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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
DOI: 10.1128/AAC.02637-18

ABSTRACT

The quinazolinones are a new class of antibacterials with in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA). The quinazolinones target cell wall biosynthesis and have a unique mechanism of action by binding to the allosteric site of penicillin-binding protein 2a (PBP 2a). We investigated the potential for synergism of a lead quinazolinone with several antibiotics of different classes using checkerboard and time-kill assays. The quinazolinone synergized with β-lactam antibiotics. The combination of the quinazolinone with commercial piperacillin-tazobactam showed bactericidal synergy at sub-MICs of all three drugs. We demonstrated the efficacy of the triple-drug combination in a mouse MRSA neutropenic thigh infection model. The proposed mechanism for the synergistic activity in MRSA involves inhibition of the β-lactamase by tazobactam, which protects piperacillin from hydrolysis, which can then inhibit its target, PBP 2. Furthermore, the quinazolinone binds to the allosteric site of PBP 2a, triggering the allosteric response. This leads to the opening of the active site, which, in turn, binds another molecule of piperacillin. In other words, PBP 2a, which is not normally inhibited by piperacillin, becomes vulnerable to inhibition in the presence of the quinazolinone. The collective effect is the impairment of cell wall biosynthesis, with bactericidal consequence. Two crystal structures for complexes of the antibiotics with PBP 2a provide support for the proposed mechanism of action.

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant public health threat. Every year 275,000 individuals in the United States are hospitalized with MRSA infections, resulting in 19,000 deaths annually (1). A major β-lactam resistance determinant of MRSA is the mecA gene, encoding penicillin-binding protein 2A (PBP 2a) (2). Activity of PBP 2a is regulated by allostery (3–5). The enzyme exhibits a sheltered active site (4, 6). However, interactions at the allosteric site by peptidoglycan, the major component of the cell wall, trigger a conformational change that opens the active site for a different peptidoglycan strand to enter it and undergo the cell wall cross-linking reaction (4, 5). In the absence of an allosteric trigger, the active site is inaccessible to β-lactam antibiotics, rendering them obsolete in treatment of infections. The need for new antibiotics or antibiotic combinations remains high in light of the global crisis on antibiotic resistance (7–9).

The quinazolinones are a new class of orally bioavailable anti-MRSA antibacterials with in vivo activity (10). Compound 1 (Fig. 1) exhibits a MIC of 2 μg/ml against MRSA NRS70, a volume of distribution of 0.3 liter/kg, a clearance of 6.87 ml/min/kg (considered low), a terminal half-life of >20 h, and absolute oral bioavailability of 50%, and it showed efficacy in the mouse peritonitis model of infection (10). We demonstrated by X-ray crystallography that compound 1 binds to the allosteric site of PBP 2a, whereby it facilitated conformational changes that resulted in opening of the active site (10). In addition, compound 1 inhibits PBP 1, an essential PBP for cell division in S. aureus (11). We recently reported on the structure-activity relationship for the quinazolinone class of antibacterials by evaluation of 77 synthetic analogs (12). A lead quinazolinone (compound 2 [Fig. 1]) has a MIC of 0.25 μg/ml against MRSA NRS70, a volume of distribution of 3.58 liters/kg (considered a large volume of distribution), a terminal half-life of 6.5 h, a clearance of 6.4 ml/min/kg (considered low), and absolute oral bioavailability of 37%, and it shows better efficacy than compound 1 in the mouse neutropenic thigh infection model (12). In this study, we investigated the synergism of compound 2 with β-lactam and non-β-lactam antibiotics. We have found that compound 2 synergized in combination with piperacillin (PIP) and tazobactam (TZB) and that it showed efficacy in the mouse neutropenic thigh model of MRSA infection.

FIG 1
FIG 1

Chemical structures of quinazolinones 1 and 2.

