The advent of antibiotics was an extremely important step in the progress of human society. Yet with the constant usage of these antibiotics, bacteria have developed resistance. One of the ways that bacteria have gained resistance is through the protein β-lactamase.
The process of evolution takes place constantly; as a result, certain bacterial strains have developed resistance to antibiotics. Antibiotics, such as penicillin (a β-lactam) stop bacterial growth by interfering with the synthesis of the peptidoglycan layer of cell walls. These antibiotics disrupt DD-peptidases that form the cross-links in peptidoglycan [1]. However, through repeated exposure to these antibiotics, bacteria have evolved to counteract this by synthesizing the protein β-lactamase. This particular enzyme attacks the β-lactam ring of antibiotics, thereby rendering the antibiotic ineffective [2].
Figure 1: Ribbon Structure of Beta Lactamase, Adapted from Fonze, E. et al (1995)
An aptamer is a short sequence of nucleic acid that binds to a certain target, usually protein, very tightly. Using the SELEX method of aptamer selection incorporates the same driving force of evolution that produced β-lactamase in bacteria. By exponentially multiplying the nucleic acid sequences that bind most tightly to the protein of interest, only the most “fit” nucleic acids remain. This tightly binding nucleic acid can subsequently interfere with protein function. Because β-lactamase confers bacterial resistance to many antibiotics, introduction of a tightly binding aptamer along with the normal antibiotic would then be sufficient to stop growth of the bacteria. Without this aptamer, the antibiotic alone would be broken down by the bacteria.
Consequently, by finding a therapeutic aptamer that binds tightly to β-lactamase, traditional antibiotics can be used instead of constantly developing new antibiotics. Development of new antibiotics is not only a lengthy process, but also very costly. Many new bacterial strains evolve every year with increased resistance towards many of the antibiotics on the market today. This fact emphasizes the need for a method that allows the use of antibiotics already in circulation. Aptamer selection against β-lactamase cannot solve the problem of multi-drug resistant bacteria, but can help to find new ways of fighting these pathogens.
Specific Aim 1: Selection of RNA Oligonucleotides against β-lactamase
Beta lactamase provides bacteria with antibiotic resistant capacity. Tightly binding RNA aptamer would inhibit β-lactamase, thereby allowing traditional antibiotics to stop growth of and eventually kill antibiotic-resistant bacteria.
Figure 2: Visual Depiction of Specific Aim
There are many types of β-lactamase that can be used for potential targets. Selection has been performed on metallo beta lactamase [3], which can break down penicillin, cephalosporin, and carbapenem. However, different findings may come of this particular proposal, as a different RNA sequence could be found. This sequence could work on different strains of bacteria than originally thought.
AbD Serotec sells five units of penicillinase (β-lactamase) from Enterobacter cloacae for $409: Catalog Number-7220-1556. Penicillinase specifically breaks down penicillin.
Another vendor, Cell Sciences, sells 1 mg recombinant E. coli β-lactamase for $235. Catalog number: CSI12795
In addition, the protein is large enough upon which to perform selection. Most of this class of proteins tends to be around 50 kD [4] in molecular weight.
[1] Kelly, J.A., et al. (1988) in Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function (Actor P., et al) American Society of Microbiology, Washington, D.C. p. 261-267.
[2] Livermore, D.M. (1991) “Mechanisms of Resistance to β-lactam Antibiotics.” Scandinavian Journal of Infectious Diseases, Supplement 78, p. 7-16.
[3] Kyu Mee Kim (2004) “Inhibition of Metallo-Beta-Lactamase by RNA” Master’s thesis Texas Tech University.
Image: Fonzé, E., P. Charlier, Y. To'th, M. Vermeire, X. Raquet, A. Dubus, and J-M. Frère. (1995) "TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant". Acta Cryst. 51:682-694.
3 comments:
This is an awesome target Sagar. It is very intuitive and could have huge commercial promise. Best of luck!
I like it. Sounds like a great idea. Make it work. :)
Here is the product information for this target. We purchased Enterobacter cloacae Type IV B-lactamase from Sigma, Cat# P-4524 and comes in 10 Units for $18.60 with a conversion factor to size to molarity. There are 0.2-0.6U/mg and B-lactamase (penecillinase) is 41 kDa (mg/mmol). Therefore, 1mg in 242ul will yield ~100uM solution. From here, we have to biotinylate with the Pierce NHS-biotin kit.
Post a Comment