FragLites and PepLites meet bromodomains

The last two Practical Fragments posts focused on bromodomains, epigenetic readers that recognize acetylated lysine residues. Today’s post could thus be considered part of a trilogy, though the focus is less on bromodomains themselves than a specific type of fragment library.

 

In 2019 we highlighted FragLites, small fragments containing pairs of hydrogen bond acceptors and/or donors along with a bromine or iodine atom. FragLites were designed to assess ligandability as well as identify what types of interactions would be favorable at various sites. The original test protein was the kinase CDK2. In an open-access paper published late last year in J. Med. Chem. by Martin Noble, Michael Waring, and colleagues at Newcastle University, FragLites are screened against two members of the bromodomain family.

 

The first bromodomain (BD1) of BRD4 is considered highly ligandable, with multiple inhibitors disclosed (see for example here). In contrast, ATAD2, a bromodomain in another subfamily, is more challenging, in part because it lacks a hydrophobic region useful for increasing affinity for small molecules. Thirty-three FragLites were individually soaked at 50 mM into crystals of either bromodomain. The halogen atom on each FragLite facilitates analysis by anomalous dispersion, allowing more sensitive detection of low-occupancy binders. This, along with Pan-Dataset Density Analysis (PanDDA), was used to identify specific protein-ligand “binding events.”

 

In total, 26 binding events at five sites were identified for BRD4; four ligands bound at more than one site. Of these, 17 FragLites bound at the orthosteric site of BRD4 (which recognizes N-acetyl lysine). In contrast, ATAD2 displayed 16 binding events total over seven sites; only three bound at the orthosteric site, consistent with its lower ligandability. ATAD2 had previously been screened crystallographically against the 776-membered DSI-poised fragment library, and this effort also identified seven ligand-binding sites, six of which were common to those discovered here, suggesting that the small FragLite set is able to identify most pockets.

 

As far as specific types of interactions, the average FragLite made 1.1 hydrogen bond, suggesting that the second donor or acceptor is often not engaged. In contrast, the bromine or iodine atom makes protein contacts in 33 of 42 binding events. In half a dozen cases no hydrogen bond to the protein was observed, with the primary interaction being a halogen bond.

 

The FragLites are small, relatively “flat” aromatic molecules, but of course most proteins interact with other proteins. To try to explore such interactions, the researchers developed a library of “PepLites:” N-terminally acetylated amino acid residues with a C-terminal bromopropargyl group. These were also screened crystallographically against the two bromodomains and produced considerably lower hit rates, with six bound to BRD4 (all at the orthosteric site) and nine bound to ATAD2 (of which five bound to the orthosteric site). Reassuringly, the N-acetylated lysine PepLite bound to both proteins in a similar manner as seen in larger peptides.

 

The researchers conclude that FragLites and PepLites “represent highly valuable components of a larger crystallographic screen, and we anticipate that this is where they will fit into most drug discovery programs.” Indeed, this is already happening; last year we wrote about how FragLites were screened against the bromodomain PHIP2 as part of a larger screen, and I was surprised this paper was not mentioned here. Laudably, all the atomic coordinates have been deposited in the Protein Data Bank, so folks are able to do their own analyses.

 

As FragLites and PepLites are screened against ever more targets, it will be fun to see what they can teach us about intermolecular interactions and starting points for new leads.

Fragments vs PBRM1 bromodomains revisited, more selectively

Last week we highlighted the discovery of a selective inhibitor for the BET family of bromodomains. The 61 human bromodomains fall into eight subfamilies, of which the BET family has probably been most heavily studied. In contrast, family VIII has received less attention, in part due to the lack of selective inhibitors. This deficit is beginning to be addressed by Shifali Shishodia, Brian Smith, and collaborators at Medical College of Wisconsin and Purdue University in J. Med. Chem.

 

The researchers were particularly interested in the aptly-named protein Polybromo-1 (PBRM1), which contains six of the 10 family VIII bromodomains. The protein has normally been considered a tumor suppressor, but it has also been implicated as a tumor promoter in prostate cancer. Chemical probes would be very useful to unravel the complicated biology. A few pan-inhibitors of family VIII have been developed, one of which we wrote about back in 2016, but none of these are selective for the PBRM1 protein.

