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Editor's Pick Mechanisms of Action: Physiological Effects

The Quinazolinone Allosteric Inhibitor of PBP 2a Synergizes with Piperacillin and Tazobactam against Methicillin-Resistant Staphylococcus aureus

Jeshina Janardhanan, Renee Bouley, Siseth Martínez-Caballero, Zhihong Peng, Mayte Batuecas-Mordillo, Jayda E. Meisel, Derong Ding, Valerie A. Schroeder, William R. Wolter, Kiran V. Mahasenan, Juan A. Hermoso, Shahriar Mobashery, Mayland Chang
Jeshina Janardhanan
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Renee Bouley
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Siseth Martínez-Caballero
bDepartment of Crystallography and Structural Biology, Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain
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Zhihong Peng
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Mayte Batuecas-Mordillo
bDepartment of Crystallography and Structural Biology, Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain
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Jayda E. Meisel
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Derong Ding
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Valerie A. Schroeder
cFreimann Life Science Center, University of Notre Dame, Notre Dame, Indiana, USA
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William R. Wolter
cFreimann Life Science Center, University of Notre Dame, Notre Dame, Indiana, USA
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Kiran V. Mahasenan
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Juan A. Hermoso
bDepartment of Crystallography and Structural Biology, Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain
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Shahriar Mobashery
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Mayland Chang
aDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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DOI: 10.1128/AAC.02637-18
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  • FIG 1
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    FIG 1

    Chemical structures of quinazolinones 1 and 2.

  • FIG 2
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    FIG 2

    Checkerboard assay to assess the interactions of compound 2 with β-lactams and non-β-lactam antibiotics against three Staphylococcus aureus strains. Synergy was seen only in MRSA strains (NRS70 and NRS123; both strains produce PBP 2a) and with β-lactams, such as oxacillin, piperacillin, imipenem, meropenem, and cefepime. NRS128 is an MSSA strain. OXA, oxacillin; PIP, piperacillin; IPM, imipenem; MEM, meropenem; FEP, cefepime; VAN, vancomycin; DOX, doxycycline; GEN, gentamicin; LZD, linezolid; AZI, azithromycin.

  • FIG 3
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    FIG 3

    Time-kill assays with compound 2 in combination with PIP and TZP. (A) Combination of compound 2 at 0.5× MIC with PIP at 0.5× MIC in MRSA NRS70 showed no significant bactericidal synergy, while the combination with PIP at 0.25× and 0.125× MIC was clearly antagonistic. The orange line represents PIP at 0.5× MIC, and the purple lines represent combination of compound 2 at 0.5× MIC with PIP at 0.5× (solid line), 0.25× (dashed line), and 0.125× (dotted line). (B) Compound 2 at 0.5× MIC (solid line) and 0.25× MIC (dashed line) in combination with TZP at 0.5× MIC in MRSA NRS70. (C) Compound 2 at 0.5× MIC in combination with TZP at 0.5× MIC in MRSA NRS123. (D) Compound 2 at 0.5× MIC (solid line) and 1× MIC (dashed line) MIC in combination with TZP at 0.5× MIC in MRSA USA300. (E) Compound 2 at 0.5× MIC in combination with 0.5× MIC of PIP in NRS100 (COL), a β-lactamase-negative MRSA strain.

  • FIG 4
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    FIG 4

    Compound 2 in combination with TZP damages MRSA NRS70, as documented by SEM. (A) Control cells without antibiotic treatment. (B) Cells treated with compound 2 at 4× MIC overnight. (C) Cells treated with TZP at 0.25× MIC. (D) Cells treated with the triple combination of compound 2 with TZP. Magnification is ×35,000. Scale bar is 2 μm.

  • FIG 5
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    FIG 5

    Compound 2 in combination with TZP shows efficacy in the neutropenic thigh infection model. Mice (n = 8 per group) were infected intramuscularly in the right thigh with MRSA strain NRS70 (105 CFU per thigh). Three doses of compound 2 (40 mg/kg), TZP (32 mg/kg of TZB and 4 mg/kg of PIP), the triple combination, or the vehicle were administered subcutaneously starting at 1 h after infection and every 8 h, for a total of three doses. The triple combination of compound 2 with TZP was significantly better than either compound 2 (P < 0.001) or TZP (P < 0.01) by itself. Values are means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney U test with two tails).

  • FIG 6
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    FIG 6

    Three-dimensional structure of the ternary complex PBP 2a-compound 2-PIP (complex 1). Shown is the molecular surface of complex 1, with compound 2 and PIP in spheres (yellow and orange for carbon atoms, respectively). Right, detailed view of residues interacting with ligands at allosteric and active sites. Polar contacts represented as dotted lines.

