Halo Library says hallo to crystals

Last week we wrote about the magical properties chlorine can impart to molecules. More generally, halogen atoms can be helpful for a variety of reasons beyond new types of interactions with proteins and improved metabolic stability. For example, fluorine NMR can be used to rapidly identify ligands, and we’ve written about a custom fluorinated library. Heavier halogens can be particularly useful for screening by crystallography, and we’ve written about two libraries (HEFLib and FragLites) containing chlorine-, bromine,- or iodine-bearing fragments. Now Francesc Ruiz, Eddy Arnold, and colleagues at Rutgers bring us the “Halo Library,” described in a new (open access) J. Med. Chem. paper.

 

The researchers assembled a library of 46 halogenated fragments. In contrast to the libraries above, which focused on either fluorine or heavier halogens, this one is multi-purpose, with about half the compounds containing fluorine, half containing bromine, and a handful of molecules containing chlorine or iodine. Most of the fragments came from their internal collection and had been screened against several targets, and the rest were commercial compounds that had been reported to bind to at least one target.

 

The Halo Library is similar in terms of molecular properties to HEFLib and FragLites, though with a slightly lower average molecular weight (172.5 Da). Like the two earlier libraries, the Halo Library is relatively “flat,” with Fsp3 = 0.2.

 

Ten years ago we highlighted work from the Arnold lab in which a library of 775 fragments was screened crystallographically against HIV reverse transcriptase (HIV-1 RT), resulting in a 4% hit rate. The researchers returned to this protein with their Halo Library, soaking crystals with individual fragments at 20 mM. This resulted in 12 hits, an impressive hit rate of 26%. Admittedly some of these halogenated fragments had been identified as binders previously, so it will be interesting to see how the library behaves on other targets.

 

In addition to the high hit rate, fragments bound to six sites not occupied by ligands in the 2013 study, and two of these sites had never been reported to be ligand binding sites, despite extensive work on this protein. (Roughly half of anti-retroviral drugs for HIV target RT, and ART regimens typically include two or three separate inhibitors.)

 

Eight of the fragments inhibited the biochemical activity of the enzyme by at least 50% at 5 mM, and three of them gave IC₅₀ values in the low mM range with ligand efficiencies as high as 0.47 kcal/mol/atom. Among these three, two bound to a single site, while the third (4-amino-3-bromopyridine) bound at four separate sites. Another library member, the “universal fragment” 4-bromopyrazole previously identified by the researchers, bound to eight sites but showed only 22% inhibition at 5 mM.

 

The binding modes of all twelve fragments are described in some detail and show the standard range of hydrogen bonds and van der Waals interactions. Halogen bonds were surprisingly rare, unlike the case of FragLites against different proteins. It would be interesting to see a summary of the types of interactions, and how many involved the halogen atoms. The identities of all the library members are provided in the Supporting Information, so you can build your own library. 

 

And on that note, this is the last week to take our survey on fragment libraries, so please make sure to vote!

Chlorine: more magic than methyl

More than a decade ago we highlighted a paper that discussed “magic methyl” groups, which can boost the affinity of a ligand for a protein by more than 100-fold. Since then we’ve noted examples where these have been used to optimize fragments. But methyl groups are just one option for fragment growing. In a recent J. Med. Chem. paper, Debora Chiodi (Scripps) and Yoshihiro Ishihara (Vividion) take a close look at chlorine – and suggest that the halogen is even more magic than methyl. (See here for Derek Lowe’s summary.)

 

Chlorine is the sixth most common element found in drugs, after carbon, hydrogen, oxygen, nitrogen, and sulfur. In terms of size it is comparable to a methyl group, but more lipophilic. It is also more electronegative, and can significantly change the electronics of a molecule. Finally, unlike methyl groups, chlorine atoms often stabilize molecules against metabolism. But what about potency?

 

The researchers examined all 50,000 papers containing matched-pair SAR published in eight medicinal chemistry journals between 2010 and 2022, a process they characterize as “painstakingly manual.” All papers in which a hydrogen to chlorine swap increased the activity by at least ten-fold were then selected for further analysis. This cutoff was used based on tradeoffs of lipophilic ligand efficiency (LLE or LipE): you want a sizable increase in potency to compensate for the fact that adding a chlorine to a molecule increases logP by nearly 1.

 

In total, the researchers found 633 articles in which the potency increased by at least 10-fold, 131 where the potency increased by at least 100-fold, and 21 where the potency increased by a whopping 1000-fold or more, far better than any methyl.

