HB Donor Fragment Selection Themes

[This post was updated on 11-Dec-2024 to reflect the publication of the 'HBDs in drug design' preprint as the K2022 article]

In this post I’ll look at a couple of fragment selection themes with a hydrogen bond donor (HBD) focus. The material has been taken from the recent ‘HBDs in drug design’ preprint (HBD3; this would subsequently be published as the K2022 article) which introduced the term ‘hydrogen bond donor-acceptor asymmetry’ and suggested that we need to think differently about HBDs and hydrogen bond acceptors (HBAs) in drug design. One example of these hydrogen bond donor-acceptor asymmetries is that HBAs are typically more strongly solvated than HBDs in aqueous media and this is especially relevant to lead optimization (as shown in the graphical abstract for HBD3 below). 

However, this post is about fragment selection, rather than fixing ADME, and so I’ll say something about differences between HBDs and HBAs in the context of binding to targets. Let’s suppose that you’d like to exploit an HBD in the binding site of your target. All you need to do is place an HBA at a point in space where it can form a good hydrogen bond (taking care to address issues like steric footprint and conformational energy) and you’ve got it sorted. However, life is not quite so simple if you’re trying to exploit an HBA in the binding site because the HBD (e.g., amide NH) that you present to it will almost invariably be accompanied by an HBA (e.g., amide carbonyl O). In contrast, it is relatively easy to design an HBA (e.g., pyridine N) into a ligand structure that is not accompanied by an HBD.   

In HBD3, I describe the HBA that accompanies pretty much every neutral HBD as ‘co-occurring’. The problem for designers is that the co-occurring HBA, which is likely to come with a larger desolvation penalty than that for the HBD, needs to be accommodated and this places constraints on design. It’s also more difficult to achieve ‘line-of-sight’ access with HBDs than is the case for HBAs (you’re likely to need line-of-sight access when targeting a polar atom at the bottom of a relatively narrow binding pocket). The following figure should give you a better idea of what I’m getting at and let’s assume that we’re trying to donate an HB to HBA sitting at the bottom of a narrow and otherwise non-polar binding pocket. Although each of the three structures has appropriate geometry for line-of-sight access, things are not likely to end well if you try to exploit this line-of-sight access in a real-life design situation.

Let’s start with the phenol and, although not pertinent to this discussion, it’s worth mentioning that hydroxyl groups are prone to conjugation in phase 2 metabolism (drugs get hydroxylated in phase 1 metabolism in order to facilitate clearance). Donation of an HB by a ligand hydroxyl to a target HBA also brings the hydroxyl oxygen (the co-occurring HBA) into proximity with the molecular surface of the target. This increases the likelihood of an energetic penalty resulting from desolvation of the phenolic oxygen. One subtle point is that donation of an HB by the phenolic hydroxyl increases the HB basicity of the oxygen which effectively increases the energetic cost of desolvating it.

The co-occuring HBA of the primary amide is an even bigger problem than for phenol because the high polarity of the carbonyl oxygen means that it carries a large desolvation penalty (bad news if you’re trying to hit an HBA at the bottom of a narrow and otherwise non-polar binding pocket). If this is not enough of a problem, you also need to worry about desolvation penalties associated with the second HBD (the primary amide has two HBDs and methyl-capping will take out the one that you need for hitting that HBA at the bottom of the binding pocket). As Lady Bracknell might have observed, “One desolvated polar atom may be regarded as a misfortune; to lose solvation of two polar atoms looks like carelessness”.

The last of the trio of structures is pyrazole linked at C4 and this avoids problems that might result from biasing the tautomeric preference. Pyrazole is a great warhead if you’re targeting a proximal HBD and HBA (as is the case when trying to hit a kinase hinge). However, pyrazole’s HBA may become a liability when trying to hit the HBA at the bottom of that otherwise non-polar binding pocket. Why not just take out pyrazole’s HBA, you might ask? The problem is that pyrroles are very electron rich and tend to be quite reactive.  One tactic is to move the co-occurring HBA from the ring to the linker (1 and 2) in a way that makes the linker electron-withdrawing and pray for a less destabilizing contact between the co-occurring HBA and the binding site. Alternatively, you can take out the co-occurring HBA and modify the linker to make it more electron-withdrawing (3). I’ve included Hammett σ values in the graphic and these will give you an idea how the substituents vary in their ability to suck electron density out of the pyrrole ring (beneficial both for making the pyrrole ring more rugged and increasing the HB acidity of its NH HBD).  I see these fragments as being of about the right size to be screened crystallographically but you might want something a bit larger than methyl if you’re using another detection method.

