Fragments vs MDM2 – retrospectively

Protein-protein interactions (PPIs) are some of the tougher targets in drug discovery: they often have relatively flat interfaces that lack obvious small-molecule binding sites. But despite large surface areas, these interfaces often have smaller hot spots, and fragment-based approaches have succeeded against targets where traditional screening approaches have failed. In a paper in this month's issue of ACS Med. Chem. Lett., David Fry and colleagues at Roche describe a retrospective fragment study against a classic PPI (this story was also presented at the CHI conference earlier this year).

Molecules that disrupt the interaction between p53 and MDM2 have been sought since the 1990s as potential anti-cancer agents. The first such inhibitor to enter the clinic was RG7112, one of the so-called nutlins developed at the Roche Nutley site, which is sadly in the long process of closing. Although this series of compounds originated from a high-throughput screen, the researchers were interested to know whether they could have been discovered from fragments. To do so, they deconstructed RG7112 into a series of smaller pieces.

RG7112 is a “tripod-like” molecule with three hydrophobic moieties (blue, red, and green, below) that bind to subpockets on MDM2, as well as a polar “cap” (pink) that projects into solvent but is nonetheless important for activity. The researchers were unable to detect the binding of any of these individual fragments by either SPR or two-dimensional (HSQC) NMR, suggesting that each moiety alone was too weak, so the researchers tried lopping off one or two pieces from RG7112. In all cases, pared-down molecules containing three out of the four moieties led to molecules with affinities in the 14 to 1000 micromolar range. The tipping-point seemed to be when they kept just two of the four component fragments: in two cases the resulting molecules showed no detectable binding, while in two other cases they did. Compound 5, in fact, was a decent hit by any measure, and crystallography revealed that this molecule binds in the same manner as RG7112.

Interestingly, compound 5 makes interactions with two adjacent subpockets, including a deep hole that seems to be critical for all MDM2 binders; p53 inserts the indole moiety of a tryptophan into this site.

However, compound 5 does teeter on the edge of what could be called a fragment: with a molecular weight of 305 it falls just outside the Rule of Three (sorry Teddy!), and with 20 non-hydrogen atoms it sits right on the border of what most people seem to include in their libraries. The researchers suggest that larger fragments may be necessary for tackling PPIs, though they recognize that doing so will increase the number of fragments necessary to sample chemical space.

This is a beautiful, thorough study, but I do worry about super-sizing fragments. There are now multiple success stories of finding and advancing fragments against PPIs, including such “teflon-targets” as Mcl-1. Perhaps the nutlins would not have been discovered starting from small fragments, but with hundreds of billions of possibilities there are certainly other, even more ligand-efficient leads out there waiting to be discovered.