 

The researchers started with an NMR screen of the second bromodomain of PBRM1, BD2, the structure of which had previously been solved by NMR. A ¹H-¹⁵N SOFAST-HMQC screen of 1968 fragments (all rule of three compliant, from Maybridge and Zenobia) in pools of 12 ultimately yielded a dozen hits, all of which are shown in the paper. Of these, compound 5 was the most potent, with dissociation constants of 45 µM by NMR titration and 18 µM by isothermal titration calorimetry (ITC). 

 

 

One of the previous pan-family VIII inhibitors described in the literature was structurally similar to compound 5, and borrowing a chlorine atom from this led to compound 11, with improved affinity. Further exploration around both phenyl rings ultimately led to compound 16, which displayed low micromolar affinity by ITC and high nanomolar activity in an inhibition assay.

 

Differential scanning fluorimetry (DSF) is commonly used to measure binding of small molecules to bromodomains, and the researchers tested some of their best compounds in a panel of bromodomains that included 9 of the 10 family VIII members. Encouragingly, compound 16 only showed a strong thermal shift (ΔT_m = 5.4 °C) to PBRM1-BD2 and moderate shifts (ΔT_m = 1.8 °C) to PBRM1-BD3 and PBRM1-BD5. No significant stabilization of the 18 other bromodomains was observed.

 

A series of shRNA experiments by the researchers revealed that the prostrate cancer cell line LNCaP was dependent on PBRM1, and compound 16 was active against these cells, albeit weakly (EC₅₀ ~ 9 µM). In contrast, the compound did not show activity against two other cancer cell lines that do not seem to be dependent on PBRM1.

 

This work is a nice example of academic fragment-based lead discovery. Although the cell activity of compound 16 is probably insufficient for a serviceable chemical probe, it does show that selectivity is possible. Hopefully these researchers, or others, will continue improving it.

Automated scaffold hopping for fragments

A post last month covered high-throughput virtual screening, but most practitioners of FBLD still start with some sort of (bio)physical screen. These initial hits can’t be expected to be optimal, since the average fragment library contains a few thousand compounds at most. Indeed, as Xavier Barril and collaborators at Universitat de Barcelona and Oxford University write, “fragment hits should be seen as beacons indicating privileged areas of chemical space to be further explored.” They describe one way to expedite exploration in a recent J. Med. Chem. paper.

 

As we noted here, most good fragments make at least one essential interaction (such as a hydrogen bond) to the protein. The approach starts with a structure of a fragment bound to the target of interest, with that essential interaction identified.

 

Next, a virtual library is searched for similar molecules, with the definition of “similar” being rather loose (>50% Tanimoto similarity). Ideally the library is large enough to produce lots of hits; the researchers used ZINC15, which contains >15 million ostensibly commercial compounds. Also, only molecules within two non-hydrogen atoms of the starting fragment are considered. In other words, a fragment with ten “heavy” atoms would yield molecules with 8-12 non-hydrogen atoms. This search is similar though perhaps more permissive than Astex’s Fragment Network (which we wrote about here).

 

All the molecules are then superposed on the initial fragment structure and only those that maintain the key interaction and binding mode are kept. Aboout 500 molecules are then selected to represent the best and most-diverse hits. These are subjected to dynamic undocking (DUck), which weeds out fragments that have weaker interactions. If desired, each of the remaining hits can be subjected to further cycles.

 

To demonstrate the approach, the researchers turned to bromodomains, a popular target class for FBLD. They started with 1XA, a fragment Teddy highlighted back in 2013 that led to a clinical compound against BRD4. The isoxazole moiety makes a hydrogen bond with the side chain nitrogen of an asparagine that normally binds to acetylated lysine residues. After one cycle, 58 molecules were selected, but unfortunately only five were actually available commercially. Compound 3 had similar affinity and ligand efficiency as 1XA, and this scaffold had not been reported as a bromodomain ligand. A crystal structure of compound 3 bound to the first bromodomain of BRD4 confirmed the predicted binding mode.