RESULTS AND DISCUSSION

Strains and MICs.Several strains of S. aureus were tested against compound 2 and vancomycin (VAN) (Table 1). MICs for compound 2 ranged from 0.03 to 1 μg/ml, while those of vancomycin were 1 to 64 μg/ml (Table 1). Of the five methicillin-sensitive strains used in the study, ATCC 29213 (13), NRS72, NRS112, and NRS128 (NCTC8325) are β-lactamase-positive strains (14). The COL strain (NRS100) is the only β-lactamase-negative MRSA strain used in the study; it constitutively expresses mecA owing to the nonfunctional mecI-mecR system (15). While the COL strain is homogenously resistant to oxacillin, N315 (NRS70) is a prototype MRSA strain with heterogeneous resistance to oxacillin; this strain has an inducible mecA gene with the wild type mecI-mecR sequences (16, 17), and it is also β-lactamase positive. In light of the fact that both resistance determinants are expressed in this strain, we selected it for use in our animal infection models.

View this table:
TABLE 1

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

Checkerboard assays.Checkerboard assays were carried out to assess the potential for synergistic activity of compound 2 with some of the commonly used antibiotics. Initially we tested three bacterial strains (MRSA strains NRS70 and NRS123 and the methicillin-sensitive S. aureus [MSSA] strain NRS128) against a panel of β-lactam and non β-lactam antibiotics. Compound 2 synergized with the β-lactams oxacillin (OXA), piperacillin (PIP), and imipenem (IPM) in NRS70 and NRS123, with a fractional inhibitory concentration (FIC) index of 0.5 (considered marginal) (Fig. 2). With cefepime (FEP) and meropenem (MEM), synergy was observed only in strain NRS70. Combinations of compound 2 with non-β-lactam antibiotics (Fig. 2) were indifferent. No synergy was observed in the MSSA strain NRS128. Subsequently we evaluated the checkerboard assay of compound 2 with an additional eight strains of MRSA and four strains of MSSA with OXA, PIP, IPM, and MEM. Compound 2 synergized with PIP in all the MRSA strains, while synergy with OXA and IPM was observed only in six of the eight MRSA strains. Synergy with MEM was seen only in one of the eight MRSA strains (see Fig. S1 in the supplemental material). The transglycosylase activity of PBP 2 is essential for peptidoglycan synthesis (18), and PIP inhibits the transpeptidase activity of PBP 2. As we outline below, compound 2 binds to PBP 1 and PBP 2a. Thus, a PIP-compound 2 combination would inhibit the transpeptidase activity of PBPs 1, 2, and 2a, which we hypothesized would have a bactericidal effect. In order to determine if this combination shows bactericidal synergy in MRSA strains, we decided to further analyze the combination using time-kill assays.

FIG 2
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.

Time-kill assays.Compound 2 was tested at 1×, 0.5× and 0.25× MIC in combination with PIP at 0.5×, 0.25×, and 0.125× MIC. The combination of compound 2 at 0.5× MIC with PIP at 0.5× MIC was better than either compound by itself but showed no significant bactericidal synergy (Fig. 3A). The combination of compound 2 and PIP at 0.25× and 0.125× MIC, respectively, was clearly antagonistic at 24 h. Bacteriostatic drugs such as compound 2 inhibit cell division without killing the bacteria, forcing them into stationary phase. Therefore, a bactericidal drug such as PIP is unable to act on nongrowing cells, a phenomenon described as drug indifference (19), which might explain this observation. Earlier studies have also shown that antibiotics, especially β-lactams, are ineffective against dormant or nongrowing S. aureus (20). These bacteria also produce β-lactamase that is inducible and hydrolyzes PIP, which inactivates the antibiotic (21). We hypothesize that at lower concentrations of PIP, β-lactamase expression would be induced and able to effectively counter the action of PIP.

FIG 3
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.

