You might remember my rantlet about the Rule of Three (RO3) at the beginning of the year. Well it seems that others consider Ro3 to be over-restrictive and this JMC article will be as welcome in some parts of Cambridge as a staffel of Ju87s.
The authors of this work describe screening of a library of 364 fragments against the aspartyl protease endothiapepsin and crystal structures of 11 hits bound to the target protein. The library was designed �without strictly applying the rule of 3� and, as it turns out, �only 4 of the 11 fragments are consistent with the rule of 3�. Not exactly a ringing endorsement for RO3 or a compelling incentive to buy a RO3-compliant fragment library.
Hopefully one point that you�ll have taken away from my earlier post is that those who gave us RO3 don�t say a whole lot about how they define hydrogen bond donors and acceptors so it can be difficult to say whether some fragments are RO3-complaint or not. I�m guessing that RO3�s proposers may not be be using the same definitions that are used to apply the Rule of 5 (RO5). However, I really don�t know and don�t really care.
Now you can see the problem. Do any of the 7 fragment hits (I refuse to call them frits since that term is more usually associated with ceramics than Drug Discovery and an association with ceramics is something that you�ll want to avoid for fragments) from the endothiapepsin screen that are reported to be inconsistent with RO3 actually fail to comply with the rule? Let�s take a look at the structures of two fragments for which binding to target was observed crystallographically. You�ll notice that I�ve retained the structure numbering from the article.
There are 2 nitrogens and 3 oxygens in 041 so with RO5 hydrogen bonding definitions this fragment would not be RO3-compliant. Personally, I wouldn�t count the amide nitrogen as an acceptor but that�s my choice. The cyclic ether oxygens in 041 will be very weak hydrogen bond acceptors and even three or four of these together will pack less of a punch than the typical amide carbonyl oxygen. I�d actually be much more worried about the reactivity of the aniline portion of this molecule but this is not the place for that discussion. Fragment 255 has 3 nitrogens and one oxygen which translates into 4 hydrogen bond acceptors if you use RO5 definitions. However, I would not count either the amide nitrogen or the bridging nitrogen of the fused heteroaromatic ring as acceptors so with my definitions the fragment would be RO3-compliant.
The assay and crystallisation were carried out at a pH of 4.6. This means that the heteroaromatic nitrogens of 255 and 291 are likely to be protonated to a significant extent under experimental conditions. It�s interesting that the solubility measurements were run at a pH of 7.4 because basic fragments such as 041, 255 and 291 should be even more soluble in assay and crystallisation buffers. It would be a different story for acids but I didn�t see any of those so I guess no harm done.
It�s good to see the output of a fragment screen being published in this manner and the crystal structures for a number of fragments bound to this target represent a welcome addition to the protein knowledge base. Given that I�ve never been a fan of RO3, I do like to see others questioning the rule although reading this paper gives the impression that RO3 has never before been questioned. I also believe that they could have addressed the issue of hydrogen bonding definitions rather than simply jumping to the conclusion that RO5 definitions (all nitrogens and oxygens are acceptors) were being used by those who gave us RO3.
On a final note, you might wonder why I keep banging on about RO3 when it�s something that I�ve never used to select fragments. It�s a good question and this is a good place to answer it. My own view is that the way many researchers have blindly adopted the rule is merely a symptom of a much bigger malaise in Drug Discovery research. Pharma appears to be dans la merde but the response of its leaders is typically to increase the frequency with which the Titanic�s complement of deck chairs is shuffled. Is this really a good time for those who represent the best chance of a future for the industry to be switching off their critical thinking skills?
Ko�ster et al, A Small Nonrule of 3 Compatible Fragment Library Provides High Hit Rate of Endothiapepsin Crystal Structures with Various Fragment Chemotypes. J. Med. Chem. 2011, in press. DOI