If you’re designing (or trying to improve the coverage of) a fragment library then another selection theme that you might want to think about is fragments that can present a high ‘density’ of HBDs to a target while minimizing the number of co-occurring HBAs. One way to do this is to use the guanidine substructure although this will cause some medicinal chemists to roll their eyes (concerns about permeability) while Ro3’s adherents would be likely to denounce you for heresy (actually not such a bad thing and I think that the late, great Denis Healey might have likened this to “being savaged by a dead sheep”). Guanidine itself is extremely basic (pKa = 13.6 | ref) which means very little of the neutral form for diffusing across membranes. However, the pKa of guanidine is also extremely sensitive to substitution and a number of approved drugs incorporate this substructure. I should also point out that, even in the neutral form of guanidine, the amide-like nitrogen atoms do not function as HBAs (even though they’d be counted as such when applying Ro5).

I’ve made a small selection of substituted guanidines that I think may of interest for screening as fragments. The pKa values that I quote in this post are from an article by two former colleagues (Peter Taylor and Alan Wait who are sadly both deceased) and this is an excellent source of measured guanidine pKa values.   Two of these (4 and 5) will be predominantly protonated at neutral pH although there’ll still be a significant amount of the neutral form that you’ll need for permeability. The other two guanidines will be predominantly neutral at neutral pH although 6 is sufficiently basic to protonate in lysosomes.  As for the pyrroles, I see these as about the right size to be screened crystallographically but you might want something a bit bigger than methyl if you plan to use a different detection method.

  

Enthalpic fragments

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Enthalpy-driven binding has been presented as a rationale for screening fragments although some have argued that thermodynamic signature is actually a 'red herring' in the context of drug discovery.  Binding of a ligand grown from a fragment hit incurs a translational entropy penalty that is similar to that of the original fragment hit and it is therefore it is hardly surprising that synthetic elaboration results in binding that is more driven by entropy.

A recent collaborative study between researchers in the Budapest Enthalpomics Group (BEG) and Prof Wilhelmina Wiplasch, well known for her seminal study ‘The Ecstasy and Agony of Recreational PAINS’, shows this view to be hopelessly naïve. The mathematical treatment used in the study is formidable and was originally developed by Prof Wiplasch during a sabbatical at the Port-au-Prince Institute of Biogerontology. Briefly, deep learning was used to model the time-dependent covariance and kurtosis of the polarizability tensor for a series of rhodanines, showing that the enthalpic nature of fragment binding is caused by their greater ligand efficiencies. “This model comprehensively outperforms all competitors”, explains Group Leader Prof Kígyó Olaj, “and we have shown for the very first time that the Sackur-Tetrode equation can be safely consigned to the dustbin of History”.

Sailor Malan's guide to fragment screening library design

Today I'll take a look at a JMC Perspective on design principles for fragment libraries that is intended to provide advice for academics. When selecting compounds to be assayed the general process typically consists of two steps. First, you identify regions of chemical space that you hope will be relevant and then you sample these regions. This applies whether you're designing a fragment library, performing a virtual screen or selecting analogs of active compounds with which to develop structure-activity relationships (SAR). Design of compound libraries for fragment screening has actually been discussed extensively in the literature and the following selection of articles, some of which are devoted to the topic, may be useful: Fejzo (1999), Baurin (2004), Mercier (2005), Schuffenhauer (2005), Albert (2007) Blomberg (2009), Chen (2009), Law (2009), Lau (2011), Schulz (2011); Morley (2013). This series of blog posts ( 1 | 2 | 3 | 4) on fragment screening library design that may also be helpful.

The Perspective opens with the following quote:

"Rules are for the obedience of fools and the guidance of wise men"

Harry Day, Royal Air Force (1898-1977)

It wasn't exactly clear what the authors are getting at here since there appears to be no provision for wise women. Also it is not clear how the authors would view rules that required darker complexioned individuals to sit at the backs of buses (or that swarthy economists should not solve differential equations on planes). That said, the quote hands me a legitimate excuse to link Malan's Ten Rules for Air Fighting and I will demonstrate that the authors of this Perspective can learn much from the wise teachings of 'Sailor' Malan.

My first criticism of this Perspective is that the authors devote an inordinate amount of space to topics that are irrelevant from the viewpoint of selecting compounds for fragment screening. Whatever your views on the value of ligand efficiency metrics and thermodynamic signatures, these are things that you think about once you've got the screening results. The authors assert, "As a result, fragment hits form high-quality interactions with the target, usually a protein, despite being weak in potency" and some readers might consider the 'concept' of high-quality interactions to be pseudoscientific psychobabble on par with homeopathy, chemical-free food and the wrong type of snow. That said, discussion of some of these peripheral topics would have been more acceptable if the authors had articulated the library design problem clearly and discussed the most relevant literature early on. By straying from their stated objective, the authors have broken the second of Malan's rules ("Whilst shooting think of nothing else, brace the whole of your body: have both hands on the stick: concentrate on your ring sight").