 

Case studies in the paper attribute potency improvements to multiple factors, including better van der Waals interactions, decreasing the basicity of a molecule, direct hydrogen bonds to the chlorine, and halogen bonding, in which the chlorine makes favorable interactions with a carbonyl oxygen. Moreover, chlorine can also improve membrane permeability (via increased lipophilicity) and pharmacokinetics. Indeed, many of the most dramatic improvements in activity are measured not against isolated enzymes but in whole cells.

 

Thus, unlike a methyl group which merely increases lipophilicity or changes the conformation of a molecule, chlorine provides several opportunities for enhanced interactions. As the researchers summarize, “the chlorine atom is able to combine the beneficial effects of a fluorine atom (e.g., electronegativity/electron-withdrawing ability, metabolic stability, increased acidity), a methyl group (e.g., lipophilicity, van der Waals interactions, steric effect), and even a bromine atom (e.g., halogen bonding), and is arguably the most versatile among these substituents.”

 

Of course, the researchers were looking for beneficial effects: chlorine is not a universal panacea. Increased lipophilicity is usually something you want to avoid in the later stages of lead optimization, and adding chlorine atoms often reduces solubility. The researchers mention examples in which adding a chlorine atom to a molecule decreased potency by more than 100-fold.

 

As for lessons, adding chlorine atoms to fragment hits is probably a good early step, as in this 2017 example. The researchers also highlight halogen-enriched fragment libraries (which we wrote about here). A ligand with an affinity of 100 µM will be easier to find than a millimolar binder, but systematically adding halogens to different positions on a molecule increases the number of fragments to include in a library. On that topic, please make sure to take our survey on libraries, which closes at the end of May.

Higher hit rates with heavier halogens

Halogen bonding is an esoteric type of molecular interaction. Any first-year chemistry student can tell you that halogens are electronegative. More advanced students learn that the electron density on a halogen attached to a carbon is not evenly distributed. Rather, an electron deficient region appears directly opposite the carbon bond on chlorine, bromine, and iodine atoms. This “σ-hole” can form attractive interactions with electron-rich moieties, such as backbone carbonyl atoms. These highly directional interactions can be useful alternatives to hydrogen bonds, especially since they allow a reduction in the number of hydrogen bond donors. But how to find them? This is the topic of a recent open-access paper in Frontiers in Chemistry by Frank Boeckler and collaborators at Eberhard Karls Universität Tübingen.

 

The researchers constructed a library of 191 commercially available halogen-enriched fragments (called HEFLibs), which we wrote about in 2019. Most fragments have a single halogen atom, though 15 have two of the same type (two chlorine atoms, for example). The initial publication had no screening data, but the new paper describes screening the library against four diverse proteins: the methyltransferase DOT1L, the oxygenase IDO1, and the kinases AAK1 and CAMK1G.

 

Ligand-detected STD NMR was used as the primary screen, with proteins present at 20 µM and fragments at 1 mM each in mixtures of two. Between 9 and 57 hits were found for each target, with unique hits for all the targets except DOT1L. Some fragments hit all four targets, including one similar to the "universal fragment" we highlighted here.

 

Interestingly, iodine-containing fragments gave higher hit rates than bromine-containing fragments, which in turn gave higher hit rates than chlorine-containing fragments. Specifically, 9 of 14 (64%) iodine-containing fragments hit at least one target, vs 51% and 35% for bromine- and chlorine-containing fragments.

 

To assess whether halogen bonding played a role, the researchers calculated maximum electrostatic potential (V_max) for each fragment; this is a measure of the size of the σ-hole. Fragment hits tended to have higher V_max values than non-hits.

 

One possible confounding influence is that aryl halides can react with cysteine residues in proteins, and indeed the researchers did find that some of their fragments are unstable in the presence of the cellular reducing agent glutathione.

 

To confirm the STD-NMR results with an orthogonal method, the researchers turned to isothermal titration calorimetry (ITC). Of 57 fragment-protein pairs tested, only ten gave K_D values less than 1 mM, and nine were against the kinases; there were even a couple single-digit micromolar binders for AAK1. ITC is less sensitive than NMR, so some of the other fragments may bind too weakly to fully characterize.