Three additional successive iterations were conducted to look for more ligands, but experimental confirmation was challenging as overall only 17 of more than 100 ligands selected for purchase were commercially available. (Compound 23 was chosen for custom synthesis as it was related to a family of high-scoring molecules.) Encouragingly, eight molecules were active in a differential scanning fluorimetry (DSF) assay, a technique that works well for BRD4. Crystal structures of two of these were obtained: compound 9 contains an isoxazole moiety like 1XA (and indeed resembles this fragment) but compound 23 is quite distinct.

Overall this looks like a valuable method for scaffold hopping. Not only might the described approach lead to novel molecules, it could provide new growth vectors that may not be accessible from the original fragment. Before jumping immediately into chemistry with your fragment hits, it may be worth trying something like this.

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.

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.

PhABits: photoaffinity-based fragment screening

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

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

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

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

In addition to BRD4-BD1, another target the researchers highlight is KRAS4B^G12D. Of the 11 unique hits, some resembled previously reported molecules, and LC-MS/MS studies suggested that they do in fact bind in the same pocket. Competition studies confirmed this, and the resulting IC₅₀ values were similar to those previously determined using HSQC NMR.

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

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.

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.

Fragment growing via virtual synthesis and screening

Practical Fragments has covered virtual screening for nearly ten years, and the tools continue to improve. More recently, researchers are using computational approaches not just to dock libraries of molecules, but to decide what compounds to make. The latest example, called AutoCouple, is described in an ACS Cent. Sci. paper by Cristina Nevado, Amedeo Caflisch, and colleagues at University of Zurich.

The researchers have a standing interest in bromodomains, epigenetic “reader” proteins that bind acetylated lysine residues. In particular, they were interested in CBP (cyclic-AMP response element binding protein). Previously the researchers had identified compound 1 through virtual screening, but although this compound had sub-micromolar affinity, it showed no cell-based activity, presumably due to the carboxylic acid, a moiety usually associated with poor cell permeability. Indeed, a CBP series we discussed earlier this year that also contained a carboxylic acid had no cellular activity.

To come up with better molecules the researchers used a program they developed and named AutoCouple because it virtually “grows” a fragment using common coupling reactions such as amide formation, Buchwald-Hartwig amination, and the Suzuki-Miyaura reaction. An initial set of 270,000 commercial compounds was computationally filtered to remove large molecules and those containing undesirable moieties. Potentially self-reactive building blocks were also removed. Ultimately 70,000 virtual compounds based on growing compound 2 (the key fragment of compound 1) were designed and docked into multiple crystal structures of CBP, and 53 were actually synthesized and tested.

Four of the 33 amides synthesized were sub-micromolar, compound 5 being one example; another 17 were low micromolar. (Five of the 10 Suzuki-derived compounds were also sub-micromolar, as was at least one of the amines.) Compound 5 was improved by using information from one of the other tested molecules to generate compound 16, with low nanomolar affinity. Crystallography confirmed that this compound binds as the docking had predicted, in a similar manner to compound 1.

Happily, not only was compound 16 more potent than compound 1, it was also active in cells. Moreover, it showed reasonable selectivity against a dozen other bromodomains.

Overall AutoCouple looks like it could be a useful tool to design and prioritize compounds for synthesis. Moreover, like the growing via merging “PINGUI” approach we highlighted earlier this year, the Python scripts appear to be freely available. It would be fun to benchmark both methods on the same targets to see how they compare.

Fragments deliver (another) inhibitor for CBP and EP300

In 2016 we highlighted a chemical probe that binds two closely related bromodomains, CBP (cyclic-AMP response element binding protein) and EP300 (adenoviral E1A binding protein of 300 kDa). These proteins bind to acetylated lysine residues in various nuclear receptors and are implicated in several types of cancer. Multiple chemical probes are always nice to have, and in a new paper in Eur. J. Med. Chem., Yong Xu and collaborators at Guangzhou Medical University, the University of Chinese Academy of Sciences, Jilin University, the University of Hong Kong, and the University of Auckland go some way towards this goal.

