Fragments vs LpxC revisited

Back in 2020 we described fragment-derived inhibitors of the highly conserved bacterial enzyme LpxC, which is essential for biosynthesis of the outer membrane in Gram-negative bacteria. In a recent (open access) paper in J. Med. Chem., a different group consisting of Ralph Holl and collaborators at Universität Hamburg and several other academic centers describe a new series.

 

The researchers started with compound 9, a molecule they had previously discovered. The substrate for LpxC is a rather large small molecule called (UDP)-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine. Compound 9 does not occupy the UDP-binding site, so the researchers initially tried building towards it with a series of simple linkers connected to a phenyl group. The (S) enantiomers tended to be more active than the (R)-enantiomers, and the most potent was compound (S)-13a, which showed sub-micromolar activity against LpxC from E. coli as well as P. aeruginosa in an enzymatic assay. (For simplicity only the E. coli data are shown here.)

 

Seeking to improve affinity, the researchers screened 650 fragments in pools of five against LpxC in the presence of compound 9 using STD NMR and WaterLOGSY. After deconvolution, this led to 97 hits. STD-based epitope mapping, which we wrote about here, was used to prioritize fragments likely to have a single, well-defined binding mode, culling the number to 19. Finally, NMR-ILOE experiments (see here) suggested that nine of this set bound in close proximity to compound 9, while the other ten did not. Four of these fragments, including the simple indole F3, were then linked to compound 9 at various positions. This is akin to SAR by NMR, but with less information about the relative binding modes so more trial and error is necessary.

 

Among the roughly two dozen molecules made, compound (S)-13j was the most potent against LpxC, with low nanomolar activity. This compound (and several others) also showed antibacterial activity against E. coli and several other strains of Gram-negative bacteria. In vitro stability studies of compound (S)-13j were promising, though the researchers noted the need for improvement. And, since the molecule contains a hydroxamic acid moiety potentially capable of binding to multiple metalloproteins, it was tested against a handful of mammalian zinc-dependent enzymes and shown to be nearly inactive.

 

Compound (S)-13j is 15-fold more potent than the simple phenyl analog (S)-13a, and molecular modeling suggested this may be due to a hydrogen bond from the protein to the indole NH. Although one could argue that it would have been possible to arrive at compound (S)-13j using standard medicinal chemistry starting from (S)-13a, this may have taken longer without knowledge of the indole fragment. Whether or not the molecules advance further, this is a nice example of using fragment screening to find a second-site binder to improve affinity of an existing lead.

Fragments vs DHODH

Rapidly proliferating cancer cells require a steady supply of nucleic acids, and cutting that off is a potential therapy. The enzyme dihydroorotate dehydrogenase (DHODH), which is important for pyrimidine synthesis, is thus an interesting drug target. In a recent ACS Med. Chem. Lett. paper, Lindsey DeRatt, Scott Kuduk, and colleagues at Janssen describe their approach.

 

The researchers had previously used virtual screening and structure-based drug design to develop compound 1, which is potent in both biochemical and cell-based assays. However, the molecule is highly effluxed by P-glycoprotein, which can limit both oral bioavailability and brain penetration. Thus, they turned to fragments.

 

An SPR screen (about which sadly no details are provided) yielded compound 2, and crystallography revealed that the amide carbonyl makes a similar contact to tyrosine 356 (Y356) as does the carbonyl in the triazolone moiety of compound 1. Merging these led to compound 4, which was considerably more potent than compound 2 but much less so than compound 1. However, further optimization led eventually to compound 25. Although less potent in an enzymatic assay than compound 1, compound 25 is equally effective in cells. It also has excellent pharmacokinetics in mice and – importantly – a considerably lower efflux ratio.

 

Interestingly, when the researchers solved the crystal structure of a related molecule bound to DHODH, they found that the carbonyl no longer interacts with Y356 but is instead flipped 180º and interacts with a different residue. The researchers conclude by stating that they are designing new molecules to reengage Y356, which could further improve potency.

 

Several lessons emerge from this brief paper. First, the flipped urea moiety is another reminder that fragments do not always maintain their orientations, as also seen here, here, and here. Second, information from the fragment was used not to improve potency but rather to address other aspects of an existing lead series, as seen here and here. And finally, one could argue that the only critical feature of the fragment remaining in the final molecule is the NH of the urea. But the fragment did cause the researchers to examine their molecules from a different perspective, resulting in a better series. Perhaps you could call this an example of fragment-assisted drug discovery. As is so often the case, fragments can inspire new ideas that may otherwise be overlooked.

Fragments improve solubility: GlaxoSmithKline’s BD2 inhibitors

The epigenetic readers known as bromodomains have been popular anticancer targets for fragment-based approaches. But rapid success in generating potent molecules has not led to equally rapid success in the clinic, in part due to toxicity. Many early molecules inhibited both of the bromodomains (BD1 and BD2) present in the four BET family proteins, and some evidence suggests that BD2-selective inhibitors would be better tolerated. Indeed, last year we wrote about AbbVie’s selective clinical compound. Now GlaxoSmithKline has just reported a new selective inhibitor in J. Med. Chem.

 

GlaxoSmithKline had previously discovered the BD2-selective molecule GSK620, which unfortunately suffered from low solubility in FaSSIF (fasted state simulated intestinal fluid). To try to improve this molecule, they turned to fragments. Although the screening details are not described here, the company’s first fragment screens against bromodomains from a decade ago provided dozens of crystallographically-characterized starting points. Compound 6 binds in a similar manner to GSK620 and has good ligand efficiency.

