New chemistries for covalent fragments

Good things come in threes, and since our last two posts have covered covalent fragments, we thought we’d continue the theme with two more papers on the topic.

The first, in MedChemComm by György Keserű and colleagues at the Hungarian Academy of Sciences, is actually a companion to a paper we covered at the end of last year. The researchers were interested in electrophilic heterocycles, and assembled a library of 84, of which 57 were commercial and the rest were synthesized. In their previous paper, the researchers focused on reactivity against proteins. This paper is focused more on aqueous stability and intrinsic reactivity with glutathione, a biologically important thiol. The paper includes handy figures and tables summarizing reactivity rate constants and half-lives (at pH 7.4). These should be useful for selecting warheads that are sufficiently reactive as to be able to label proteins, but not so reactive as to label many proteins nonselectively.

The researchers also did computational studies to try to understand different trends in reactivity. And if you’re interested in testing the compounds yourself, they note that the “library is available for screening against relevant targets upon request from the authors.”

The second paper, by Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University, was published in J. Chem. Inf. Model. Last year we highlighted their diversity-oriented target-focused synthesis (DOTS) approach, which combines virtual screening with automated synthesis to rapidly generate new compounds for testing. They have now expanded this approach (called CovaDOTS) to focus on covalent modifiers.

Conceptually, CovaDOTS is akin to fragment linking, in which one of the “fragments” is the nucleophilic residue in the target protein. The process starts with a known noncovalent ligand which is computationally grown by attaching it to commercially available building blocks that contain reactive warheads. These new molecules are then linked with the side chain of an amino acid (cysteine and serine in the paper), and the assemblies are docked against the protein to find molecules that fit well. Ultimately, the best would be resynthesized and tested.

The researchers applied CovaDOTS to three proteins for which covalent and non-covalent ligands had been previously characterized crystallographically. In the case of two kinases, EGFR and ERK2, the program performed well, with the “correct” (i.e., published) ligand observed in the top 14% and 4.4% of hits. For the serine peptidase PREP, the published ligand was the top hit of 303 molecules scored. In all three cases, the predicted binding mode also closely resembled the experimentally determined structure.

One limitation of CovaDOTS is that, as currently implemented, it considers only commercially available building blocks that also “possess both a warhead and an activated function compatible with the selected chemical reaction(s).” It would be interesting to combine the approach with the “make-on-demand” molecules we discussed a few months ago. And of course, it will be interesting to see real-world examples of how the program performs, in addition to these retrospective case studies.

This ends the June 2019 covalent fragment trilogy, but I think it’s fair to say that covalent fragments have a bright future. Look forward to many sequels!

Screening irreversible covalent fragments, computationally and experimentally

Last week we discussed a paper that characterized commercially available irreversible fragments and screened them against ten proteins. The “warheads” used were either acrylamides or chloroacetamides. This week we’re continuing the theme of irreversible fragments with two papers, each using different warheads.

The first paper, published in Bioorg. Med. Chem. Lett. by Alexander Statsyuk and collaborators at University of Houston, Schrödinger, and Northwestern University, uses a computational approach.

Research we previously highlighted from the Statsyuk lab found that methyl vinylsulfones have a narrower range of reactivities than – for example – acrylamides. This property is important  to ensure that differences in binding to a target are caused by (specific) noncovalent interactions rather than mere differences in warhead reactivity. For the current campaign, the researchers constructed a virtual library in which 1648 commercially available carboxylic acids were coupled to H₂N-CH₂-CH=CH-SO₂-CH₃.

The researchers used the CovDock program from Schrödinger to dock the fragments. (Another computational tool for doing so is DOCKovalent, which we described back in 2014.) The specific target chosen was cathepsin L, a cysteine protease which has been implicated in a variety of diseases from cancer to osteoporosis. The virtual screen yielded 33 high-scoring compounds, five of which were synthesized based on price and diversity. Unfortunately, most of these had “solubility issues,” but compound 11 did show time-dependent inhibition of cathepsin L. The researchers also found that the racemic methyl substituent could be removed (compound 13), suggesting that compound growing might be productive. Compound 13 was also selective against three other cysteine proteases.

