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.

07 March 2020

Fragments vs SARS-CoV-2 Protease: open science in action

Last year we highlighted work done by a consortium called Open Source Antibiotics to find fragment hits against antibacterial targets. A similar effort has now launched to discover leads for COVID-19. And those involved have done so with breathtaking speed and openness.

A group of researchers including Dave Stuart, Martin Walsh, and Frank von Delft (Diamond Light Source) has performed a fragment screen against crystals of the main protease (MPro) of SARS-CoV-2, the virus that causes COVID-19. Even before fully analyzing all of the data, let alone publishing it on bioRxiv, they are making it available here, with promises of frequent updates.

MPro is a cysteine protease essential for viral viability. The first crystal structure of the protein was solved in January and posted on bioRxiv late last month. The Diamond researchers synthesized the gene and used it to produce protein that crystallized and diffracted to high (1.39 Å) resolution. Importantly, they found a crystal form in which the active site was empty and thus well-suited to fragment soaking. In just three days the XChem researchers grew, soaked and analyzed 600 crystals. Since then they have screened over 1000 fragments and found 7 that bind in the active site. These will be released in the protein data bank on March 11, though the coordinates and electron density maps can already be downloaded from XChem and viewed interactively here. An overlay shows a large and attractive pocket with multiple opportunities for protein-ligand interactions.

Frank sent an email on March 6 describing this achievement to a number of researchers, and within minutes Brian Shoichet (UCSF) said that he would be using the fragments as controls in a large library docking screen he is doing. Just a few hours later Andrew Hopkins (Exscientia) said that he has SPR and enzymatic assays up and running and is willing to screen compounds sent to him. Then John Chodera (Memorial Sloan-Kettering Cancer Center) volunteered to do free-energy calculations.

As anyone who has worked in drug discovery, fragment-based or not, will recognize, there is still a long road ahead to turn these fragments into effective drugs. But this global team has sprinted off the starting line. Please join them in the race if you can.

02 March 2020

FBLD meets DEL

FBLD, of course, starts with small libraries of small fragments. DNA-encoded chemical libraries (DEL) usually start from the opposite extreme. Massive numbers of molecules are combinatorially synthesized attached to DNA, screened against a target using affinity selection, and hits identified by sequencing the DNA. A recent paper in J. Med. Chem. by Christopher Wellaway and colleagues at GlaxoSmithKline uses information from both approaches to generate a high-quality candidate.

The researchers were interested in bromodomain and extraterminal (BET) family proteins – the same targets we discussed last week. GlaxoSmithKline had already put molecules into the clinic, but they were looking for structurally different backup candidates, so they performed a DEL screen on the BD1 domain of BRD4. A library of 117 million compounds yielded potent compound 10, and crystallography revealed that the 2,6-dimethylphenol moiety bound in the acetyl-lysine-binding pocket.

Phenols are often metabolic liabilities, and indeed compound 10 was rapidly cleared in mice. However, GlaxoSmithKline has a long and successful history of fragment screening against bromodomains; Teddy first described some of their seminal work back in 2012, when the world didn’t end. Compound 16 had been found in a previous screen as a hit against BRD4, and crystallography revealed that the pyridone binds in a similar fashion to the phenol moiety. (Similar pyridones had been reported by others, for example this one.) Merging the molecules led – after a bit of tweaking – to compound 20a. In addition to BRD4, this molecule binds another bromodomain, BAZ2A, which the researchers wanted to avoid. Structure-based design led them to the more selective compound 20i.

Although compound 20i is potent in cells, it still has moderate clearance in rats. Unsubstituted benzimidazole rings have been reported to be unstable, so the researchers systematically explored a series of substitutions, ultimately arriving at compound 24 (I-BET469). Not only is this compound potent and soluble, it is remarkably stable, with “no detectable turnover in rat, dog, and human microsomal and hepatocyte preparations.” Oral bioavailabilities approach 100%, and the compound proved to be effective in acute and chronic mouse inflammation models. Although selectivity against non-BET family bromodomains members is good, compound 24 does strongly bind to both BD1 and BD2 domains of all four BET family members, and as we saw last week this may lead to toxicity.

Nonetheless, this is a lovely example of using a fragment to replace a problematic moiety in a larger molecule, as we’ve seen previously for chymase, Factor VIIa, and Factor XIa. Throughout the optimization the researchers paid close attention to molecular properties such as lipohilicity and molecular weight, and this resulted in a molecule with excellent pharmacokinetics despite the presence of potentially unstable moieties such as the morpholine. If nothing else, this will be a useful in vivo chemical probe.

24 February 2020

Fragments in the clinic: ABBV-744

Bromodomains, which recognize acetylated lysine residues, are popular cancer targets due to their role in gene regulation. A plethora of potent inhibitors have been reported, many of them derived from fragments, and some have even gone into the clinic. The story behind one of these, ABBV-744, was recently published in Nature by Yu Shen and colleagues at AbbVie.

