Checking halogen bonds

Halogen bonds (or X-bonds) are one of the less appreciated protein-ligand interactions. As we discussed in 2022, the polarized nature of a carbon-halogen bond creates a partially-positively charged “σ-hold” at the bit of the halogen furthest from the carbon, and this can make favorable interactions with lone pairs on oxygen or sulfur atoms (or nitrogen, but in most proteins this is limited to histidine residues and is rare.) Halogens can also interact with aromatic π-systems such as the side chains of phenylalanine, tryptophan, histidine, and tryptophan. Since many fragments contain halogen atoms by design, halogen bonds may occur frequently. But how do you decide whether “a halogen in proximity of a possible acceptor” actually contributes to binding? In a new (open-access) paper in Protein Science, Ida de Vries, Robbie Joosten, and colleagues at Oncode Institute and The Netherlands Cancer Institute provide a new metric.

 

The researchers examined structures of halogen-containing ligands bound to proteins in PDB-REDO, a database of carefully vetted and refined structures from the Protein Data Bank. They only included structures solved to better than 2.5 Å resolution and omitted structures where halogens had high B-factors, which may be the result of radiation damage. This led to 8423 structures in which a halogen possibly interacted with an oxygen or sulfur atom and 8096 potential halogen-π interactions, which were analyzed in detail.

 

A halogen bond to an oxygen or sulfur atom can be described by the interatom distance and two angles: θ1 (carbon-halogen-oxygen/sulfur) and θ2 (halogen-oxygen/sulfur-carbon). Halogen-π-system bonds can be defined by distance to the centroid of the π-system and θ1, the carbon-halogen-centroid angle. (The paper has a nice diagram.) These parameters were calculated and annotated for all the structures.

 

Median distances were 3.5 Å between halogen and oxygen/sulfur, regardless of the halogen. Median θ1 angles were smaller than the 150º-180º expected, particularly for fluorine atoms, while median θ2 angles were more consistent with theory, at 90º-120º.

 

For halogen-π-systems, median distances were 4.8 Å for all halogens except iodine, which came in slightly higher. But θ1 angles were still smaller than expected, mostly between 110º-140º.

 

Armed with this tranche of high-quality data, the researchers established a Halogen Bond Score, or HalBS. For any potential halogen bond in a new crystal structure or other structural model, the distance, θ1, and, if applicable, the θ2 values are calculated, and if any of these diverge too far from the median values, HalBS flags them. Importantly, the researchers acknowledge that “the current HalBS cannot be used as a direct validation metric but can provide an indication of genuine halogen bonds and ‘not so proper’ halogen bonds.”

 

With this caveat HalBS could be useful, and the researchers have made the source code available at https://github.com/PDB-REDO/HalBS (though the link doesn’t seem to work for me). As they note, more data, such as might be provided by widespread deposition of large crystallographic fragment screens, could further refine HalBS. Of course, the existence of a halogen bond exists says little about how much binding energy it contributes, but it’s a start.

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!

FragLites and PepLites meet bromodomains

The last two Practical Fragments posts focused on bromodomains, epigenetic readers that recognize acetylated lysine residues. Today’s post could thus be considered part of a trilogy, though the focus is less on bromodomains themselves than a specific type of fragment library.

 

In 2019 we highlighted FragLites, small fragments containing pairs of hydrogen bond acceptors and/or donors along with a bromine or iodine atom. FragLites were designed to assess ligandability as well as identify what types of interactions would be favorable at various sites. The original test protein was the kinase CDK2. In an open-access paper published late last year in J. Med. Chem. by Martin Noble, Michael Waring, and colleagues at Newcastle University, FragLites are screened against two members of the bromodomain family.

 

The first bromodomain (BD1) of BRD4 is considered highly ligandable, with multiple inhibitors disclosed (see for example here). In contrast, ATAD2, a bromodomain in another subfamily, is more challenging, in part because it lacks a hydrophobic region useful for increasing affinity for small molecules. Thirty-three FragLites were individually soaked at 50 mM into crystals of either bromodomain. The halogen atom on each FragLite facilitates analysis by anomalous dispersion, allowing more sensitive detection of low-occupancy binders. This, along with Pan-Dataset Density Analysis (PanDDA), was used to identify specific protein-ligand “binding events.”

 

In total, 26 binding events at five sites were identified for BRD4; four ligands bound at more than one site. Of these, 17 FragLites bound at the orthosteric site of BRD4 (which recognizes N-acetyl lysine). In contrast, ATAD2 displayed 16 binding events total over seven sites; only three bound at the orthosteric site, consistent with its lower ligandability. ATAD2 had previously been screened crystallographically against the 776-membered DSI-poised fragment library, and this effort also identified seven ligand-binding sites, six of which were common to those discovered here, suggesting that the small FragLite set is able to identify most pockets.

 

As far as specific types of interactions, the average FragLite made 1.1 hydrogen bond, suggesting that the second donor or acceptor is often not engaged. In contrast, the bromine or iodine atom makes protein contacts in 33 of 42 binding events. In half a dozen cases no hydrogen bond to the protein was observed, with the primary interaction being a halogen bond.

