Combinatorial Libraries As a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens

Fleeman, Renee; LaVoi, Travis; Santos, Radleigh; Morales, Arturo J.; Nefzi, Adel; Welmaker, Gregory S.; Medina-Franco, José L.; Giulianotti, Marc A.; Houghten, Richard A.; Shaw, Lindsey N.

doi:10.1021/jm501628s

Cited by 71 publications

(75 citation statements)

References 54 publications

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“…To determine whether transcriptional changes observed within the mutant strain resulted in detectable physiological sensitivities, we assessed MICs for various antibacterial agents, as described by our group previously (77). Briefly, overnight cultures were diluted 1:1,000 into fresh medium in 96-well plates, and antibiotics at various concentrations were added at amounts no greater than 2% of the final volume.…”

Section: Methodsmentioning

confidence: 99%

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus

et al. 2017

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Staphylococcus aureus is a major human pathogen that causes infection in a wide variety of sites within the human body. Its ability to adapt to the human host and to produce a successful infection requires precise orchestration of gene expression. While DNA-dependent RNA polymerase (RNAP) is generally well characterized, the roles of several small accessory subunits within the complex have yet to be fully explored. This is particularly true for the omega ( or RpoZ) subunit, which has been extensively studied in Gram-negative bacteria but largely neglected in Grampositive counterparts. In Escherichia coli, it has been shown that ppGpp binding, and thus control of the stringent response, is facilitated by . Interestingly, key residues that facilitate ppGpp binding by are not conserved in S. aureus, and consequently, survival under starvation conditions is unaffected by rpoZ deletion. Further to this, -lacking strains of S. aureus display structural changes in the RNAP complex, which result from increased degradation and misfolding of the ␤= subunit, alterations in ␦ and factor abundance, and a general dissociation of RNAP in the absence of . Through RNA sequencing analysis we detected a variety of transcriptional changes in the rpoZ-deficient strain, presumably as a response to the negative effects of depletion on the transcription machinery. These transcriptional changes translated to an impaired ability of the rpoZ mutant to resist stress and to fully form a biofilm. Collectively, our data underline, for the first time, the importance of for RNAP stability, function, and cellular physiology in S. aureus. IMPORTANCEIn order for bacteria to adjust to changing environments, such as within the host, the transcriptional process must be tightly controlled. Transcription is carried out by DNA-dependent RNA polymerase (RNAP). In addition to its major subunits (␣ 2 ␤␤=) a fifth, smaller subunit, , is present in all forms of life. Although this small subunit is well studied in eukaryotes and Gram-negative bacteria, only limited information is available for Gram-positive and pathogenic species. In this study, we investigated the structural and functional importance of , revealing key roles in subunit folding/stability, complex assembly, and maintenance of transcriptional integrity. Collectively, our data underline, for the first time, the importance of for RNAP function and cellular harmony in S. aureus.KEYWORDS RNA polymerase subunit omega, RpoZ, Staphylococcus aureus, gene regulation T ranscription in all forms of life is a tightly controlled process, necessitated by the essentiality of correct temporal and spatial expression of genes for survival. All transcriptional activity within a cell is maintained by the DNA-dependent RNA polymerase (RNAP). This multiprotein complex is structurally and functionally similar in distant forms of life, displaying only minor variations in composition, e.g., the presence/ absence of certain subunits (1, 2). In bacteria RNAP consists of four main subunits, i.e.,

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Section: Methodsmentioning

confidence: 99%

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus

et al. 2017

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“…MIC and minimal bactericidal concentration (MBC) assays were performed in this study as documented by us previously (18,(23)(24)(25). Briefly, A. baumannii strains were grown in tryptic soy broth overnight cultures at 37°C with shaking.…”

Section: Methodsmentioning

confidence: 99%

“…Biofilm eradication determination assays were performed as described by us previously (25,26), as follows. Each of the A. baumannii strains was grown overnight in MHB.…”