The story starts with a protein-detected NMR screen (highlighted here), which ultimately led to ABBV-075 (highlighted here). This molecule binds tightly to the four BET-domain family members (BRD1, BRD3, BRD4, and BRDt). However, ABBV-075 causes gastrointestinal toxicity as well as a reduction in platelets when tested in mice. Indeed, these effects are seen when BRD4 alone is genetically silenced in mice, suggesting on-target toxicity. However, each of the BET proteins has two separate bromodomains, called BD1 and BD2, and the researchers thought that a selective inhibitor of the BD2 domain might be better tolerated.

Screening about 2500 compounds from the ABBV-075 program revealed that compound 1 was still quite potent against the BD1 domain of BRD4 but lost activity against the BD2 domain. Further optimization ultimately led to ABBV-744, which is at least two orders of magnitude more selective against the BD2 domains of all four BET-domain proteins over the respective BD1 domains. It also shows no activity against a panel of kinases and other bromodomains, and is orally bioavailable. A crystal structure of the molecule bound to either BD1 or BD2 reveals that the key interactions seen in ABBV-075 are maintained, but that the added amide moiety makes interactions only available in BD2, while the larger diphenylmethyl ether moiety is better accommodated in BD2 due to a slightly larger pocket (containing a valine rather than an isolueucine residue).

ABBV-744 is active against multiple acute myeloid leukemia and prostate cancer cell lines, and the paper thoroughly explores the biology of a selective BD2 inhibitor. Most striking is that in a mouse xenograft model, ABBV-744 shows similar activity at 1/16 of its maximum tolerated dose (MTD) as ABBV-075 shows at its MTD. Even at doses well above efficacious exposure levels, ABBV-744 shows only limited platelet reduction and no gastrointestinal toxicity in mice. As mentioned at FBLD 2018, this molecule has entered clinical development, while ABBV-075 has quietly been dropped from AbbVie’s pipeline.

This is a lovely example of biology-guided medicinal chemistry that is reminiscent of the BCL-family inhibitors, which started with less specific molecules and culminated with the approval of BCL2-selective venetoclax. Although the fragment origins of ABBV-744 are clear, they are not mentioned in the paper and – like the KRAS inhibitors and AZD5991 – could be easily overlooked. In all these cases small starting points have delivered potentially huge drugs, and Practical Fragments wishes everyone involved the best of luck.

17 February 2020

Fragments vs MNK1 and MNK2: take three

Mitogen-activating protein kinase-interacting kinases 1 and 2, or MNK1 and MNK2, are implicated in several cancers while seeming to be dispensable for normal cells, making them attractive oncology targets. Indeed, we’ve previously written about two FBDD-derived clinical compounds against these kinases, eFT508 and ETC-206. In a J. Med. Chem. paper published last month, Alvin Hung and colleagues at A*STAR describe a third series.

The researchers started by screening 1700 fragments in a biochemical assay, resulting in 11 molecules that inhibited both MNK1 and MNK2 with decent ligand efficiency. Four of these had a meta-substituted pyridine, as in compound 6. Making a few analogs led to compound 13, with low micromolar potency against both enzymes.

Crystallography proved unsuccessful, but making the reasonable assumption that the pyridyl nitrogen binds to the kinase hinge region led to two models, only one of which was consistent with the SAR. Decoration of the phenyl ring led to compound 21, with submicromolar activity. Although still early, the researchers collected both in vitro and in vivo ADME data on this molecule, which turned out to be quite promising.

Next the researchers turned to the pyridyl ring and found that appending small (5-membered) heterocycles could also boost potency, as in compound 36. At this point, after multiple attempts with previous compounds, crystallography finally yielded a structure that confirmed the proposed binding mode. Combining elements from compounds 21 and 36 directly led to only a slight boost in activity, but further tweaking ultimately led to compound 47, the most potent member of the series. Unfortunately this molecule was unstable in mouse liver microsomes, but a related compound showed good mouse pharmacokinetics as well as impressive selectivity in a panel of 104 kinases.

No pharmacodynamic studies are described, and perhaps this series was deprioritized to focus on ETC-206, which was also developed at A*STAR. Indeed, the later compounds in this paper reveal a frustrating struggle between potency and stability often seen in medicinal chemistry. This is captured in a nice timeline that shows a rapid improvement in potency over about 8 months, followed by a slight drop as the researchers tried to improve exposure. Although no Goldilocks molecule is reported, this paper is nonetheless a lovely example of fragment to lead optimization done for the most part without the aid of crystallography.

