Introduction
The severe increase in antibiotic resistance and cross-resistance among many pathogenic bacterial strains[1] presents a significant health threat.[2] The marginal attention to severances of the results from antibiotics usage led to rapid increase in the appearance of multi-drug resistant pathogenic bacterial strains.[3] Moreover, the extremely slow progress (actually negligible) in developing new antibiotics by the pharma companies worldwide causes extreme contemporary issues.[4] Consequently, it seems that along with the traditional attempts to improve current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. For example, the common preference for broad-spectrum antibiotics should be challenged, as it triggers the development of antibiotic resistance in a large variety of bacteria, including those that are not involved in the particular disease. In addition, many antibiotics do not distinguish between pathogens and some of the bacteria of the microbiome, thus may alter the microbiome population.[5] Furthermore, most antibiotics are chemically substituted non-digestible and non-biodegradable small molecules that upon leaving the patient’s body and reaching the environment, may escape the purification systems and cause ecological and environmental complications,[6] which in turn contribute to further resistance development. Here we describe our studies towards reducing and/or controlling antibiotics resistance, focusing on the identification of species-specific and environmentally-friendly protein synthesis inhibitors, which may be used as lead compounds for the development of novel drugs.
Expected Lower Rate of Resistance
The ribosome is the biological macromolecule responsible for protein synthesis according to the genetic code in all the living cells. In prokaryotes it consists of two ribosomal subunits, namely the large ribosomal subunit, called 50S (according to its sedimentation coefficient), and the small ribosomal subunit, or 30S (Fig. 1).
Fig. 1: The skeleton of the ribosome from Staphylococcus aureus:[7] the large ribosomal subunit is represented in blue, the small ribosomal subunit in pink.
Owing to the vital role played by the ribosomes, many antibiotics target them.[8] Most ribosomal antibiotics obstruct the key ribosomal functions, namely decoding and peptide bond formation. As these sites are rather limited in number and chemical options, we initiated studies, based on our previous attempts to identify particular ribosomal structural motifs,[9] aiming at the development of novel antibiotics. Particularly we focus on “pathogen-specific antibiotics”, in contrast to the current preference of broad range antibiotics. For this aim, we identified species-specific ribosomal structural motifs that may be exploited for the design of novel antibiotics of controllable chemistry.
In principle, for each bacteria several such sites could be identified, and for each of these sites a specific matching molecule could be designed as a lead compound. Hence, for each pathogen we will have in hand a collection of lead compounds, each of which can be further developed into an antibiotic drug. These are planned to be used one at the time. Thus, the usage of species-specific instead of broad range antibiotics, alongside the expected variety of these antibiotics, which can be used consequently, are bound to lead to a slower pace of resistance appearance.
We are focusing on surface exposed sites, with chemical structure and composition allowing the design of matching compounds with favorable chemistry. These sites were discovered mainly by comparing the sequences and the structures of the large ribosomal subunit of the pathogenic bacteria of interest, to those of other non-pathogenic bacteria. Our approach became feasible thanks to the recent year’s availability of cryo-electron microscopy for the determination of high-resolution structures of large biomolecules like the ribosomes. Once the explicit pathogen unique sites were identified, solvent exposed regions of rRNA (Fig. 2) were selected. These were targeted by antisense oligonucleotides in a cell free in vitro transcription-translation activity assay, using luciferase as reporter gene to check protein synthesis inhibition.
Fig. 2: The skeleton of the large ribosomal subunit from Staphylococcus aureus.[10] Potential target sites for antisense therapy are shown in cyan.
The interactions between the inhibiting antisense oligonucleotides and their designed targets were then assessed by running fluorescein labelled versions of the oligonucleotides in an agarose gel, along with both the ribosomal subunits from the bacteria of interest, using a fluorescein labelled Shine-Dalgarno sequence[11] as positive control. In this way we observed selective binding of our designed antisense oligonucleotides to the large ribosomal subunit. These interactions, paired with a selective binding of the Shine-Dalgarno sequence to the small ribosomal subunit, represent a proof that the designed sequences are indeed binding to their predicted target sites.
Conclusions
Our studies are leading towards the design of “pathogen-specific antibiotics”, in contrast to the current preference of broad-spectrum antibiotics usage. The discovery of the unique species target that can lead to antibiotics that will be used sequentially, alongside the speeding up of the procedures for the identification of the actual clinical pathogen (by a few companies) open new, untraditional path towards species specific antibiotics, of which sequential application should reduce and slow down the appearance of resistance.
References
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[2] Organization, W. H. (2014), “Antimicrobial resistance global report on surveillance: 2014 summary”, World Health Organization.
[3] Kurosu M., Siricilla S. and Mitachi K. (2013), “Advances in MRSA drug discovery: where are we and where do we need to be?”, Expert Opin. Drug Discov. 8(9), 1095-1116.
[4] Ventola, C.L. (2015), “The antibiotic resistance crisis: part 1: causes and threats”, Pharmacy and therapeutics 40(4), 277-283.
[5] Willing B.P., Russell S.L. and B. Brett Finlay B.B. (2011), “Shifting the balance: antibiotic effects on host-microbiota mutualism”, Nature Reviews Microbiology 9, 233-243.
[6] Grennia P., Ancona V. and Barra Caracciolo A (2018), “Ecological effects of antibiotics on natural ecosystems: A review”, Microchemical Journal 136, 25-39.
[7] Belousoff M.J., Eyal Z., Radjainia M., Ahmed T., Bamert R.S., Matzov D., Bashan A., Zimmerman E., Mishra S., Cameron D., Elmlund H., Peleg A.Y., Bhushan S., Lithgow T. and Yonath A. (2017) “Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome”, American Society for Microbiology 8(3), 00395-17.
[8] Tenson T. and Mankin A. (2006), “Antibiotics and the ribosome”, Mol. Microbiol. 59, 1664-1677.
[9] Davidovich C. (2010), “Targeting Functional Centers of the Ribosome”, Doctoral Thesis, Weizmann Institute of Science, Rehovot, Israel.
[10] Eyal Z., Matzov D., Krupkin M., Wekselman I., Paukner S., Zimmerman E., Rozenberg H., Bashan A. and Yonath A. (2015), “Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus”, PNAS 112, 5805-5814.
[11] Jayaraman K., McParland K., Miller P. and Ts’o P.O. (1981), “Selective inhibition of Escherichia Coli protein synthesis and growth by nonionic oligonucleotides complementary to the 3’ end of 16S rRNA”, Proc. Natl. Acad Sci USA 78, 1537-1541.