Section: Methodsmentioning

confidence: 99%

“…In order to test potential resistance toward the quinazolines, a serial passage assay was performed alongside control compounds (sulfamethoxazole and trimethoprim), as described by us previously (25). A. baumannii strain 1403 was grown overnight in LB medium at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Cytotoxicity assays were performed using human HepG2 cells (human liver epithelial with hepatocellular carcinoma), as described by us previously (18,25). Cells were cultured in Dulbecco's modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, for 3 days at 37°C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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Characterizing the Antimicrobial Activity of N ² , N ⁴ -Disubstituted Quinazoline-2,4-Diamines toward Multidrug-Resistant Acinetobacter baumannii

Fleeman

Horn

Barber

et al. 2017

Antimicrob Agents Chemother

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We previously reported a series of N 2 ,N 4 -disubstituted quinazoline-2,4-diamines as dihydrofolate reductase inhibitors with potent in vitro and in vivo antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. In this work, we extended our previous study to the Gram-negative pathogen Acinetobacter baumannii. We determined that optimized N 2 ,N 4 -disubstituted quinazoline-2,4-diamines are strongly antibacterial against multidrug-resistant A. baumannii strains when the 6-position is replaced with a halide or an alkyl substituent. Such agents display potent antibacterial activity, with MICs as low as 0.5 M, while proving to be strongly bactericidal. Interestingly, these compounds also possess the potential for antibiofilm activity, eradicating 90% of cells within a biofilm at or near MICs. Using serial passage assays, we observed a limited capacity for the development of resistance toward these molecules (4-fold increase in MIC) compared to existing folic acid synthesis inhibitors, such as trimethoprim (64-fold increase) and sulfamethoxazole (128-fold increase). We also identified limited toxicity toward human cells, with 50% lethal doses (LD 50 s) of Յ23 M for lead agents 4 and 5. Finally, we demonstrated that our lead agents have excellent in vivo efficacy, with lead agent 5 proving more efficacious than tigecycline in a murine model of A. baumannii infection (90% survival versus 66%), despite being used at a lower dose (2 versus 30 mg kg Ϫ1 ). Together, our results demonstrate that N 2 ,N 4 -disubstituted quinazoline-2,4-diamines have strong antimicrobial and antibiofilm activities against both Gram-positive organisms and Gram-negative pathogens, suggesting strong potential for their development as antibacterial agents.

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Optimizing Antimicrobial Peptide Dendrimers in Chemical Space

et al. 2018

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We used nearest‐neighbor searches in chemical space to improve the activity of the antimicrobial peptide dendrimer (AMPD) G3KL and identified dendrimer T7, which has an expanded activity range against Gram‐negative pathogenic bacteria including Klebsiellae pneumoniae, increased serum stability, and promising activity in an in vivo infection model against a multidrug‐resistant strain of Acinetobacter baumannii. Imaging, spectroscopic studies, and a structural model from molecular dynamics simulations suggest that T7 acts through membrane disruption. These experiments provide the first example of using virtual screening in the field of dendrimers and show that dendrimer size does not limit the activity of AMPDs.

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Combinatorial Libraries As a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens

Cited by 71 publications

References 54 publications

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus

Characterizing the Antimicrobial Activity of N ² , N ⁴ -Disubstituted Quinazoline-2,4-Diamines toward Multidrug-Resistant Acinetobacter baumannii

Optimizing Antimicrobial Peptide Dendrimers in Chemical Space

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Combinatorial Libraries As a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens

Cited by 71 publications

References 54 publications

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus

Characterizing the Antimicrobial Activity of N 2 , N 4 -Disubstituted Quinazoline-2,4-Diamines toward Multidrug-Resistant Acinetobacter baumannii

Optimizing Antimicrobial Peptide Dendrimers in Chemical Space

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Characterizing the Antimicrobial Activity of N ² , N ⁴ -Disubstituted Quinazoline-2,4-Diamines toward Multidrug-Resistant Acinetobacter baumannii