10 February 2020

Toward SAR by SFX

As our poll last year revealed, X-ray crystallography has inched out ligand-detected NMR to become the most popular fragment-finding method. One criticism often leveled at crystallography is biological relevance: very few proteins in nature are found in the crystalline state. Moreover, crystallography is a dish usually served cold, with crystals typically frozen in liquid nitrogen. The reason for this is that the powerful X-ray beams used to elucidate the structure of molecules also rip them apart, and freezing them slows the damage. But protein-ligand complexes at low temperature may not always reflect our more temperate world.

One approach to collecting crystallographic data at room temperature is to do so very quickly, before radiation damage can occur. This is done using serial femtosecond crystallography (SFX), in which many crystals are individually examined using brief, intense beams from X-ray free-electron lasers (XFELs). The X-ray pulses last less than 20 femtoseconds, a time so breathtakingly short that light only travels the width of a typical human cell. In a recent IUCrJ paper, Robin Owen, Michael Hough, and collaborators at the Diamond Light Source, the University of Essex, and elsewhere describe a high-throughput version.

The protein crystals themselves can be quite small, just 1-20 µm across, compared with the > 50 µm crystals typically used in crystallography. These microcrystals are mounted in silicon “chips” containing 25,600 little apertures; the X-ray beam can then be swept across each of the positions.

The researchers demonstrated that they could collect high-quality data for three proteins that are particularly sensitive to radiation damage: two heme peroxidases and a copper nitrite reductase. For all three proteins, they were able to determine high-quality structures of bound fragment-sized ligands. Indeed, some of the ligands were even smaller than all but the smallest fragments: imidazole (five non-hydrogen atoms) and nitrite (three non-hydrogen atoms). The latter case was particularly impressive given that the nitrite displaces a bound water molecule, so the difference between empty and liganded protein is even more subtle.

The first word of this blog is “Practical,” so how does this technique stack up? The researchers used 2-4 chips for each structure, and data collection took about 14 minutes per chip. Despite the miniaturization, sample consumption is not trivial: 1.4 to 6 mg of protein and 4-40 µmol of ligand for each data set. However, the researchers showed that could get by with less data – in some cases significantly so – and state that a 4-5-fold improvement in throughput would be straightforward. Using processing software such as PanDDa could further improve results. I suspect it is only a matter of time before we see the first FBLD by SFX screen. It will be fun to see how useful it turns out to be compared with established methods.

03 February 2020

Fragments vs RIP2: from flat fragment to shapely selectivity

Last week we highlighted the utility of shapely fragments. However, as the latest review of fragment-to-lead success stories again shows, starting with a “flat” fragment does not condemn a lead to flatland. This is illustrated in a recent J. Med. Chem. publication by Adam Charnley and colleagues at GlaxoSmithKline.

The researchers were interested in receptor interacting protein 2 kinase (RIP2), which is implicated in various inflammatory diseases. A fluorescence polarization screen of 1000 fragments at 400 µM yielded 49 hits with inhibition constants ranging from 5-500 µM. Thirty of these confirmed in a thermal shift assay, and 20 were characterized crystallographically bound to the enzyme. Hit-to-lead chemistry was pursued for five series; the most successful started with compound 1a.

The crystal structure revealed that the carboxamide of compound 1a makes interactions with the hinge region of the kinase, with the phenyl group in the back pocket. A search of related molecules available in-house led to compound 2a, with a satisfying boost in potency. Interestingly, the crystal structure of this molecule bound to RIP2 revealed that the binding mode of the pyrazole moiety had flipped to keep the phenyl ring in the back pocket (compound 1a in cyan, 2a in gray). Enlarging the phenyl group to better fill the pocket led to compound 2k.

This molecule had relatively poor selectivity against several other kinases, but introducing a ring as in compound 8 improved the situation. Crystallography suggested that installing a bridged ring would pick up further interactions with the protein, and although the resulting molecule did not have better affinity, selectivity improved. Finally, a hydroxyl group was introduced (compound 11) to try to pick up interactions with a non-conserved serine residue. This addition did not improve biochemical activity, and in fact a crystal structure revealed that the hydroxyl group was pointing towards solvent, but the activity in human whole blood improved. Importantly, compound 11 was remarkably selective for RIP2: just 1 of 366 other kinases tested at 1 µM showed >70% inhibition.

This is a lovely fragment-to-lead success story that reiterates several important lessons. First, a generic (in this case commercial) and nonselective fragment can lead to novel, selective series. Second, as has been seen multiple times, fragment binding modes can flip unexpectedly, especially during early optimization. Finally, despite the relative flatness of fragment 1a (Fsp3 = 0, though the two aromatic rings are slightly twisted), it could be optimized to a more shapely lead, and the increased complexity is likely responsible for the impressive selectivity. Left unreported is the stability and pharmacokinetics of compound 11: the hydroxyl and all those sp3-hybridized carbons are likely metabolic hotspots. As is so often the case in lead discovery, what solves one problem can too often create another.