The section on design principles for fragment libraries opens with a slightly gushing account of the Rule of 3 (Ro3). This is unfortunate because this would have been the best place for the authors to define the fragment library design problem and review the extensive literature on the subject. Ro3 was originally stated in a short communication and the analysis that forms its basis is not shared. As an aside, you need to be wary of rules like these because the cutoffs and thresholds may have been imposed arbitrarily by those analyzing the data. For example, the GSK 4/400 rule actually reflects the scheme used to categorize continuous data and it could just have easily been the GSK 3.75/412 rule if the data had been pre-processed differently. I have written a couple ( 1 | 2 ) of blog posts on Ro3 but I'll comment here so as to keep this post as self-contained as possible. In my view, Ro3 is a crude attempt to appeal to the herding instinct of drug discovery scientists by milking a sacred cow (Ro5). The uncertainties in hydrogen bond acceptor definitions and logP prediction algorithms mean that nobody knows exactly how others have applied Ro3. It also is somewhat ironic that the first article referenced by this Perspective actually states Ro3 incorrectly. If we assume that Ro5 hydrogen bond acceptor definitions are being used then Ro3 would appear to be an excellent way to ensure that potentially interesting acidic species such as tetrazoles and acylsulfonamides are excluded from fragment screening libraries. While this might not be too much of an issue if identification of adenine mimics is your principal raison d'etre, some researchers may wish to take a broader view of the scope of FBDD. It is even possible that rigid adherence to Ro3 may have led to the fragment starting points for this project being discovered in Gothenburg rather than Cambridge. Although it is difficult to make an objective assessment of the impact of Ro3 on industrial FBDD, its publication did prove to be manna from heaven for vendors of compounds who could now flog milligram quantities of samples that had previously been gathering dust in stock rooms.



This is a good point to see what 'Sailor' Malan might have made of this article. While dropping Ro3 propaganda leaflets, you broke rule 7 (Never fly straight and level for more than 30 seconds in the combat area) and provided an easy opportunity for an opponent to validate rule 10 (Go in quickly - Punch hard - Get out). Faster than you can say "thought leader" you've been bounced by an Me 109 flying out of the sun. A short, accurate (and ligand-efficient) burst leaves you pondering the lipophilicity of the mixture of glycol and oil that now obscures your windscreen. The good news is that you have been bettered by a top ace whose h index is quite a bit higher than yours. The bad news is that your cockpit canopy is stuck. "Spring chicken to shitehawk in one easy lesson."

Of course, there's a lot more to fragment screening library design than counting hydrogen bonding groups and setting cutoffs for molecular weight and predicted logP. Molecular complexity is one of the most important considerations when selecting compounds (fragments or otherwise) and anybody even contemplating compound library design needs to understand the model introduced by Hann and colleagues. This molecular complexity model is conceptually very important but it is not really a practical tool for selecting compounds. However, there are other ways to define molecular complexity in ways that allow the general concept to be distilled into usable compound selection criteria. For example, I've used restriction of extent of substitution (as detailed in this article) to control complexity and this can be achieved using SMARTS notation to impose substructural requirements. The thinking here is actually very close to the philosophy behind 'needle screening' which was first described in 2000 by researchers at Roche although they didn't actually use the term 'molecular complexity'.


As one would expect, the purging of unwholesome compounds such as PAINS is discussed. The PAINS field suffers from ambiguity, extrapolation and convolution of fact with opinion. This series ( 1 | 2 | 3 | 4) of blog posts will give you a better idea of my concerns. I say "ambiguity" because it's really difficult to know whether the basis for labeling a compound as a PAIN (or should that be a PAINS) is experimental observation, model-based prediction or opinion. I say "extrapolation" because the original PAINS study equates PAIN with frequent-hitter behavior in a panel of six AlphaScreen assays and this is extrapolated to pan-assay (which many would take to mean different types of assays) interference. There also seems to be a tendency to extrapolate the frequent-hitter behavior in the AlphaScreen panel to reactivity with protein although I am not aware that any of the compounds identified as PAINS in the original study were shown to react with any of the proteins in the AlphaScreen panel used in that study. This is a good point to include a graphic to break the text up a bit and, given an underlying theme of this post, I'll use this picture of a diving Stuka.