The researchers started with a virtual screen of 272,741 fragments (MW < 300 Da) docked against CBP. The top 5000 were clustered into related subsets and analyzed manually. Of thirteen fragments purchased and tested in an AlphaScreen assay, two had IC₅₀ values better than 40 µM. Compound 6 was slightly less potent, but showed good selectivity against three other bromodomains.

The docking model of compound 6 suggested that more bulk between the indole and the carboxylic acid could be beneficial. Several molecules were made and tested, with compound 25e being the most potent. A related molecule was characterized crystallographically bound to CBP; this suppored the predicted binding mode.

Next, various small lipophilic elements were added to try to pick up additional interactions, ultimately leading to compound 32h, with low nanomolar affinity. This compound, which is equally active against EP300, also showed promising selectivity: it had no activity in a panel of six other bromodomains, including BRD9, which is inhibited by the chemical probe (CPI-637) mentioned above. Unfortunately compound 32h has no activity in cells, which the researchers speculate is due to the carboxylic acid. Masking this moiety with a tert-butyl ester causes a modest reduction in the biochemical activity but does lead to low micromolar activity in several cell assays.

Although much remains to be done, this is a nice example of advancing a computationally-derived fragment with limited structural information. I suspect we’ll see more of these, particularly for well-understood target families.

Fragments in the clinic: ABBV-075 / Mivebresib

Bromodomains bind to acetylated lysine residues in proteins to control gene transcription. These epigenetic regulators have received considerable attention as drug targets, particularly for oncology. Last year we highlighted work out of AbbVie in which fragments found in an NMR screen were advanced to two series of molecules that potently inhibit the four members of the BET family of bromodomains. A more recent publication in J. Med. Chem. by Keith McDaniel and his colleagues at the company describes how one of the fragments was transformed into the clinical compound ABBV-075, or mivebresib.

Compound 6 was not the most potent fragment identified, but crystallography confirmed that it binds in the acetyl lysine binding pocket. The earlier work described how the pyridazinone moiety was replaced with a pyridone and another phenyl ring was added to make molecules such as compound 9, with sub-micromolar activity.

Further modification of the pyridone led to compound 19, with a nearly 20-fold boost in affinity. Crystallography revealed that the pyrrolopyridone makes a bidentate interaction with a critical asparagine residue in BRD4, and also displaces a “high-energy” water molecule.

Next, the researchers sought to pick up additional interactions, and it turned out that introducing a nitrogen off the central ring was synthetically straightforward and would point substituents towards a pocket in the protein. This led to low nanomolar inhibitors such as compound 25, and crystallography revealed that one of the sulfonamide oxygen atoms makes a hydrogen bond with a backbone amide. Happily, the improvement in potency was also accompanied by an improvement in stability in liver microsome assays.

Unfortunately, although the pharmacokinetics in mice were reasonable, these compounds showed high clearance in rats. Analysis of the metabolites revealed that this was largely due to oxidation of the unsubstituted phenyl ring, so the researchers took the classic route of introducing halogen atoms to both deactivate the ring and block metabolism sites. This ultimately led to ABBV-075.

In addition to excellent potency in biochemical, biophysical, and cell-based assays, ABBV-075 showed excellent antitumor effects in a mouse xenograft assay when dosed orally at the low concentration of just 1 mg/kg. In addition to BRD4, the compound binds tightly to the other BET family members but is selective against most of the other bromodomains. It also demonstrates good pharmacokinetic properties in mice, rats, dogs, monkeys, and humans. ClinicalTrials.gov lists a Phase 1 study currently recruiting.

This is a lovely, textbook example of how structurally-enabled fragment growing combined with careful pharmacokinetic-based optimization can lead to a clinical candidate. Obviously there is a long and uncertain road ahead for the molecule prior to approval, but getting this far is a victory in itself.

Review of 2017 reviews

The year is done, and the darkness

Falls from the wings of Night.

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

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

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

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

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

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

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

Targets

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

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

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

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

Techniques

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

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

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

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

Other topics

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

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

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

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

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

And may 2018 be filled with music, and light.