27 January 2020

Three dimensional fragments revisited

A long-running debate in the fragment world centers on the utility of “three dimensional” fragments. Proponents argue that these (often aliphatic) fragments may be more novel, have better physicochemical properties, and have more vectors for elaboration than “flatter” (mostly aromatic) molecules. Skeptics retort that hit rates are likely to be lower for these more complex molecules, and good luck making analogs. Two papers published late last year add more data to the debate.

The first paper, published in J. Med. Chem. by William Pomerantz and collaborators at the University of Minnesota and Eli Lilly, describes the results of a fragment screen against the bromodomain BRD4(D1), a popular member of the BET family. The 467 fragment library was enriched for shapely fragments as assessed by plane of best fit (PBF), which is the “average distance of a non-hydrogen atom from a plane drawn through the compound such as to minimize the average.” For example, "flat" benzene has a PBF of 0 while the cofactor NADPH has a PBF of 1.53.

The library was screened using ligand-observed (CPMG) NMR, and 34 hits were confirmed using protein-observed fluorine (PrOF) NMR. All of these were competitive with the known ligand (+)-JQ1, consistent with binding at the acetylated lysine recognition site. The average PBF of the hits was 0.44, essentially the same as the library itself (0.46). This is higher than the average PBF (0.36) of all fragments crystallized with BRD4 in the protein data bank.

Structures of all the hits are provided, and some of them are indeed quite unusual. The researchers characterized a substituted thiazepane crystallographically and were able to optimize this to a 32 µM binder with good ligand efficiency. This fragment was also selective against a handful of other bromodomains.

The researchers had previously screened BRD4(D1) under identical conditions with a more traditional, “flatter” library with an average PBF of 0.26. Interestingly, in that case the hits were less shapely than the library as a whole, with an average PBF of 0.17. The confirmed hit rate was also higher: 20% vs 7%. That said, the fragments in the traditional library tended to be smaller (averaging 180 Da vs 241 Da), so the molecular complexity of this library was likely to be lower, which could account for the higher hit rate.

The second paper, published in Bioorg. Med. Chem. Lett. by Ulrich Grädler and collaborators at Merck KGaA, EMD Serono, Edelris, and Proteros, focuses on cyclophilin D (CypD), which has been implicated in cardiovascular disease and multiple sclerosis. Unlike BRD4, this is a tough target: an HTS screen of 650,000 compounds in a biochemical assay yielded just 178 hits, none of which confirmed. Undeterred, the researchers screened 2688 fragments by SPR at 2 mM, resulting in 58 confirmed hits, all quite weak (millimolar). Crystallography was attempted on most of them, yielding six structures, including such shapely specimens as compounds 3 and 7.

Compound 3 binds in the lipophilic S2 pocket of CypD, overlapping with the aniline moiety of previously reported compound 2. Fragment merging led to compound 14, with nearly 40-fold improved affinity over compound 2. A similar strategy merging compound 3 with fragment 8 led to low micromolar compound 27, two orders of magnitude more potent than the starting fragments. Perhaps most impressively, fragment linking compound 3 with compound 7, a shapely fragment which binds in the S1’ pocket, led to submicromolar compound 39, with affinity more than 10,000-fold higher than either fragment.

So in the end, fanciers of shapely fragments and detractors alike can feel vindicated by these papers. Hit rates might be lower for three dimensional fragments, but the resulting hits are likely to be less precedented. In the case of CypD, a shapely fragment led to three different series for a target that had resisted HTS. Of course, there is still some way to go: no cell, permeability, or stability data are provided for any of the molecules, and medicinal chemists may blanch at the seven stereocenters in compound 39. But these are interesting starting points, and it will be fun to see where they end up.

20 January 2020

Fragments in the clinic: AMG 510

Few cancer targets are as prominent as KRAS, a molecular switch that has been called “the beating heart of cancer.” Mutations that cause the switch to be stuck in the “on” state occur in roughly a quarter of human tumors. The protein’s role in driving cancer has been known for nearly forty years, but for most of that time it has been considered undruggable.

Until now.

When bound to GTP, KRAS is turned on, promoting cell proliferation until the GTP is hydrolyzed to GDP. Activating mutants impair this hydrolysis. An obvious approach to targeting KRAS would be to develop small molecules that bind in the nucleotide-binding pocket, as has been done successfully for kinases. Unfortunately, the extremely high affinity of KRAS for GTP, along with the high intracellular concentration of GTP, makes this impossible.

One activating mutant replaces a glycine with cysteine (G12C), providing a convenient handle for covalent inhibitors. In 2013, Kevan Shokat and colleagues at University of California San Francisco described in Nature how they used Tethering to identify fragments such as 6H05 that bound to this cysteine through a disulfide bond. Because disulfides are not stable in cells, they replaced this “warhead” with an acrylamide moiety and made a few other tweaks to arrive at compound 12. Extensive optimization described by Yi Liu and collaborators at Kura, Wellspring, and Janssen in a 2018 Cell paper led to molecules such as ARS-1620, which showed potent biochemical and cell-based activity and promising results in mouse xenograft models.