  • FIG 7
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    FIG 7

    Proposed mechanism of action of the triple combination of compound 2 and TZP. Inhibition of β-lactamase (BlaZ) by tazobactam (TZB) protects piperacillin (PIP) from hydrolysis (*PIP, hydrolyzed PIP). Intact PIP by itself cannot bind to PBP 2a as the active site is closed (PDB code 1VQQ). Compound 2 binds to PBP 2a and triggers the allosteric response of the enzyme (PDB code 4CJN), opening the active site to binding by PIP (ternary complex [this study]), which shuts down cell wall biosynthesis by this enzyme. In addition, compound 2 binds to PBP 1 to interfere with cell wall biosynthesis. PIP can also inhibit the bifunctional PBP 2 and prevent cell wall cross-linking by the enzyme. The concurrent inhibition of PBP 1, PBP 2a, PBP 2, and β-lactamase results in bactericidal synergy. The functional domains are indicated as TP (transpeptidase) and GT (glycosyltransferase).

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  • TABLE 1

    MIC values of compound 2 against a panel of Staphylococcus aureus strainsa

    Strain (reference)HA/CASCC typePFGE typeMIC (μg/ml)
    Compound 2VAN
    VRS4bHAIVUSA1000.2564
    NRS22cHAIIUSA6000.258
    NRS386HA/CAIVUSA7000.1251
    NRS387PediatricIVUSA8000.1251
    NRS483CAIVUSA10000.251
    NRS484CAIVUSA11000.1251
    NRS714HAIVUSA8000.1252
    NRS249HAIV—k0.1252
    NRS70 (16, 17)HAIIUSA1000.251
    NRS123 (38)CAIVUSA4000.1252
    VRS1b (39)HAIIUSA100164
    VRS2b (39)HAIIUSA1000.2532
    NRS384 (16, 40)CAIVUSA3000.031
    NRS100 (15)I0.252
    NRS119d (41)HAIV0.1251
    ATCC 29213e,f (13)0.031
    NRS72e,g (42)0.061
    NRS77h0.1251
    NRS112e,i0.031
    NRS128e,j (14)0.51
    • ↵a CA, community acquired; HA, hospital acquired; SCC, staphylococcal cassette chromosome; PFGE, pulsed-field gel electrophoresis.

    • ↵b Vancomycin-resistant strain.

    • ↵c HeteroVISA strain. The strain was deposited as a heterogeneous vancomycin-intermediate S. aureus phenotype. VISA strains show an MIC of 4 to 8 μg/ml for vancomycin.

    • ↵d Linezolid-resistant strain (MIC of linezolid, 32 μg/ml).

    • ↵e β-Lactamase-positive MSSA strain.

    • ↵f MSSA standard quality control strain used in the laboratory.

    • ↵g MSSA476; hypervirulent and community acquired; USA400.

    • ↵h MSSA (RN1); derived from NCTC8325; blaZ negative.

    • ↵i MSSA (MN8); high-density pathogenic variant.

    • ↵j MSSA derived from NCTC8325; blaZ positive.

    • ↵k —, isolated from France; PFGE type is not known.

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      Table S1 and Fig. S1 to S3

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The Quinazolinone Allosteric Inhibitor of PBP 2a Synergizes with Piperacillin and Tazobactam against Methicillin-Resistant Staphylococcus aureus
Jeshina Janardhanan, Renee Bouley, Siseth Martínez-Caballero, Zhihong Peng, Mayte Batuecas-Mordillo, Jayda E. Meisel, Derong Ding, Valerie A. Schroeder, William R. Wolter, Kiran V. Mahasenan, Juan A. Hermoso, Shahriar Mobashery, Mayland Chang
Antimicrobial Agents and Chemotherapy Apr 2019, 63 (5) e02637-18; DOI: 10.1128/AAC.02637-18

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The Quinazolinone Allosteric Inhibitor of PBP 2a Synergizes with Piperacillin and Tazobactam against Methicillin-Resistant Staphylococcus aureus
Jeshina Janardhanan, Renee Bouley, Siseth Martínez-Caballero, Zhihong Peng, Mayte Batuecas-Mordillo, Jayda E. Meisel, Derong Ding, Valerie A. Schroeder, William R. Wolter, Kiran V. Mahasenan, Juan A. Hermoso, Shahriar Mobashery, Mayland Chang
Antimicrobial Agents and Chemotherapy Apr 2019, 63 (5) e02637-18; DOI: 10.1128/AAC.02637-18
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    • ABSTRACT
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KEYWORDS

MRSA
allosterism
piperacillin
quinazolinone
synergy
tazobactam

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