The second paper, by David House, Katrin Rittinger and collaborators at GlaxoSmithKline, the Francis Crick Institute, and Cellzome, was published in J. Am. Chem. Soc. The researchers were interested in protein ubiquitination, in which the small protein ubiquitin is conjugated to various other proteins to cause a variety of effects depending on the context. The biology is fiendishly complex, but the final step is done by an E3 ubiquitin ligase, of which there are more than 600 in human cells. Needless to say, selective chemical probes would be useful.

The researchers were specifically interested in LUBAC, an RBR E3 ubiquitin ligase which conjugates ubiquitin to proteins with an N-terminal methionine to modulate cellular pathways important in cancer and inflammation. The ligase itself actually consists of three protein subunits, with HOIP containing the catalytic cysteine residue. Although a couple inhibitors had been previously reported, the researchers found these to be nonspecific. Thus, they built and screened their own fragment library. For a warhead, they chose the 4-aminobut-2-enoate methyl ester, which Statsyuk had previously shown has a narrow range of reactivity and is about 10-fold less reactive than the vinylsulfones discussed above. The researchers constructed a small set of 104 fragments, grouped them into pools of 4 or 5, and screened these against 2 µM HOIP at 20 µM each using intact protein mass spectrometry. Compound 5 was one of the best hits.

Compound 5 functionally inhibited HOIP and was selective against about a dozen other cysteine-containing enzymes. The researchers obtained a crystal structure of the molecule bound to HOIP, which confirmed covalent binding to the active site cysteine. Limited SAR studies led to a slightly more potent analog (containing a six-membered ring instead of a five-membered ring), and this molecule showed pathway inhibition in a cell-based assay with EC₅₀  = 37 µM. Activity-based profiling in two cell lines revealed only 8 or 11 proteins that were significantly modified by the compound in addition to HOIP.

The molecules in both of these papers still require considerable work to become chemical probes, let alone development candidates. Nonetheless, they are useful starting points, and together demonstrate the increasing interest and utility of irreversible covalent fragments.

Characterizing and screening commercially available irreversible covalent fragments

A few years ago we highlighted the utility of irreversible fragments. Because these molecules form covalent bonds with their targets, they can be more effective than similarly sized noncovalent molecules at inhibiting proteins. However, compared with conventional fragments, the quality and quantity of commercial irreversible fragments is limited. This is changing, as described (open access!) by Nir London (Weizmann Institute of Science) and a large, multinational group of collaborators in J. Am. Chem. Soc.

The researchers assembled a collection of 993 fragments from Enamine, all of which contained a cysteine-reactive warhead, either a chloroacetamide (76%) or an acrylamide (24%). The molecules were largely rule of three compliant, even with the warhead included.

A major concern with screening irreversible fragments is that binding to the target protein can be dominated by the inherent reactivity of the warheads rather than non-covalent (and presumably target-specific) interactions from the fragment. Indeed, a previous study found that the reactivities of acrylamides ranged over more than three orders of magnitude. To assess fragments for this, the researchers developed a rapid, plate-based spectrophotometric assay based on labeling the reduced form of Ellman’s reagent. Not surprisingly, the chloroacetamides tended to be more reactive than the acrylamides, but overall the reactivity range across both classes was a relatively modest ~100-fold.

Next, the researchers screened their library against ten cysteine-containing proteins. Fragments were screened in pools of five (200 µM each) with 2 – 10 µM protein for 24 hours at 4 °C. As with Tethering, intact protein mass spectrometry was used to identify hits, which were found for seven of the ten proteins. Hit rates ranged from 0.2 to 4%.

Not surprisingly for fragments, some hits were promiscuous: they strongly labeled two or more proteins. However, these represented less than 3% of the library. Surprisingly, promiscuity did not correlate with reactivity, and in fact some of the most reactive fragments did not label any of the proteins. This suggests that non-covalent interactions are playing a role in promiscuity, and indeed many of the frequent hitters were aminothiazoles – which have previously been found to be promiscuous.

The researchers also screened their fragments (at 10 µM) against three cell lines, and here they did see a correlation with reactivity, with the most reactive fragments tending to be more toxic.

Next, the researchers began optimizing hits against two targets. The first, OTUB2, is a deubiquitinase (DUB) implicated in diverse diseases from amyotrophic lateral sclerosis to diabetes to cancer. The primary screen yielded 47 hits which labeled at least 50%, of which 37 were quite selective. Co-crystal structures were solved for 15 fragment-protein complexes, and two shared a hydrazide moiety (as in PCM-0102954) which made multiple hydrogen bonds with the protein. Two rounds of SAR-by-catalog eventually led to OTUB2-COV-1, which inhibited the enzyme with a respectable k_cat/K_I = 3.75 M^-1 s^-1. Despite containing a chloroacetamide, the molecule labeled just 26 of 2998 cysteines in proteins detected in a cell-based proteomic assay.