In 2014, Carmot Therapeutics began a collaboration with Amgen to discover covalent inhibitors of the G12C mutant of KRAS. Carmot’s technology, Chemotype Evolution, entails rapid synthesis and testing of large libraries around an existing molecule such as a fragment (see here for a nice animation). In this case, fragments chosen included simple acrylamides, the thought being that – unlike the disulfides in Tethering – these could be carried through into the final molecule.

Initial hit 2 was truncated and cyclized to compound 4, which had comparable activity but with fewer atoms and rotatable bonds. Additional iterations of Chemotype Evolution and medicinal chemistry ultimately led to compound 1, with nanomolar activity in cells containing KRASG12C. Crystallography revealed that the tetrahydroisoquinoline moiety bound in a previously cryptic pocket formed by movement of a histidine side chain. These interactions contributed to the high affinity of the molecule; more details are provided in a paper published last year in ACS Med. Chem. Lett. by Victor Cee, myself, and our collaborators at Amgen and Carmot.

Unfortunately, although compound 1 was more potent in cell assays than ARS-1620, it had low oral bioavailability and rapid clearance in mice and rats. However, as described in J. Med. Chem., Brian Lanman and colleagues at Amgen superimposed ARS-1620 with compound 1 and realized that it would be possible to access the cryptic pocket from the former compound. This strategy proved successful, ultimately leading to AMG510, which entered the clinic in August 2018. And although it is still early, a paper in Nature by Jude Canon, J. Russell Lipford, and collaborators at Amgen and elsewhere describes promising responses to the drug by a handful of patients with non-small-cell lung carcinoma.

Amgen is not the only company to have capitalized on the Tethering results: Mirati used the information to develop their clinical-phase MRTX849. And Janssen has also entered the clinic with JNJ-74699157.

There are multiple lessons here. First, as we’ve seen previously, a single fragment can lead to multiple clinical compounds. Second, progress often requires considerable changes to the initial fragment. This story is clearly a case of fragment-assisted drug discovery, and in the interest of space I’ve had to omit most of the lovely medicinal chemistry, not to mention biology and biophysics, detailed in these five papers.

Another lesson is that covalent fragments can enable lead discovery for targets not accessible through other means. But this enablement may require non-conventional molecules; the initial fragments violate the rule of three, and the disclosed clinical compounds have molecular weights in excess of 500 Da. Despite these challenges, remarkably rapid progress is possible: less than five years elapsed between the first Nature publication and the entry of AMG 510 into the clinic.

Most important, this research has led to possibly life-extending molecules – one of the responders described in the second Nature paper had been on drug for 42 weeks. Practical Fragments wishes her or him, and everyone else involved with the trials, the best of luck.

13 January 2020

FBLD by Cryo-EM

X-ray crystallography is the most popular fragment-finding method according to our latest poll. This is in part due to the previously unmatched level of detail a crystal structure can provide, and in part due to the increasing speed and automation for data collection and processing. But many proteins can never be coaxed into crystals, and while it is possible to advance fragments in the absence of structure, it is rarely easy.

In the past few years, cryogenic electron microscopy (cryo-EM) has come to rival crystallography in terms of resolution (see here for details). The first mention of the technique on Practical Fragments was in 2013, when Teddy wrote that he “could not figure out how you would use [it] in screening/FBHG. However, the point of emerging technology is to emerge…” And emerge it has. At FBLD 2018 researchers from Astex presented the first cryo-EM structure of a bound fragment, and in a new paper in Drug Disc. Today Harren Jhoti and collaborators at Astex and Isohelio provide details for two proteins.

The first target, β-galactosidase (Bgal), is a model protein that has previously been characterized by cryo-EM. The researchers solved the bound structures of three small molecules, two of them fragment-sized, to resolutions of 2.2-2.3 Å. The quality of the maps is such that they could easily be mistaken for crystallographic data: the density is clear and includes ordered water molecules. Induced conformational changes are evident, stereochemistry is unambiguous for all the ligands, and one piperidine ring even shows a hole in the middle. Even more impressive, the structures were solved using automated software.

But three structures do not a screen make. For this, the researchers chose the oncology target pyruvate kinase 2 (PKM2). They designed a small library of 68 highly soluble fragments and screened these at 5 mM. The structures of two complexes are shown, and while the resolution is lower than for Bgal, clear contacts with the protein are evident. Next, the researchers screened cocktails of four fragments, each at 25 mM. Fragments in mixtures were chosen to have diverse shapes, and two structures are shown demonstrating that the technique can distinguish the binders.