Therefore, we decided to add tazobactam (TZB), a β-lactamase inhibitor, to the combination. PIP is a broad-spectrum penicillin and in combination with TZB, the spectrum of activity increases to include β-lactamase-producing organisms (21). PIP-TZB (TZP) was maintained at an 8:1 ratio, which is the clinically used ratio of the drug (22). For the time-kill assays, TZP concentrations were maintained at 0.5× MIC (32 to 4 μg/ml) for the strains tested. With the NRS70 strain, bactericidal synergy (>3-log reduction in CFU/milliliter) was observed when compound 2 at a concentration as low as 0.25× MIC was combined with TZP (Fig. 3B). For NRS123, bactericidal synergy was observed at 0.5× MIC (Fig. 3C). For USA300, bactericidal synergy was observed only at 1× MIC of compound 2 in combination with TZP (Fig. 3D). In all three cases, a ≥3-log reduction in colony counts was observed. The addition of TZB inhibits β-lactamase, allowing PIP to bind and inhibit PBP 2. Additionally we tested NRS100, a β-lactamase-negative MRSA strain, in time-kill assays (Fig. 3E). Synergy was observed with 0.5× MIC of compound 2 and 0.5× MIC of PIP, without the addition of TZB. Hence, this experiment supports the assertion that TZB serves as a β-lactamase inhibitor, as would be expected.

Scanning electron microscopy.The morphological changes in the bacterial cells due to treatment with the triple combination of compound 2 with TZP were evaluated by scanning electron microscopy (SEM). MRSA NRS70 was imaged first (Fig. 4A), before the cells were exposed to the antibiotics. At the tested concentrations, no discernible damage to bacteria was observed with the bacteriostatic agent compound 2 (Fig. 4B) or with TZP (Fig. 4C) by itself. However, cell lysis was observed in the samples treated with the triple combination (Fig. 4D, white arrows).

FIG 4
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.

Quinazolinone binding assays for PBP 1 and PBP 2a.We had previously shown that compound 1 inhibits the allosteric site and also binds to the PBP 1 active site by intrinsic fluorescence and Bocillin FL assays, respectively (10). Binding of compound 2 to the allosteric site of PBP 2a was assessed by intrinsic fluorescence quenching of purified PBP 2a. For this assay, the active site of PBP 2a was covalently modified first with long exposure to high concentration of oxacillin (23). The dissociation constant (Kd) for binding of compound 2 to the allosteric site of PBP 2a in the oxacillin-PBP 2a acylenzyme complex was determined to be 7.4 ± 1.0 μg/ml, which was similar to that for compound 1, 6.9 ± 2.0 μg/ml (10). We also investigated inhibition of PBP 1 by compound 2 using Bocillin FL (24), a fluorescent penicillin. In this assay, compound 2 competes with Bocillin FL, a covalent modifier of the active sites of PBPs. Compound 2 inhibits PBP 1N with a 50% inhibitory concentration (IC50) of 38.6 ± 9.9 μg/ml, which is above the MIC of compound 2. We had previously found that compound 1 inhibits PBP 1 of membrane preparations of S. aureus ATCC 29213, an MSSA strain, with an IC50 of 78 ± 23 μg/ml (10). The higher IC50s relative to MIC can be explained by competition with a covalent modifier (Bocillin FL) of the active site compared to the noncovalent inhibitors compounds 1 and 2. In addition, the membrane environment or the presence of partner proteins might be necessary to give an IC50 near the MIC range. Nevertheless, the results show that compound 2 binds to the allosteric site of PBP 2a and to PBP 1.

Neutropenic thigh infection.We had shown that the triple combination of compound 2 with TZP was bactericidal by time-kill assays (Fig. 3 to D). In order to assess the efficacy of the triple combination in vivo, we conducted a murine neutropenic thigh infection study. The compounds were administered subcutaneously 1 h after the infection by strain NRS70, and two additional doses were given at 9 h and 17 h; the doses were selected from pharmacokinetic studies. The infected thighs were harvested and the bacterial density was quantified. The bacterial count in compound 2-treated mice was 0.78 log lower (P > 0.05) than in vehicle-treated mice, whereas the TZP-treated group was 1.02 log lower (Fig. 5). The triple combination-treated group had the bacterial density lowered by 2.12 logs compared to the vehicle group (P < 0.001), 1.34 logs lower than compound 2 by itself (P < 0.001), and 1.1 logs lower than TZP by itself (P < 0.01). These results clearly indicate that the triple combination of compound 2 with TZP is efficacious in vivo.