 

 

Compound 6 shows equal potency against BD1 and BD2 of BRD4, but merging the five-membered core with GSK620 led to BD2-selective compound (S)-11. (Although compound 6 contains a pyrrole, the NH was inconveniently positioned and thus the researchers explored other five-membered heterocycles during scaffold hopping; the paper describes furan and pyrazole series.) Further optimization of the furan, in part based on earlier SAR, ultimately led to GSK743.

 

This molecule showed greater than 1000-fold selectivity for the BD2 bromodomain of BRD4 over the BD1 domain, and >300-fold selectivity for the BD2 domains of BRD2, BRD3, and BRDT as well as selectivity against a large panel of other bromodomains. More extensive profiling revealed it to be clean against CYP3A4, hERG, and other potential off-targets, and it was also negative in an Ames test for mutagenicity. Pharmacokinetics and oral bioavailability were also reasonable in both rat and dog. The compound had potent antiproliferative activity against acute myeloid leukemia cell lines. Finally, FaSSIF solubility was at least 20-fold better than for GSK620.

 

This is a nice example of fragment-based scaffold hopping, akin to another example from GlaxoSmithKline we highlighted last year. Whether GSK743 ultimately advances will probably depend on how other molecules in the class perform. Biology will have the final say, but fragments – combined with elegant medicinal chemistry – provided the tools to answer the questions.

From noncovalent fragment to reversible covalent CatS inhibitor

We noted just a couple weeks ago that covalent fragment-based approaches have been on a tear. Much of the recent focus has been on irreversible inhibitors, but as we discussed back in 2013 there is much to be said for reversible covalent molecules too. These are the subject of a new paper in J. Med. Chem. by Markus Schade and colleagues at Grünenthal GmbH.

 

The researchers were interested in cathepsin S (CatS), one of 11 members of a family of cysteine proteases. The enzyme has been implicated in a laundry list of diseases, from arthritis to neuropathic pain to Sjögren’s Syndrome, and indeed a few inhibitors entered clinical trials in the early twenty-first century. However, selectivity turns out to be essential: inhibiting the related cathepsin K can lead to cardiovascular problems and stroke. Molecules that appear selective often  contain a basic nitrogen and so can accumulate in lysosomes, achieving sufficiently high local concentrations to inhibit CatK.

 

CatS is a small (24 kDa) enzyme, ideal for protein-observed NMR. An ¹⁵N-HSQC screen of 1858 noncovalent fragments yielded 18 hits, all of which showed similar chemical shift perturbations (CSPs) suggesting binding in the S2 pocket. X-ray crystallography was successful for three fragments, confirming that they do indeed bind in the S2 pocket. Appealingly, this region of the protein is structurally different from the other cathepsins, suggesting a route to selectivity.

 

The sulfonamide moiety of compound 1 (blue) binds in a very similar fashion to the sulfone of a previously reported reversible covalent inhibitor, compound 16 (red). Growing compound 1 to compound 37 led to a significant boost in potency, and crystallography revealed that the binding mode remained the same.

At this point the researchers sought to remove a few heteroatoms as well as introduce the nitrile warhead from compound 16, yielding compound 39b. Surprisingly, this molecule was no more potent than the non-covalent precursor. However, fragment growing into the S3 pocket yielded a massive boost in potency in the form of compound 44. Further SAR and crystallography revealed that much of the increased affinity is due not to specific interactions in the S3 pocket but rather to a hydrogen bond between the newly introduced amide proton and a main chain carbonyl of CatS. Compound 44 is also highly selective against CatK and CatB, showing negligible inhibition of either at 10 µM. Unfortunately, cellular potencies of representative compounds were down by more than three orders of magnitude, likely due to low permeability.

 

While there is still some way to go to establish whether these molecules will succeed where others have failed, this is nonetheless a nice case of fragment-assisted lead discovery And while one can certainly argue that it would have been possible to derive compound 44 from compound 16 through classical medicinal chemistry, fragments clearly helped.

Merge and grow: Fragment-based activators of SOS1

The RAS family of proteins is implicated in roughly one third of cancers, and as such has been a long-standing target for drug discovery. Earlier this year we highlighted how covalent fragment-based approaches were instrumental in discovery of direct KRAS inhibitors. A recent paper in J. Med. Chem. by Stephen Fesik and colleagues at Vanderbilt University takes a more unusual approach.

RAS proteins are activated when guanine exchange factors (GEFs) such as Son of Sevenless 1 (SOS1) exchange GDP for GTP. Clinical compounds bind to a mutant form of KRAS and block this process. Previous high-throughput screening in Fesik’s group had found molecules that bind to and activate SOS1-mediated nucleotide exchange. While it might seem counterintuitive to activate a known oncogene, these molecules can actually block downstream RAS signaling by inducing a feedback mechanism. Here, the researchers used fragment screening to look for a new series.

The catalytic core of SOS1 is ~65 kD, relatively large for the protein-detected NMR methods beloved of the Fesik group, so they produced proteins in which the methyl groups of Ile, Val, Leu, and Met were ¹³C-labeled. Selective Ile to Ala mutations allowed them to assign the various methyl groups. An ¹H-¹³C HMQC screen of nearly 14,000 fragments yielded 59 hits (~0.1%), all quite weak: only five had dissociation constants better than 1 mM. Crystal structures were obtained for 16, revealing that all of them bind in the same site previously identified (see also here for similar work from a different group).

