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.

Ivermectin postmortem: PAINful experiences with a good drug

Ivermectin is a miracle drug. It cures infections caused by several types of parasitic roundworms, including those that cause river blindness and elephantiasis, two highly unpleasant diseases. Half of the 2015 Nobel Price for Physiology or Medicine was awarded to researchers who discovered the drug.

 

More recently, ivermectin was touted as a treatment for SARS-CoV-2. Unfortunately, after more than 90 human clinical trials, the preponderance of evidence shows that it is ineffective against COVID-19. A new (open-access) Perspective in J. Med. Chem. by Olaf Andersen, Jayme Dahlin, and collaborators at Weill Cornell Medicine, the National Institutes of Health, University of North Carolina Chapel Hill, and University of California San Francisco explores why it proved so misleading, with lessons for other drug repurposing efforts.

 

In 2011, ivermectin was one of 480 compounds tested in a biochemical assay at 50 µM and appeared to disrupt the interaction between HIV integrase and a mammalian protein involved in viral trafficking. In April 2020, low micromolar concentrations of ivermectin were reported to have anti-SARS-CoV-2 activity in a cellular assay. Given a terrifying new disease with no treatments, an approved drug that showed even tenuous activity looked like a lifeline. Researchers around the world began studying ivermectin. PubMed citations jumped from 459 in 2019 to 734 in 2021.

 

The new paper examines some of this past work and dives deeply into the biological effects of ivermectin. The molecule is poorly soluble in water (around 1-2 µM) and partitions into cellular membranes, where it activates a chloride channel receptor in worms, paralyzing them. At this it is quite potent: when taken as directed, human plasma concentrations of ivermectin are around 60 nM, but because the drug is highly protein bound the free concentration is only around 6 nM.

 

The astute reader may notice that the apparent antiviral activity was observed at low micromolar concentrations of ivermectin, three orders of magnitude higher than physiologically relevant, and the initial biochemical assay was conducted at an even higher concentration. In fact, that work was done using an AlphaScreen, which is notoriously sensitive to artifacts. The new paper demonstrates that ivermectin forms aggregates at low micromolar concentrations, and that these aggregates interfere with the AlphaScreen. (We previously wrote about how many of the early reports of compounds active against SARS-CoV-2 proteins were in fact aggregators.)

 

If ivermectin perturbs membrane proteins in worms, what does it do in human cells? The researchers tested the drug against panels of G protein-coupled receptors (GPCRs) and found that in one assay format it inhibited more than a quarter of 168 GPCRs at 10 µM, much higher than physiologically relevant but comparable to the in vitro experiments with SARS-CoV-2. Further studies revealed that ivermectin changes the properties of membranes at low micromolar concentrations, as assessed by multiple methods including electrophysiological assays. Several ivermectin analogs were also tested and found to have similar activity, consistent with nonspecific effects. Impressively, these experiments were done blinded to the experimenter.

 

You might think that messing with membranes would not be good for cells, and you would be right. The researchers found that ivermectin decreased cell viability at low micromolar concentrations in a variety of assay formats through multiple mechanisms, both cytotoxic and apoptotic. Importantly, the concentrations at which ivermectin was active against cells were similar to the concentrations where it showed activity against SARS-CoV-2. The researchers also analyzed 766 PubChem assays and found that ivermectin is active in nearly a third of those assessing cellular toxicity. Killing a host cell is certainly one way of killing a virus, but likely not a useful one.

 

In summary, the original data suggesting that ivermectin is a developable antiviral agent was flawed. The researchers describe this as “a saga of the damage that can be done by assay interference compounds” and a “cautionary tale for the dangers of ‘pandemic exceptionalism.’” They continue:

 

The fact that a repurposed drug is well-characterized clinically, or that there is an ongoing pandemic, may justify performing clinical and mechanistic experiments in parallel, but not skipping mechanistic studies, where the key experiments could have been done in a matter of a few weeks/months.

 

This J. Med. Chem. paper is a meticulous, comprehensive study; with 71 pages of supporting information there is far more to cover than I can do justice to in a blog post. The paper also includes a useful flowchart for derisking nonspecific membrane perturbation. It is well worth reading, particularly for those new to drug discovery. As Richard Feynman warned, “the first principle is that you must not fool yourself -- and you are the easiest person to fool.”

Exploiting avidity for finding fragments

As our poll last year demonstrated, there is no shortage of methods to find fragments. But that doesn’t mean new approaches aren’t welcome, particularly when they also apply to fragment growing. This is the promise of a recent paper in J. Med. Chem by Thomas Kodadek and collaborators at University of Florida Scripps and Deluge Biotechnologies. (Tom and first author Isuru Jayalath also presented this at the DDC meeting earlier this year.)

 

The researchers were inspired by the concept of avidity, the observation that multiple copies of a ligand bound to a multiprotein assembly can form a more stable complex than monomeric ligands bound to monomeric proteins. Could this phenomenon be exploited to find weak fragments?

 

A previous DNA-encoded library screen on streptavidin had identified 28 macrocycles, all of which contained one of two closely related fragments. The affinity of the more potent fragment came in at 706 µM using SPR. The researchers coupled this fragment to TentaGel beads, 10 µm wide polystyrene spheres covered in polyethylene glycol (PEG) chains terminated by amine groups. The PEG makes the beads water soluble. The beads were soaked in a solution of fluorescently labeled streptavidin, washed, and analyzed. Importantly, streptavidin exists as a tetramer, so each tetramer could bind up to four bead-bound fragments.

 

Streptavidin bound avidly to the beads, even when incubated at low (50 nM) concentrations. A control protein did not bind, nor did streptavidin bind to beads modified with a negative control fragment. Moreover, a monomeric version of streptavidin did not bind to the beads, illustrating the importance of avidity. Finally, adding the natural ligand biotin kept streptavidin from binding to the beads.

 

TentaGel beads have long been used in combinatorial synthesis, so the researchers built a small library in which the initial fragment was coupled to 48 carboxylic acids. These were then incubated with labeled streptavidin, and some of the beads showed more intense fluorescence, suggesting more protein binding. SPR analysis revealed that these new molecules had improved affinity, with the best coming in at 90 µM as a monomer. Thus, the primary screen can rank order affinities.

 

This is great for oligomeric proteins, but what about the large number of targets that are monomeric? Many recombinant proteins are expressed as fusions with glutathione S-transferase (GST), which facilitates purification. Importantly, GST exists as a homodimer in solution. The researchers screened a GST fusion of the oncology target Rpn13 against a small library of 94 fragment-coupled beads and found five hits. SPR studies confirmed weak (K_D > 2 mM) binding for two hits to pure Rpn13 (ie, without the GST fusion), and this binding could be competed with a known peptide ligand of Rpn13.

 

Screening beads in individual wells is one thing, but to really increase throughput it would be nice to be able to screen mixtures of different beads. To do so, the researchers developed a photocleavable linker between bead and fragment. The linker also contained an alkyne group that could be modified with a brominated imidazopyridinium moiety. This tag is UV active, ionizes well, and the bromine’s unique isotopic signature helps distinguish true hits from noise. Beads containing more than 50 different compounds, including the two fragment hits we mentioned above, were incubated with labeled streptavidin. Beads to which protein bound were separated by fluorescence-activated cell sorting (FACS), clicked with the tag, cleaved from the beads, and analyzed by mass spectrometry. Only the two known binders were identified, demonstrating the specificity of the approach.

 

This is a neat paper well worth reading. I particularly like the fact that the method can be done with minimal equipment. I look forward to seeing how it works against more targets.