Rule of 3 takes some flak

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?

Literature cited

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

FBLD versus DOS

The relative merits of Fragment Based Ligand Design (FBLD) and Diversity-Oriented Synthesis (DOS) were recently debated in a Nature Forum. This debate has already been reviewed both in Practical Fragments and In The Pipeline.

I believe by setting up the debate like this, the editorial staff of the journal show a poor understanding of Lead Discovery (LD). In essence a comparison is being made between apples and oranges. FBLD (also known as FBLG with the LG for lead generation) is an integrated LD framework and a comparison with conventional high throughput screening (HTS) and associated Hit-to-Lead (H2L) chemistry would have made more sense. DOS is essentially an approach to extending the chemical space covered by screening collections and filling �holes� in the existing chemical space. A DOS approach could be easily used to enhance existing fragment libraries (especially if using molecular shape to quantify similarity) while the output of a fragment screen could be used as input to design of DOS libraries. The �Core and Layer� approach that I�ve used in design of generic fragment libraries (and even one library for cell screening) can accurately be described as diversity-oriented.

The case for FBLD is made by Philip Hajduk who makes the important points that a relatively small number of fragments can be used to cover a relatively large chemical space and that synthetic resource is always directed towards the target of interest. I like to say that leads from FBLD are assembled from proven molecular recognition elements and would add that fragments allow you to search chemical space with a better-controlled resolution than do more elaborated molecules. I don�t happen to agree with his assertion that �there is ample evidence that larger molecules are more likely than smaller ones to succeed as drugs in clinical trials� but this does not weaken the first two points that he makes. It's worth remembering that you usually need protein crystal structures in FBLD both for the target (at the outset of screening) and for complexes with weakly-bound fragments. If you don't obtain these quickly you're going to be working on Project Passchendaele.

DOS is championed by Warren Galloway and David Spring. They note that there are situations in which FBLD is not currently applicable, for example in phenotypic screens (see Derek Lowe's comments In The Pipeline) or for probing certain protein-protein interactions. I agree with this point and believe that we�ll always need a variety of assays for successful LD, especially as Drug Discovery is expected to get even more challenging in the future. If you�re trying to enhance the ability of screening libraries to hit targets then it makes sense to use molecular diversity criteria to extend coverage in a more systematic manner. I don�t see why the term DOS should only apply when molecular size exceeds an arbitrary cut off and believe the real issue is more about how than whether DOS should be used to enhance screening libraries.

The advocates of DOS need to take a close look at how they define diversity. If the conserved core of a DOS library cannot be accommodated in a binding site then, barring nuclear fusion, none of the compounds in the library will fit either. From the point of view of this target the library has no diversity regardless of the number of compounds in it.

I was disappointed that molecular complexity (check this link for an alternative view) was not raised by either party in this debate since it�s a unifying concept that brings together different strategies for compound library design. Very complex molecules leave the H2L chemists with little or no room to manoeuvre. This is less of a problem if the screening hit nails the target with nanomolar potency and has jaw-dropping bioavailability. However, reality is more likely to be micromolar with one or more ADMET issues needing to be addressed. Advocates of DOS really do need to start thinking a bit more about molecular complexity in the context of screening compounds for biological activity. I always encourage folk designing a DOS library to make a relatively large sample of the library prototype so that it can be included in the fragment screening collection.

So what�s the verdict? I believe that FBLG is here to stay although it is not yet clear how widely applicable the approach is. I also believe that some form of DOS can be used to enhance any screening collection provided that:

(1) Diversity is seen in the context of the existing collection
(2) The importance of hit exploitability is recognised

I�d be interested to hear what other people think about this topic so feel free to comment. I�ll also set this up as a discussion for the FBDD LinkedIn group since commenting there is a bit easier. Also don�t forget that the journal allows you to comment on the article directly.


Literature cited

Hajduk, Galloway & Spring, A question of library design (Forum Drug Discovery). Nature 2011, 470, 42-43 | DOI

Nicholls et al, Molecular Shape and Medicinal Chemistry: A Perspective. J. Med Chem. 2010, 53, 3862-3886 | DOI

Hann, Leach & Harper, Molecular Complexity and Its Impact on the Probability of Finding Leads for Drug Discovery. J. Chem. Inf. Comput. Sci., 2001, 41, 856�864 | DOI

Rule of Three considered harmful?

