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.

(Not) getting misled by crystal structures: part 5 – conformational heterogeneity

It’s been a while since the last installment in our “getting misled” series. One of the key issues with crystallography is that ligands are almost always modeled as binding in a single conformation. This does not necessarily reflect reality, as we discussed here. Indeed, as described here and here, subtle changes can cause ligands to dramatically change their binding modes, which could reflect the fact that the initial ligand itself had multiple binding modes, and the change simply shifts the equilibrium. In an effort to proactively seek out disparate binding conformations, Henry van den Bedem and a group of collaborators from Stanford, UCSF, Schrödinger, and Université Paris-Saclay have created a new program, which they describe in J. Med. Chem. (See here for In The Pipeline’s discussion.)

The open-source program, called qFit-ligand, starts with an existing protein-ligand structure and an electron density map. It first breaks the ligand into rigid fragments (such as rings) and rotatable bonds. Each rigid group is then allowed to move around and rotate to fit the density. Of course, this might entail the rest of the molecule moving as well to avoid bumping into the protein; up to five positions are stored for each rigid group. Combinations that best match the electron density are retained: for a ligand with three rigid groups, 15 conformations would be considered. Importantly, the entire process is automated.

The researchers validated qFit-ligand against a set of 73 reasonably high-resolution structures from the protein data bank (PDB) that had included two different binding conformations; they started with just one of the reported conformations and used their program to find the second. qFit-ligand was very effective at identifying cases where a terminal portion of the molecule had flipped or rotated, though less so for more difficult cases such as displacement of the entire ligand.

Next, the researchers turned to the D3R dataset of 145 high-quality, manually curated crystal structures, where qFit-ligand correctly identified 7 of the 10 structures with alternate conformations, and even identified an alternative conformation for a ligand that had not previously been detected.

The researchers then examined a large set of crystal structures that had been flagged as potentially dubious, and found several could be improved by including alternate conformations. Similarly, an examination of all 126 crystal structures of BRD2-4 bromodomain-ligand complexes in the PDB revealed that 12 almost certainly had previously undetected alternate binding conformations; another 24 likely did.

qFit-ligand strikes me as a powerful tool for getting beyond the static picture usually presented by crystallography. Because the program is automated, the researchers note, it should be complementary to high-throughput approaches such as PanDDa (which we described here). Of course, using qFit-ligand effectively assumes that everyone is aware of the potential for both false positives and negatives. As the researchers conclude, “communication between structural biologists, computational chemists, and medicinal chemists remains a requisite for successful, rational design.”

Fragment events in 2019

Happy New Year! Lots of exciting events scheduled this year, many of them in the first half.

March 20-22: Although not exclusively fragment-focused, the Sixth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and will be held in the nice city of Nice Cannes. You can read my impressions of the 2018 event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here.

March 24-26: The Royal Society of Chemistry's Fragments 2019 will be held in the original Cambridge. This is the seventh in an esteemed conference series that alternates years with the FBLD meetings. You can read my impressions of Fragments 2013 and Fragments 2009.

April 9-10: CHI’s Fourteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting here, here, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here. Also, Ben Davis and I will teach an FBDD short course on April 8, and it comes with dinner!

April 28-May 1: If you're looking for an even more intensive course, the 15th EFMC Short Course on Medicinal Chemistry is entitled "Small Becomes Big in Medicinal Chemistry: Fragment-based Drug Discovery." This will be held near Leiden, and as the number of participants is limited to 35, you should register early.

May 19-21: Structures are often critical for FBLD. The Second Annual Industrial Biostructures America Conference, which will be held in La Jolla, CA, is sponsored by Proteros and will cover a range of structural techniques.

September 1-4: BrazMedChem2019 will be held in the Brazilian city of Pirinopolis, and I know there will be some FBLD-relevant content.

November 12-15: The third FBDD Down Under will take place in Melbourne, and given the success of the first, it should be excellent.

Fourth Quarter: If you can't make it to Nice, NovAliX will also be holding a biophysics meeting for the first time in Japan, likely Osaka or Tokyo, sometime between mid-October and early December. Stay tuned for further details.

Know of anything else? Add it to the comments or let us know!