Scientists from Merck Research Laboratories have identified a synthetic molecule that was able to interfere with bacterial growth by interfering with a regulatory RNA sequence.

The findings are a proof of principle that targeting regulatory RNA could lead to the development of new antibiotics, and possibly drugs targeting other types of diseases.

The results appeared in the Oct. 1, 2015, issue of Nature.

Corresponding author John Howe, principal scientist at Merck, told BioWorld Today that when they started their experiments, he and his colleagues were looking for a way to inhibit riboflavin synthesis, but not particularly for a compound that would inhibit regulatory RNA.

"We thought, frankly, that we'd find an inhibitor of one of the enzymes," he said.

The synthesis of riboflavin, otherwise known as vitamin B2, in principle makes a good antibacterial target because humans have given up on making their own – it is one of the essential nutrients that need to be taken up through the diet.

Bacteria, on the other hand, do make their own. They are also able to take up riboflavin from the environment, but especially for gram negative bacteria, where such uptake is purely passive, environmental uptake is "not sufficient to meet their needs," Howe said.

In their experiments, Howe and his team first did a phenotypic screen with a compound of about 57,000 synthetic small molecules. Howe explained that there are "a lot more attractive opportunities to do structure-activity relationships" for synthetic compounds than for natural analogs.

Previous work looking at targeting riboflavin synthesis has relied on such analogs of natural products. In practice, though, inhibitors based on analogs have also inhibited enzymes that are critical for metabolic processes in eukaryotic cells.

The team was looking for compounds that would inhibit bacterial growth in the absence of riboflavin, but not in its presence – a clue that the molecule was working by interfering with riboflavin synthesis.

In mouse studies, they identified such a compound, which they named ribocil. Treating mice with an Escherichia coli infection with the molecule led to 1,000- to 10,000-fold reduction in bacterial levels, "and no mortality or morbidity."

Howe and his team then set out to decipher ribocil's precise mechanism. The team used resistance studies to identify the molecule's binding site – and found that binding site was not, as they had expected, on one of the enzymes of the riboflavin biosynthetic pathway. Instead, ribocil bound not just to RNA, but to noncoding RNA that was part of a so-called riboswitch.

Such riboswitches regulate the translation of mRNA. In the case of ribocil, its binding inhibits translation of the mRNA for ribB, an enzyme that catalyzes an early step in riboflavin synthesis.

Targeting regulatory RNA, which, Howe said, controls about 5 percent of bacterial gene expression, broadens the range of druggable targets that antibiotics could go after.

And regulatory RNA is not only, or even primarily, important in bacteria. One of the biggest genomics projects of recent years, the Encyclopedia of DNA Elements (ENCODE) project, despite what its name might imply, does not limit itself to DNA. Instead, project leaders wrote in a 2012 review article on "Landscape of transcription in human cells," the ENCODE projects has also "sought to catalogue the repertoire of RNAs produced by human cells as part of the intended goal of identifying and characterizing the functional elements present in the human genome sequence." (See BioWorld Today, Sept. 24, 2009.)

In their paper, Howe and his colleagues noted that "human genetic disorders including fragile X syndrome, myotonic dystrophy, and Huntington disease, all result from expanded nucleotide repeats located in transcripts, which fold into stable RNA hairpin structures" and could in principle be targeted by synthetic molecules.

In their supplementary discussion, the team also noted that ribocil, because its inhibitory activity was much greater in E. coli than in either Pseudomonas aeruginosa or Acinetobacter baumannii. They concluded that by targeting riboswitches, it might be possible to target specific bacterial types.

More specific antibiotics would have fewer untoward effects on the gut microbiome. When the microbiome is wiped out by current broad-spectrum antibiotics, the antibiotic treatment can create a niche for colonization with harmful bacteria such as Clostridium difficile. (See BioWorld Today, Sept. 22, 2015.)

Howe declined to specifically discuss Merck's commercial plans in areas related to the paper's subject, but did note that the company hoped to stimulate discussion by publishing the data.

"There's more and more regulatory roles" being identified for RNA, he said. But the area is still new enough so that there is "a lot of theory, but not as many proof-of-principle papers yet."