Throughput is still an issue: each round of data collection and processing – whether individual compound or cocktail – took about a day. However, the researchers expect that improvements in software and hardware should enable 400 fragments to be screened in less than a month. Indeed, given that much of the focus of cryo-EM has been on generating novel structures, one can imagine various shortcuts for screening, such as collecting smaller numbers of images to look for any evidence of bound ligands. I wonder too if something like PanDDA could be developed for cryo-EM.

Both proteins described have been previously characterized by crystallography, but these are still early days, so it is only a matter of time before we see fragment structures in proteins that haven’t been crystallized. That will open thousands of proteins heretofore inaccessible for structure-based design. The researchers conclude by predicting that “cryo-EM will have a transformative impact on the pharmaceutical industry in the near future.” I would not bet against this.

06 January 2020

Fragment events in 2020

Updated as of 29 March - we'll continue to update as we receive more information, particularly for the June meetings.

June 2-4: CHI is launching an inaugural Expanding Chemical Space program as part of their World Pharma Week in Boston, and it looks like there will be several talks on FBDD.

June 15-17:  Although not exclusively fragment-focused, the Eighth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and returns to Boston this 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.

April 13-17 August 24-28: CHI’s Fifteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego April 14-15 August 25-26. This is part of the larger Drug Discovery Chemistry meeting, and includes a short course on FBDD on April 13 August 24. 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 20-23: FBLD 2020 will be held for the first time in the original Cambridge (UK). This will mark the eighth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2018FBLD 2016FBLD 2014,  FBLD 2012FBLD 2010, and FBLD 2009. Registration is open, and abstracts are accepted through April 30.

December 17-18: What better way to close the year than in Hawaii? The second Pacifichem Symposium devoted to fragments will be held in Honolulu. Pacifichem conferences are 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. Abstract submission is open but closes April 15.

Know of anything else? Please leave a comment or let us know!

31 December 2019

Review of 2019 reviews

The year ends, and with it the awkward teenage phase of the twenty-first century. As we have done since 2012, we're using this last post of the year to highlight conferences and reviews over the previous twelve months.

There were some good events, including CHI’s Fourteenth Annual Fragment-based Drug Discovery meeting in San Diego in April, their Discovery on Target meeting in Boston in September, and the third Fragment-based Drug Design Down Under 2019 in Melbourne in November, which also saw the launch of the Centre for Fragment-Based Design. Our updated schedule of 2020 events will publish next week.

Turning to FBLD reviews, Martin Empting (Helmholtz-Institute for Pharmaceutical Research Saarland) and collaborators published a general overview in Molecules. This is a nice up-to-date summary, covering library design, methods to find, confirm, and rank fragments, and optimization approaches. It’s also open access so you can read it anywhere.

Protein-protein interactions can be particularly challenging drug targets, and these are covered in a Eur. J. Med. Chem. review by Dimitrios Tzalis (Taros Chemicals), Christian Ottmann (Technische Universiteit Eindhoven) and colleagues. The focus is on clinical compounds, and several of these – including venetoclax, ASTX660, mivebresib, onalespib – are discussed in detail. The article is particularly useful in discussing late-stage optimization of pharmacokinetic and pharmacodynamic properties. It also provides a nice summary of physicochemical properties for fragment hits and derived candidates.

Target selectivity is always important, and this is the focus of a review in Exp. Opin. Drug Disc. by Rainer Riedl and collaborators at the Zurich University of Applied Sciences and the Università degli Studi dell’Insubria. Although the broader topic is de novo drug design, fragment-based methods are prominent, and include case studies we’ve discussed on nNOS, pantothenate synthetase, and MMP-13.

In terms of specific targets, Fubao Huang, Kai Wang, and Jianhua Shen at the Shanghai Institute of Materia Medica provide an extensive review of lipoprotein-associated phospholipase A2 (Lp-PLA2) in Med. Res. Rev. This serine hydrolase has been studied for four decades but – as the researchers note – “divergence seems to be ubiquitous among Lp-PLA2 studies.” At least this is not for lack of good chemical tools, fragment-derived (see here, here, and here) and otherwise.

Although NMR has fallen behind crystallography in our latest poll, that is certainly not reflected in terms of reviews. In particular, 19F NMR is covered in three papers. CongBao Kang (A*STAR) manages to pack a lot (including 261 references!) into a concise review in Curr. Med. Chem. Topics include protein-observed 19F NMR, in which one or more fluorine atoms are introduced into a protein genetically, enzymatically, or chemically, as well as ligand-observed methods, in which fluorine-containing small molecules are directly observed or used as probes that are displaced by non-fluorine-containing molecules.

