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.

Fragments vs herpesviridae

The name herpes makes most people think of painful ulcers in the mouth, or worse. But herpesviruses are actually a family of viruses that can also cause chicken pox, mononucleosis, and other diseases. Some 95% of adults are infected by at least one type of herpesvirus, and these can become deadly if people become immunocompromised, such as during an organ transplant. A drug that would inhibit all forms of herpesviruses would be useful, and the first steps are described in a recent ACS Med. Chem. Lett. paper by Michael Plotkin and colleagues at Merck.

 

The details of the primary screen are sparse, though the researchers did say they physically screened more than 100,000 compounds to identify molecules such as compound 5, a modest inhibitor of the DNA polymerases from both cytomegalovirus (CMV) and varicella zoster virus (VZV). (For most compounds the paper reports biochemical activity towards both of these polymerases as well as antiviral activity for CMV, VZV, herpes simplex virus 1 (HSV-1), and HSV-2, but for simplicity I’ll only show data for CMV here. The compounds generally have comparable activity towards different viruses.)

 

Hydrogen bond acceptors such as the ketone in compound 5 were found to be essential for activity, and exploring a variety of analogs led to compound 12, which in addition to submicromolar biochemical activity against the DNA polymerases also showed antiviral activity against CMV and other herpesviruses.

 

The paper goes into considerable detail on the lead optimization. The (S) enantiomer of compound 12 was an order of magnitude more potent than the (R) enantiomer. Modifications made to both of the phenyl rings ultimately led to compound 44, with low nanomolar biochemical activity against the polymerases and sub-micromolar antiviral activity against CMV, VZV, HSV-1, and HSV-2. Importantly, the researchers note that they did not have crystal structures during optimization, a useful reminder that structural information is not always necessary.

 

Compound 44 had modest oral bioavailability in rodents, but closely related compound 42 containing a trifluoromethyl group in place of the bromine was better, albeit with slightly lower biochemical potency. This molecule led to high survival rates in mice when dosed either before or after being exposed to HSV-1. In separate studies, the compound reduced CMV viral load. For both HSV-1 and CMV compound 42 compared favorably to acyclovir and ganciclovir, two commonly used drugs.

 

Although there is still some way to go to a drug, the researchers end by promising to describe “further progress of this series.” I look forward to reading about this.

Fragments in Brazil

Most of the fragment events we’ve highlighted are in the US, Europe, and Australia, but that does not fully reflect where all the good science is happening. In a recent ACS Med. Chem. Lett. paper, Carolina Horta Andrade, Maria Cristina Nonato, and Flavio da Silva Emery introduce CRAFT: the Center for Research and Advancement in Fragments and molecular Targets.

 

Established in 2021, CRAFT is a collaboration between the University of Saõ Paulo and the Federal University of Goiás. The center is focused on endemic diseases of Brazil. As the researchers note, only one of the 60 or so fragment-derived drugs that have entered the clinic is an anti-infective, so there is clearly significant need. CRAFT also has an educational and training component reminiscent of the European FragNet and the Australian Centre for Fragment-Based Design.

 

One focus of CRAFT is fragment library design, including underexplored heterocyclic systems. Importantly, the researchers are investigating new synthetic methodologies to be able to functionalize different regions of the fragments. They are also exploring fragments similar to or derived from natural products.

 

Targets are of course essential, and CRAFT is investing in protein production and characterization, such as the enzyme DHODH from Leishmania; we’ve written recently about a fragment approach to the mammalian counterpart.

 

Finally, CRAFT is investing in structure-based design, ligand-based design, and phenotypic screening. And in 2024 no venture would be complete without use of machine learning.

 

Academic laboratories often struggle with downstream drug discovery efforts such as drug metabolism and pharmacokinetics. CRAFT recognizes this and has partnered with the Welcome Centre for Anti-Infectives Research to train participants in DMPK.

 

The researchers “invite the global scientific community to collaborate with us in addressing neglected diseases.” I hope they succeed. Five years ago we highlighted the consortium Open Source Antibiotics, but that site seems to be updated infrequently. The COVID Moonshot has been more successful but is arguably less urgent given the billions of dollars of industry money that poured into research on SARS-CoV-2. From an ethical perspective society should invest more on combating tropical diseases. And as the planet warms, these diseases will increasingly move out of the tropics.