FIG 5
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).

Concentrations of compound 2 at 8 h after the last dose were 9.24 ± 3.19 μg/ml in plasma and 3.94 ± 3.19 μg/g in the thighs in mice (n = 8 mice per group) that were given compound 2. In the triple-drug combination, concentrations of compound 2 were 9.92 ± 4.40 μg/ml and 2.85 ± 2.00 μg/g in plasma and thighs, respectively. Concentrations of PIP and TZB in all groups were below the limit of quantification (0.1 μg/ml for PIP and 0.5 μg/ml for TZB) at 8 h after the last dose, consistent with the reported half-lives of 1 h (25).

Crystal structures of antibiotic complexes with PBP 2a.We have not succeeded in crystallizing PBP 1 to date, but we have done so for PBP 2a. The crystal structure of the ternary complex PBP 2a-compound 2-PIP (complex 1) was solved at a 2.5-Å resolution (Table S1). Best crystals were obtained by overnight soaking of native PBP 2a crystals into a mother liquor containing 1 mM compound 2 and then soaking for 3.5 h into a mother liquor containing 1 mM compound 2 and 4 mM PIP. The structure of complex 1 identifies compound 2 within the allosteric site and PIP covalently bound to the active site (Fig. 6 and Fig. S2A). Acylation of the catalytic S403 by PIP is observed together with movement (compared with the apo state) of α2-α3 loop containing the gatekeeper residue Y446, with the attendant creation of a network of hydrogen bonds with the active-site residues to accommodate PIP (Fig. 6). Previously observed changes at β3 and the N terminus of α2 (where catalytic serine is located) are also seen, in particular the twist in the β3 strand that has been observed in all other complexes with β-lactams (4, 26), which was attributed to the sequence of events leading to serine acylation by the antibiotic (26). We observed density in the allosteric site that can be mapped to compound 2. The bulk of the compound is seen within the density, with the exception of the ethynylphenyl group at 5 o’clock (Fig. S2A), which is a mobile element within the complex.

FIG 6
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.

Different crystallographic experiments were performed in which soaking times were decreased for both compound 2 and PIP. The crystal structure of PBP 2a-PIP (complex 2) (Fig. S2B) was obtained after 10 min of soaking with compound 2 (2 mM) and then 15 min of additional soaking with PIP (4 mM [see Materials and Methods]). In complex 2 there was no electron density for compound 2 at the allosteric site; however, PIP was clearly identified attached to the catalytic S403 (Fig. S2B) in both monomers (chains A and B) of the asymmetric unit. This result indicates that even short incubation times with compound 2 were sufficient to promote opening of the active site of PBP 2a to allow binding by PIP. In order to further validate the effect of compound 2 in PIP inhibition, the crystallization experiment yielding complex 2 was performed by keeping the concentration and incubation times for PIP the same (4 mM and 15 min), but in the absence of compound 2. Diffraction data sets collected for these crystals were consistent with that of the apo conformation for PBP 2a, with the unoccupied active site in the closed conformation. This finding further validates that the presence of compound 2 is necessary for access to the active site by PIP, per the allosteric model.

Complex 2 presents unique structural features with relevant mechanistic implications. As previously mentioned, both chains show PIP attached to S403. However, the protein backbone for complex 2 presents larger deviations from the apo structure and from complex 1 (Fig. S3). Consistent with the concept of allosteric triggering by compound 2, chain A in complex 2 presents the lid covering the active site, the α2-α3 loop, and a segment of the helix α3 completely disordered (residues 417 to 457) (Fig. S3). PIP in complex 2 shows a small number of hydrogen bond interactions (Fig. S3) compared with that of PIP in complex 1 (Fig. 6). There is no twist in the β3 strand, despite S403 of complex 2 being seen acylated by PIP (Fig. S4). Therefore, complex 2 represents an intermediate state during allosteric rearrangement and stabilization of the covalently bound β-lactam at the active site. This complex reveals that the observed twist in the β3 strand on acylation is likely produced during accommodation of the antibiotic within the active site after acylation.