Fragments vs BRD4, two ways

Bromodomains, epigenetic targets that recognize acetylated lysine residues, have received considerable attention from the fragment community. (I devoted all of last July to the topic, and covered it more recently here.) Of the dozens of bromodomain-containing proteins, the four BET-family members have been highly studied, and the second bromodomain of BRD4 (BRD4-BRDII) in particular has been implicated in cancer and inflammation. In two new papers, researchers from AbbVie describe inhibitors of this target.

In the first Bioorg. Med. Chem. Lett. paper, George Sheppard and colleagues briefly describe a protein-detected (¹³C-HSQC) NMR screen of BRD4 in which the methyl groups of isoleucine, leucine, valine, and methionine were ¹³C-labeled. About 18,000 fragments were screened in pools of 30, and hits were then tested individually in NMR and time-resolved fluorescence resonance energy transfer (TR-FRET) assays. Despite extensive work on this target by multiple groups, these screens were able to identify several new fragments, such as the related compounds 1 and 2.

Crystallography of each fragment bound to BRD4 revealed that they bind in the acetyl lysine recognition site and make contacts with the conserved asparagine residue as well as a nearby water molecule. Merging the fragments led to compound 5, with a slight increase in affinity.

Comparison with other BRD4 inhibitors suggested a growth strategy, leading to compound 15, with nanomolar activity in the TR-FRET assay and two cell-based assays. The compound was orally bioavailable but had relatively high clearance, so further medicinal chemistry focused on changing the original core. This ultimately led to compound 38, with improved oral bioavailability, lower clearance, good selectivity against non-BET bromodomains, and activity in a mouse xenograft assay.

The second paper, by Le Wang, John Pratt, and colleagues in J. Med. Chem., starts with a different fragment from the original screen, compound A1. This molecule was even weaker than the fragments described above, but crystallography confirmed that it binds in the same acetyl lysine binding pocket.

Again, comparison with known inhibitors provided ideas for fragment growing, rapidly leading to compound A11. Further medicinal chemistry – which is extensively described in the paper – led to compound A30a, which bears considerable resemblance to the series reported in the previous paper.

Crystal structures of compounds bound to the protein suggested that it might be possible to make a macrocycle, which would in theory increase the affinity by locking the molecule in a low energy conformation. This proved to be synthetically challenging but ultimately worthwhile in the form of compound A74b. (Incidentally, this is the first case I can recall where a fragment led to a macrocycle. It won’t be the last.) Not only was this molecule more potent than the open form, it also showed excellent oral bioavailability and pharmacokinetics, good selectivity against non-BET bromodomains, and even better activity in a mouse xenograft model. 

One lesson from these papers is that fragments can generate new ideas even for heavily pursued targets. A second is that, as we saw in the recent poll, crystallographic information can be critical for advancing fragments to leads. The discovery of new moieties along with clear data on their binding modes can be a powerful combination for creative medicinal chemists.

Fragments vs BRPFs: A chemical probe

Bromodomains, a type of functional module within proteins, recognize acetylated lysine residues in other proteins to act as epigenetic “readers.” Humans have 61 of them spread across 46 different proteins, and figuring out what recognizes what and in which context has been a major undertaking for the past several years. Fragment-based approaches have proven very successful: Practical Fragments devoted the entire month of last July to bromodomains. In a new paper in J. Med. Chem., a large group of academic and industry investigators led by Paul Fish describe their successful efforts to find a chemical probe for the four members of the BRPF family.

The story actually starts with a fragment screen against a different bromodomain, PCAF, which we discussed last year. Compound 5b was a hit against that target, but bromodomain fanciers will recognize this as a privileged pharmacophore against the target class, and it turned out to be even more potent against BRPF1.

Crystallography confirmed that compound 5b bound in the acetyl-lysine pocket as expected, and also revealed a potential vector for growing, as exemplified by compound 6. Introducing a methyl group led to a 10-fold boost in affinity for compound 7, which as the researchers point out is near the limit of what you can expect for hydrophobic interactions. Further growing ultimately led to NI-42, with low nanomolar potency.

