Fragments vs DOT1L, two ways

This past July Practical Fragments was devoted almost entirely to bromodomains, an important type of epigenetic protein. Protein lysine methyltransferases (PKMTs) are another significant class: 51 human enzymes that transfer a methyl group from the cofactor S-adenosylmethionine (SAM) to the side chain amine of lysines, typically in histones. In two recent papers in ACS Med. Chem. Lett., researchers from Novartis describe how they discovered inhibitors of DOT1L, a target for certain leukemias.

The first paper, by Frédéric Stauffer and colleagues, started with a fragment screen of the DOT1L catalytic domain using surface plasmon resonance (SPR). This led to the discovery of compound 1, which is teetering on the edge of molecular obesity (at least for a fragment) but did show activity in a functional assay as well as binding by NMR. Moreover, a co-crystal structure revealed that it binds in a new pocket near the SAM binding site, primarily through hydrophobic and stacking interactions.

Replacing the potentially unstable pyrrole with a quinoline led to compound 3, and subsequent structure-based design led to compound 5. Interestingly, while the methoxy substituent on compound 5 was installed to form a hydrogen bond with the protein, this instead caused a shift in binding mode – a reminder that fragments don’t always retain their original orientations during optimization. This new binding mode provided a vector to grow through a narrow channel into another pocket, ultimately resulting in compound 8, with low nanomolar activity.

The second paper, by Christoph Gaul and colleagues, started with a high-throughput screen (HTS). One low micromolar hit turned out to be a (felicitous) regioisomeric impurity from a commercial supplier. Crystallography revealed that this binds in the same pocket as the fragment in the previous paper, and subsequent medicinal chemistry led to low nanomolar inhibitors such as compound 3’. Unfortunately these turned out to have low permeability, probably due to the high number of hydrogen bond donors and acceptors. Fragmenting compound 3’ led to compound 4’, with a dramatic loss in potency, but structure-based design ultimately led to potent molecules such as compound 12’. This compound is also selective against other PKMTs, cell active, and orally bioavailable in rats.

These two papers provide a nice window into the complexity of lead discovery. In contrast to other examples, the fragment made largely hydrophobic interactions, while the HTS hit made numerous hydrogen bonds. Both hits bound in a new pocket, a reminder that secondary ligand binding sites are common. And in both cases, extensive medicinal chemistry was necessary and led to molecules that scarcely resemble their starting points. Interestingly, a previously described clinical candidate against this target, EPZ-5676, was identified by yet another approach: structure-based design starting from the cofactor SAM. All of which is to say that there are lots of ways to find inhibitors, and they don’t always fall into neat categories.

Fragments vs GSK3β via DOS

Diversity-oriented synthesis, or DOS, enables the rapid and systematic synthesis of multiple related compounds from small sets of molecules and reactants. By creatively choosing the chemistry, DOS practitioners can selectively generate all diastereomers and produce more complicated molecules than are usually found in commercial screening collections. While much of the attention has been focused on larger molecules, DOS offers clear applications for addressing the chemistry challenges of FBLD. This is illustrated nicely by a recent paper in ACS Med. Chem. Lett. by Alvin Hung, Damian Young, and collaborators at the Broad Institute, Harvard, the Albert Einstein College of Medicine, A-STAR, and Baylor College of Medicine.

The researchers started with a very small (86 fragment) library, which Damian is in the process of expanding to 3000 compounds. Differential scanning fluorimetry was used to screen the molecules against the kinase GSK3β, which is implicated in cancer and Alzheimer’s disease. Three related fragments slightly increased the melting temperature of the enzyme, of which the simplest was compound 1S.

One nice feature of DOS is that – by design – analog synthesis is straightforward. Thus the researchers made a dozen or so derivatives to flesh out the SAR. This revealed that the enantiomer, compound 1R, stabilized the protein even more than the initial hit. STD and WaterLOGSY NMR confirmed binding, and isothermal titration calorimetry (ITC) revealed modest but measurable affinity. Synthesis of a few additional analogs led to compound 15R, with low micromolar affinity as assessed both by ITC and an enzymatic assay. Ligand efficiency was also good, though the ligand efficiency by atom number (LEAN) values of the molecules do not quite meet Teddy’s Safran Zunft Challenge – a wager due to be settled at FBLD 2016 in a few weeks.

