01 March 2021

Fragments vs MEK1: allosteric binders

MEK1 is a central player in the MAP kinase signaling cascade, which is often dysregulated in cancer. As such the enzyme has been the focus of considerable research and the target of four approved drugs. Interestingly, these drugs bind not to the hinge region targeted by most kinase inhibitors but rather to an allosteric pocket adjacent to the ATP binding site. The drugs also look somewhat alike. Seeking something completely different, Paolo Di Fruscia, Fredrik Edfeldt, Helena K├Ąck, and colleagues at AstraZeneca turned to fragments. They have recently published their results in ACS Med. Chem. Lett.
As we discussed in 2016, the AstraZeneca fragment library is quite large at 15,000 molecules. The researchers used a computational screen to narrow this down to a more manageable 1000 compounds for ligand-detected NMR screening. AMP-PNP, a nonhydrolyzable version of ATP, was included to block the hinge region, biasing the screen for fragments that bind the allosteric site. (See here for earlier work looking for ATP-competitive molecules.) A total of 142 fragments were identified and further characterized by SPR, and 46 showed dissociation constants better than 1 mM and similar affinities in both the presence and absence of AMP-PNP, suggesting they do indeed bind in the allosteric site.
Crystallography was attempted on all the fragments, but only two produced structures. Reassuringly, both bound in the allosteric site. But with only limited structural information, the researchers tested analogs of the fragment hits within their corporate collection. This identified compound 10, which is more potent than initial fragment 3. Moreover, compound 10 lends itself well to library synthesis.

All library members were initially made and tested as racemates. When the two enantiomers of the best hit were separated, compound 23 was found to be a sub-micromolar binder, roughly 100-fold better than the other enantiomer. At this point the researchers finally obtained a crystal structure of compound 23, confirming that it did bind in the allosteric pocket. Compound 23 is also still fragment-sized, just three heavy atoms larger than compound 3.
The astute reader will notice that the word “inhibitor” has not appeared until now, and indeed despite the encouraging affinity no mention is made in the paper of inhibition – a rather important feature! At a conference in 2019 Paolo did describe further optimization to a functional molecule, so hopefully we will see a second publication detailing this work.
Like the NPBWR1 story last month, this is another nice example of advancing fragments in the absence of structural information. It is also a good case study of fragments yielding completely different chemical matter in a crowded field.

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