Small-Molecule Inhibitors of Staphylococcus aureus RnpA-Mediated RNA Turnover and tRNA Processing

Abstract

c New agents are urgently needed for the therapeutic treatment of Staphylococcus aureus infections. In that regard, S. aureus RNase RnpA may represent a promising novel dual-function antimicrobial target that participates in two essential cellular processes, RNA degradation and tRNA maturation. Accordingly, we previously used a high-throughput screen to identify smallmolecule inhibitors of the RNA-degrading activity of the enzyme and showed that the RnpA inhibitor RNPA1000 is an attractive antimicrobial development candidate. In this study, we used a series of in vitro and cellular assays to characterize a second RnpA inhibitor, RNPA2000, which was identified in our initial screening campaign and is structurally distinct from RNPA1000. In doing so, it was found that S. aureus RnpA does indeed participate in 5=-precursor tRNA processing, as was previously hypothesized. Further, we show that RNPA2000 is a bactericidal agent that inhibits both RnpA-associated RNA degradation and tRNA maturation activities both in vitro and within S. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect the in vitro activities of unrelated bacterial or eukaryotic ribonucleases and did not display measurable human cytotoxicity. Finally, we show that RNPA2000 exhibits antimicrobial activity and inhibits tRNA processing in efflux-deficient Gram-negative pathogens. Taken together, these data support the targeting of RnpA for antimicrobial development purposes, establish that small-molecule inhibitors of both of the functions of the enzyme can be identified, and lend evidence that RnpA inhibitors may have broad-spectrum antimicrobial activities.

Crystal structure of the ribonuclease-P-protein subunit from Staphylococcus aureus

“…An alternative protocol for protein production was employed compared with the NMR structure paper. The gene coding for S. aureus RnpA (4-117) in the pET-30 Ek/LIC::RnpA vector (Eidem et al, 2015) was polymerasechain-reaction (PCR) amplified using forward and reverse primers shown in Table 1 containing the NcoI and the XhoI restriction enzyme site (underlined), respectively. A tryptophan (W) residue was introduced by the forward primer to the N-terminus of RnpA for the convenience of monitoring protein purification with a UV detector.…”

“…Alternatively, RnpA is also a key element in the messenger RNA (mRNA) degradosome discovered in both Staphylococcus aureus and Bacillus subtilis (Roux et al, 2011). Because RnpA plays a critical role in two essential cellular processes in the pathogen S. aureus it has been postulated that targeting RnpA-mediated RNA metabolism may represent a promising new antibiotic therapeutic strategy for treatment of S. aureus infections (Eidem et al, 2012(Eidem et al, , 2015Olson et al, 2011;Willkomm et al, 2003Willkomm et al, , 2010Drainas, 2016;Berchanski & Lapidot, 2008). Crystal structures for P RNA, RnpA, and the full RNase P complex have been determined from non-pathogenic bacterial species such as Thermotoga maritima (Kazantsev et al, 2003(Kazantsev et al, , 2005Reiter et al, 2010;Torres-Larios et al, 2005) and B. subtilis (Daniels et al, 2014;Stams et al, 1998) as well as a recent NMR solution structure for T. maritima RnpA (Zeng et al, 2018).…”

SOS Response and Staphylococcus aureus: Implications for Drug Development

“…Instead, S. aureus has an mRNA degradosome complex formed by diverse proteins, including RNase enzymes (RNase J1, RNase J2, RNase Y, and RnpA), enolase, phosphofructokinase, polynucleotide phosphorylase (PNPase), and DEAD box RNA helicase. RnpA, a component of this complex, has been reported as a target to inhibit bacterial survival and pathogenesis [70,71]. However, the role of mRNA degradosome complex in SOS response regulation remains to be elucidated in this pathogen.…”

“…RNase E deicient strains exhibit a reduction in SOS activation, revealing that RNase E inhibitors could be possibly used as drug adjuvants [68]. Although RNase E orthologs have been identiied in a range of other bacteria and in bacteria and chloroplasts [69,70], RNA turnover is not regulated by an RNase E ortholog protein in S. aureus [70]. Instead, S. aureus has an mRNA degradosome complex formed by diverse proteins, including RNase enzymes (RNase J1, RNase J2, RNase Y, and RnpA), enolase, phosphofructokinase, polynucleotide phosphorylase (PNPase), and DEAD box RNA helicase.…”

Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

“…As such, ribonucleases provide central points of control over post-transcriptional network function. Ribonuclease regulation of RNA function, and the mechanisms of action of ribonuclease regulators are not well understood, but are receiving increased scrutiny as potential points of drug intervention 4 5 .…”