Mechanism of action and concluding remarks.Our proposed mechanism of action is summarized in Fig. 7. TZP is a clinically approved combination drug against β-lactamase-producing organisms. However, the combination is ineffective against MRSA strains that harbor PBP 2a, since PIP—and most other β-lactams—cannot inhibit this PBP. Compound 2 is a quinazolinone antibiotic that targets staphylococcal PBPs and inhibits bacterial growth. Although the quinazolinone by itself is bacteriostatic in vitro, when compound 2 at concentrations as low as 0.25× MIC is combined with PIP (an inhibitor of PBP 2) and TZB (a β-lactamase inhibitor), bactericidal synergy is observed (Fig. 3B). Compound 2 also binds to the allosteric site of PBP 2a and triggers opening of the active site, which then becomes accessible for binding by PIP. The activation of PBP 2a has been documented in microbiological experiments and by X-ray crystallography in the present work. We also document that compound 2 binds to PBP 1, which further complements the inhibition of PBP 2 by PIP. In the MRSA NRS70 strain used in our in vivo experiment, TZB functions to protect PIP from being hydrolyzed by the class A β-lactamase.

FIG 7
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).

We found that the triple combination of quinazolinone 2 with TZP shows efficacy both in vitro and in a clinically relevant mouse infection model. With this strategy, β-lactam antibiotics can be resurrected from obsolescence for clinical use in synergistic combinations (27) with subinhibitory concentrations of newer antibiotics. This strategy mitigates the complications associated with emergence of resistance in preserving antibiotics for clinical use.

MATERIALS AND METHODS

Reagents.The antibiotics used in the study included oxacillin, cefepime, imipenem, meropenem, vancomycin, gentamicin, azithromycin, doxycycline, and tazobactam (Sigma-Aldrich, St. Louis, MO), piperacillin (TCI, Portland, OR), and linezolid (AmplaChem Inc., Carmel, IN). The lead quinazolinone (compound 2) was synthesized as the sodium salt in our laboratory using methodology reported earlier (12).

Microorganisms.MRSA strains NRS22, NRS70 (N315), NRS123 (MW2), NRS100 (COL), NRS119, NRS249, NRS384 (USA300), NRS386, NRS387, NRS483, NRS484, NRS714, VRS1, VRS2, and VRS4 and MSSA strains NRS72, NRS77, NRS112, and NRS128 were obtained through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA). ATCC 29213 was purchased from American Type Culture Collection (ATCC; Manassas, VA) (Table 1).

MIC determination.The MICs of compound 2 against these organisms were determined in cation-adjusted Mueller-Hinton II broth (CAMHB-II; Becton, Dickinson and Co., Sparks, MD) using the microdilution technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (28). The experiments were done in triplicate.

Checkerboard assay.Compound 2 was tested against a panel of β-lactams and non-β-lactam antibiotics with 10 MRSA strains and 5 MSSA strains, to assess synergistic interactions using the checkerboard assay in 96-well plates (Corning Incorporated, Corning, NY), as described earlier (29). The fractional inhibitory concentration (FIC) index was calculated for each combination per the methodology of Eliopoulos and Moellering (30). An FIC index of ≤0.5 was considered synergistic, >0.5 to <2 indifferent, and >2 antagonistic. The experiments were done in triplicate.

