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Small molecule inhibitors of Staphylococcus aureus RnpA alter cellular mRNA turnover, exhibit antimicrobial activity, and attenuate pathogenesis.

Olson PD, Kuechenmeister LJ, Anderson KL, Daily S, Beenken KE, Roux CM, Reniere ML, Lewis TL, Weiss WJ, Pulse M, Nguyen P, Simecka JW, Morrison JM, Sayood K, Asojo OA, Smeltzer MS, Skaar EP, Dunman PM - PLoS Pathog. (2011)

Bottom Line: In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro.We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus.Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

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Identification of small molecule inhibitors of RnpA-mediated RNA degradation.(A) Representative screening effort results; dark blue arrow indicates substrate alone (negative control); grey arrow indicates enzyme (positive control); light-blue arrows indicate compounds that inhibited RnpA activity by ≥50%. (B) An agarose gel-based assay was used to distinguish bona-fide RnpA inhibitors from primary screening artifacts. Shown is the gel mobility of molecular weight marker, spa mRNA in the absence (−) or presence (+) of 20 pmol RnpA and RnpA-mediated spa mRNA degradation in the presence of increasing concentrations of RNPA1000, as described in Materials and Methods. (C) Structure of RnpA-inhibitory molecule RNPA1000.
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ppat-1001287-g003: Identification of small molecule inhibitors of RnpA-mediated RNA degradation.(A) Representative screening effort results; dark blue arrow indicates substrate alone (negative control); grey arrow indicates enzyme (positive control); light-blue arrows indicate compounds that inhibited RnpA activity by ≥50%. (B) An agarose gel-based assay was used to distinguish bona-fide RnpA inhibitors from primary screening artifacts. Shown is the gel mobility of molecular weight marker, spa mRNA in the absence (−) or presence (+) of 20 pmol RnpA and RnpA-mediated spa mRNA degradation in the presence of increasing concentrations of RNPA1000, as described in Materials and Methods. (C) Structure of RnpA-inhibitory molecule RNPA1000.

Mentions: The above results indicate that S. aureus RnpA is an essential enzyme that exhibits in vitro ribonuclease activity and either alone, as a component of RNase P or in concert with other cellular components participates in bulk RNA degradation. Moreover, the protein is well conserved across Gram-positive bacteria but lacks amino acid conservation with mammalian proteins, making it an attractive target for novel antibiotic drug development. Accordingly, we set out to exploit the protein's in vitro ribonuclease activity as a means to identify RnpA inhibitory agents; a fluorescence-based high through-put assay was used to screen 29,066 commercial compounds (ActiProbe-25K and Natural product libraries; Timtec; Newark, DE) for small molecule inhibitors of RnpA-mediated in vitro RNA degradation (Figure 3A). In total, fourteen molecules inhibited the enzyme's RNA turnover activity by ≥50%. A gel-based secondary assay confirmed that five of these molecules were bona-fide inhibitors of RnpA-mediated RNA degradation (Figure 3B). One of these compounds, RNPA1000 (Figure 3C; IC50  = 100–125 µM), did not affect the activity of the commercially available E. coli RNase HI, RNase A, RNase I or in-house purified S. aureus RNase J1 at any concentration tested (0–750 µM), but did mildly inhibit E. coli RNase III activity (IC50  = 500–750 µM; data not shown). These and other data (see below) suggest that RNPA1000 may have specificity for S. aureus RnpA, yet as with any small molecule we cannot rule out the possibility that the agent may also affect other S. aureus enzymes. To assess whether RnpA-inhibitory agents exhibit potential as antimicrobials, a series of experiments were performed to evaluate whether RNPA1000 inhibited S. aureus growth and could limit S. aureus pathogenesis in a systemic model of infection.


Small molecule inhibitors of Staphylococcus aureus RnpA alter cellular mRNA turnover, exhibit antimicrobial activity, and attenuate pathogenesis.

Olson PD, Kuechenmeister LJ, Anderson KL, Daily S, Beenken KE, Roux CM, Reniere ML, Lewis TL, Weiss WJ, Pulse M, Nguyen P, Simecka JW, Morrison JM, Sayood K, Asojo OA, Smeltzer MS, Skaar EP, Dunman PM - PLoS Pathog. (2011)

Identification of small molecule inhibitors of RnpA-mediated RNA degradation.(A) Representative screening effort results; dark blue arrow indicates substrate alone (negative control); grey arrow indicates enzyme (positive control); light-blue arrows indicate compounds that inhibited RnpA activity by ≥50%. (B) An agarose gel-based assay was used to distinguish bona-fide RnpA inhibitors from primary screening artifacts. Shown is the gel mobility of molecular weight marker, spa mRNA in the absence (−) or presence (+) of 20 pmol RnpA and RnpA-mediated spa mRNA degradation in the presence of increasing concentrations of RNPA1000, as described in Materials and Methods. (C) Structure of RnpA-inhibitory molecule RNPA1000.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037362&req=5

ppat-1001287-g003: Identification of small molecule inhibitors of RnpA-mediated RNA degradation.(A) Representative screening effort results; dark blue arrow indicates substrate alone (negative control); grey arrow indicates enzyme (positive control); light-blue arrows indicate compounds that inhibited RnpA activity by ≥50%. (B) An agarose gel-based assay was used to distinguish bona-fide RnpA inhibitors from primary screening artifacts. Shown is the gel mobility of molecular weight marker, spa mRNA in the absence (−) or presence (+) of 20 pmol RnpA and RnpA-mediated spa mRNA degradation in the presence of increasing concentrations of RNPA1000, as described in Materials and Methods. (C) Structure of RnpA-inhibitory molecule RNPA1000.
Mentions: The above results indicate that S. aureus RnpA is an essential enzyme that exhibits in vitro ribonuclease activity and either alone, as a component of RNase P or in concert with other cellular components participates in bulk RNA degradation. Moreover, the protein is well conserved across Gram-positive bacteria but lacks amino acid conservation with mammalian proteins, making it an attractive target for novel antibiotic drug development. Accordingly, we set out to exploit the protein's in vitro ribonuclease activity as a means to identify RnpA inhibitory agents; a fluorescence-based high through-put assay was used to screen 29,066 commercial compounds (ActiProbe-25K and Natural product libraries; Timtec; Newark, DE) for small molecule inhibitors of RnpA-mediated in vitro RNA degradation (Figure 3A). In total, fourteen molecules inhibited the enzyme's RNA turnover activity by ≥50%. A gel-based secondary assay confirmed that five of these molecules were bona-fide inhibitors of RnpA-mediated RNA degradation (Figure 3B). One of these compounds, RNPA1000 (Figure 3C; IC50  = 100–125 µM), did not affect the activity of the commercially available E. coli RNase HI, RNase A, RNase I or in-house purified S. aureus RNase J1 at any concentration tested (0–750 µM), but did mildly inhibit E. coli RNase III activity (IC50  = 500–750 µM; data not shown). These and other data (see below) suggest that RNPA1000 may have specificity for S. aureus RnpA, yet as with any small molecule we cannot rule out the possibility that the agent may also affect other S. aureus enzymes. To assess whether RnpA-inhibitory agents exhibit potential as antimicrobials, a series of experiments were performed to evaluate whether RNPA1000 inhibited S. aureus growth and could limit S. aureus pathogenesis in a systemic model of infection.

Bottom Line: In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro.We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus.Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

Show MeSH
Related in: MedlinePlus