Fragments F-4 and F-7 bound in similar positions as each other and also as the HTS-derived compounds, so the researchers merged them to yield molecules such as compound 1b, with improved affinity and ligand efficiency.

Crystallography suggested that a nearby aspartic acid residue could be engaged through fragment growing, leading to molecules such as compound 2d. In addition to low micromolar affinity, this molecule also activated SOS1-mediated nucleotide exchange. In a cell-based assay, the compound caused enhanced phosphorylation of downstream target ERK at low concentrations and decreased phosphorylation at high concentrations, similar to what had been seen for the earlier series of molecules. Presumably, the biphasic response is due to a negative feedback loop that ultimately clamps down RAS signaling.

This is a nice example of structurally enabled fragment-merging and growing, assisted by knowledge of other ligands. While the compounds are probably not sufficiently potent to serve as chemical probes, they could be useful starting points. Activating the RAS pathway may or may not be a good approach for treating cancer, and we need suitable chemical tools to answer this question.

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.

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 KRAS^G12C. 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.

Fourteenth Annual Fragment-based Drug Discovery Meeting

CHI’s Drug Discovery Chemistry (DDC) meeting took place last week in San Diego. I think this was the largest yet, with >825 attendees, a third from outside the US, and nearly 70% from industry. The initial DDC meeting in 2006 had just four tracks, of which FBDD is the only one that remains. This one had nine tracks and four one-day symposia, so it was obviously impossible to see everything. Like last year, I’ll just stick to broad themes.

Success Stories

As always, clinical compounds received deserved attention. Among two I’ve covered recently, Paul Sprengeler described eFFECTOR’s MNK1/2 inhibitor eFT508, while Wolfgang Jahnke discussed Novartis’s allosteric BCR-ABL1 inhibitor ABL001. As previously mentioned, ABL001 is a case study in persistence: the project started in stealth mode and was put on hold a couple times until seemingly intractable problems could be overcome.

Another story of persistence, albeit with a less happy outcome, was presented by Erik Hembre, who discussed Lilly’s BACE1 program. Teddy wrote about their first fragment-derived molecule to enter the clinic, LY2811376, back in 2011. Unfortunately this molecule showed retinal toxicity in three-month animal studies, so the researchers further optimized their molecule to LY2886721, which made it to phase 2 studies before dropping out due to elevated liver enzymes. Reasoning that a more potent molecule would require a lower dose and thus lower the risk of toxicity, the researchers used structure-based drug design to get to picomolar LY3202626, which also made it to phase 2 before being scuttled due to the apparent invalidation of BACE1 as an Alzheimer’s disease target.

Talks on BCL2 and MCL1 inhibitors from Vernalis, AstraZeneca, and Servier all involved fragments in some capacity, but unfortunately they were in the protein-protein interaction track which was held concurrently with the FBDD session I was chairing. Suffice it to say you can expect to hear more about the phase 1 compounds AZD5991 and S654315.

A few earlier-stage success stories included Till Maurer’s discussion of the Genentech USP7 program (see here), Santosh Neelamkavil on Merck’s Factor XIa inhibitors, and Rod Hubbard on Vernalis DYRK1A, PAK1, and LRRK2 inhibitors. We have previously written about how displacing “high-energy” water molecules can be useful, and this tactic was used by Sven Hoelder at the Institute of Cancer Research for their BCL6 inhibitors. Last week we highlighted halogen bonds, which proved important for transforming molecules that simply bind to MEK1 to molecules that bind and inhibit the protein, as described by AstraZeneca’s Paolo Di Fruscia.

Methods

The MEK1 story Paolo told began with a very weak (0.45 mM) fragment that the team was able to advance to 300 nM in the absence of structure, though they did eventually obtain a crystal structure that supported further optimization. On the topic of crystallography, Marc O’Reilly discussed the Astex MiniFrag approach, which we recently wrote about here. Only a couple of these fragments contain a bromine atom, but Marc did mention that, of the 10,051 X-ray complexes solved at Astex, a number show halogen bonds, including some to the hinge region in kinases.

At FBLD 2018 Astex’s Chris Murray showed the first cryo-EM structure of a fragment bound to a protein, and Marc confirmed that they have now obtained structures of fragments bound to two targets, with fragments as small as 120 Da and resolution as good as 2.3 Å. They are increasing automation, with turnaround times of less than 24 hours in some cases. Santosh also mentioned that Merck is applying cryo-EM to fragments.

Frank McCormick (UCSF) highlighted multiple fragment-finding methods used to discover inhibitors against RAS family proteins, which are responsible for more than a million cancer deaths each year. In addition to stalwarts such as crystallography and NMR, these include less common methods such as Tethering and the second harmonic generation (SHG) approach for detecting conformational changes used by Biodesy. RAS was reported as a cancer driver almost forty years ago, but only now are the first direct inhibitors entering the clinic – a testimony to both the challenging nature of the target and how far we’ve come.

SHG and Tethering were also highlighted elsewhere: Charles Wartchow described how SHG identified 392 hits from a collection of 2563 fragments against an E3 ligase bound to a target protein at Novartis, while Michelle Arkin described her use of Tethering at UCSF to find molecules that could stabilize a complex of 14-3-3 bound to a specific client protein (see here).

An effective sponsored talk was presented by Björn Walse of SARomics Biostructures and Red Glead Discovery, who described weak affinity chromatography (WAC). Once they saw the schedule for DDC, they looked for a target that would be presented shortly before their presentation, and chose the protein USP7 as a test case. Beginning in January, they screened a library of 1200 fragments to obtain 34 hits, of which 7 confirmed in a thermal shift assay. This led to an SAR-by-catalog experiment, and 11 of the 31 fragments tested showed activity, as did a Genentech positive control compound.