Of course, potency is one thing, but when you’re dealing with dozens of related proteins you really need specificity to understand the biology. Happily, NI-42 was quite selective for members of the BRPF family. Importantly, it has only 4.5 µM activity against BRD4, which can dominate cell phenoytpes when inhibited. The selectivity is actually remarkable given the close structural similarity of NI-42 to PFI-1, which hits BRD4 and which Teddy wrote about here. The researchers suggest that the difference between the N-H in PFI-1 and the N-methyl in NI-42 drives the selectivity – so one could argue that the probe actually makes use of two magic methyls.

NI-42 also showed good cell permeability and target engagement in cells, adequate solubility, decent pharmacokinetics, and respectable oral bioavailability in mice. Although a screen of 211 cancer cell lines did not reveal stunning activity, cell models for other diseases are being evaluated. Also, the researchers have generated an inactive but closely related control simply by replacing both methyl groups with ethyl groups.

This is a lovely fragment optimization story. It is also a useful reminder that, as with phosphodiesterases and kinases, a nonselective fragment can ultimately yield a selective chemical probe.

Review of 2016 reviews

This year is finally coming to an end, and as we've done for the past four years, Practical Fragments will highlight some of the reviews that we didn't cover previously.

In terms of what we did cover, there were several excellent events, including the eleventh annual CHI FBDD Conference in San Diego, an inaugural meeting in Houston, and of course the first-ever major fragment event in Boston, FBLD 2016.

The twentieth anniversary of SAR by NMR was also commemorated by the eighth book devoted to FBLD, as well as a massive two volume work on lead generation. We also covered a special issue of Molecules and reviews on clinical candidates and library design.

Another review on library design was published recently in Drug Disc. Today by Ian Gilbert, Paul Wyatt, and colleagues at the University of Dundee. The researchers have built a set of 356 diverse compounds consisting of “capped” scaffolds, such that any hits could be rapidly expanded. Undergraduates did much of the actual library assembly, learning skills such as parallel chemistry and how to work with polar compounds. There is lots of nice detail in this paper, including on library storage conditions.

Targets

Practical Fragments often highlights successful fragment to lead programs, and these were the focus of a Perspective in J. Med. Chem. by Christopher Johnson (Astex) and collaborators: all 27 cases published in 2015 in which the affinity of a fragment was improved at least 100-fold to a 2 µM or better lead. Many of these were covered in Practical Fragments, including BTK, DDR1/2, ERK2, MELK, Mtb TMK, PKCθ, RET, FactorXIa, MMP-13, BCATm, PDE10A, soluble epoxide hydrolase, tankyrase, ATAD2, MCL-1, RAD51, XIAP/cIAP, and mGluR5. The paper also draws general conclusions about target types, molecular weights, cLogP values, and LE.

Targeting tuberculosis (TB) is the subject of two reviews from University of Cambridge researchers, one in Drug Discov. Today by Vitor Mendes and Tom Blundell and one in Parasitology by Anthony Coyne, Chris Abell, and colleagues. Fragment-based approaches have been more or less successful against several TB proteins, including pantothenate synthetase, CYP121, BioA, EthR, and thymidylate kinase, while other targets – such as shikimate kinase and CYP144 – have proven more difficult.

July was bromodomain month at Practical Fragments, and this target class is the subject of a review in Drug Discov. Today: Technol. by Dimitrios Spiliotopoulos and Amedeo Caflisch at the University of Zurich. The focus is on computational fragment screening methods, with examples for BRD4 and CREBBP. And while we’re on the topic of computational methods, Olgun Guvench of SilcsBio has a brief review in Drug Discov. Today on computational functional group mapping.

Rounding out target-focused reviews, Paramjit Arora and colleagues at New York University focus on protein-protein interactions (PPIs) in a Trends Pharm. Sci. paper. This covers multiple approaches to finding PPI inhibitors, including fragment-based, and also touches on hotspots and structure-based design.