 

Unfortunately, crystallography has been unsuccessful so far, so it remains unclear whether any of the hits are actually making halogen bonding interactions with the proteins. Halogens are good at filling lipophilic pockets, so it is perhaps likely that less specific van der Waals interactions are the key affinity drivers. But the Boeckler group has been pursuing halogen bonding for more than a decade, so I look forward to seeing more on this topic.

 

And in the meantime, happy Pi Day!

A bestiary of fragment hits

What do fragment hits look like, and how do they bind? Fabrizio Giordanetto, David Shaw, and colleagues at D. E. Shaw Research were interested in these questions, and their answers are provided in a recent J. Med. Chem. paper (open access, and also covered well by Derek Lowe).

The researchers started by searching the protein data bank (PDB) for the word “fragment” and selecting higher resolution structures (at least 2.5 Å) with a ligand containing 20 or fewer non-hydrogen atoms. Those of you who have done bibliometric searchers will appreciate that a lot of manual curation is required, and the initial list of 5115 complexes was ultimately winnowed down to 489, with 462 unique fragments and 126 unique proteins, about two-thirds solved to ≤2.0 Å resolution.

In contrast to a previous study, only a minority (18%) of proteins contained more than one binding site, suggesting that secondary (possibly allosteric) sites may be less common than hoped.

As to the fragments themselves, 21 bound in more than one pocket (not necessarily on the same protein), including the universal fragment 4-bromopyrazole. The fragments ranged in size between 6 and 20 non-hydrogen atoms, with 81% having 10 to 16, consistent with our poll last year. Given these sizes, it is perhaps not surprising that the vast majority of fragments conformed to the rule of three.

Roughly two thirds of the fragments were uncharged, while 22% contained a negative formal charge (usually a carboxylic acid) and 11% contained a positive formal charge such as an aliphatic amine. Interestingly, more than 90% of the fragment hits were achiral. Since our 2017 poll found that most fragment libraries contain chiral compounds, these results might suggest lower hit rates for these compounds. Fragment hits also tended to have lower Fsp³ scores than those in a popular commercial library, which is consistent with the observation that “less shapely” fragments give higher hit rates.

Digging more deeply into the chemical structures themselves, nearly a third of the fragments contained a phenyl ring, while 6% contained a pyridine and 5% contained a pyrazole. Thiophenes, indoles, indazoles, piperidines, furans, and pyrrolidines were present in 2-3% of fragment hits. But there was also plenty of diversity: more than half of fragments contained a unique ring system.

So that’s what fragment hits look like. How do they bind? Deeply, for starters: about three quarters of the fragment hits buried more than 80% of their solvent-accessible surface area, and 21 fragments were completely engulfed within a protein.

Not surprisingly, more than 90% of complexes showed at least one polar interaction, such as a hydrogen bond or a coordination bond to a metal ion. Many complexes contained more than one, and one had seven! Interestingly, these polar interactions also tended to be buried. Nitrogen and oxygen atoms from the fragments were equally likely to form hydrogen bonds. Interactions with bound water molecules were considered for high-resolution (≤1.5 Å) structures, and nearly half of these contained a water molecule with at least two hydrogen bonds to the protein and one to a fragment.

Beyond these conventional sorts of polar interactions, there were also less traditional interactions such as arene-mediated contacts, which occurred in nearly half of cases. As we’ve noted, these are often under-appreciated but can be useful for improving affinity. The subject of halogen bonds came up recently, but these turned out to be quite rare, appearing in just 3% of cases. Sulfur-mediated contacts and carbon hydrogen bonds were more common, appearing in 11-12% of complexes, respectively.

All of this has important implications for fragment library design. As the researchers note, this set of 462 fragments could be used as the basis for a library, and laudably all the structures are provided in the supporting information. Generalizing beyond these specific molecules, roughly a quarter of the atoms in the fragment hits are polar (nitrogen or oxygen) and thus more likely to form classic hydrogen bonds. The researchers “strongly suggest” maintaining this ratio in designing new fragments.

The researchers also suggest presenting “a minimum set of individual polar pharmacophoric elements, as opposed to distributing several pharmacophores on a given fragment,” which is essentially the minimal pharmacophore strategy described here.

The one category of data I would have liked to see was affinity. Many binding measurements were probably not reported, and experimental error can be particularly confounding for weaker interactions, but even a subset of the data should allow some conclusions about the strength of various molecular interactions. Hopefully this will be the basis of a follow-up publication.

Helpful halogens in fragment libraries

A couple weeks ago we highlighted a small fragment collection (MiniFrags) designed for crystallographic screening. We continue the theme this week with two more papers on the topic, with an emphasis on halogens.

