Given the huge influence of Ro5, it was only natural that others would attempt to cash in on some of this influence. If Ro5 is indeed a Sacred Cow then perhaps it is not too unfair to suggest that some have sought to milk it. We illustrate this phenomenon with a discussion of The Rule of 3 which follows on from our previous post on Ro5.
Ro3 was published in 2003 as a short item in a journal discussion forum. The rule applies to the selection of compounds for fragment screening (see review article and posts from StrictlyMedicinal and Whistling in the Wind ). We know little about this subject and have no experience whatsoever in the area. However we maintain the finest traditions of the pharmaceutical industry by not letting this inhibit us from having opinions on the subject.
Ro3 effectively scales down Ro5 for fragment screening libraries. The rule suggests that hits from fragment screening have MW<300Da, ClogP <=3, HB-donors <=3 and HB-acceptors <=3. The rule's creators also suggest keeping rotatable bonds and polar surface area below 3 and 60Ang**3 respectively. As presented, Ro3 raises questions about how the hydrogen bonding groups are defined. You will recall that Ro5 defines all N and O as acceptors and all NH and OH as donors. This can be can be criticized (should tertiary amide nitrogen be classed as an acceptor?) but at least the donors and acceptors are specified with sufficient precision to allow all but the most innumerate to establish whether a molecule breaks the rule. If Ro3 is using Ro5 definitions (all donors are also acceptors) of hydrogen bonding, then the restriction of <=3 donors is completely redundant because it is enforced by <=3 acceptors.
Now let’s take a look at tetrazole, the classic carboxylic acid isostere found in the Angiotensin II receptor antagonist candesartan. There are 4 nitrogen atoms in the tetrazole ring (the name is a bit of a giveaway) and Ro5 would count 4 acceptors and 1 donor. So if Ro3 uses the Ro5 hydrogen bonding model, tetrazoles would miss the fragment screening fun and as would a number of acidic sulfonamides. Looking at tetrazole a bit differently you might say that the nitrogen with the hydrogen isn’t really an acceptor because its lone pair participates in the aromaticity of the ring while the lone pairs of the other nitrogens are mere spectators. However a tetrazole will ionize under physiological conditions to give an anion in which all 4 nitrogens can now function as hydrogen bond acceptors. Confused? Don’t worry, so are we!
So we still haven’t decided whether Ro3 allows us to put 5-phenyltetrazole into the screening library that we’re building. It’s a must win project (aren't they all?) and all known ligands are anionic. Acidic sulfonamides may be similarly forbidden and we’re not finding Ro3 a whole lot of help right now. Our view is that if you’re going to publish a rule based on counting things you do need say exactly what those things are. Especially when people are going to cite your rule in the literature and market screening libraries based on your rule. Are we being overly pedantic? We will let you, the reader, decide.
Perhaps we can answer some of the questions by doing some literature searching. Most of Ro3’s creators were re-united in a 2006 publication which does cite Ro3, suggesting that it still represents their views to some extent. Now go back and check the link again and take a really, really good look at it. Count the nitrogens in the fragment in the graphical abstract with the IC50 of 0.33mM. One, two, three, four! Everyone get four? Excellent! What an attentive class you've been! If Ro5 definitions of hydrogen bonding are used it would appear that Ro3's creators exploiting a fragment that violates their own rule. How very naughty that would be! As an aside, we hope that you’ve noticed that this tetrazole is different from the candesartan tetrazole because it is linked thru nitrogen and can’t ionize. We don’t think that this nitrogen will actually function as an acceptor although it will augment the acceptor ability of the other nitrogen atoms. But this is not the Ro5 model of hydrogen bonding. The view from The Grassy Knoll might be that the real purpose of Ro3 is to spread confusion and dissuade others from using fragments that its creators would prefer to reserve for their own use. However much we enjoy conspiracy theories, we do not subscribe to this extreme view that we believe to be overly paranoid.
