Antisense Peptide Nucleic Acids in Antibacterial Drug Discovery
Authors: Martin Burnham, Yinduo Ji
Journal: Molecular Therapy (2004)

The recent emergence of multidrug-resistant bacterial pathogens, particularly methicillin-resistant and vancomycin intermediate-resistant Staphylococcus aureus, is generating enormous public health concern [[1]Pfaller M.A. et al.Bacterial pathogens isolated from patients with blood-stream infection: frequency of occurrence and antimicrobial susceptibility patterns.Antimicrob. Agents Chemother. 1998; 42: 1762-1770PubMed Google Scholar]. It seems that every week we read of yet another example of the ominous increase in the resistance to antibiotics of a bacterial foe our parents had defeated. A cause of particular anxiety has been the ability of the staphylococci to overcome most of the antibiotics in our arsenal. Furthermore, the slowdown in the discovery of new classes of antibiotics that started in the 1970s is making us even more vulnerable to emerging resistance [2Bush K. Why it is important to continue antibacterial drug discovery.ASM News. 2004; 70: 282-287Google Scholar, 3Shlaes D. Projan S. Edwards J. Antibiotic discovery: state of the state.ASM News. 2004; 70: 275-281Google Scholar]. There is an urgent need for the development of new agents to combat multidrug-resistant pathogens. The recent bioterror attacks in this country also point to the need for the development of new drugs against microbes that could be developed as agents of biological warfare. To address this challenge, and with the hopes of reviving their lagging profits, drug companies are setting about this task in a methodical way. The identification and exploration of new drug targets is a major component of the effort to develop antibacterial agents against multi-resistant pathogens [4Zalacain M. et al.A global approach to identify novel broad-spectrum anti-bacterial targets among proteins of unknown function.J. Mol. Microbiol. Biotechnol. 2003; 6: 109-126Crossref PubMed Scopus (76) Google Scholar, 5Ji Y. The role of genomics in the discovery of novel targets for antibiotic therapy.Pharmacogenomics. 2002; 3: 315-323Crossref PubMed Scopus (22) Google Scholar]. The availability of the complete genome sequences of many bacterial pathogens has allowed us to undertake a systematic and comprehensive evaluation of the requirement of each and every gene for the growth of bacteria in culture and for their survival during infection. Genomics, transcriptomics, pro- teomics and other more obscure "omics" technologies have been applied exhaustively to the long list of human pathogens in an effort to identify new drug targets. At the same time, chemists have ramped up their combinatorial syntheses of new molecules into the millions and drug companies have automated their drug discovery platforms to find target inhibitor hits from amongst this myriad of compounds in mere days. These hits are then prioritized for the development of suitable lead compounds for further optimization on the basis of their potency and chemical properties. In this issue of Molecular Therapy, Nekhotiaeva et al. report that peptide nucleic acids (PNAs) attached to short carrier peptides can be used as antisense agents to downregulate the expression of specific proteins in Gram+ S. aureus. PNA is an uncharged and stable DNA mimic (see figure). To facilitate the entry of the PNAs into the bacteria, the authors attached them to cationic carrier peptides that facilitate transport across the cell wall. Previously, the same authors showed that carrier peptide-linked PNAs directed toward the start-codon region of specific genes could lead to the down- regulation of their expression in Gram− Escherichia coli [[6]Good L. Nielsen P.E. Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA.Nature Biotechnol. 1998; 16: 355-358Crossref PubMed Scopus (202) Google Scholar]. The extension of this strategy to Gram+ bacteria allows its application to the study of staphylococci and streptococci, which are of major clinical importance both in terms of the frequency of disease and of the cost to the healthcare budget. These results provide a potentially useful tool for the validation of antibacterial drug targets, and for the discovery and development of new antibacterial agents against a broad spectrum of pathogens. If difficult hurdles can be overcome, PNAs linked to the appropriate carrier peptides might even be exploited as therapeutic agents themselves one day. Marty Rosenberg's group at GlaxoSmithKline was able to show that antisense RNAs expressed from inducible plasmid vectors could be used in S. aureus to identify and evaluate essential genes, both in culture and in natural infection [[7]Ji Y. et al.Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA.Science. 2001; 293: 2266-2299Crossref PubMed Scopus (353) Google Scholar]. Similar results were reported by another group [[8]Forsyth R.A. et al.A genome-wide strategy for the identification of essential genes in Staphylococcus aureus.Mol. Microbiol. 2002; 43: 1387-1400Crossref PubMed Scopus (404) Google Scholar]. Subsequently, the GlaxoSmithKline workers constructed a library of strains expressing antisense molecules to the majority of essential genes in S. aureus. Their aim was to use inducible antisense downregulation of potential targets of interest to demonstrate enhanced sensitivity to antibacterial compounds, thereby identifying the putative site of action of compounds under study [9Yin D. et al.Identification of antimicrobial targets using a comprehensive genomic approach.Pharmacogenomics. 2004; 5: 101-113Crossref PubMed Scopus (42) Google Scholar, 10Yin D. et al.Validation of antibacterial mechanism of action using regulated antisense RNA expression in Staphylococcus aureus.FEMS Microbiol. Letters. 2004; 231: 177-184Crossref PubMed Scopus (71) Google Scholar]. However, it is generally only an article of faith that a potential antibacterial target that has been demonstrated to be essential in laboratory strains will be equally critical in strains of the pathogen that cause disease. This is because clinical strains are usually far less amenable to gene vector mobilization and maintenance techniques used in the laboratory. The peptide-linked PNA antisense agents described in the new work could potentially be used to demonstrate the essential nature in clinical strains of a particular target shown to be important in laboratory strains—for example by demonstrating detrimental effects on viability following disruption of normal levels of the target. If so, then the method will provide workers with the ability to gain greater confidence in the value of applying the costly panoply of drug discovery and development resources against the particular target protein in question. In the case of the development of a broad spectrum antibiotic, it is important to know the variation in susceptibility to the antibiotic and the essentiality of the drug target in both naturally-occurring strains of a particular bacterial pathogen, as well as in other bacterial species—some of which will also not be amenable to genetic engineering procedures. Here too, peptide-linked PNA antisense agents could be enormously useful if the techniques used for their application to S. aureus and E. coli are conveniently adapted to other species. Similarly, peptide-linked antisense PNAs show promise as convenient tools to confirm that the target of any compound found to possess antibacterial activity against a laboratory strain used in a screening method is the same discrete protein present in clinical strains of the pathogen, strains that may very well possess different growth characteristics and antibiotic susceptibilities. A PNA approach that makes use of an antisense library to essential genes could also be exploited to identify the potential mode of action of natural or synthetic antibacterial compounds in bacterial species refractory to certain molecular biology techniques. In such a case, the PNAs could be used to identify, confirm, and track the mechanism of action of a given compound. A tantalizing question is whether peptide-PNA antisense antibiotics will ever reach the clinic. There are some obvious challenges that arise, such as finding suitable target sequences across the broad spectrum of bacterial pathogens, and overcoming resistance to the potential PNA-based antibiotic arising from low frequency spontaneous mutation in the target sequences. There is also no doubt that such agents would also present their own peculiar problems with regards to their delivery, pharmacology, and pharmacokinetics. However, we are optimists. We suspect that a century from now, medical practitioners will take a swab of a patient's infection, identify a target sequence and synthesize an appropriate antisense antibiotic in a machine on their desktop to administer to the patient.