Fragments vs Gram-negative bacterial PPAT

Of the 30+ fragment-derived drugs that have entered the clinic, only one is an antibiotic. In part this reflects a shift away from this therapeutic area by many companies. Novartis, though, has continued to invest, as demonstrated by two consecutive papers in J. Med. Chem.

The researchers were interested in the enzyme phosphopantetheine adenylyltransferase (PPAT, or CoaD), which catalyzes the penultimate step of coenzyme A biosynthesis from ATP and 4'-phosphopantetheine. Although the enzyme is present in all organisms, the bacterial form is highly conserved across prokaryotes and significantly different than the human form. It is also essential for bacterial growth, thus making it an attractive target.

In the first paper, Robert Moreau and colleagues start big: a high-concentration screen (at 500 µM) of 25,000 fragments as well as NMR-based screens of their core 1408 fragment library. Triaging both hit sets led to a cornucopia of 39 crystal structures of bound fragments; the chemical structures of a dozen are provided in the paper, with IC₅₀ values from 31 to >2500 µM. Perhaps surprisingly, all of these bound at the pantetheine binding site of the enzyme, suggesting that this is a “hotter” hot spot than the ATP-binding site.

Three of the fragments are described in more detail. The first was optimized from 273 µM to 4.3 µM, but subsequent advancement was unsuccessful. The second fragment, with an IC₅₀ of 230 µM against E. coli PPAT, could be optimized to mid-nanomolar inhibitors; unfortunately these were much less active against PPAT from P. aeruginosa, so this series was also abandoned. But the third fragment discussed, compound 6, proved to be more tractable.

Initial optimization based on other hits led to compound 32, and addition of a methyl to the benzylic linker provided a satisfying 30-fold improvement in potency for compound 33. This “magic” methyl appeared to help desolvate the adjacent NH as well as pre-orient the molecule in the bound conformation. Further growing from this position led to compound 53, which provided a further 7-fold improvement in potency. Crystallography revealed a hydrogen bond between the nitrile nitrogen and a protein backbone amide. Unlike the previous series, this compound was active against PPAT from both E. coli and P. aeruginosa.

The second paper, by Colin Skepper and colleagues, describes further optimization of the molecules to picomolar binders. There’s a lot of lovely medicinal chemistry in both papers, but unfortunately all the molecules displayed at best only modest antibacterial activity. One problem is that Gram-negative bacteria have two membranes: an outer one which blocks lipophilic molecules and an inner one which blocks hydrophilic molecules. Compounds that can make it past these barriers also face an array of diverse efflux pumps, and these seemed to be the downfall of this project. The core of the molecule makes multiple hydrogen bonds to PPAT; about twenty different heterocycles were tested, but most of these had significantly lower potency, and the active ones were efflux pump substrates.

These difficulties in part explain why companies have been moving away from antibiotics. This was not a minor effort: each paper listed more than twenty authors. The second ends somewhat wistfully. “Although none of the series disclosed… yielded a clinical candidate, it is our hope that these studies will help pave the way toward the discovery of new Gram-negative antibacterial agents with novel modes of action.” It is a worthy – if arduous – quest.