Multiple bromodomains, multiple methods, and even more fragment hits

All this month Practical Fragments has been focused on bromodomains, highlighting chemical probes against BRD9, CBP and EP300, and family VIII bromodomains. Today’s post covers three earlier-stage programs on three different bromodomains.

In Acta Pharm. Sinica, Bing Xiong, Nai-xia Zhang, and colleagues at the Chinese Academy of Sciences discuss their work on BRD4, an anti-cancer target about which we’ve written previously. The researchers describe the construction of a fragment library designed for NMR screening; this is a good resource for people undertaking similar efforts. Interestingly, of 800 compounds purchased, only 539 were soluble to at least 100 µM in aqueous buffer. These were pooled into 56 groups of 8-10 compounds and screened at 200 µM (total fragments) using STD and T1ρ. This yielded 10 hits, of which three had measurable IC₅₀ values from 110 to 440 µM. Five of the hits were characterized in more detail using two dimensional NMR (¹H-¹⁵N HSQC), and three by X-ray crystallography. Some of these fragments are less-precedented as bromodomain ligands, and could be useful starting points for further work.

In contrast to BRD4, for which multiple ligands have been reported, the bromodomain on BRPF1 is less explored. In a recent paper in J. Med. Chem., Jian Zhu and Amedeo Caflisch (University of Zürich) provide 20 new co-crystal structures, all of which have been deposited in the protein data bank. The researchers performed a computational screen of 24,133 molecules using a program called SEED, which was able to crank through the entire set in just a day. Crystal soaking was attempted with thirteen of the top 30 hits, resulting in five structures, of which three bound in the manner predicted. Crystal structures of another 15 analogs and other bromodomain inhibitors were also determined. Some of the molecules are reasonably potent, with double-digit micromolar affinities and good ligand efficiencies.

Finally, while most bromodomains have a conserved asparagine residue that makes hydrogen bonds to the substrate (or inhibitor), 13 of the 61 known human bromodomains do not, and these tend to be more difficult targets. The second bromodomain of the pleckstrin homology domain-interacting protein (PHIP(2)), which has been implicated in melanoma, is one of these “atypical” bromodomains. Researchers at the Structural Genomics Consortium (SGC) led by Frank von Delft (Diamond Light Source) and Paul Brennan (University of Oxford) took a crystallography-first approach toward this target, as they report in an open-access paper in Chemical Science.

The researchers started by assembling what they call a “poised fragment library”. This is essentially a library designed for rapid follow-up chemistry, in which each library member can be deconstructed into individual components, which can be systematically varied. For example, a fragment might consist of two moieties connected by an amide bond, so that analogs could be easily made using parallel synthesis. The initial 2347 fragments were a subset of the 11,677 fragments available in-house or through collaborators, but the researchers also identify a set of 10,448 commercially available poised fragments. Commendably, they also provide full identities of both sets of fragments, which could be useful for folks building or adding to their own collections.

The Diamond Light Source is able to crystallographically screen 1000 fragments per week, but in this case only 406 diverse fragments were tested. Rather than using the nearly universal DMSO as a solvent, the researchers dissolved their fragments in ethylene glycol, since DMSO actually binds to bromodomains. Previous solution-phase screens of PHIP(2) at the SGC had come up empty, so the crystallographic screen was done at the very high concentration of 200 mM. Not surprisingly, this yielded just four hits.

Each of the hits bound in the acetyl-lysine recognition pocket, and three of them even showed high-micromolar activity in an AlphaScreen assay, with impressive ligand efficiency values. A few dozen analogs were made, which led to slight increases in activity in all cases, and measurable activity for analogs of the fragment which had shown no activity by itself. Although there is still a long way to go to find chemical probes for PHIP(2), at least there are now good starting points.

And that concludes bromodomain month. The number of papers and chemical probes that have come out just this year are a testament to the power of fragments to tackle this class of targets, perhaps equaled only by kinases. And while I'm not aware of any clinical candidates targeting bromodomains that started as fragments, I'm sure these will be coming soon.

Fragments deliver a chemical probe for Family VIII bromodomains

Today’s post continues the theme of July as bromodomain month at Practical Fragments. The 61 human bromodomains (found in 46 proteins – some proteins have more than one) have been divided into eight families based on their sequences. Family VIII contains ten members, some of which are involved in keeping stem cells from differentiating. Two papers describe chemical probes that target some or most members of this family.