All methods can generate false positives and false negatives (see for example here and here), some of which were described in an excellent talk by Engi Hassaan of Philipps University. Engi discussed how improving the sensitivity of an STD assay by decreasing salt concentration identified more fragments that had previously been found by crystallographic screening. She also presented a case study of how introducing a tryptophan residue into a small protein to facilitate purification led to problems down the road when the tryptophan side chain blocked a key pocket in the crystal lattice. Gregg Siegal (ZoBio) also highlighted a case where a fragment bound to the dimer interface in a crystal structure, whereas in solution the fragment bound to the active site, as observed by NMR.

Finally, among computational methods, Pawel Sledz (University of Zurich) gave a nice overview of the SEED and AutoCouple methods, while Paul Hawkins (OpenEye) described rapid searching of more than 10 billion chemical structures using ROCS (rapid overlay of chemical features). SkyFragNet is looking closer with each passing year.

There is much more to say, so please feel free to comment. Several good events are still coming up this year, and mark your calendar for 2020, when DDC returns to San Diego April 13-17!

Better properties from fragments: c-Abl kinase activators

Last year we described the discovery of asciminib, an allosteric inhibitor of the kinase BCR-Abl that binds in the enzyme’s myristoyl-binding pocket. As we also highlighted nearly a decade ago, molecules that bind in this pocket can either inhibit or activate the enzyme. Although inhibitors have the most obvious therapeutic potential as anti-cancer agents, activators of the ubiquitously expressed c-Abl protein could potentially treat chemotherapy-induced neutropenia. In a recent J. Med. Chem. paper, Sophie Bertrand and coworkers at GlaxoSmithKline describe their efforts in this area.

The researchers started with a high-throughput screen of 1.3 million compounds. Among the hits was fragment-sized compound 2, which showed good binding and activation in biochemical assays but only modest activity in cells. Building off the left side of the molecule improved biochemical potency, but cell activity still lagged. SAR studies on the dichlorophenyl moiety suggested that this hydrophobic group was probably optimal, and a crystal structure of an analog bound to the enzyme confirmed this. Replacing the central thiazole with other aromatic rings also did little to improve cell activity.

The researchers acknowledge “that the chemistry strategy was largely pursuing compounds with rather poor physical properties,” notably low solubility, high lipophilicity, and high aromatic character. As co-author Robert Young has noted previously, physical properties matter. Happily, a fragment screen identified compound 28.

Adding the acetyl group from the HTS hit generated compound 29, with improved activity compared to the fragment. Moreover, this molecule had better solubility and permeability compared to the more lipohilic, thiazole-containing compound 2. Compound 29 also showed significantly improved activation of c-Abl in a cellular assay. Crystallography revealed that it bound in a similar fashion as compound 2, but with a twisted, more “three-dimensional” shape.

Further optimization, in part informed by previous work done on the thiazole series, ultimately led to compound 52, the most active compound synthesized. Another molecule in the pyrazoline series showed good pharmacokinetic properties in mice. Unfortunately, in vivo efficacy studies had to be halted early due to unexpected (and not clearly understood) toxicity.

This paper nicely illustrates several points. First, the power of fragment-assisted drug discovery, in which information from both HTS and FBLD is combined for lead optimization. Second, the inherently fuzzy line between FBLD and other discovery approaches: had compound 28 been tested in the HTS collection, it likely would have been a hit. Third, the importance of physicochemical properties. And finally, the inadequacy of potency and physicochemical properties alone to produce a developable compound. You can optimize your molecule to the best of your ability but still be sideswiped by nasty surprises such as toxicity. It is helpful to be clever in drug discovery, but you need to be lucky too.

Readers beyond bromodomains: Fragments vs YEATS

Epigenetic readers recognize modified amino acids in histone proteins to cause changes in gene expression. Readers containing bromodomains, which recognize acetylated lysine residues, have received particular attention, and fragment-based approaches have led to at least a couple bromodomain inhibitors entering clinical development. But the numerous bromodomains are not the only epigenetic readers to recognize acetylated lysine residues. In a recent paper in J. Med. Chem., Apirat Chaikuad, Stefan Knapp, and collaborators at Goethe-University Frankfurt and University of Oxford describe their efforts targeting a different family.

YEATS domains are present in four human proteins, three of which have been linked to cancer. Unlike bromodomains, YEATS domains recognize lysine residues modified with acyl derivatives beyond acetyl, such as propionyl, butyryl, and crotonyl. The biological significance of these modifications is not clear, and no inhibitors of these proteins had been reported when the work began.

The researchers focused on the oncogenic eleven-nineteen-leukemia protein (ENL). They solved the first apo crystal structure of ENL (ie, without a bound ligand), which revealed that although the binding pocket was pre-formed, there was some flexibility in the side chain residues. They also noted distinct differences in how the acylated lysine is recognized, including the absence of an asparagine residue that is conserved in all bromodomains, and a more-open pocket that can accommodate larger acyl chains.