We now examine how Ro3 treats molecular weight. Before Ro3’s creators present their rule they note a MW range of 100-250Da for fragment libraries screened using high throughput X-ray crystallography. They then refer to some analysis of fragment hits which suggested that these obeyed a Rule of 3. This raises more questions than it answers. Did they actually screen anything with MW greater than 300Da or even the 250Da that they first mention? If so, did the larger fragments actually fail to hit or were they just too insoluble? Are the cutoffs absolute or do they, by analogy with Ro5, allow a defined fraction of acceptable fragments to lie above the rule's limit?
Cynically we wonder how much of detail of the Ro3 has been imposed in attempt to milk Ro5. We read of NMR screening libraries having average MW of 200Da and wonder whether a 10% Ro5-like cutoff of 250Da might be more appropriate. Unfortunately the Rule of 2.5 doesn't quite have the same bite while doing some quite horrid things to hydrogen-bonding groups that would render electrons quite irrelevant. When your rule is based on 5, unit differences are less noticeable. And of course the main problem with setting up integer-based rules for fragment selection is that The Rule of 2 has already been taken by the formidable Lady Bracknell. So it really could never have been anything other than the rule of 3. Misfortune, carelessness or nascent dairy industry? We are simple folk and it is not for us to say.
This concludes our technical review of Ro5 and in the next posting in the series we will examine some of its 'sociological' fallout. We hope that you have enjoyed the commentary so far.
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6 comments:
Like Lipinski's rules, the Rules of the Ro3 aren't really rules at all - more guidelines, and hence you should never feel too bad about bending them once in a while.
Even if one is going to call the rules guidelines, it is still necessary to define 'once in a while'. Ro5 actually does this by setting the cutoffs not the maximum values observed for oral drugs but to values above which 10% of oral drugs are found. Whatever you think of the Ro5 view of physical chemistry, it is difficult to criticise the precision with which this view is defined.
In contrast Ro3 neither defines hydrogen bonding nor quantifies 'once in a while'. We have argued that Ro3 is incompletely defined in the form in which it was orginally presented. This does not seem to have inhibited a number of researchers citing it in their publications. The distinction between rules and guidelines is completely peripheral to the arguments presented in the post.
Good analysis...in the conference which I attended, they discussed the development of Aliskiren (Tecturna). In the Q & A session, somebody pointed out that in modern DD, the molecule might have been dropped because it violates RO5. Roderick Hubbard from Vernalis then made this point about it being just a guideline, and also about lead development being a much more haphazard and relative process than is envisioned. I just think that one needs to analyse pros and cons for individual cases, as is usually the case.
I had a quick look at the structure of aliskeren (http://merops.sanger.ac.uk/cgi-bin/smisum?mid=J19.503)and reckon that it doesn't break the rule of 5 which counts donors by nitrogen or oxygen rather than hydrogen. A primary amide contributes a single donor even though there are two hydrogen atoms capable of functioning as donors. Two of the acceptors are aromatic ether oxygen atoms which are at the weak end of the range of acceptors. However the point I would re-iterate is that whatever you think of the Ro5 HB model, it is fully defined. The same cannot be said for Ro3.
The point aboput being able to break the rules sometimes is a completely separate issue. If you have a rule/guideline that can be broken 10% of the time (like Ro5) it can be argued that it encodes useful information. If it can be broken 50% of the time then it's going be a real crapshoot. If the rule can be broken 100% of the time it is completely irrelevant where you set the cut and the rule encodes no information at all.
Isn't the rule of thumb with Ro5 and Ro3 that violating one restraint is semi-fine, violating two is bad, and three or more is just silly? (fragments should have be small enough that we can expand them into high affinity ligands and soluble enough that we can have high enough concentrations to fish them out of the assay - Ro5 violators could be natural compound leads which usually aren't that compatible with organic synthesis).
/morten g
The point being made in this post is that the hydrogen bond donors and acceptors are not defined for Ro3. Also the Ro3 paper doesn't say a whole lot about how violations of Ro3 should be treated. My interpretaton of Ro3 is that it is treated as absolute (i.e. no violations pemitted) but the authors of Ro3 might do something different where they work.
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