The first paper, which actually came out last year in Science Advances, is from a multinational group including Thomas Günther (Universität Freiburg), Stefan Knapp and Susanne Müller (both University of Oxford) and collaborators at Pfizer. The researchers started by screening libraries of acetyl lysine mimetics that had yielded inhibitors against other bromodomains. These came up empty; even promiscuous bromodomain inhibitors failed to hit Family VIII members. As is so often the case, when all else fails, the researchers turned to fragments. A thermal shift assay revealed that salicylic acid – the polypharmacological metabolite of aspirin – binds to the bromodomain PB1(5). Isothermal titration calorimetry (ITC) confirmed this result, providing a dissociation constant of 250 µM.

The researchers were also able to obtain a crystal structure of PB1(5) bound to salicylic acid in the acetyl lysine binding site common to all bromodomains, with the carbonyl making the usual hydrogen bond with a conserved asparagine. But whereas most other bromodomain binders make a water-mediated bridge to a conserved tyrosine, the phenol makes a direct hydrogen bond. The benzene ring also binds deeper in the pocket, displacing four highly conserved water molecules.

The subsequent medicinal chemistry optimization of this fragment is described in a paper published earlier this year in J. Med. Chem. by Dafydd Owen and colleagues at Pfizer, along with collaborators at the University of Oxford, DiscoveRx, Eurofins, the University of Massachusetts Worcester, and Johann Wolfgang Goethe University. Testing commercial and proprietary analogs of salicylic acid quickly revealed that uncharged enamides such as compound 2 were more effective at stabilizing PB1(5) against thermal denaturation than salicylic acid, and crystallography confirmed a similar binding mode.

Two rounds of library synthesis were conducted, first with 130 amines and then with 320 amines, with physicochemical properties of target compounds chosen in advance such that cLogP would range between 1 and 4. Seven family VIII bromodomains were screened in parallel, and compounds were identified with differing specificities. Some of the compounds were unstable in water, but introducing steric hindrance around the amine improved stability and led to compounds such as PFI-3. This is potent against the family VIII bromodomains PB1(5), SMARCA2A, and SMARCA4 and did not hit at least 40 other bromodomains tested. A related compound is active against more of the family VIII bromodomains while still maintaining good selectivity against other bromodomains.

Both of these probes are able to bind to family VIII bromodomains in cells and were used to explore the proteins’ biological roles. A variety of cellular phenotypic assays showed minimal changes, and the compounds do not appear to be toxic. They did attenuate myocyte or adipocyte differentiation, while PFI-3 caused embryonic stem cells to differentiate. One gets the impression that the researchers were hoping for more profound effects, but that’s why you make chemical probes in the first place. Whether or not these compounds will ultimately prove useful as drug leads, they should help to unravel some fiendishly complex biology.

Fragments in the clinic: 2016 edition

There’s a new FBDD review out today in Nat. Rev. Drug Discovery. I know - there are lots of reviews each year - but this one is written by a who's who list of luminaries, including Steve Fesik (Vanderbilt), Rod Hubbard (Vernalis and University of York),  Wolfgang Jahnke (Novartis), and Harren Jhoti (Astex). I'm also an author so I'm undoubtedly biased, but I think it provides a nice overview of the field, especially for those who don't have time to read the recent book.

The review distills hard-won wisdom from two decades of work and covers practical decisions needed when using fragments: library design, screening methods, protein-ligand interactions, hit to lead strategies, and applications. Another useful feature is what I believe to be the most complete and up-to-date list of fragment-derived drugs that have entered clinical development. Where possible these include chemical structures, so definitely check it out.

The drugs themselves are listed below. Although it has not even been two years since the last compilation, it is exciting to see several promotions and new entrants. This table includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). A few compounds from earlier lists have been removed because their fragment origins could not be confirmed. Drugs reported as still active in clinicaltrials.gov, company websites, or other sources are in bold, and those that have been discussed on Practical Fragments are hyperlinked to the most relevant post.