Next, the researchers chose a set of nineteen fragments containing a central amide bond to mimic acetylated lysine. None of these showed activity in a thermal shift assay, but when the ligands were soaked (at 5-40 mM) into crystals of ENL, electron density consistent with binding was observed for ten of them, and two could be modeled with some confidence. (For the other nine compounds, the crystals no longer diffracted.) These two fragments also showed binding by isothermal titration calorimetry (ITC). This is a useful reminder of the need for orthogonal assays, and the power of crystallography to detect weak hits. Compound 19, a rather super-sized fragment, was similar to compounds identified in a high-throughput screen that the researchers reported here and here.

Using this information, the researchers made a handful of analogs and found that compound 20 had high nanomolar affinity as assessed by ITC. Like last week’s story, this effort could probably be considered more fragment-assisted than fragment-based. But whatever the precise genealogy, hopefully molecular descendants of compound 20 will help to elucidate the biological poetry of the YEATS domains.

Fragments vs PI3Kδ via deconstruction and regrowth

Ligand deconstruction, in which a larger molecule is dissected into component fragments that are subsequently optimized, can be useful for developing new chemical series. This is nicely illustrated in a paper recently published in J. Med. Chem. by Kenneth Down and colleagues at GlaxoSmithKline.

The researchers were interested in phosphoinositide 3-kinase δ (PI3Kδ), a popular target for a variety of indications from oncology to inflammation. They had already developed GSK2292767 as a clinical candidate, but they wanted a backup with a different chemotype. Crystallography revealed that the indazole moiety was interacting with the hinge region of the protein. Trimming off the top of the molecule (compound 4) led to a loss of both potency and specificity against three related members of the lipid kinase family, not surprising given the fact that indazole is a privileged fragment for kinases in general.

To generate a new series, the researchers sought to replace the indazole hinge binder using modeling and previously published information. Starting with a selection of more than 30 possible hinge binders, they synthesized 324 molecules and found that compound 11 was more potent and ligand efficient than compound 4, as well as reasonably selective against other PI3K isoforms. Growing this fragment-sized molecule led to compound 16, with low nanomolar potency against PI3Kδ, greater than 100-fold selectivity against three related PI3K isoforms and 29 additional kinases, good permeability, and activity in a cellular assay.

The careful observer will note that the dihydropyran hinge binder in compound 11 is shorter than the indazole in compound 4, and indeed crystal structures of compounds 11 and 16 complexed to PI3Kδ revealed that the pyridine sulfonamide fragment is shifted in the active site compared to the original drug molecule, accommodated by various conformational shifts in the protein.

This paper is a good illustration of what has been called fragment-assisted drug discovery. Nowhere in the article do the researchers use the phrase “fragment-based,” though they do refer to the pyridine sulfonamide as a “privileged fragment.” In the end, the proof of practicality is in the chemical matter, so we’ll need to wait until more is revealed about this series.

Thirteenth Annual Fragment-based Drug Discovery Meeting

CHI’s Drug Discovery Chemistry (DDC) meeting was held last week in San Diego. The event continues to grow, and this year hosted some 800 attendees, three quarters from the US and two thirds from biotech or pharma. The first DDC meeting in 2006 had just four tracks, of which FBDD is the only one that remains. The current event had nine tracks and three one-day symposia. There was always something interesting happening, and usually several – at one point three talks involving fragments were going simultaneously. Like last year, I’ll just try to give a few impressions.

What struck me most was the number of success stories, several involving clinical compounds. Last year we highlighted Pfizer’s discovery of a chemical probe against ketohexokinase (KHK); Kim Huard described how this was optimized to PF-06835919, the first and only KHK inhibitor to enter the clinic, which is now in phase 2 trials for NAFLD.

Another phase 2 compound was described by Paul Sprengeler (eFFECTOR Therapeutics). A handful of fragments designed from published work were characterized crystallographically bound to the kinase MNK1, and careful structure-based design resulted in eFT508, an MNK1/2 inhibitor which is being tested against various cancers.

A few years back we highlighted Genentech’s work on the kinase ERK2. In a lovely example of fragment-assisted drug discovery, Huifen Chen told “the convoluted journey of an ERK2 fragment series (with an HTS detour)”. SAR from the fragment series was used to inform the optimization of an HTS series originating from partner Array BioPharma, and was particularly useful for fixing some pharmacokinetic liabilities. Huifen emphasized the importance of using information from multiple strategies, ultimately leading to GDC-0994, which entered phase 1 trials for cancer.

Rounding up the list of clinical compounds, I heard through the grapevine that AbbVie’s dasabuvir, approved for hepatitis C, had fragments in its ancestry. I’d be interested to know more; though since success usually has many fathers, precise parentage can be tricky to ascertain.

Earlier stage success stories included the discovery of BI-9321, a highly selective inhibitor of NSD3-PWWP-1, which binds to methylated lysine residues in proteins. Jark Böttcher described how a collaboration between Boehringer Ingelheim and the Structural Genomics Consortium started with NMR and DSF-based screens of 1899 fragments to identify the cell-active chemical probe.

Jenny Viklund (Sprint Bioscience) described the discovery of potent, selective inhibitors of MTH1, a potential anti-cancer target. The project was successful, but unfortunately the molecules did not have the desired effect in cancer cell lines; this and other evidence helped to devalidate the target. Although undoubtedly disappointing, knowing what not to pursue is still important, and who knows – perhaps the target will turn out to be important in the future.

Finally, Steve Fesik (Vanderbilt) described a number of success stories against the KRAS protein, one of the holy grails of oncology. He also described how a fragment screen against a similarly hot target, the transcription factor MYC, failed utterly – the numerous compounds reported in the literature turned out to be artifacts or DNA intercalators. However, colleague Bill Tansey found that MYC interacts with the protein WDR5, and this protein-protein interaction turned out to be tractable, ultimately yielding potent inhibitors. This is a useful reminder that even if your target is not directly ligandable, biology is complicated enough that you may be able to modulate it through one of its partners.

