fluorimetry (DSF) showed the molecule caused a slight decrease in thermal stability. Both of these results suggest some sort of pathological mechanism, but the researchers did multiple experiments to rule out aggregation. A dose-response suggested a dissociation constant well above 1 mM, and none of 54 analogs tested proved any better. Soaking crystals of PrP with 20 of these was also unsuccessful.

Next, the researchers performed a thermal shift assay of just over 30,000 compounds (not necessarily fragments), which yielded both stabilizers and destabilizers. Unfortunately, none of 93 tested by two-dimensional NMR (HSQC) revealed any sign of binding. A DNA-encoded library (DEL) screen of 256,000 macrocycles also didn’t yield any confirmed hits (though more on that below), nor did a computational screen of just under 7 million molecules.

Given this experience, the researchers cautiously conclude that their “results may hint toward relative rarity of PrP binders in chemical space.” They do suggest several alternative approaches, such as screening the more biologically-relevant membrane-bound form of the protein. It may also be worth doing a high-concentration crystallographic screen. Finally, the researchers note that one of the DEL scaffolds “was judged to be a likely covalent binder and was not pursued further.” This decision may be worth revisiting. Indeed, covalent approaches have led to clinical compounds against another formerly undruggable target, KRAS.

According to the blog post, the researchers have pivoted to oligonucleotide therapeutics, where they seem to be making good progress. This makes sense, and I wish them luck. But I hope someone returns to PrP itself with new tools. If they do, the assays established and described here will prove invaluable.

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.

2020

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 here, here, 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

2021
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 here, here, 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 here, here, 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.

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.