Casting light on target-guided synthesis

Target-guided synthesis, in which a protein templates the formation of its own inhibitor, is a concept first proposed decades ago. There are roughly two flavors. Dynamic combinatorial chemistry (DCC) involves reversible formation of the product, and we wrote in 2017 about some of the challenges. Kinetic target-guided synthesis (KTGS) involves irreversible chemistry, for which the options are limited. The classical click chemistry azide-alkyne cycloaddition is so slow that reactions usually take days, which can be a problem for delicate proteins. A recent (open-access) paper in Angew. Chem. Int. Ed. by Cyrille Sabot et al. describes a bright way to accelerate things.

 

The researchers turned to photochemistry, specifically diazirine chemistry. Illuminating 3-trifluoromethyl-3-phenyldiazirines leads to loss of nitrogen and formation of highly reactive carbenes. The carbenes are so hot that they can react indiscriminately with proteins, as we described here. However, the reaction with thiols is faster than the reaction with other functional groups on proteins, so the researchers reasoned that a library of thiols could out-compete the protein.

 

The carbonic anhydrase bCA-II was chosen as a model protein. Sulfonamide-containing molecules such as compound 5 are known to be good inhibitors. This “anchor” molecule was incubated at 60 µM with seven different diazirines, each at 400 µM, in the presence or absence of 30 µM bCA-II and then irradiated with 365 nM light for a few minutes. Most of the reactions produced similar amounts of product in the presence or absence of bCA-II, but compound 1b yielded about threefold more of compound 2d in the presence of bCA-II, suggesting the reaction was being templated by the protein. 

 

Control experiments lend credence to this hypothesis. First, adding a known competitive bCA-II inhibitor reduced the formation of compound 2d to background levels. Second, other proteins did not cause a similar enhancement in the formation of compound 2d. Finally, conducting the experiment with phenylmethanethiol (ie, a variant of compound 5 lacking the sulfonamide moiety essential for interaction with bCA-II) did not cause an enrichment of the photochemical product in the presence of the enzyme.

 

Chiral HPLC was used to show that compound 2d was slightly enriched for the (R)-enantiomer, with an enantiomeric excess of around 10%, when the reaction was conducted in the presence of bCA-II but not in the absence. The two enantiomers were synthesized and tested, and the (R) form did indeed have slightly better activity (300 nM vs 330 nM).

 

This is a thoughtful, well-conducted investigation. But it makes me even less sanguine about the practicality of KTGS for finding new chemical matter, for several reasons. First, the efficiency of the reaction is poor: the researchers calculate the yield of compound 2d at around 1% of the enzyme concentration, so low that they used single-ion monitoring (SIM) mass spectrometry to detect it. Because of this low efficiency, the concentration of enzyme used needs to be quite high.

 

The most serious strike against KTGS is the fact that all of the diazirines generated potent (sub-micromolar) inhibitors. One of them was even slightly better than compound 2d but did not show enrichment in the presence of bCA-II. False negatives seem to be a major problem, as we’ve written previously.

 

One caveat to my caveats is that compound 2d is only marginally more potent than the starting compound 5. NMR experiments conducted with diazirine 1b suggest binding to the protein, though the affinity was not quantified. Perhaps a different fragment linking system, in which both fragments have measurable affinity for the target, would be better suited to demonstrate the utility of KTGS. For now, this paper does a nice job highlighting its drawbacks.

Nucleophilic fragments vs SARM1: in situ inhibitor assembly

Recently Practical Fragments wrote about nucleophilic fragments that could react with proteins or cofactors. Previously we’ve also written about in situ chemistry, in which a protein catalyzes the formation of an inhibitor. An interesting marriage of these concepts has just been published (open access) in Mol. Cell by Robert Hughes (Disarm Therapeutics), Thomas Ve (Griffith University) and a group of international collaborators.

 

The researchers were interested in the protein SARM1, which is implicated in the axon degeneration associated with several neurodegenerative disorders. Last year the researchers published a Cell Rep. paper (also open access) in which a biochemical screen of roughly 200,000 molecules led to the discovery of isoquinoline as a 10 µM inhibitor of SARM1. Optimization led to 5-iodoisoqinoline, dubbed DSRM-3716, a 75 nM fragment-sized inhibitor. The paper goes on to demonstrate that the molecule not only prevents axonal degeneration but can even promote recovery of injured axons. The new paper explores the mechanism of action.