The researchers also found 36 fragment hits against NUDT7, a protein potentially associated with diabetes, and many of these stabilized the protein in a differential scanning fluorimetry (DSF) assay. Crystal structures were obtained for several, and compound PCM-0102716 showed an overlap with the non-covalent molecule NUDT7-REV-1 derived from a previous crystallographic fragment screen. When the researchers merged these, the resulting NUDT7-COV-1 showed low micromolar inhibition and rapid labeling (k_cat/K_I = 757 M^-1 s^-1). This is all the more impressive given that the original noncovalent hit showed no activity. NUTDT7-COV-1 also showed target engagement in a cell assay, and hit only 37 of 2025 detected cysteine residues in a proteomics screen.

This is a nice, thorough paper, though I suspect people in industry will be wary of the chloroacetamides that form the bulk of the library. Nonetheless, chemical structures and reactivity data for all the fragments are reported in the supporting information, making this a useful resource for anyone wishing to dip their toes into covalent fragment screening.

Is thermodynamic data useful for drug discovery?

Just over a decade ago Ernesto Freire suggested that small molecules whose binding energy is dominated by the enthalpic – rather than the entropic – term make superior drugs. He also suggested that such molecules may be more selective for their target. But the backlash came quickly, and a couple years ago we wrote that focusing on thermodynamics probably isn’t particularly practical. A new perspective in Drug Disc. Today by Gerhard Klebe (Philipps-University Marburg) revisits this topic.

Klebe suggests that enthalpy was initially embraced “because readily accessible and easily recordable parameters are much sought after for the support of the nontrivial decision over which molecules to take to the next level of development.” (I would be interested to know whether sales of isothermal titration calorimetry (ITC) instruments spiked around 2010.) Unfortunately, both theoretical and practical reasons make thermodynamic measurements less useful than hoped.

First, and as we noted previously, “in an ITC experiment… the balance sheet of the entire process is measured.” In particular, water molecules – which make up the bulk of the solution – can affect both enthalpic and entropic terms. Klebe describes an example in which the most flexible of a series of ligands binds with the most favorable entropy to the target protein; this is counterintuitive because the ligand adopts a more ordered state once bound to the protein. It turned out that in solution the ligand traps a water molecule that is released when the ligand binds to the protein, thus accounting for the favorable entropy.

Indeed, water turns out to be a major confounding factor. We’ve previously written about “high-energy” water; Klebe notes that an individual water molecule can easily contribute more than 2 kcal/mol to the overall thermodynamic signature. And of course, proteins in solution are literally bathed in water. The structure of this water network, which may change upon ligand binding, is rarely known experimentally, but optimizing for it can improve affinity of a ligand by as much as 50-fold. Conversely, attaching a polar substituent to a solvent-exposed portion of a molecule to improve solubility sometimes causes a loss in affinity, and Klebe suggests this can be due to disruption of the water sheath.

Beyond these theoretical considerations, experimental problems abound. We’ve previously discussed how spurious results can be obtained when testing mixtures of ligands in an ITC experiment, but even with single protein-ligand complexes things can get complicated. Klebe shows examples where the relative enthalpic and entropic components to free energy change dramatically simply because of changes in buffer or temperature. This means that the growing body of published thermodynamic data needs to be treated cautiously.

So what is to be done? First, thermodynamic data should always be treated relatively: “we should avoid classifying ligands as enthalpy- or entropy-driven binders; in fact, we can only differentiate them as enthalpically or entropically more favored binders relative to one another.”

Klebe argues that collecting data on a variety of ligands for a given target under carefully controlled conditions will be useful for developing computational binding models. This is important, but not the kind of work for which people usually win grants, let alone venture funding.

He also suggests that, by collecting thermodynamic data across a series of ligands, unexpected changes in thermodynamic profiles might reveal “changes in binding modes, protonation states, or water-mediated interactions.” Maybe. But it takes serious effort to collect high-quality ITC data. Are there examples where you’ve found it to be worthwhile?