One of the rewards of following a field for years is being able to revisit classic papers to see how they’ve held up. Two years ago we re-examined molecular complexity. This year marks the tenth anniversary of the publication of the “rule of three”, and Harren Jhoti and colleagues from Astex have marked the occasion with a brief but trenchant letter in Nature Reviews Drug Discovery.
Think back, if you will, to 2003. Abbott researchers had published their seminal SAR by NMR paper seven years previously, but fragment-based efforts were still widely scattered, with each organization more or less figuring things out on its own. It was in this primordial environment that Jhoti and colleagues published a short “discussion forum” in Drug Discovery Today. It framed its premise as a question (A ‘rule of three’ for fragment-based lead discovery?) and suggested that fragments have the following characteristics:
- Molecular weight (MW) < 300
- ClogP ≤ 3
- # of hydrogen bond donors ≤ 3
- # of hydrogen bond acceptors ≤ 3
In the new publication, the researchers note that most of the focus has been on the first two critera. Indeed, as has been pointed out, there is some ambiguity as to how one defines hydrogen bond donors and acceptors.
Since proposing the Rule of Three, Astex has been moving towards ever smaller compounds; the majority of their fragments now have fewer than 17 non-hydrogen atoms, with a molecular weight < 230 Da. One consequence is that the other properties automatically fall into line: a quick search of ~100,000 compounds with ≤ 16 heavy atoms reveals that 86% have ClogP ≤ 3, while out of 370,000 compounds with MW ≤ 300, only 72% have ClogP ≤ 3.
This push towards smallness has been questioned, particularly in the context of protein-protein interactions, where some have suggested that larger fragments may be required. Jhoti (and others) counter with two arguments.
On a theoretical level, all proteins are made up of amino acids, so there shouldn’t be anything special about protein-protein interactions:
Fragments are – or should be – simple enough to probe the basic architecture of all proteins yet have sufficient complexity to allow them to be elaborated into lead compounds.
On a practical level, after screening more than 30 targets, the researchers find that many fragments that hit protein-protein interactions also hit other targets.
The researchers are in favor of “three-dimensional” fragments, but not at the cost of increased size. They note that the perception that fragment libraries are dominated by “flat molecules” may be distorted by the fact that many fragment success stories (including nearly half of clinical-stage compounds) involve kinases, which have a predilection for planar adenine-like fragments. That said, they acknowledge that many fragment libraries are sub-optimal, leading to heartbreak during optimization. As they note with restraint, “not all fragment libraries are alike.”
Finally, there is a nice analysis of what to do with fragments that don’t reproduce in orthogonal assays. They typically observe 30%-40% correlation between fragment hits from ligand-observed NMR and X-ray crystallography, but note that this isn’t bad given that an NMR hit can be detected at just 5% binding, while crystallography typically needs at least 70% occupancy. Thus, NMR can detect fragments with solubilities less than their dissociation constants, which is unlikely in the case of crystallography. Although it is reassuring when multiple techniques confirm, the danger is that:
This strategy implicitly places a reliance on the least sensitive technique. This is of particular concern as the most potent fragment is often not the best starting point for hit-to-lead chemistry.
Not surprisingly to those familiar with Astex, the researchers put a premium on crystallographic information.
Closing with the rule of three, I think part of what bothers some folks is the notion of “rules” in general; nature has never read an issue of Nature, and drug discovery will never be as reductionist as physics is. Indeed, the researchers acknowledge that the rule of three “is just a guideline that should not be overemphasized.” The rule of three is a play on Chris Lipinski’s (equally contentious) rule of five; perhaps the “guideline of three” would have been less controversial. But the spirited discussion ensuing over the years has generated light as well as heat, which the authors welcome: