Fragments vs VE-PTP: biophysics in action

Protein kinases attach a phosphate group onto amino acid side chains in proteins. Phosphorylation regulates myriad aspects of cell signaling, and thus kinases are common drug targets. Indeed, roughly one third of fragment-derived clinical compounds target kinases. Protein phosphatases remove phosphate groups and thus also make potentially valuable drug targets. Unfortunately, they are very difficult to selectively inhibit, and indeed no fragment-based drugs have entered the clinic. A new paper in Biochemistry from Wataru Asano, Yoshiji Hantani, and colleagues at Japan Tobacco takes the first steps towards rectifying this.

 

Phosphatases are so difficult to drug because most of them have small, highly charged active sites that have evolved to bind phosphate. This moiety and strongly anionic analogs are not very cell permeable or orally bioavailable. Moreover, the small size of the active site makes selectivity challenging, and the fact that many phosphatases contain an active-site cysteine makes them particularly susceptible to assay artifacts.

 

The researchers were interested in vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays a role in vascular homeostasis and angiogenesis. They chose 25,000 fragment-sized molecules (with < 20 heavy atoms) from their HTS collection, all with aqueous solubility > 300 µM, and screened these at 250 µM in a mass-spectrometry-based functional assay. Those that inhibited enzyme activity by at least 40% were retested in dose-response format and also characterized by SPR. Many highly acidic compounds such as sulfonic acids were found, but the researchers were particularly intrigued by Cpd-1, which is only modestly acidic with a calculated pKa of 3.9.

 

Cpd-1 inhibited VE-PTP, but although SPR showed binding, this was not saturable. Thus, the researchers turned to NMR, using multiple protein-observed as well as ligand-observed methods to demonstrate that the molecule binds to the active site of the enzyme. This was confirmed with a crystal structure, which also revealed an “unhappy” water molecule nearby, leading to Cpd-2. This molecule was characterized by crystallography, SPR, and ITC. The molecule proved to be unexpectedly selective for VE-PTP over four other PTPs. The researchers hypothesize that binding to PTPs is often dominated by conserved electrostatic contacts, and because Cpd-2 is less highly charged it relies on other, more specific interactions.

 

This is a nice example of using a variety of biophysical techniques to find and advance fragments. The researchers do a good job of describing the strengths and weaknesses; for example, it was impossible to determine the dissociation constant of Cpd-1 by SPR due to non-specific binding with the protein, reminiscent of a Pin1 story from several years ago.

 

There is still a long way to go, with no cell activity or permeability described for Cpd-2. Still, the paper ends boldly: “we believe that this compound will be developed as a potential drug for VE-PTP-related diseases.” Here’s wishing them success.

Fragments vs CDC25B phosphatase – from behind

Protein phosphatases, which remove phosphate groups from proteins, fall into the category of low-hanging but firmly attached fruit: many make great targets, but getting lead-like inhibitors is tough. Indeed, the enzymes seem to be particularly susceptible to PAINS (see for example here and here). A major challenge is the phosphate-binding site, which has a predilection for highly negatively charged (and non-druglike) moieties. In a paper just published in ACS Chem. Biol., Tomasz Cierpicki and his group at the University of Michigan neatly sidestep this issue.

The researchers were interested in the dual-specificity protein phosphatase CDC25B, which is important in cell cycle regulation and thus a potential anti-cancer target. They started with a ¹H–¹⁵N HSC NMR screen of 1500 fragments in pools of 20, with each fragment present at 0.25 mM. This yielded a single hit: 2-fluoro-4-hydroxybenzonitrile.

Because the researchers were using protein-observed NMR and had previously assigned the backbone resonances, they were able to use chemical shift perturbations to identify the binding site. Surprisingly, this turned out to be not the active site at all, but rather a region about 15 Å away. They were able to confirm this site using X-ray crystallography, which further revealed that the fragment binds in a small pocket near where the substrate protein CDK2 binds.

The researchers noticed a nearby sulfate ion (from the crystallization buffer) and, after first doing a brief SAR by catalog survey, they tried to link this to their hit. Although this certainly didn’t improve physicochemical properties, it did result in tighter binding, and crystallography confirmed that the new molecule bound as designed. This molecule also inhibited the phosphatase, albeit modestly (IC₅₀ 1-2 mM). The result suggests that blocking this protein-protein interaction is effective at blocking activity.

It remains to be seen how much affinity there is to be had at this site. Still, I do have a soft spot for phosphatase inhibitors that bind outside the active site. At the very least this paper provides a new direction for an old – and very difficult – class of targets.

Metallophilic fragments – or PAINS?

Several years ago we described fragment libraries designed to chelate metal ions. The idea is that these could serve as affinity anchors to target metal-containing proteins. However, in designing any fragment library, it is essential to avoid pan-assay interference compounds (PAINS), molecules that act through pathological mechanisms. A new paper in Bioorg. Med. Chem. Lett. by Amy Barrios and collaborators at the University of Utah and University of California San Diego illustrates one of these mechanisms.

The researchers were interested in a protein tyrosine phosphatase (PTP) called LYP. PTPs contain catalytic cysteine residues and are thus particularly prone to false positives caused by oxidation or adventitious metals such as zinc. The researchers screened a library of 96 metal-chelating fragments against LYP in the presence or absence of zinc to find fragments that could either rescue the enzyme or inhibit it, either cooperatively with zinc or on their own. Not surprisingly, they were able to find several fragments that could rescue LYP from added zinc, presumably by coordinating the metal and removing it from the active site.