Success sometimes requires breaking rules, as illustrated by the rule-of-5-defying drug venetoclax. Indeed, as noted by AbbVie’s Phil Cox, 18 of the 76 oral drugs approved since 2014 are bRo5s (beyond rule of 5). But if you’re going to break rules you should expect a harder path, and Phil described factors that correlate with success. Pete Kenny will be delighted to know that this has resulted in a new metric, AB-MPS, which is defined as the sum of the number of rotatable bonds, aromatic rings, and the difference of the ClogD from 3; values less than 12 are correlated with a higher probability of being orally bioavailable among AbbVie’s bRo5s.

Former guest blogger Brian Stockman described NMR-based functional screens he is doing with undergraduates at Adelphi University. Library acquisition can be challenging for a small organization, but happily Dean Brown at AstraZeneca has established an Open Innovation program for neglected diseases – if you’re interested and eligible you can receive a high-quality 1963-fragment library plated and ready for screening.

Of course there was plenty to learn about fragment-finding methods too, both in talks and in a discussion session led by Rod Hubbard (University of York and Vernalis). Microscale thermophoresis (MST) continues to be controversial, with researchers from a couple companies commenting that it’s fantastic the 20% of the time it works, while another company had success rates of ~95%. Thermal shift assays were also contentious, though Fredrik Edfeldt’s (AstraZeneca) method of adding urea or D₂O (see here) to improve the sensitivity created significant buzz.

Cryo-electron microscopy continues to make rapid strides for structurally characterizing difficult targets, such as membrane proteins. Christopher Arthur (Genentech) did not downplay the many technical hurdles, particularly in sample preparation, but he thought that 2 Å resolution structures would be routine within the next decade. Although they have yet to analyze fragment binding, this is only a matter of time.

Ben Cravatt (Scripps) discussed ligand discovery on a proteome-wide scale using electrophilic fragments. His group has currently discovered more than 2000 ligandable cysteine residues in human cells – an exciting if daunting number of potential new targets.

And in the category of now for something completely different, Josh Wand (University of Pennsylvania) described nanoscale encapsulation – in which individual proteins are confined in reverse micelles suspended in liquid pentane; the low viscosity increases tumbling time and thus resolution for NMR, while the miniscule volume increases the concentration of protein and any accompanying fragments. This allows detection of extraordinarily weak interactions (dissociation constants of several hundred millimolar or worse). The technique is limited to very polar fragments because less polar ones would diffuse into pentane, but it would be interesting to see if a fluorocarbon replacement for the hydrocarbon allowed a wider range of fragments to be tested.

I could keep writing but I’ll stop here, hopefully before you stop reading; please leave comments. There are still several good events coming up this year, and mark your calendar for next year, when DDC returns to San Diego April 8-12, 2019!

Fragments vs USP7, two ways, both allosteric

Proteins in cells are constantly synthesized and degraded in a complex, highly regulated manner managed in part by the ubiquitin proteasome system. Simplistically, a ubiquitous small protein called ubiquitin is conjugated to other proteins, targeting them for destruction, and some of the proteins thus targeted control the stability of still other proteins. But ubiquitination is not destiny: ubiquitin can be removed by more than 100 deubiquitinating enzymes, or DUBs.

As I said, this is complex. But complexity has never stopped folks from pursuing drug targets, and multiple groups are interested in a particular DUB called USP7, which is implicated in cancer and other indications. USP7 is one of more than 50 members of a subfamily of DUBs that use cysteine as a catalytic residue. Selectivity is an obvious challenge, and since cysteine is chemically reactive, any screening result carries a high risk of being an artifact. Two recent papers describe how fragment-based approaches led to potent, selective inhibitors.

The first, published in J. Med. Chem. by Paola Di Lello, Vicki Tsui, and coworkers at Genentech, started with an NMR fragment screen. This identified molecules such as compound 1, which NMR data suggested bound near the active-site cysteine. This and other fragments were used to conduct virtual screens of the much larger Genentech library, and 21 of these were then tested experimentally. Most of these either didn’t bind, bound to multiple sites, or caused protein aggregation, but four of them, including compound 2, showed clear binding to a specific site on USP7 and also inhibited the enzyme in a biochemical assay.

Surprisingly, protein-detected NMR suggested that these four molecules did not bind in the active site as expected but rather in an adjacent “palm site”, a hypothesis that was confirmed by a crystal structure of compound 2 bound to USP7. This led the researchers to reexamine other hits from the original NMR screen, where they identified several aminopyridinephenols, such as compound 13.

Meanwhile, a biochemical HTS against USP7 had identified 76 hits, but most of these turned out to be artifacts, and none of them yielded co-crystal structures with the enzyme. The fragment findings led the researchers to revisit some of the weaker hits that had been overlooked, such as compound 15. This led to a crystal structure showing binding in the palm site, and further medicinal chemistry ultimately led to molecules such as compound 28 (GNE-6640), with nanomolar activity in both biochemical and cell-based assays. A separate paper in Nature characterizes the biology in more detail, revealing that molecules in this series interfere with ubiquitin binding and are highly selective for USP7.

