scholarly journals Identification of Small Molecule Inhibitors of Staphylococcus aureus RnpA

Antibiotics ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 48 ◽  
Author(s):  
Jennifer M. Colquhoun ◽  
Lisha Ha ◽  
Andrew Beckley ◽  
Brinkley Meyers ◽  
Daniel P. Flaherty ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Small Molecule ◽  
Messenger Rna ◽  
Rna Degradation ◽  
Screening Assay ◽  
Whole Cell ◽  
Cellular Processes ◽  
Dual Functional ◽  
Worm Model

Staphylococcus aureus RnpA is thought to be a unique dual functional antimicrobial target that is required for two essential cellular processes, precursor tRNA processing and messenger RNA degradation. Herein, we used a previously described whole cell-based mupirocin synergy assay to screen members of a 53,000 compound small molecule diversity library and simultaneously enrich for agents with cellular RnpA inhibitory activity. A medicinal chemistry-based campaign was launched to generate a preliminary structure activity relationship and guide early optimization of two novel chemical classes of RnpA inhibitors identified, phenylcarbamoyl cyclic thiophene and piperidinecarboxamide. Representatives of each chemical class displayed potent anti-staphylococcal activity, limited the protein’s in vitro ptRNA processing and mRNA degradation activities, and exhibited favorable therapeutic indexes. The most potent piperidinecarboxamide RnpA inhibitor, JC2, displayed inhibition of cellular RnpA mRNA turnover, RnpA-depletion strain hypersusceptibility, and exhibited antimicrobial efficacy in a wax worm model of S. aureus infection. Taken together, these results establish that the whole cell screening assay used is amenable to identifying small molecule RnpA inhibitors within large chemical libraries and that the chemical classes identified here may represent progenitors of new classes of antimicrobials that target RnpA.

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

2015 ◽  
Vol 59 (4) ◽  
pp. 2016-2028 ◽  
Author(s):  
Tess M. Eidem ◽  
Nicole Lounsbury ◽  
John F. Emery ◽  
Jeffrey Bulger ◽  
Andrew Smith ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Antimicrobial Activities ◽  
Rna Degradation ◽  
Dual Function ◽  
Content Type ◽  
Trna Processing ◽  
Cellular Assays

ABSTRACTNew agents are urgently needed for the therapeutic treatment ofStaphylococcus aureusinfections. In that regard,S. aureusRNase 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 small-molecule 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 ofin vitroand 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 thatS. aureusRnpA 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 bothin vitroand withinS. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect thein vitroactivities 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.

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

2018 ◽  
Vol 74 (10) ◽  
pp. 632-637
Author(s):  
Lisha Ha ◽  
Jennifer Colquhoun ◽  
Nicholas Noinaj ◽  
Chittaranjan Das ◽  
Paul M. Dunman ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Staphylococcus Aureus ◽  
Rna Processing ◽  
Bacterial Species ◽  
Rna Degradation ◽  
Ribonuclease P ◽  
Protein Subunit ◽  
Aromatic Residues ◽  
Cellular Processes ◽  
P Protein

Staphylococcus aureus ribonuclease-P-protein subunit (RnpA) is a promising antimicrobial target that is a key protein component for two essential cellular processes, RNA degradation and transfer-RNA (tRNA) maturation. The first crystal structure of RnpA from the pathogenic bacterial species, S. aureus, is reported at 2.0 Å resolution. The structure presented maintains key similarities with previously reported RnpA structures from bacteria and archaea, including the highly conserved RNR-box region and aromatic residues in the precursor-tRNA 5′-leader-binding domain. This structure will be instrumental in the pursuit of structure-based designed inhibitors targeting RnpA-mediated RNA processing as a novel therapeutic approach for treating S. aureus infections.

scholarly journals CDK10 in Gastrointestinal Cancers: Dual Roles as a Tumor Suppressor and Oncogene

2021 ◽  
Vol 11 ◽  
Author(s):  
Zainab A. Bazzi ◽  
Isabella T. Tai
Keyword(s):  
Cell Proliferation ◽  
Tumor Suppressor ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Screening Assay ◽  
Cellular Processes ◽  
Molecule Inhibitor ◽  
High Throughput Screening Assay ◽  
Kinase Activity Assay

Cyclin-dependent kinase 10 (CDK10) is a CDC2-related serine/threonine kinase involved in cellular processes including cell proliferation, transcription regulation and cell cycle regulation. CDK10 has been identified as both a candidate tumor suppressor in hepatocellular carcinoma, biliary tract cancers and gastric cancer, and a candidate oncogene in colorectal cancer (CRC). CDK10 has been shown to be specifically involved in modulating cancer cell proliferation, motility and chemosensitivity. Specifically, in CRC, it may represent a viable biomarker and target for chemoresistance. The development of therapeutics targeting CDK10 has been hindered by lack a specific small molecule inhibitor for CDK10 kinase activity, due to a lack of a high throughput screening assay. Recently, a novel CDK10 kinase activity assay has been developed, which will aid in the development of small molecule inhibitors targeting CDK10 activity. Discovery of a small molecular inhibitor for CDK10 would facilitate further exploration of its biological functions and affirm its candidacy as a therapeutic target, specifically for CRC.

