Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid

Abstract

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to “druggable” target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ70 (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition.Methodology/Principal FindingsBy synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)3K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ70 in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ70 were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM.ConclusionsThe present result suggested that RNAP primary σ70 is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.

Antisense Growth Inhibition of Methicillin-Resistant Staphylococcus aureus by Locked Nucleic Acid Conjugated with Cell-Penetrating Peptide as a Novel FtsZ Inhibitor

“…Therefore, highperformance delivery of LNA is mandatory. (KFF) 3 K conjugated with PNA or phosphorodiamidate morpholino oligomer dramatically increased cell permeability and uptake of antisense oligonucleotides in a series of bacteria, including S. aureus, E. coli, and Salmonella enterica (16,24,30). To date, few studies have been performed to improve the permeability of LNA into bacteria through conjugation with the peptide (KFF) 3 K. In the present study, we synthesized the conjugated PLNA with (KFF) 3 K and LNA using the Cys-SMCC-C6 linker.…”

“…Many previous studies have shown that the translation start codon region of mRNA is the effective region for antisense inhibition (13-15). Recently, some studies have demonstrated that antisense agents targeting non-start codon regions have efficient antisense effects, such as PNA targeting of rpoD mRNA of MRSA and LNA targeting of the IRES of hepatitis C virus (12,16). This implies that possible targeting sites are generally nucleotide sequences free of any double-stranded secondary structures, which can be theoretically forecasted by RNA structure software (17).…”

Bacterial Transcription as a Target for Antibacterial Drug Development

“…The 2.2 -CH interaction involves ␤= residues R275, R278, R281, L282, I291, N294, and M298 and 70 residues Q403, Q406, E407, I410, and M413 (37,38,176,177), but this is a nontraditional drug target as it lacks any deep clefts, which are considered desirable binding sites for small molecules. Nevertheless, a peptide-based study showed that peptides mimicking 2.2 could inhibit RNAP-binding to prevent in vitro transcription initiation, and prevention of production by antisense peptide nucleic acids could inhibit the growth of clinically relevant multidrug-resistant S. aureus (178,179).…”

Targeted antisense radiotherapy and dose fractionation using a 177Lu-labeled anti-bcl-2 peptide nucleic acid-peptide conjugate

“…Those patients whose tumors are found to overexpress bcl-2 might then be good candidates for treatments that include down-regulation of bcl-2, such as targeted immunotherapy, radioimmunotherapy, or antisense therapy. In preclinical settings, peptide nucleic acid (PNA) has been evaluated for antisense therapy against a wide range of diseases including bacterial and viral infections, lymphoma, Alzheimer's disease and allograft survival [9][10][11][12][13][14][15][16]. PNA is a DNA-like molecule with high stability, binding affinity and specificity to complementary mRNA targets [17].…”