One view of the fragment screening mission is that we are trying to present diverse molecular recognition elements to targets of interest. In the context of screening library design, we tend to think of molecular recognition in terms of pharmacophores, shapes and scaffolds. Although you do need to keep lipophilicity and molecular size under tight control, the case can be made for including compounds that would usually be considered to be beyond norms of molecular good taste. In a fragment screening situation I would typically want to be in a position to present molecular recognition elements like naphthalene, biphenyl, adamantane and (especially after my time at CSIRO) cubane to target proteins. Keeping an eye on both molecular complexity and aqueous solubility, I'd select compounds with a single (probably cationic) substituent and I'd not let rules get in the way of molecular recognition criteria. In some ways compound selections like those above can be seen as compliance with Rule 8 (When diving to attack always leave a proportion of your formation above to act as top guard). However, I need to say something about sampling chemical space in order to make that connection a bit clearer.

This is a good point for another graphic and it's fair to say that the Stuka and the B-52 differed somewhat in their approaches to target engagement. The B-52 below is not in the best state of repair and, given that I took the photo in Hanoi, this is perhaps not totally surprising. The key to library design is coverage and former bombardier Joseph Heller makes an insightful comment on this topic. One wonders what First Lieutenant Minderbinder would have made of the licensing deals and mergers that make the pharma/biotech industry such an exciting place to work.  

The following graphic, pulled from an old post, illustrates coverage (and diversity) from the perspective of somebody designing a screening library.  Although I've shown the compounds in a 2 dimensional space, sampling is often done using molecular similarity which we can think of inversely related to distance. A high degree of molecular similarity between two compounds indicates that their molecular structures are nearby in chemical space.  This is a distance-geometric view of chemical space in which we know the relative positions of molecular structures but not where they are.  When we describe a selection of molecular structures as diverse, we're saying that the two most similar ones are relatively distant from each other. The primary objective of screening library design is to cover relevant chemical space as effectively as possible and devil is in the details like 'relevant' and 'effectively'. The stars in the graphic below show molecular structures that have been selected to cover the chemical space shown. When representing a number of molecular structures by a single molecular structure it is important, as it is in politics, that what is representative not be too distant from what is being represented. You might ask, "how far is acceptable?" and my response would be, as it often is in Brazil, "boa pergunta". One problem is scaffolds differ in their 'contributions' to molecular similarity and activity cliffs usually provide a welcome antidote to the hubris of the library designer.         

I would argue that property distributions are more important than cutoff values for properties and it is during the sampling phase of library design that these distributions are shaped. One way of controlling distributions is to first define regions of chemical space using progressively less restrictive selection criteria and then sample these in order, starting with the most restrictively defined region. However, this is not the only way to sample and might also try to weight fragment selection using desirability functions. Obviously, I'm not going to provide a comprehensive review of chemical space sampling in a couple of paragraphs of a blog post but I hope to have shown that the sampling of chemical space is an important aspect of fragment screening library design. I also hope to have shown that failing to address the issue of sampling relevant chemical space represents a serious deficiency of the featured Perspective. 

The Perspective concludes with a number of recommendations and I'll conclude the post with comments on some of these. I wouldn't have too much of a problem with the proposed 9 - 16 heavy atom range as a guideline although I would consider a requirement that predicted octanol/water logP be in the range 0.0 - 2.0 to be overly restrictive. It would have been useful for the authors to say how they arrived at these figures and I invite all of them to think very carefully about exactly what they mean by "cLogP" and "freely rotatable bonds" so we don't have a repeat of the Ro3 farce. There are many devils in the details of the statement:"avoid compounds/functional groups known to be associated with high reactivity, aggregation in solution, or false positives". My response to "known" is that it is not always easy to distinguish knowledge from opinion and "associated" (like correlated) is not a simple yes/no thing. It is not cleat how "synthetically accessible vectors for fragment growth" should be defined since there is also a conformational stability issue if bonds to hydrogen are regarded as growth vectors.   

This is a good point at which to wrap things up and I'd like to share some more of Sailor Malan's wisdom before I go. The first rule (Wait until you see the whites of his eyes. Fire short bursts of 1 to 2 seconds and only when your sights are definitely 'ON') is my personal favorite and it provides excellent, practical advice for anybody reviewing the scientific literature. I'll leave you with a short video in which a pre-Jackal Edward Fox displays marksmanship and escaping skills that would have served him well in the later movie. At the start of the video, the chemists and biologists have been bickering (of course, this never really happens in real life) and the VP for biophysics is trying to get them to toe the line. Then one of the biologists asks the VP for biophysics if they can do some phenotypic screening and you'll need to watch the video to see what happens next...