Drug	Company	Target
Approved!
Vemurafenib	Plexxikon	B-Raf(V600E)
Venetoclax	AbbVie/Genentech	Selective Bcl-2
Phase 3
PLX3397	Plexxikon	FMS, KIT, and FLT-3-ITD
Verubecestat	Merck	BACE1
AZD3293	AstraZeneca/Astex/Lilly	BACE1
Phase 2
AT7519	Astex	CDK1,2,4,5,9
AT9283	Astex	Aurora, JAK2
AZD5363	AstraZeneca/Astex/CR-UK	AKT
Erdafitinib	J&J/Astex	FGFR1-4
Indeglitazar	Plexxikon	pan-PPAR agonist
LY2886721	Lilly	BACE1
LY517717	Lilly/Protherics	FXa
Navitoclax (ABT-263)	Abbott	Bcl-2/Bcl-xL
NVP-AUY922	Vernalis/Novartis	HSP90
Onalespib	Astex	HSP90
Phase 1
ABL001	Novartis	BCR-ABL
ABT-518	Abbott	MMP-2 & 9
ABT-737	Abbott	Bcl-2/Bcl-xL
ASTX660	Astex	XIAP/cIAP1
AT13148	Astex	AKT, p70S6K, ROCK
AZD3839	AstraZeneca	BACE1
AZD5099	AstraZeneca	Bacterial topoisomerase II
BCL201	Vernalis/Servier/Roche	BCL-2
DG-051	deCODE	LTA4H
IC-776	Lilly/ICOS	LFA-1
LP-261	Locus	Tubulin
LY2811376	Lilly	BACE1
PF06650833	Pfizer	IRAK4
PLX5568	Plexxikon	kinase
SGX-393	SGX	BCR-ABL
SGX-523	SGX	Met
SNS-314	Sunesis	Aurora

The current list contains more than 30 clinical-stage drugs but is certainly incomplete, particularly in Phase I. If you know of any others (and can mention them) please leave a comment.

Fragments deliver a chemical probe for CBP and EP300

As we mentioned last week, July is bromodomain month at Practical Fragments. Today we’ll start by looking at two closely related bromodomains, one found in cyclic-AMP response element binding protein (CBP) and another from adenoviral E1A binding protein of 300 kDa (EP300). Both proteins have been implicated in a variety of diseases, particularly cancer, so a chemical probe would be very valuable.

Alexander Taylor and collaborators at Constellation Pharmaceuticals, Genentech, and WuXi, describe such a probe in a recent paper in ACS Med. Chem. Lett. The researchers screened about 2000 fragments in a thermal shift assay using 0.8 mM of each fragment. Compounds that increased the melting temperature of the CBP bromodomain by at least 1° C were validated first by time-resolved fluorescence resonance energy transfer and then by ¹⁵N HSQC NMR, ITC, and X-ray crystallography. Compound 1 was one of the more attractive hits, in particular because it was considerably less active against BRD4, whose inhibition causes all sorts of changes to cells.

Crystallography of the racemic compound clearly showed that only one of the enantiomers bound, and this was confirmed in functional assays when both enantiomers were tested separately. The active enantiomer makes some of the same interactions typical of all bromodomains with the natural ligand (N-acetylated lysine). Fragment growing was attempted off the aromatic ring, and although several vectors were tolerated, most decreased selectivity against BRD4. However, close examination of the structures revealed a promising vector that led to compound 14, with good selectivity against BRD4. Further optimization ultimately led to CPI-637, with low nanomolar activity against both CBP and EP300 as well as good cell-based activity. Crystallography revealed that this compound binds in a similar manner as the initial fragment.

The selectivity of CPI-637 against other bromodomains is also good (> 700-fold less active against BRD4), though it does hit BRD9 with sub-micromolar activity. Just as with the initial fragment, the opposite enantiomer of CPI-637 is considerably less active. Although no pharmacokinetic data are provided, at the very least this should be a useful probe for cell-based studies.

Switching gears to another aspect of CBP, the multidomain protein p300/CBP-associated factor (PCAF) has a bromodomain that may bind to CBP, though the biology is not entirely clear. PCAF is known to bind an acetylated HIV protein, and has been proposed as a target for AIDS. Obviously this is another opportunity for a chemical probe! The first steps are reported in a paper by Stefan Knapp and collaborators at Goethe University Frankfurt, University of Oxford, Leiden University, ZoBio, and University of Cambridge, published in J. Med. Chem (and open-access).

