Wednesday, October 14, 2015

Hitting the RNA switch: A new method of antibiotic design

Greetings,

Once again, we returning back to the discussion of antibiotics. The last time I discussed about antibiotics was a post on use of predatory bacteria as a possible alternative for antibiotics and Teixobactin which was isolated from a soil microbe with potential activity. Though there are several lead compounds in pharmaceutical research which will possibly make a good antibiotic, the claim has been that there is no new class of antibiotic. There have been a couple of publications in the past on chemicals of interest that are completely different in their approach compared to traditional antibiotics. However, to date we don't have any of these in the shelf.

The latest exciting news of potential lead compound acting through a totally different mechanism is thus a great research of interest for the microbial community. Howe etal (Representing Merck) has described a new antibiotic lead named as Ribocil. As Thomas Hermann describes “Finding an antibiotic with a new target has always been one of the holy grails of antibiotics discovery. It looks like that’s what the Merck group has now accomplished.”

As early as the 1950's antibiotics were discovered through screening. Several thousand natural compounds were purified and tested against bacterial isolates to look for potent activity. One such example is streptomycin. Merck used a similar approach. Only that instead of the natural compound, they screened their synthetic compounds library via a method of high throughput phenotypic screen. This lead to identification of ~57,000 synthetic small molecules which potentially inhibited E coli. The researchers then argued that riboflavin pathway was specific to bacteria and hence an inhibitor would be of great interest in this category. So they screened the E. coli strain (MB5746) defective in wild-type lipopolysaccharide (LPS) levels and drug efflux was selected. to identify if the riboflavin pathway was involved in action the researchers setup a rescue assay. The principle being that if riboflavin synthesis is effected, by adding riboflavin the bacteria would be able to grow despite the presence of antibiotic.

Fig 1: Structure of Ribocil.
Source
One such chemical identified in the process was Ribocil. The next step is to identify the mechanism. First, in an independent experiment, they created resistant E coli mutants. Most commonly, it is done by serial subculture of strains at sub lethal MIC levels. These strains were labelled as Ribocil. By comparing whole genome sequences between resistant mutants and parental strain, several mutations were identified. What do you expect? A mutated enzyme. However, it was seen that all the mutations were in a gene called RibB which indeed was an enzyme in the synthesis pathway. Just that mutation was not in the coding region. It was in a non coding RNA region- the Riboswitch. Quoting from the paper, "Based on the functional role of the FMN riboswitch and that all ribocilmutations map to this regulatory element, we hypothesized that ribocil inhibits riboflavin biosynthesis directly by mimicking the FMN ligand and binding to the FMN riboswitch to inhibit ribB expression".

Fig 2: Structure of an mRNA showing Riboswitch. Source
Let us digress a little. What is a Riboswitch? There are several mechanism by which protein transcription is controlled. It depends on environmental condition as to what are the proteins that needs to be produced and what concentrations. One of them is regulation of mRNA itself. Ribsowitches are a region most commonly seen in 5'-UTR regions. They have a specific folding which allows its interaction with other proteins. The loops can be opened and mRNA readable for protein synthesis through the control of this region hence acting as a switch. In this case, as Howe puts it, "So instead of regulating the enzyme itself, ribocil is regulating the production of the enzyme". For the first time ever, a riboswitch-binding molecule that is not a close structural analogue of a metabolite ligand is discovered.

The paper of course goes on a long list of experiments done to validate the binding characteristics, structural X crystallography studies and animal experiments to show its ability to be an antibiotic. The team further tweaked the Ribocil structure to enhance activity. This is a start. As Gerry Wright comments, “I’ve no idea if ribocil will end up being a drug candidate, but the work is a proof of principle, which is very important, and it makes us look to new areas of biology as targets for antibiotics.”

This study has brought up some interesting discussion, on how this study is insightful. For example, HIV is a hard to beat virus. HIV encoded non coding RNA's has been studied for a longtime (Link). It has been suggested that a similar approach maybe tried for HIV to identify potential therapeutics. A article published in 2014, has shown that a site in HCV called as  Internal Ribosomal Entry Site (IRES) can be attacked by using small synthetic molecules (Link).

On an important note, the chemical is still a long way though from making it to the shelf and mutations arise easily.

ResearchBlogging.org
Howe JA, Wang H, Fischmann TO, Balibar CJ, Xiao L, Galgoci AM, Malinverni JC, Mayhood T, Villafania A, Nahvi A, Murgolo N, Barbieri CM, Mann PA, Carr D, Xia E, Zuck P, Riley D, Painter RE, Walker SS, Sherborne B, de Jesus R, Pan W, Plotkin MA, Wu J, Rindgen D, Cummings J, Garlisi CG, Zhang R, Sheth PR, Gill CJ, Tang H, & Roemer T (2015). Selective small-molecule inhibition of an RNA structural element. Nature PMID: 26416753

Hermann T (2015). Non-coding RNA: Antibiotic tricks a switch. Nature PMID: 26416738

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