 

The FragLites are small, relatively “flat” aromatic molecules, but of course most proteins interact with other proteins. To try to explore such interactions, the researchers developed a library of “PepLites:” N-terminally acetylated amino acid residues with a C-terminal bromopropargyl group. These were also screened crystallographically against the two bromodomains and produced considerably lower hit rates, with six bound to BRD4 (all at the orthosteric site) and nine bound to ATAD2 (of which five bound to the orthosteric site). Reassuringly, the N-acetylated lysine PepLite bound to both proteins in a similar manner as seen in larger peptides.

 

The researchers conclude that FragLites and PepLites “represent highly valuable components of a larger crystallographic screen, and we anticipate that this is where they will fit into most drug discovery programs.” Indeed, this is already happening; last year we wrote about how FragLites were screened against the bromodomain PHIP2 as part of a larger screen, and I was surprised this paper was not mentioned here. Laudably, all the atomic coordinates have been deposited in the Protein Data Bank, so folks are able to do their own analyses.

 

As FragLites and PepLites are screened against ever more targets, it will be fun to see what they can teach us about intermolecular interactions and starting points for new leads.

A slew of sites for fragments in HIV Reverse Transcriptase

The protein HIV-1 reverse transcriptase (RT) has been something of an Achilles heel for HIV; 13 approved drugs inhibit this enzyme! However, HIV is more adaptable than Achilles, and can develop resistance to drugs, creating a need for new molecules. With this in mind, Eddy Arnold and colleagues at Rutgers University performed an extensive fragment campaign against this target; their work was recently published in J. Med. Chem.

The researchers assembled a library of 775 fragments, 500 from Maybridge and most of the rest from Sigma-Aldrich and Acros. These were combined into 143 pools of 4 to 8 fragments, each at 100 mM in DMSO. Crystals of RT grown with the drug rilpivirine were soaked with each of the pools; rilpivirine stabilizes the protein and yields crystals that diffract to high resolution. The researchers also added 80 mM arginine and 6% trimethylamine N-oxide (TMAO) to the soaking solutions; arginine helped solublize some of the more hydrophopic fragments and improved electron density, while TMAO improved diffraction.

Overall, the researchers found 34 fragments that bound to HIV RT, a hit rate just over 4%. Interestingly, halogenated fragments seemed to give a much higher hit rate: 7 of 29 fluorine-containing fragments produced structures, as did 4 of the 17 brominated fragments and one of the two chlorinated fragments. I don’t recall seeing halogens previously over-represented among fragment hits, though last year we did write about halogen-enriched fragment libraries. The sample sizes reported here are small, but if the findings hold up in other studies, fluorine fetishism may be further justified.

But just as interesting as the composition of the fragment hits is the number of binding sites in the protein: 16, with names ranging from the descriptive (“NNRTI Adjacent” and “Incoming Nucleotide Binding”) to the concise (“399”) to the downright thuggish (“Knuckles”). In the case of three of these sites, some of the fragments also inhibited enzymatic activity.

There is a lot of nice information here, and eight co-crystal structures have been deposited in the protein data bank. Still, I am left a bit dizzy at the sheer number of sites. In fact, one fragment (4-bromopyrazole) bound to all of the 16 sites! What are we to make of this – is this a privileged fragment or a promiscuous binder? And as for the sites with no known functional activity, are these useful? What do you think?

Halogenated fragments stabilize mutant p53

Practical Fragments recently discussed using fluorinated fragments for ¹⁹F NMR, but there are other halogens out there – are these useful for constructing fragment libraries? SGX Pharmaceuticals had a collection of fragments enriched with bromine atoms, the thought being that this atom would facilitate crystallography. Halogens can also make productive interactions with proteins, including so-called “halogen bonds” to backbone carbonyl atoms or pi-systems. With this in mind, Andreas Joerger at Cambridge University and Frank Boeckler at Eberhard-Karls University and their colleagues have assembled and screened a “halogen-enriched fragment library.” Their results are reported in a recent issue of J. Am. Chem. Soc.

The library consists of 79 non-reactive, soluble aromatic compounds containing bromine or iodine. Because these elements are so large, the researchers used a modified rule of 3 – instead of a molecular weight limit of 300, they limited the fragments to no more than 22 heavy atoms (see also our recent post on this topic here). They then screened this library against the Y220C mutant form of p53, which contains a surface crevice that destabilizes the protein and contributes to cancer cell survival. Thermal shift assays were used as the primary screen, with hits being confirmed by 2D NMR and ITC. This resulted in the discovery of compound 3, which crystallography confirmed was making a halogen bond to a backbone carbonyl.

Modifying the amine substituent improved potency modestly, and building off the phenyl ring towards a nearby pocket improved the potency further, albeit at a cost in ligand efficiency. Still, this compound (PhiKan5196) does represent the most potent Y220C binder reported, and represents an order of magnitude improvement over previous work. Moreover, the molecule induces apoptosis in p53 Y220C containing human cancer cell lines but not in matched wild-type p53 cell lines. (Unfortunately the compound also appears to be generally cytotoxic.)

This library is an interesting approach in part because it is somewhat heretical: for various reasons most library designers exclude molecules containing bromine or, especially, iodine. That said, the thyroid hormones do contain iodine aplenty, and MEK kinase seems to have a predilection for bromine or iodine as well. What do you think? Are halogenated fragments a useful tool for certain targets, or an unproductive diversion?