The researchers screened two separate fragment libraries using either thermal shift assays (at 1 mM fragment) or TINS. Hits were confirmed using SPR and crystallography, resulting in seven structures. As expected, all the fragments bound at the site where N-acetylated lysine normally binds. The PCAF bromodomain appears to be quite rigid, with little movement in structures with the different bound fragments. A few elaborated molecules were tested, with the best showing low micromolar affinity as assessed by ITC; crystal structures with these molecules are also reported and deposited in the protein data bank. It will be fun to see whether their potency can be improved.

We’ll have another post on bromodomains next week, but first stay tuned later this week for an updated list of fragment-derived drugs that have entered the clinic.

Fragments deliver a chemical probe for BRD9

Bromodomains have nothing to do with bromine. Rather, they are small (~110 amino acid) domains that recognize acetylated lysine residues, a common modification on histones, and are thus key epigenetic “readers”. Humans have more than 60 of them, and as you can imagine selectivity is not assured. However, fragments have proven very useful in targeting these proteins. Since the first mention of bromodomains on Practical Fragments back in 2011 the number of posts has been growing rapidly, so for the first time ever we’ve decided to devote an entire month to the topic.

In other words, July is bromodomain month! We’ll start with two papers against the bromodomain BRD9, part of the SWI/SNF chromatin remodeling complex that seems to be important for acute myeloid leukemia.

The first paper, in J. Med. Chem. (and open access), is published by Laetitia Martin and collaborators at Boehringer Ingelheim, University of Oxford, and Cold Spring Harbor. The researchers used three orthogonal biophysical screening methods: differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and microscale thermophoresis (MST). A library of 1697 fragments was screened at 0.4 mM (DSF), 0.1 mM (SPR) or 0.5 mM (MST), and hits were then validated using ¹⁵N HSQC NMR. The 77 hits that confirmed were taken into crystallography, producing 55 structures.

Validation rates in the NMR secondary screen were excellent for DSF (94%) and SPR (84%) but less so for MST (31%). That said, of the 38 validated hits from MST, 29 were not found in either of the other techniques, and 14 of these produced crystal structures. This is a useful reminder that while screening cascades can whittle down many hits, they do run the risk of throwing out the proverbial babies along with the bathwater.

In parallel with the biophysical screens, a virtual screen of ~73,500 fragments was conducted using Glide to identify 208 fragments that were then tested using SPR and DSF. This led to 23 hits, 11 of which produced crystal structures.

Two of the more potent fragments were the structurally related compound 3 (from the biophysical screen) and compound 4 (from the virtual screen). Optimization started with compound 4 by adding electron donating groups to the phenyl ring to try to improve a stacking interaction observed in the crystal structure. This led to compound 10, and building out the other ring to make it more similar to fragment 3 led to BI-9564.

BI-9564 has low nanomolar activity in both a biochemical assay as well as isothermal titration calorimetry (ITC). It is also quite selective: among 48 other bromodomains, it only hits the closely related BRD7 and CECR, and it is >10-fold more potent on BRD9. None of a panel of 321 kinases were inhibited with IC₅₀ < 5 µM, and only 2 of 55 GPCRs were inhibited. The compound is also cell active, reasonably soluble, has good pharmacokinetics in mice, and orally bioavailable. In short, BI-9564 is an excellent chemical probe – and is in fact being offered as such.

While we’re on the subject of BRD7 and BRD9, it’s worth noting another recent paper, this one in ChemBioChem from Ke Ruan and colleagues at the University of Science and Technology of China. The researchers screened their library of 890 fragments against BRD7 using three different ligand-detected NMR techniques: STD, WaterLOGSY, and CPMG. Fragments were screened in pools of 10 with each fragment present at 400 µM. This yielded just 10 hits, of which 5 confirmed when tested individually. Protein-observed NMR was then performed on these, suggesting that they all bind in the acetyl-lysine recognition sites; they have similar affinities for both BRD7 and BRD9, with dissociation constants between 22 and 600 µM. Crystallography confirmed the binding mode for one of the fragments bound to BRD9. Interestingly, this showed quite a bit of plasticity in the protein compared to the un-liganded structure. Indeed, the BI researchers suggest that different degrees of protein flexibility between BRD7 and BRD9 could account for the selectivity differences observed for BI-9564.

Stay tuned next week for more fragment-screening against a different class of bromodomains!