DOT1L revisited: fragment linking to picomolar binders

Last September we highlighted two papers out of Novartis in which various fragment-based methods delivered two separate series of low nanomolar inhibitors against the histone methyltransferase DOT1L, an epigenetic anticancer target. The next installment of this story, by Christoph Gaul and Novartis colleagues, has just published in ACS Med. Chem. Lett.

DOT1L uses the cofactor S-adenosylmethionine (SAM), but the previously identified fragments bind in a site adjacent to the cofactor’s binding site. Seeking to gain affinity the researchers decided to look for fragments that bind in the same pocket as SAM. SAM and ATP both contain an adenine ring, and plenty of fragment libraries have been built around the fact that ATP is a kinase cofactor. About 200 kinase-targeted fragments were tested in a high-concentration biochemical screen. Compound 2 was active, and crystallography revealed that it forms multiple hydrogen bonds with DOT1L. More importantly, crystallography also revealed that compound 2 could bind simultaneously with inhibitors occupying the site adjacent to the cofactor binding site.

Modeling suggested that it would be possible to link compound 2 with second-site binder compound 3. This proved successful, with compound 4 having low nanomolar affinity. For aficionados of linking, the free energy of binding for compound 4 is comparable to the sum of the free energies of binding for compounds 2 and 3. This level of additivity is similar to results on different targets at Ariad and Evotec. Interestingly, the two fragments (2 and 3) conform to a previous suggestion from Evotec that – to maximize the likelihood of success in fragment linking – one fragment should be polar and make multiple hydrogen bonds with the protein, while the second fragment should make largely lipophilic interactions.

But the researchers didn’t stop there. Adding a methyl group to the secondary amine on compound 4 improved affinity by more than an order of magnitude, and further tweaking the right-hand side of the molecule led to compound 7, with low picomolar affinity. This compound also showed low nanomolar activity in several different cell-based screens, and was completely inactive against a panel of 21 other methyltransferases. Selectivity against kinases is not shown, and given the origin of the left-hand fragment this could be an issue, potentially complicating the interpretation of biological assays. That said, polypharmacology can be useful, particularly in oncology.

Fragment linking has a reputation for being difficult, but this is another example that it can work. The high affinity of compound 7 is particularly remarkable considering the number of rotatable bonds. Of course, plenty of challenges likely remain: no data are provided for in vitro stability, let alone pharmacokinetics. Although the structure of compound 7 may raise eyebrows, it is worth remembering that even larger, more lipophilic molecules have become successful drugs.