The first, published in J. Med. Chem. by Martin Noble, Michael Waring, and collaborators at Newcastle University, describes a library of “FragLites.” These small (< 14 non-hydrogen atom) fragments are designed to explore “pharmacophore doublets,” such as a hydrogen bond acceptor (HBA) next to a hydrogen bond donor (HBD). For example, the universal fragment 5-bromopyrazole contains an HBA separated by one bond from an HBD. The researchers constructed a set of compounds with either two HBAs or an HBA and an HBD separated by 1 to 5 bonds. Importantly, all compounds also contained either a bromine or iodine atom, the idea being that anomalous dispersion could be used to help identify the fragments using crystallography. A total of 31 FragLites are described, with between 1 to 9 examples for each type of connectivity.

As a test case, these were screened against the kinase CDK2, which has previously been screened crystallographically. FragLites were soaked into crystals at 50 mM, and 9 of the FragLites were found to bind in a total of 6 sites, 4 of which had not been previously observed. The anomalous signal provided by the halogens was important: when the researchers used only normal scattering they identified just 10 of the 16 binding events even when using the powerful PanDDA background correction method. The anomalous signal also helped clarify the binding modes.

The ATP-binding site is where 7 of the 9 FragLites bound, with all but one of them making hydrogen bonding interactions to the hinge region. While not surprising, this does demonstrate that the FragLites can be used experimentally to identify the best binding site. Interestingly, (2-methoxy-4-bromophenyl)acetic acid bound in the active site as well as three other secondary sites; one of these sites hosted three copies of the ligand! It will be interesting to see whether this fragment is generally promiscuous in other proteins too.

As the researchers note, the composition of the FragLite library can be optimized. For example, both of the HBA-HBD fragments with 1-bond separation were identified as hits, while only 3 of the 9 HBA-HBD fragments with 2-bond separation were. Is this due to the choice of fragments, the target tested, or both? The approach is conceptually similar to the Astex minimal pharmacophore concept, so it will be useful to include other types of pharmacophores too (a single HBA or HBD, for example).

A related paper was published in Front. Chem. by Frank Boeckler and colleagues at Eberhard Karls Universität Tübingen. Long-time readers will recall his earlier halogen-containing library designed for identifying halogen bonds: favorable interactions between halogens and Lewis bases such as carbonyl oxygen atoms. Perhaps because they have relatively stringent geometric requirements (2.75 – 3.5 Å, and a bond angle of 155-180°), halogen bonds are often ignored; the FragLite paper doesn’t even mention them.

The new Boeckler paper describes the construction of a library of 198 halogen-containing fragments, all of which are commercially available and relatively inexpensive. Most of these are rule-of-three compliant, though quite a few also contain more than three hydrogen bond acceptors. Also, given that each fragment contains a halogen, the molecular weights are skewed upward. Solubility was experimentally determined for about half of the fragments, but the highest concentration tested was only 5 mM, and even here several were not fully soluble.

Although no screening data are provided, the researchers note that their “library is available for other working groups.” In the spirit of international cooperation, I suggest a collaboration with the FragLite group!

Halogen Hydrogen Bonding...Designable or Not?

The use of brominated fragments for X-ray screening is well known; it was the basis for former company SGX (now part of Lilly).  The purported advantage of brominated fragment is that you can identify the fragment unambiguously using anomolous dispersion.  In this paper, they are focused on using fragments to identify surface binding sites on HIV protease.  Prior work has focused on creating a new crystal form (complexed with TL-3, a known active site inhibitor) that has four solvent accesible sites: the exosite, the flap, and the two previous identified sites.  They took 68 brominated fragments and soaked these crystals: 23 fragments were found.  However, most of these actives were uninteresting.  Two compounds were found to be interesting, one bound in the exosite and one in the flap site. 

So, what's interesting in this paper?  Well, they (re)discover that brominated fragments can bind all over with a variety of affinities.  However, the bromine allows you to unambiguously identify those fragments through anomolous dispersion.  This is NOT interesting.  They discover that although it is a subject of much debate lately: specific interactions of the ligand with the target dominate the "bromine interaction".  This IS interesting.  They do not discuss this in much detail, but their grand extrapolations of this method to general applicability I don't buy. 

 I think the key take away from this paper is whether the halogen hydrogen bond undesignable and just a subject of serendipity?