Protein-observed 19F NMR (PrOF NMR) is covered in Acc. Chem. Res. by William Pomerantz and colleagues at the University of Minnesota. Although the first example was published 45 years ago, only in the past few years has the technique been used for studying protein-ligand interactions. The researchers note that introducing fluorines into aromatic residues is ideal because they are relatively rare, simplifying interpretation, and overrepresented at protein-protein interactions, maximizing utility. Several case studies are described, and even proteins as large as 180 kDa are amenable to the technique.

Ligand-based fluorine NMR screening is simpler and more common than techniques that focus on proteins, and this topic is thoroughly reviewed by Claudio Dalvit (Lavis) and Anna Vulpetti (Novartis) in J. Med. Chem. After a section on theory, the researchers discuss library design, including a long section on quality control (which involves assessing solubility, purity, and aggregation of the molecule in a SPAM filter). Direct and competition-based screening approaches are covered in detail; for the latter, a new method for determining binding constants is provided. The paper concludes with more than a dozen case studies. Clearly much has changed in the ten years since I wondered “why fluorine-labeled fragments are not used more widely.” This perspective is a definitive guide to the topic.

Moving to less common methods for characterizing fragments, György Ferenczy and György Keserű (Research Center for Natural Sciences, Budapest) cover thermodynamic profiling in Expert Opin. Drug Disc. After discussing several case studies, they conclude that “thermodynamic quantities are not suitable endpoints for medicinal chemistry optimizations” due to the complexity of contributing factors. This is consistent with another recent paper on the subject (see here), though the information provided is still interesting for understanding molecular interactions.

And although you might have thought the 2017 VAPID publication was the last word on the limitations of ligand efficiency (LE), Pete Kenny has published a splenetic jeremiad on the topic in J. Cheminform. (see also his blog post on the topic, which includes a sea serpent). This is largely a retread of a 2014 article on the same topic (reviewed by Teddy in his inimitable manner here). Pete also describes a more complicated alternative to LE involving residuals, though unfortunately he provides no evidence that it provides more useful information. Pete is of course correct to remind us that metrics have limitations, but assertions that LE “should not even be considered to be a metric” are overwrought.

Two articles discuss virtual chemical libraries. In J. Med. Chem., W. Patrick Walters (Relay Therapeutics) describes efforts to measure, enumerate, and explore chemical space. He notes that false positives could quickly overwhelm a virtual screen of a hundred million molecules, but as we saw earlier this year, progress is being made. Indeed, Torsten Hoffmann (Taros Chemicals) and Marcus Gastreich (BioSolveIT) focus on navigating the vastness of chemical space in Drug Disc. Today. They note that the Enamine REAL Space is up to 3.8 billion commercially accessible compounds, more than double the number of stars in the Milky Way. But this pales in comparison to the 1020 potential compounds in Merck’s MASSIV space. Just storing the chemical structures of these in compressed format would require 200,000 terabytes – and searching them exhaustively is beyond current technology.

Ratmir Derda and Simon Ng (University of Alberta) discuss “genetically encoded fragment-based discovery” in Curr. Opin. Chem. Biol. This involves starting with a known fragment that is then coupled to a library of peptides and screened to find tighter binders. The researchers provide a number of case studies, though adding even a small peptide to a fragment will generally have deleterious effects on ligand efficiency. And – Rybelsus not withstanding – oral delivery of peptides is challenging.

Finally, Vasanthanathan Poongavanam, Xinyong Liu, and Peng Zhang, and collaborators at Shandong University, University of Bonn, University of Southern Denmark, and K.U. Leuven review “recent strategic advances in medicinal chemistry” in J. Med. Chem. Among a wide range of topics from drug repurposing to antibody-recruiting molecules is a nice, up-to-date section on target-guided synthesis. As I opined a couple years ago, I still doubt whether this will ever be generally practical, but from an intellectual standpoint I’m happy to see work continue on the approach.

And with that, Practical Fragments says goodbye to the teens and wishes you all a happy new year. Thanks for reading and commenting. May 2020 bring wisdom, and progress.

16 December 2019

Fragments in the clinic: S64315 / MIK665

Earlier this year we highlighted the discovery of AZD5991, a phase 1 compound from AstraZeneca that inhibits the anti-apoptotic cancer target Mcl-1. Those efforts made use of a fragment previously published by a different research group. Mcl-1 has been a popular target for some time; the first mention on Practical Fragments dates to 2010. The story behind another investigational drug is described in a couple papers from earlier this year.

The first, in ACS Omega by Rod Hubbard and colleagues at Vernalis, University of York, and Servier, describes fragment screening efforts against both Bcl-2 and Mcl-1. The proteins are related both structurally and functionally, and Bcl-2 is the target of venetoclax – the second fragment-derived drug approved. Some of the early fragment hits bound to both proteins, but selective and potent inhibitors were ultimately developed. In the interest of space only those against Mcl-1 will be discussed here.