Another fragment effort on this target was reported by Timothy Harrison and collaborators at Almac and Queen’s University, Belfast in Nat. Chem. Biol. An SPR screen of 1946 fragments against the catalytic domain of USP7 led to compounds such as fragment B. This was combined with molecules from other groups that had been reported in the literature, leading to compound 1. Subsequent medicinal chemistry, informed by crystallography, led to compound 4, with low nanomolar biochemical and cell-based activity and excellent selectivity. The enantiomer is much less active, and compound 4 should be a useful chemical probe to further understand the biology of USP7.

Remarkably, not only do the two series of molecules bind some distance away from the active site cysteine (yellow, upper right), they bind in completely different, non-overlapping sites!

These papers illustrate the importance of allosteric sites for tackling specific members of large protein families. They are also both cases of “fragment-assisted drug discovery.” Unlike many success stories we’ve highlighted, it is difficult or impossible to find the initial fragment in the final molecules. Heck, Genentech’s best molecules bind in a completely different site from where the first fragment hits bound. Being open to such possibilities, and using all available data from every possible source, are keys to success.

Fragments vs Trypanosoma parasites

Last month we highlighted how fragments could be used to discover inhibitors of protein-protein interactions (PPIs). Today we continue the theme of fragments vs PPIs, in this case the interaction between PEX14 and PEX5, proteins which are important for glucose metabolism in disease-causing protists such as Trypanosoma.

The research, published recently in Science, was done by a large multinational team led by Grzegorz Popowicz, Michael Sattler (both at Helmholtz Zentrum München), and Ralf Erdmann (Ruhr University Bochum). They started by solving the NMR structure of the N-terminal domain of PEX14 from T. brucei, the organism that causes sleeping sickness. Previous work had shown that PEX5 binds to this domain, with two aromatic side chains of PEX5 binding in adjacent hydrophobic pockets. With this information in hand, the team performed a virtual screen of several million (non-fragment-sized) molecules. Eight of the best-scoring hits were tested, and four showed binding in an NMR assay, with compound 1 having the highest affinity.

Next, the researchers screened a library of 1500 fragments (each at 1 mM in pools of 5) using ¹H, ¹⁵N HMQC NMR. This led to 12 hits with affinities better than 2 mM. Strikingly, all of these fragments contained fused bicylic aromatic ring systems, three of which were substituted naphthyls. Appending these onto compound 1 led to compound 4, with low micromolar affinity. Introducing an amine to interact with a glutamic acid residue in PEX14 led to compound 5, with high nanomolar affinity. This compound also showed activity against several species of pathogenic Trypanosoma. Further tweaking led to a molecule with activity in a mouse model of infection.

This example of fragment-assisted drug discovery (FADD) is reminiscent of other cases (described here, here, and here) in which fragments were used to replace elements of a previously identified molecule. While it is possible that traditional medicinal chemistry could have achieved the same result, fragments probably helped winnow down the number of molecules to be synthesized. It is also nice to see this technology applied to understudied diseases. 

Fragments vs BCL6, two ways

Disrupting protein-protein interactions (PPIs) tends to be challenging: interfaces are often large and flat, with few deep pockets in which small molecules can bind. Also, much like unhappy families, PPIs are usually dissimilar, meaning that HTS collections yield fewer, less attractive hits. Both of these challenges are well-addressed by fragments, and indeed last year saw the approval of venetoclax, which targets a PPI. Two new papers in J. Med. Chem. report inhibitors of another PPI.

B-Cell lymphoma 6 (BCL6) binds to other proteins to regulate gene expression. As its name suggests, it was identified in diffuse large B-cell lymphoma (DLBCL), and is thus an interesting anticancer target. Also, structures of the protein in complex with a peptide suggested that it may not be impossible to find small molecule inhibitors. Yusuke Kamada and colleagues at Takeda set out to do just this.

An SPR-based screen of 1494 fragments, each at 1 mM, identified 64 hits which confirmed in dose-response titrations. However, only seven compounds confirmed in an STD-NMR experiment. Of these, compound 1 was characterized crystallographically bound in the peptide groove.

A few tweaks to the fragment led to compound 4, with improved potency. Meanwhile, an HTS screen had identified the very weak compound 5, and crystallography showed that it bound at the same site as compound 4. Merging the two molecules led to compound 7, with mid-nanomolar biochemical potency and low micromolar activity in a cell-based assay. This is a classic case of fragment-assisted drug discovery (FADD), and a good illustration of how FBLD and HTS can be complementary.

The second paper, by William McCoull and a large team of collaborators from AstraZeneca and Pharmaron, goes somewhat further. The researchers conducted an SPR-based screen of 3500 fragments along with a virtual screen. Both found hits, and led to molecules with the same core as compound A2. Crystallography revealed that these also bind in the peptide groove, and combining elements of both molecules while tweaking the properties led to compound A5, with improved affinity.

The next step was growing compound A5 to try to make a hydrogen bond interaction with the protein. That led to compound A8, with submicromolar affinity. A crystal structure of a related compound bound to BCL6 revealed that two chemically distant portions of the molecule were in close proximity, suggesting a macrocyclization strategy similar to what we described last week. This proved highly successful, improving the affinity by more than two orders of magnitude for compound A11. NMR studies of the linear and cyclized compounds revealed that the conformation of the latter was indeed closer to that seen in the crystal structure.

The medicinal chemistry continues extensively from here. In particular, compound A11 showed some activity against the kinase CK2, but this could be engineered out. Multiple additional changes were explored, with many compounds showing low nanomolar activity in a biochemical assay and some showing high nanomolar activity in a cell-based assay. Unfortunately, none were very effective at inhibiting the proliferation of lymphoma cell lines. The authors state, “we conclude that the BCL6 hypothesis as a means of treatment for DLBCL is still unproven and we have elected not to progress this series of BCL6 inhibitors further into development.”