 

SARM1 is an NADase: it cleaves the critical cofactor nicotinamide adenine dinucleotide (NAD⁺). While using NMR to study the mechanism of inhibition, the researchers found that DSRM-3716 reacts with NAD⁺ to form the new compound shown. In this sense, DSRM-3716 acts as a prodrug, somewhat analogous to sulfanilamide antibiotics which act as PABA mimics to block folate biosynthesis.

 

What’s behind the inhibition of SARM1? A series of crystallographic and cryo-EM studies of SARM1 reveal that the protein can self-associate into multimers which are either inactive or active depending on the relative orientations of the individual proteins. NAD⁺ normally binds at the interface between two SARM1 proteins. The compound made from NAD⁺ and DSRM-3716 binds here as well, blocking further activity. The crystal structures also revealed a clear halogen bond (see here) with the iodine in DSRM-3716, explaining the increased activity over isoquinoline itself.

 

Unlike the nucleophilic fragments we wrote about last month, isoquinoline probably won’t raise too many eyebrows among medicinal chemists, as the moiety is found in a handful of approved drugs. The researchers also demonstrated that DSRM-3716 itself is selective for SARM1 in a panel of other enzymes that use NAD⁺.

 

This is a lovely case of high-throughput screening in which the hit turns out to be a fragment. Indeed, the highly charged compound that actually inhibits SARM1 would not be cell-permeable, but that's just fine since it is formed inside cells. It is worth noting that nearly 1000 approved drugs could be classified as fragments in terms of molecular weight. In the case of CNS drugs, small is beautiful, and it will be fun to watch how far DSRM-3716 derivatives will be able to advance.

In situ click chemistry on RNA

In templated or in situ reactions, bonds form between two fragments that are brought together in the context of a larger molecule such as a protein. We have written previously about dynamic combinatorial chemistry (DCC), which depends on reversible bond formation where the larger molecule shifts the equilibrium toward the linked fragments. For irreversible bond formation, the larger molecule effectively catalyzes the formation of an inhibitor (or at least a binder). In a recent Angew. Chem. Int. Ed. paper Jyotirmayee Dash and colleagues at the Indian Association for the Cultivation of Science describe an application of the latter that uses RNA as the template.

The target of interest was TAR RNA, a short region of viral RNA essential for HIV replication. We have previously highlighted a few examples of fragment screening against RNA (here, here, and here), including TAR, but most of the hits were weak.

The researchers used azide-alkyne cycloaddition, the quintessential click chemistry reaction. They built a small library of four alkynes (only one of which was fragment-sized) and 11 azides. All of these were incubated together (4 µM of each alkyne and 1 µM of each azide) in the presence of 5 µM biotin-labeled TAR RNA for 72 hours. (The reaction is typically slow at room temperatures unless catalyzed by metal ions.) Magnetic streptavidin-coated beads were then used to capture the RNA and any bound ligands, which were identified by HPLC-MS. Control experiments were run with TAR DNA or a mutant form of TAR RNA lacking an essential bulge. The result was one fairly potent compound (below) that was specific for TAR RNA, as well as a couple other molecules that were both weaker and less specific.

The affinity of compound 3ba for TAR RNA was measured by isothermal titration calorimetry (ITC) as well as by a fluorescence assay, which were in good agreement. Importantly, the ITC data suggested 1:1 binding, which is particularly important given that the ligand contains two 2-aminothiazoles, a moiety that has been called a PrAT for its promiscuous behavior. Finally, the ligand could displace the Tat peptide at low micromolar concentrations, suggesting that it is binding at the biologically relevant site of TAR.

I do have a few quibbles. It would have been interesting if the researchers had reported the affinity of the azide and alkyne themselves to see how much of a boost they got by linking. And since the most potent molecule is not always selected from target-guided synthesis, it would have been interesting to make and test other possible cycloaddition products to see if they missed anything useful.

Still, it is nice to see a submicromolar RNA binder come out of an in situ screen. Targeting RNA with small molecules has recently become trendy, and it will be fun to see how far approaches like these can go.