Both proteins required considerable protein engineering, which is described in detail. Ultimately one form of human Mcl-1 was used for crystallography, while mouse protein was used for NMR screening due to its better stability. A total of 1064 fragments were screened at 0.5 mM each (in pools of eight) using ligand-observed NMR; 39 confirmed using STD NMR, WaterLOGSY, and CPMG. Additionally, fluorescence polarization, 2-dimensional (HSQC) NMR, ITC, and SPR were used to validate hits. Crystallography proved challenging in the beginning but ultimately helped drive optimization of more potent molecules. The large number of different assays employed is consistent with our recent poll results.

Protein-observed NMR was particularly useful in providing information on the quality of both the ligand and protein (reminiscent of the “validation cross” discussed here). Before crystallography was able to play a meaningful role, “NMR-guided models,” combining partial protein assignments with flexible docking, were used to drive SAR.

While the first paper focuses on protein optimization and biophysics, the second (in J. Med. Chem.), by András Kotschy and collaborators, focuses on chemistry. Fragment 1a was one of several hits pursued, initially by looking for analogs, but most of these had comparable (weak) activity. In the absence of a crystal structure a systematic chemistry campaign was conducted, varying elements of the core and sidechains. Many of these molecules had comparable activity against both Mcl-1 and Bcl-2, but replacing the nitrogen linker with an oxygen led to selectivity against the former. The addition of hydrophobic substituents led to compound 10c, with submicromolar activity.

Anticipating poor cell permeability for a negatively charged, lipophilic molecule, the researchers introduced a positively charged methylpiperazine moiety at various positions around the molecule, ultimately leading to compound 18a. In addition to potent Mcl-1 binding, this molecule is active in cells and shows reasonable pharmacokinetic properties in mice. Further optimization to S64315 does not appear to have been published yet, though the structure was disclosed earlier this year, and the fragment origins remain clear.

Together these papers provide a thorough description of drugging a difficult target. They also provide insights into the investment required. The Mcl-1 project began around 2007, and it took a decade before S64315 entered the clinic. Enabling drug discovery against protein-protein interactions required multiple biophysical techniques in addition to all the standard components of pharmaceutical research. The researchers note that “establishing such a platform can take some time and resource – a tool compound is usually needed to validate the assays, but the assays are needed to identify the tool compound.” In the end they have succeeded, and Practical Fragments wishes them – and the patients being treated – the best of luck.

09 December 2019

A new library of fluorinated Fsp3-rich fragments

Among fragment-finding methods, ligand-based NMR ranks near the top in terms of popularity. Of its many variations, fluorine (19F) NMR appears to be gaining in popularity. Fluorine NMR has several advantages, including high sensitivity and the fact that many fragments can be screened simultaneously because of the wide chemical shift range for fluorine. Although more commercial fluorine-enriched libraries are available now than when we first wrote about the approach a decade ago, the diversity of these libraries is still somewhat limited. This problem has been tackled by Mads Clausen at the Technical University of Denmark and an international team of collaborators in a new Angew. Chem. Int. Ed. paper.

The researchers wanted to create a fluorinated fragment library that would be not just diverse but also contain a high fraction of sp3-hybridized carbons (high Fsp3). Some of the early claims around “three dimensional” fragments have been questioned, and there seems to be little if any correlation between the shapeliness of fragments and that of derived leads, but if you’re going to make new fragments in academia it makes sense to explore interesting molecular architectures.

Starting from just six simple building blocks, each containing a trifluoromethyl group, the researchers generated nine different cores which were further derivatized at multiple positions to yield 115 diverse fragments. Consistent with diversity-oriented synthesis, no more than five synthetic steps were used for any molecule. All molecules were made as racemates in order to further increase the diversity of the library.

The resulting “3F Library” is mostly rule-of-three compliant, though given that the trifluoromethyl moiety alone adds 69 Da the fragments do tend to be larger, with an average molecular weight of 284 Da. They are, however, less lipophilic than two commercial fluorinated fragment libraries. And with an average Fsp3 = 0.7 and 3.3 chiral centers they are also quite shapely as assessed by principal moment of inertia.

Building a library is nice, but will it provide hits? To find out, the researchers screened the 102 fragments that passed quality control against four targets. They used a transverse (T2) relaxation assay (specifically, CPMG) in which fragments bound to a protein tumble more slowly, causing a reduction in 19F signal intensity. Hit rates ranged from 3% to 11%, and about two thirds of these confirmed in STD or WaterLOGSY assays. As seen by the examples shown here, the fragments are quite diverse.

Whether these hits will lead to more potent molecules remains to be seen. Laudably the paper ends with the statement: “we hope that the 3F library will find use for other researchers and we encourage anyone interested in screening the fragments to contact us.” If you are looking for interesting new fragments that are tailored for follow-up chemistry, I encourage you to take the team up on their offer.