The most potent inhibitor of the enzyme was 1,2-dihydroxynaphthalene, with an IC₅₀ of 2.5 µM. However, the researchers quickly discovered that it was time-dependent, showing greater potency the longer it was incubated with the enzyme. This is a classic sign of something funny going on, and the researchers realized that molecules like this can oxidize spontaneously. In fact, the oxidized product (1,2-naphthoquinone) is even more potent, and also time-dependent (IC₅₀ = 1.1 µM after 2 hours). Not only can napthoquinones directly modify cysteine residues, they can generate reactive oxygen species that can in turn modify cysteine residues – this very molecule was reported as doing so more than five years ago.

This is the kind of mechanism you want to avoid, and it is likely to rear its ugly head whenever compounds of this ilk are screened, particularly against a protein with an active-site cysteine. Hopefully this publication will serve as a warning to folks who may be using this screening library.

One could argue that this paper falls into the category of “you probably already knew this.” However, even if you knew it, many others likely did not. At the ACS meeting symposium on PAINS earlier this month, Kip Guy urged people to publish their PAINS stories. They may not be the sexiest papers, but if they inform others what to avoid they may be among the most useful.

Substrate activity screening for phosphatase inhibitors

Regular readers will be aware that there are lots of ways to find fragments, but one approach we haven’t covered yet is substrate activity screening, or SAS. A new paper in J. Med. Chem. by Jon Ellman and coworkers at Yale uses this technique to find inhibitors of striatal-enriched protein tyrosine phosphatase (STEP), which is implicated in cognitive decline in a variety of diseases.

Many enzymes can accept a wide range of substrates, and these are often fragment-sized. The basic idea behind SAS is that, since substrates (by definition) bind to a target, finding new substrates gets you new binders, and for some target classes it is straightforward to transform substrates into inhibitors. Of course, you could screen for inhibitors from the start, but the nice thing about looking for substrates is that you are far less likely to encounter artifacts. This is because artifacts normally muck up assays; it’s harder to envision a spurious substrate.

Phosphatases clip phosphates from their substrates. Protein tyrosine phosphatases (PTPs), for example, dephosphorylate tyrosine residues in proteins; they essentially perform the opposite reaction of protein tyrosine kinases. Like kinases, though, finding selective inhibitors can be challenging. The researchers started by building a small library of 140 phosphorylated fragments (previously described here) and looking for those that were particularly good substrates. One of the best for STEP was substrate 8, which looks quite different from phosphotyrosine.

Replacing the substrate phosphate group with a bioisostere (difluoromethylphosphonic acid) that could not be hydrolyzed by the enzyme gave compound 12, which had an inhibition constant (K_i) similar to the Michaelis constant (K_m) of substrate 8. Subsequent optimization led to compound 12s, with a low micromolar K_i and at least 18-fold selectivity against four other PTPs.

Unfortunately, the highly acidic phosphate bioisosteres in these molecules limit membrane permeability: although compound 12s inhibits STEP activity in rat neuronal cell cultures, it is not permeable in a model of the blood-brain barrier. Perhaps some of the less polar phosphate bioisosteres discovered in a previous virtual screen could help.

SAS is an interesting method, and I’m curious as to why more people aren’t using it. Of course, it does require generating bespoke libraries of fragment substrates, but once you have these they are useful for many members of a target class. What do you think?

Virtual phosphate fragments

Phosphate groups are handy little things: easy for enzymes to put on and take off, they pack a lot of charge in a small volume, thereby providing plenty of binding energy for electrostatic interactions. Not surprisingly, they are ubiquitous in biology. Unfortunately, the same things that make them attractive for an organism make them problematic for drugs: they are easily removed, and their highly negative charge gives molecules containing phosphates a real problem getting across membranes. What’s a chemist to do?

This was the dilemma faced by Ruth Brenk, Ian Gilbert, and colleagues at the University of Dundee. They were interested in inhibiting the enzyme 6-phosphogluconate dehydrogenase (6PGDH) from the parasite that causes sleeping sickness. (See here for previous work from the same group using fragment methods to discover inhibitors against a different enzyme from the same organism.) The enzyme 6PGDH, as its name suggests, binds phosphate-containing substrates and has a very polar active site. Nanomolar inhibitors have been reported in the literature, but these contain phosphates and are not active in cell assays.

As reported in a recent issue of Bioorganic and Medicinal Chemistry, the researchers computationally filtered a set of commercially available compounds to find those that were less than 320 Da and were negatively charged, thereby potentially mimicking a phosphate. They then used DOCK 3.5.54 to see which of the resulting 64,000 molecules might bind in the active site of 6PGDH, resulting in 5836 possible hits. Subsequent triaging led to the purchase of 71 compounds. These were tested for inhibition of the enzyme at 200 micromolar concentration. Ten of these compounds inhibited the enzyme more than 80% at this concentration, of which 3 gave clean IC50 curves. These three molecules are all 5-membered carboxylic-acid-containing heterocycles, and although the IC50s are modest (ranging from 28 to 45 micromolar), they have good ligand efficiencies (up to 0.66 (kcal/mol)/atom). A computational search for analogs resulted in a few more active molecules with similar properties.

Whether these fragments can be advanced remains to be seen. The calculated solubilites, Log P, total polar surface area, and intestinal absorption parameters are more attractive than previous inhibitors, but the history of phosphate mimics is not encouraging. Most prominently, the protein PTP-1B, which recognizes phosphotyrosine residues, was once one of the hottest drug targets around, spawning a cottage industry of groups developing phosphotyrosine mimetics. Fragment methods were particularly effective, and numerous potent small molecules were published. But none of them were sufficiently drug-like, and to my knowledge none are in the clinic. Still, it is worth trying: 6PGHD may be more druggable, and approaches like this are likely to provide an answer.