Biophysics

It is impossible to imagine FBLD without biophysics, and this is the topic of an authoritative review in Nat. Rev. Drug Disc. by Jean-Paul Renaud (NovAliX), Chun-wa Chung (GlaxoSmithKline), U. Helena Danielson (Uppsala University), Ursula Egner (Bayer), Michael Hennig (leadXpro), Rod Hubbard (University of York) and Herbert Nar (Boehringer Ingelheim). In addition to covering all the major techniques, the paper does a great job of delving into some of the more obscure and emerging methods, providing an excellent discussion of the throughput and requirements for each technique as well as the kinds of information obtained. Although the review is broader than FBLD, the application of biophysical techniques to fragments is a major theme. The researchers also remind us that, “contrary to the belief that all drug discovery challenges are best solved through the introduction of new technologies, substantial advances can also be driven by innovative application.”

Individual biophysical techniques also received plenty of attention over the year, including three on NMR. The first, by Alvar Gossert and Wolfgang Jahnke (Novartis) in Prog. Nucl. Magn. Reson. Spectrosc., is a 44-page practical guide to identifying and validating protein ligands. This contains a wealth of information on most of the NMR methods you will ever likely encounter; it includes a handy chart summarizing the molecular weight and concentration limits for each technique, suggested workflows, and thorough discussions of potential pitfalls. The review may appear daunting to the novitiate – it is replete with equations and pulse sequences – but the writing is clear. In the end, much comes down to the concept of the “validation cross”, a rubric for assessing the integrity of both ligand and protein, and evaluating binding effects on both ligand and protein.

Two additional reviews, both from William Pomerantz and colleauges at the University of Minnesota, focus specifically on protein-observed ¹⁹F NMR. The first, a Perspective in J. Med. Chem., is a good general introduction. Despite being the 13^th most abundant element on our planet, only five natural products are confirmed to contain fluorine. Introducing this element into proteins – as has been done in more than 70 cases – can be a useful approach for discovering new ligands. And if you want to start doing this yourself, a paper in Nature Protocols provides practical details.

Turning to other biophysical techniques, surface plasmon resonance (SPR) continues to be very popular, and is reviewed by Alain Chavanieu and Partine Pugnière in Expert Opin. Drug Discov. The paper provides a good general overview on using SPR for FBLD, covering the theory, history, various screening strategies, comparison to other methods, recent applications to a variety of different targets, and a suggested workflow.

Calorimetry is less commonly used for fragment screening, even though it can provide thermodynamic data. Michael Recht and collaborators at the Palo Alto Research Center and Zenobia discuss both enthalpy arrays as well as more conventional isothermal titration calorimetry (ITC) in a Methods Enzymol. chapter.

Chemistry

But while biophysics is important, FBLD would be nowhere without chemistry. In MedChemComm, Stefan Kathman and Alexander Statsyuk (then Northwestern, now University of Houston) review one chemical approach, covalent tethering. This touches on the original reversible (thermodynamically-controlled) disulfide tethering approach developed back at Sunesis but is primarily focused on irreversible (kinetically-controlled) methods. The paper does an excellent job summarizing challenges, potential pitfalls, design rules, and recent successes. As of early this year the Statsyuk lab had sent their 100-member covalent fragment library to nine different research groups, three of which had already identified hits. The review ends with some provocative questions, and it will be fun for practitioners to answer them as covalent approaches garner increasing attention.

Another chemical technique we’ve touched on is substrate activity screening (SAS), and this is reviewed in ChemMedChem by Pieter Van der Veken and collaborators at the University of Antwerp. All published examples are summarized, including the modified approach developed by the Van der Veken lab; some unpublished data are also discussed. The paper also includes a good general section on the subtleties and complexities of transforming substrates into inhibitors.

Finally, if all this is a bit too much, a good general review on FBLD was published in Pharmacol. Ther. by Martin Scanlon and colleagues at Monash University. This concise but thorough paper covers theory, history, library design, hit finding and characterization, and select clinical success stories. The longest section is devoted to chemical strategies for elaborating fragments, and includes some of the less commonly used methods such as target-guided synthesis, Tethering, and off-rate screening.

And that’s it for this year. Thanks for reading, and especially for commenting. Take care, do important work, and may 2017 be better than we can reasonably hope.