The human body is constantly exposed to bacterial strains. To combat the bacterial strains that cause disease or infection, patients are often prescribed a variety of different medications, many of which have numerous harmful side effects. Dr. Howe and colleagues from Merck Research Labs set out to discover a new method to eliminate bacteria that have infected the human body in a safer and more efficient way.
Dr. Howe and colleagues from Merck Research Labs set out to discover a new method to eliminate bacteria that have infected the human body in a safer and more efficient way.
An essential compound to the bacterial life cycle and growth is riboflavin, more commonly known as vitamin B2. When this compound is absent from a bacteria’s internal cellular environment but present in the surrounding environment, the bacteria ingests it to conserve energy. However, in most cases bacteria will not have access to the compound from their surroundings, which will thus force them to independently synthesize the compound. With this knowledge in mind, the research team hypothesized that if the selfproduction of riboflavin in bacteria could be somehow inhibited or suppressed, the bacteria would starve to death, thereby halting the spread of infection within the body.
The researchers began by studying the process by which riboflavin synthesis is regulated. Their findings led to the close examination of certain stretches within messenger RNA (mRNA, the blueprint that tells the cells how to make proteins) called riboswitches. These structures are termed “noncoding RNAs,” as they do not code for proteins. However, riboswitches play an important role within the cell, particularly in the regulation of riboflavin synthesis. These riboswitches are composed of two main parts, the first being a sequence that physically binds a specific protein (generally, what is binding will be termed a “ligand”), and the second being an “expression platform.” A ligand will bind to the specific binding site within the riboswitch, which will cause a conformational change within the expression platform, resulting in a physical change of shape of the mRNA molecule. Consequently, this is where the term riboswitch originates; a “switch” occurs upon the binding of a specific ligand. Keeping in mind that riboswitches reside in mRNA molecules, any structural change within that molecule will usually prevent the cell from reading the blueprint, thereby inhibiting the production of the protein.
Their findings led to the close examination of certain stretches within messenger RNA (mRNA, the blueprint that tells the cells how to make proteins) called riboswitches.
Contextualizing this information in light of the bacterial riboflavin synthesis process, the specific riboswitch that is responsible for the regulation of riboflavin is known as the FMN (flavin mononucleotide) riboswitch. On a molecular level, the riboflavin compound is converted into FMN, which will then directly interact with the riboswitch and suppress the production of proteins that produce riboflavin. The phrase “negative feedback loop” refers to the process by which a product of a certain multistep synthesis process interacts with a molecule in one of the early synthesis steps to entirely shut down the production of said product; a process which exemplifies efficiency. When the bacterium’s internal riboflavin levels are high enough, these riboflavin molecules will interact with an early player in the riboflavin synthesis process (in this case, the riboswitch) and completely shut it down. Howe’s research team harnessed this concept to devise synthetic elements that could be used as novel drugs to inhibit the production of bacterial riboflavin and thus kill the bacteria.
In the hunt for finding another molecule that would inhibit the production of riboflavin, the researchers happened upon a riboflavin derivative named roseoflavin. This compound is similarly converted into roseoflavin mononucleotide RoFMN, and it similarly inhibits the synthesis pathway. The only problem that roseoflavin posed was the compound’s lack of specificity. In other words, roseoflavin was found to not only inhibit its own production, but also the production of around 40 more enzymes that are involved in other synthesis processes, which occur not only within the bacteria but in humans as well. Because roseoflavin lacks the specificity component of riboflavin, it would have the potential to cause harmful side effects in humans. As a result, roseoflavin was not considered for therapeutic purposes.
In the hunt for finding another molecule that would inhibit the production of riboflavin, the researchers happened upon a riboflavin derivative named roseoflavin.
As the research advanced, Howe’s team screened a preselected group of synthetic mimics of riboflavin (synthetic molecules that would resemble riboflavin in structure and function). After numerous trials, the researchers found a molecule named ribocil that was able to effectively and specifically suppress riboflavin production. In order to make sure that ribocil was in fact targeting the riboswitches, the research team found strains of bacteria that were resistant to ribocil (characterized by failing to inhibit riboflavin production). To their surprise, the researchers discovered that all strains contained mutations in the riboswitch nucleotide sequence that prevented the binding of ribocil. This discovery led to the hypothesis that ribocil directly interacts with the FMN riboswitch by mimicking the FMN molecule, thereby suppressing the expression of the ribB gene (a gene that codes for a vital protein in the production of riboflavin). What makes ribocil such a great candidate for drug therapies is the fact that it will specifically bind to the bacterial riboswitch and inhibit only that riboswitch, without interfering with the production of other human proteins (which was seen with roseoflavin). Similarly, ribocil’s high specificity of binding to the correct FMN riboswitch allows it to target only virulent bacterial strains. This is once more compared to the action of roseoflavin, which was found to inhibit not only human proteins, but also cell machinery of bacteria that are beneficial to the body. Ribocil can easily enter bacterium without any support proteins, which compounds like roseoflavin require. Thus, the researchers concluded that the administration of ribocil would theoretically be an effective therapeutic antibacterial approach.
It is important to note that although ribocil was found to work with a specific bacterial riboswitch, one should refrain from making generalizations regarding other riboswitches because a multitude of other factors may impact their inhibition. All in all, this discovery of the ability to synthetically regulate riboswitches opens doors for further research on how to harness these unique RNA elements with hope of novel medical approaches for combating bacterial infections.
Howe, J., Wang, H., & Fischmann, T. (2015). Selective smallmolecule inhibition of an RNA structural element. Nature, 000. doi:10.1038/nature15542.