The calm before the click in chitinase

In situ click chemistry is a topic we’ve covered before on Practical Fragments. Essentially, two ligands bind near one another on a target protein and react to form a linked molecule. There are several published examples, but it is not clear why it sometimes works and sometimes doesn’t. A new paper in Proc. Acad. Nat. Sci. USA by a team of Japanese and US researchers led by Satoshi Ōmura and Toshiaki Sunazuka at the Kitasato Institute in Tokyo addresses this question.

The researchers had previously discovered potent inhibitors of an antibacterial target enzyme called Serratia marcescens chitinase B, or SmChiB, using in situ click chemistry. In the presence of SmChiB, azide 2 reacts with alkyne 3 to yield triazole 4, which binds 26-fold more tightly than azide 2:

 

In the new paper, the goal was to use crystallography and computational chemistry to investigate how the reaction proceeds. To avoid azide 2 reacting with alkyne 3 in the crystal and so better visualize starting points, the researchers prepared the closely related alkene 5 mimic of alkyne 3. Unfortunately, due to its (unmeasurably poor) affinity, alkene 5 did not yield a co-crystal structure on its own.

The researchers were able to obtain a co-crystal structure of SmChiB bound to triazole 4 (green carbons below). Surprisingly, a co-crystal structure of azide 2 showed the molecule bound in a quite different orientation. However, a co-crystal structure of the ternary complex of azide 2 (cyan below) and alkene 5 (magenta) bound simultaneously to SmChiB revealed a close overlay of azide 2 with the corresponding fragment in triazole 4. Alkene 5 in the ternary complex adopted two conformations (the electron density is memorably described as resembling “a two-horned goat head”). As shown in the figure below, one of these orientations places the alkene moiety in close proximity to the azide moiety, primed for clicking.

Next, the researchers used this ternary structure to run high-level density functional theory calculations to determine the energetics of the click reaction and compared these with the same reaction run in water. The values were quite similar (if anything, the protein had a slightly higher activation barrier), suggesting that the protein was not directly catalyzing the reaction with specific amino acid side chains. Rather, the reaction was being accelerated simply by the preorganization of the azide and alkyne.

On the one hand, these results aren’t really a surprise: I think most people assumed that in situ chemistry works by bringing the reactants together rather than anything more exotic (with the odd exception). On the other hand, it is nice to see experiment match theory.

More generally, the results help to explain why in situ click chemistry is so challenging. The crystal structure of azide 2 and alkene 5 shows the relevant moieties quite close to each other, yet the reaction is still somewhat inefficient. Finding two fragments that not only bind near one another but are also oriented properly is likely to be a rare event.

Protein-templated click chemistry – just add copper

We’ve written previously about protein-templated chemistry (here and here), in which a protein catalyzes the formation of a more potent inhibitor from two lower affinity fragments. Of course, proteins aren’t the only things that can catalyze reactions: copper is well-known to promote the cycloaddition between azides and alkynes. Like peanut butter and chocolate, it turns out that copper in the context of a protein can be even better than either alone, as reported in a recent issue of Angew. Chem. by an international team of researchers from Japan and the US.

The researchers were interested in using in situ click chemistry to discover inhibitors of histone deacetylases (HDACs), and they decided to see if they could use an activity assay to detect the formation of inhibitors formed in situ. They incubated two different hydroxamic-containing alkynes (known HDAC inhibibitors) with 15 different azides in the presence of HDAC8 and looked for enhanced inhibition of the enzyme. Of these 30 combinations, they found a single hit: the reaction of compound 1b with compound 2o (see figure).


However, there were several oddities. First, the linked compound (anti-3) is no more potent than the initial hydoxamic-containing fragment. Second, only the anti isomer was formed, despite the fact that the syn isomer is almost 10-fold more potent. Finally, the yields of anti-3 were much higher than typically observed in these sorts of experiments. This made the researchers suspicious, and after a series of experiments they determined that trace amounts of copper, most likely introduced in the synthesis of 1b, had incorporated into the active site of HDAC8 and were serving to accelerate the reaction. A small amount of copper in the absence of protein was unable to catalyze the reaction, nor was the protein alone when copper was carefully removed.

There are a number of interesting implications from this paper, but one in particular is rather sobering: in situ assembly screening does not necessarily yield the most potent inhibitor. I suspect this is a general feature of kinetically-guided methods of inhibitor discovery, but what do you think?