This makes sense, though I wouldn’t abandon all hope. For two other PPIs, BCL2 and MCL1, robust cell activity required picomolar affinity in a biochemical assay. Whether this level of potency is achievable for BCL6 remains an open question.

Fragments vs PRC2 revisited: a chemical probe

Earlier this year we highlighted a paper from Novartis in which ligand deconstruction was used to deconstruct an HTS hit against the epigenetic target methyltransferase polychrome repressive complex 2 (PRC2). In a new J. Med. Chem. paper, Ying Huang and Novartis colleagues report a similar approach on a different HTS hit, ultimately yielding a promising chemical probe.

Compound 7 was identified as one of about 1400 hits from a high-throughput biochemical screen of 1.4 million molecules (described here at PLOS ONE). Crystallographic studies revealed that, like the previous molecule, this one also binds in the site on the EED subunit of PRC2 that normally recognizes trimethylated lysine 27 on histone H3.

Crystallography also suggested that the left half of the molecule didn’t seem to be making productive interactions with the protein. Lopping this off actually increased the activity and dramatically improved the ligand efficiency. Fragment-sized compound 8 was then subjected to extensive medicinal chemistry, ultimately resulting in EED226. Crystallography revealed that the binding modes of the initial hit and the final molecule are quite similar.

In addition to good biochemical activity, EED226 also shows good cell potency and impressive selectivity against other histone methyltransferases, kinases, and unrelated safety targets. It also shows excellent oral bioavailability in mice and acceptable pharmacokinetic properties. The compound caused complete tumor regression in a mouse xenograft model.

A separate paper in Nat. Chem. Biol. further characterizes EED226. A chemoproteomics study of a labeled version of EED226 revealed that it is remarkably selective for the PRC2 complex in human cell lysates. Also, EED226 is active against mutant cell lines that are resistant to other PRC2 inhibitors currently in the clinic, which are competitive with the cofactor rather than the trimethylated lysine residue. In fact, EED226 can bind to the PRC2 complex simultaneously with these other inhibitors, so dosing both together could give improved efficacy and slow the emergence of resistance.

As with the previous post on this target, the discovery of EED226 is a nice example of fragment-assisted drug discovery (FADD). Unlike that case, in which fragmentation led to an initial loss in potency, here trimming back the molecule paid immediate dividends.

Artists often talk about finding a sculpture within a stone by cutting away excess material. It is rewarding to see that chemists can use the same strategy.

Fragments vs PRC2: ligand deconstruction

Ligand deconstruction is a strategy for early-stage drug discovery in which a known hit is dissected into component fragments and one or more of them is optimized. When successful, it can lead to new and improved chemical series. One such example was just published in J. Med. Chem. by Andreas Lingel and colleagues at Novartis.

The researchers were interested in finding inhibitors of the protein methyltransferase polychrome repressive complex 2 (PRC2). Although some drugs have entered the clinic against this anticancer target, all of these are competitive with the cofactor S-adenosylmethionine (SAM), and resistant mutants are already being detected. Thus, the team sought a molecule that would act through a different mechanism.

PRC2 is actually a complex of four different proteins. The SET domain of the protein EZH2 contains the catalytic machinery, but a protein called EED stabilizes the protein complex and is necessary for activity. EED also recognizes trimethylated lysine residues on histone substrates, allosterically activating methyltransferase activity.

A high-throughput biochemical screen identified compound 1, which has low micromolar activity and is noncompetitive with the SAM cofactor and substrate peptide. Subsequent NMR and crystallography experiments revealed that compound 1 binds to EED, with the tertiary amine binding in the same pocket that normally recognizes trimethyllysine. However, compound 1 is quite complex, with three stereocenters. Thus, the researchers sought to deconstruct it to something simpler. They began by chopping off two of the rings – an unconventional disconnection but one supported by crystallography, which revealed that the terminal rings were not closely associated with the protein.

The resulting fragment 2 was down more than an order of magnitude in potency but had improved ligand efficiency. Crystallography confirmed that it binds in a very similar fashion to compound 1. Initial SAR was conducted around the methoxybenzyl moiety, which is buried within the enzyme. Most changes were not tolerated, but compound 9 did show somewhat improved activity.

Next, the researchers sought to optimize the positively charged portion of the molecule. Replacing the amine with a guanidine improved the affinity but at a cost to cell permeability. This led to a search for less conventional replacements, ultimately yielding the 2-aminoimidazole moiety in compound 16. Not only did this regain the activity of the initial molecule, it also shows good permeability and cell activity. Crystallography revealed that it too binds in a similar fashion to the original hit.

This is a nice example of fragment-assisted drug discovery (FADD), in which concepts from FBDD were used to simplify and optimize a hit from HTS. There is of course much more to do with this series, not the least of which is figuring out exactly how the molecules actually inhibit PRC2. Trimethyllysine-containing peptides that bind to EED normally activate the enzyme, yet the small molecules that bind to the same site somehow allosterically inhibit activity. Despite multiple crystal structures, the researchers frankly acknowledge that they were “not able to decipher the molecular basis for this phenomenon.” A number of conformational changes occur when EED binds to ligands, and perhaps these propagate through the protein complex. A picture may be worth 1000 words, but we may have to wait for the movie to learn the full story.