Proteins synthesized by rabbit reticulocyte membrane-bound ribosomes

1982 ◽  
Vol 60 (5) ◽  
pp. 580-585 ◽  
Author(s):  
Réal Lemieux ◽  
Claude Godin
Keyword(s):  
Protein Synthesis ◽  
Ionic Strength ◽  
Messenger Rna ◽  
Rna Degradation ◽  
Main Protein ◽  
Membrane Bound ◽  
Phenol Treatment

Rabbit reticulocyte membrane-bound ribosomes liberated by deoxycholate treatment contain degraded forms of ribosomal and messenger RNA. This degradation occurs after the liberation of the ribosomes from the membranes by the detergent because intact ribosomal and messenger RNA can be extracted from washed membranes by phenol treatment. Increasing the ionic strength of the detergent buffer prevents this RNA degradation and allows the recovery of membrane-bound ribosomes capable of protein synthesis. Comparison of the proteins synthesized in vitro by the polyribosomes shows that the main protein produced by both free and membrane-bound ribosomes is globin. However, the two types of polyribosomes could be distinguished by the nonglobin proteins they produce.

Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4641-4641
Author(s):  
Hidenori Hattori ◽  
Kulandayan K Subramanian ◽  
Hongbo R. Luo
Keyword(s):  
Small Molecule ◽  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Physiological Role ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Functional Screening ◽  
Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

scholarly journals β-Lactams Increase the Antibacterial Activity of Daptomycin against Clinical Methicillin-Resistant Staphylococcus aureus Strains and Prevent Selection of Daptomycin-Resistant Derivatives

2012 ◽  
Vol 56 (12) ◽  
pp. 6192-6200 ◽  
Author(s):  
Shrenik Mehta ◽  
Christopher Singh ◽  
Konrad B. Plata ◽  
Palas K. Chanda ◽  
Arundhati Paul ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mutant Selection ◽  
Methicillin Resistant ◽  
Content Type ◽  
Oxacillin Resistance ◽  
Worm Model ◽  
Mrsa Strains ◽  
Selection Of

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) has emerged to be one of the most important pathogens both in health care and in community-onset infections. Daptomycin (DAP) is a cyclic anionic lipopeptide recommended for treatment of skin infections, bacteremia, and right-sided endocarditis caused by MRSA. Resistance to DAP (DAPr) has been reported in MRSA and is mostly accompanied by a parallel decrease in oxacillin resistance, a process known as the “seesaw effect.” Our study provides evidence that the seesaw effect applies to other β-lactams and carbapenems of clinical use, including nafcillin (NAF), cefotaxime (CTX), amoxicillin-clavulanic (AMC), and imipenem (IMP), in heterogeneous DAPrMRSA strains but not in MRSA strains expressing homogeneous β-lactam resistance. The antibacterial efficacy of DAP in combination with β-lactams was evaluated in isogenic DAP-susceptible (DAPs)/DaprMRSA strains originally obtained from patients that failed DAP monotherapy. Bothin vitro(MIC, synergy-kill curve) andin vivo(wax worm model) approaches were used. In these models, DAP and a β-lactam proved to be highly synergistic against both heterogeneous and homogeneous clinical DAPrMRSA strains. Mechanistically, β-lactams induced a reduction in the cell net positive surface charge, reverting the increased repulsion provoked by DAP alone, an effect that may favor the binding of DAP to the cell surface. The ease ofin vitromutant selection was observed when DAPsMRSA strains were exposed to DAP. Importantly, the combination of DAP and a β-lactam prevented the selection of DAPrvariants. In summary, our data show that the DAP–β-lactam combination may significantly enhance both thein vitroandin vivoefficacy of anti-MRSA therapeutic options against DAPrMRSA infections and represent an option in preventing DAPrselection in persistent or refractory MRSA infections.

scholarly journals THE DEAD-BOX PROTEIN CSHA FROM Staphylococcus aureus Mu 50 EXHIBITS RIBONUCLEASE ACTIVITY

2021 ◽  
Vol 44 (02) ◽  
Author(s):  
HANH THI DIEU NGUYEN ◽  
NGOC AN NGUYEN ◽  
GIA BUU TRAN ◽  
TAN VIET PHAM
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Degradation ◽  
Ribonuclease Activity ◽  
Aureus Strain ◽  
Dead Box ◽  
Rna Remodeling ◽  
Bacterial Rna ◽  
Rna Degradosome ◽  
The Dead

DEAD-box proteins play important roles in many RNA processes ranging from RNA synthesis to RNA decay. Furthermore, it has been reported that some bacterial DEAD-box proteins known to be components of the RNA degradosome do not cleave RNA substrates directly. However, the role of DEAD-box proteins in RNA degradation is poorly understood. The present study demonstrated that the DEAD-box protein CshA from the vancomycin-resistant Staphylococcus aureus strain Mu50 possesses RNA degradation activity, ribonuclease activity. Despite having RNA-dependent ATPase activity, CshA did not exhibit RNA helicase activity in vitro. Instead, CshA catalyzed the degradation of single-stranded RNAs of various duplex RNA substrates to form blunt-end RNA products. Thus, we suggest that the ribonuclease activity of the DEAD-box protein CshA may contribute to RNA remodeling in the bacterial RNA degradosome. To our knowledge, this study is the first to report that a DEAD-box protein from a pathogenic bacterium is implicated in multiple ATP-independent activity on RNA, such as degradation.

scholarly journals An efficient way to screen inhibitors of energy-coupling factor (ECF) transporters in bacteria uptake assay

2021 ◽  
Author(s):  
Spyridon Bousis ◽  
Steffen Winkler ◽  
Jörg Haupenthal ◽  
Francesco Fulco ◽  
Eleonora Diamanti ◽  
...  
Keyword(s):  
Cost Effective ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Screening Assay ◽  
Vitro Assay ◽  
Whole Cell ◽  
Fast Solution ◽  
Uptake Assay ◽  
Biological Environment

Herein, we report a novel whole-cell screening assay using Lactobacillus casei as model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity relationships.

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