A key selling point of DOS is that, by accelerating chemistry, it enables optimization even without structural information. In this case the researchers suspected that the fragment binds in the hinge region of the kinase, and subsequent crystallography revealed that this was indeed so. Interestingly though, the quality of the crystal structure was insufficient to unambiguously place compound 1R; perhaps it binds in multiple conformations. The crystal structure of compound 15R, on the other hand, was clear.

Of course, there is still a long way to go for this series, and it remains to be seen how broadly applicable DOS will be for FBLD. I look forward to seeing additional examples.

Improving FBLD at AstraZeneca

FBLD started early at AstraZeneca (AZ). The first conference Practical Fragments covered was held at their erstwhile Alderley Park site; Pete Kenny is an AZ alum, and the company has put at least four fragment-derived drugs into the clinic. Clearly their scientists have learned plenty about what works and what doesn’t, and much of this wisdom is distilled into an excellent recent review in Drug Discovery Today. The authors include Nathan Fuller, Joe Patel, and Lorena Spadola, the first two of whom are organizers for the upcoming FBLD 2016 – for which there is still just barely time to register.

Things weren’t easy in the beginning: of the 63 FBLD targets screened between 2002 and 2008, a mere 10% led to tractable lead series with interpretable SAR. This improved to 37% of the 19 campaigns conducted between 2009 and 2011, and to 64% of the 11 projects between 2012 and 2014.

What accounts for these improvements? Target selection certainly played a role. In earlier years many targets were dropped due to portfolio reasons or lack of validation – nearly half for the period 2009-2011. Often FBLD was tried in desperation when all else failed, and chemists were not always available for fragment-to-lead efforts. Today, fragment screening is considered for all water-soluble targets at AZ, and fully integrated teams are brought into the process earlier. In 2012 the company established a team of medicinal chemists dedicated to FBLD – a strategy that has also been used at other companies.

But many of the improvements are technological rather than organizational. Biophysical screens are displacing high-concentration biochemical screens, which are particularly prone to false positives and false negatives. 1D and 2D NMR remain mainstays, but SPR and X-ray crystallography are increasingly being used in primary screens.

Another major effort was revamping the fragment library, which currently stands at 15,000 members. Each fragment was experimentally confirmed to be soluble to at least 0.5 mM in water and 100 mM in DMSO, and the rule of three was used more as a guideline than a rule. The collection was designed to include a good proportion of “three-dimensional” fragments, as assessed by plane of best fit (PBF) and principal moment of inertia (PMI). About a quarter of the fragments are proprietary, and the company also has another 750,000 molecules within their corporate collection that could be classified as fragments, greatly facilitating follow-up studies.

A 15,000 member library is atypically large, but in practice smaller subsets of the library are deployed: 384 for crystallographic screening, 1152 for NMR screening, and 3072 for SPR screening. Each subset is optimized for the technique. For example, because the crystallographic subset is so small, it is designed to sample chemical space as efficiently as possible. This is done by maximizing the diversity of the fragments and choosing the smallest fragments possible – less than 17 non-hydrogen atoms, as at Astex. In contrast, the NMR and SPR subsets contain fragments having up to 21 non-hydrogen atoms, and the SPR set also contains close analogs of some fragments to improve confidence and provide preliminary SAR. There is some overlap between the sets to facilitate confirmation; for example, a 768-member “ligandability set” is shared between the NMR and SPR screening libraries. Finally, AZ has built a customized set of 800 covalent fragments.

For the most part, fragment hits from each subset tend to have similar properties as the subset in general, suggesting that each sub-library is well-suited for its technique. Importantly, this is true even for three-dimensional fragments, which comprise nearly half of the hits across 19 targets. The researchers also examined how effectively fragments were able to fill the volume of a given binding pocket for five targets with multiple crystal structures. They found that shapely fragments were at least as good as – and sometimes better – at filling the pockets, even with fewer three-dimensional fragments.

Finally, the article summarizes eight projects in which fragment hits were progressed. Dissociation constants for the hits ranged from 50 to 3230 µM; these were advanced to leads with affinities ranging from 1.5 to 180 nM. In half these cases the ligand efficiency improved, and in all cases the three dimensionality increased as defined by PBF. Two of the targets, phosphoglycerate dehydrogenase and mInhA, are discussed in some detail, complete with chemical and crystal structures. Hopefully all will be covered more fully in upcoming publications.

There’s lots more in this paper than I can summarize in a blog post, including multiple figures and tables, so definitely check it out.