I should start this post by saying that I�ve never actually used the Rule of Three for fragment selection. Part of the reason for this is simply a matter of timing since I�d been designing fragment libraries before the Rule of Three came along. However, I believe that there are reasons that you need to take a very close look at the Rule of Three if you�re planning to build a fragment library strategy around it. The rule was introduced in late 2003:

�We carried out an analysis of a diverse set of fragment hits that were identified against a range of targets. The study indicated that such hits seem to obey, on average, a �Rule of Three�, in which molecular weight is < 300, the number of hydrogen bond donors is =3, the number of hydrogen bond acceptors is =3 and ClogP is =3. In addition, the results suggested NROT (=3) and PSA (=60) might also be useful criteria for fragment selection. These data imply that a �Rule of Three� could be useful when constructing fragment libraries for efficient lead discovery.�

My first criticism of the Rule of Three is that the authors do not say how they define hydrogen bond acceptors. I�ll illustrate this point with reference to the phenylhydantoin below which along with the accompanying properties was retrieved from eMolecules. As far as I�m concerned, this compound would have been perfectly acceptable for inclusion in a fragment library before the Rule of Three was published and the publication of the rule would not make change my mind. If, however, you asked me whether the compound complied with the Rule of Three, I�d have to admit that I simply don�t know. The number of hydrogen bond donors is not an issue because there is only one of these in the molecule. The number of acceptors is more problematic. I would only count the oxygen atoms in this molecule as acceptors and, since there are two of these, the molecule would be compliant with the Rule of Three. However the well-known Rule of Five treats all nitrogen and oxygen atoms as acceptors so if you use those criteria you�ll count a total of four acceptors and conclude that the compound is not compliant with the Rule of Three. This is not a problem for me because I don't use the Rule of Three but spare a thought for the person assembling a commercial fragment library.


My second criticism of the Rule of Three concerns how it was actually derived. The authors describe performing �an analysis of a diverse set of fragment hits� without actually saying anything about what this analysis entailed. If they were analysing hits from their own fragment screens then the characteristics of the hits will reflect the criteria by which compounds were selected for fragment screening. If they were sampling from a more extensive database of screening hits, I�d still want to know how the fragment hits were distinguished from the other hits.

My third criticism is as much about how cut offs get used as it is of the Rule of Three. There�s a diagram of a funnel that you often see in virtual screening reviews. We also use funnels (or filters as we prefer to call them) in screening library design and in fact this activity is not a whole lot different from working up a virtual screen. Typically we apply filters and sample (e.g. using molecular diversity criteria) from what makes it through. Note that I say �filters� rather than �a filter�. The Core and Layer (CaL) approach to library design has been described both in this blog and in a journal article. In CaL the filters used prioritise compounds get less restrictive as more compounds are added to the library. The reason for doing this is that it gives better control of chemical space coverage since it forces the selection of the smallest and least complex molecules first. A molecular diversity maximiser such as BigPicker, will tend to pick larger, more complex molecules because these tend to be more dissimilar to each other.

I am also prepared to accept compounds that have measured/calculated logP values in excess of 3 provided that the appropriate precautions (select ionisable compounds and/or use measured solubility values) have been taken to minimise the risk of poor solubility. You don�t want a whole library of compounds with logP values in excess of 4 but having some will increase the range of targets that you can nail. I am more concerned about the distribution of logP and molecular size in a library than I am with their maximum values and believe using multiple cut offs allows better control of these distributions.

You'll find plenty of material on the internet that deals with the Rule of Three although inconsistencies can be observed. It is not clear whether or not the Rule of Three includes the restrictions on NROT and PSA. As I read it in the original article, I don't think it does but I'm not sure and think it could have been made clearer. This webpage (accessed 11-Jan-2011) appears to suggest that Maybridge FBDD team think that the NROT and PSA criteria are included in the Rule of Three. However, another webpage (accessed 11-Jan-2011) seems to suggest that the FBDD team at Chembridge think otherwise. Cambridge Medchem Consulting (accessed 11-Jan-2011; I expect that this page will get updated once the error is discovered) appear to share the Chembridge view that the NROT and PSA criteria are not included in the Rule of Three although they use < instead of = when stating the Rule which makes a big difference when the number in question is 3. Yet another variation on the Rule of Three can be found in the BioScreening.net glossary (accessed 12-Jan-2011) in which the hydrogen bond criteria are stated as "number of H-bond donors and acceptors less than, or equal to 3", which could be taken to imply that the sum of donors and acceptors cannot exceed 3.

I should of course let you know where the title of this post comes from since I borrowed most of it from a computer science paper that is over forty years old. I can�t even claim originality for adapting the title of the earlier paper because my friends at OpenEye have beaten me to that as well. Furthermore my first two criticisms of the Rule of Three have already been made by other bloggers (accessed 11-Jan-2011).

I hope that this post will at least make people ask a few questions when presented with rules like these in the future. I'll also set up a discussion in the LinkedIn Medicinal Chemistry group which will facilitate posting of comments.

Literature cited

Congreve, Carr, Murray & Jhoti, A �Rule of Three� for fragment-based lead discovery? Drug Discov. Today 2003, 8, 876-877 | DOI

Lipinski, Lombardo, Dominy &Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 1997, 23, 3-25 | DOI

Blomberg, Cosgrove, Kenny & Kolmodin, Design of compound libraries for fragment screening. JCAMD, 2009, 23, 513-525 | DOI

Dijkstra, go to statement considered harmful. Communications of the ACM, 1968, 11, 147-148 | DOI