Time-kill assay.The synergistic combinations found using the checkerboard assays were further validated with time-kill assays performed in triplicate in CAMHB-II, as per the methodology of Eliopoulos and Moellering (30). The assay was done in 5-ml tubes, and compound 2 was tested at 1×, 0.5× and 0.25× MIC in combination with PIP at 0.5×, 0.25×, and 0.125× MIC. Compound 2 at the above concentrations was also tested with the piperacillin-tazobactam (TZP) combination at a 8:1 ratio. The TZP combination was tested at 0.5× and 0.25× MIC of the combination for the strains used. A control tube with no antibiotic was also included. Synergy was defined as a ≥2-log10 decrease in CFU/ml between the combination and its most active constituent after 24 h and if the colony count in the combination was ≥2 log10 CFU/ml below the starting inoculum.

Electron microscopy studies.Bacteria in mid-exponential growth phase were treated overnight with 4× MIC of compound 2, 0.25× MIC of TZP, and the triple combination at the same concentrations. Following incubation, the cells were washed with PBS (three times), applied to poly-l-lysine (Santa Cruz Biotechnology, Dallas, TX)-coated microglass coverslips (Electron Microscopy Sciences, Hatfield, PA), and incubated for 15 min. The cells were fixed for 1 h with 2% gluteraldehyde, followed by washing with sodium cacodylate buffer at pH 7.4. The samples were fixed with 1% osmium tetroxide for 1 h and rinsed (three times) in buffer. The samples were then put through a graded ethanol series for dehydration, followed by critical-point drying. The slides were then mounted on scanning electron microscopy (SEM) stubs and sputter coated with iridium to a thickness of 5 nm. Microscopy and imaging were done using a Magellan 400 XHR scanning electron microscope (FEI, Hillsboro, OR).

PBP 2a expression and purification.The mecA gene was cloned and was expressed, followed by purification of PBP 2a to homogeneity as reported previously (4).

PBP 1 expression and purification.A gene for PBP 1 was cloned without the sequence for the first 64 amino acids based on our sequence alignment with the related Streptococcus pneumoniae PBP 2x. This gene was amplified by PCR and ligated into the multiple-cloning site of pET24a(+) designated pET24a-PBP1N; the recombinant plasmid was used to transform Escherichia coli BL21(DE3) for overexpression, followed by induction and protein purification of the N-terminally truncated PBP 1. An overnight culture of E. coli BL21(pET24a-PBP1N) (10 ml) was used to inoculate 1 liter of LB-kanamycin (50 μg/ml) and grown at 37°C with shaking (190 rpm) until the optical density at 600 nm (OD600) reached 0.5 (approximately 3 h). Expression was induced with a final concentration of 0.5 mM isopropyl-β-d-thioglactopyranoside (IPTG) and shaking at 16°C overnight (approximately 18 h). Cells were harvested by centrifugation at 4,700 × g for 35 min at 4°C, and the pellet was resuspended in 25 mM HEPES buffer, pH 8.0, and 150 mM NaCl (buffer A). The cells were lysed by mechanical means with 10  2-min cycles of sonification on ice and the debris was pelleted by centrifugation at 20,200 × g and 4°C for 45 min. The supernatant (lysate) was loaded onto an S-Sepharose column (2.5 by 50 cm; 160-ml High S support resin) equilibrated with buffer A at 3 ml/min. Proteins were eluted with a linear gradient of 0.15 to 1.00 M NaCl using buffer B (25 mM HEPES, 1 M NaCl [pH 7.0]) at 1 ml/min in 500 ml. The fractions containing the recombinant PBP 1 (determined by SDS-PAGE) were combined and concentrated using a 10,000-molecular-weight (MW)-cutoff centrifugal filter device to a final volume of 3 ml. The concentrate was loaded onto a Sephacryl S-200 size exclusion column (2.5 by 100 mm; 300-ml Sephacryl S-200) with a syringe. Proteins were eluted with 300 ml of buffer B at 1 ml/min. The fractions containing pure PBP 1 (determined by SDS-PAGE) were combined and concentrated using a 10,000-MW-cutoff centrifugal filter device.

Bocillin FL competition assays.Active-site binding Bocillin FL to PBP 1 was assayed as previously described (10). Briefly, a 1 μM solution of purified PBP 1 was preincubated with various concentrations of compound 2 for 10 min at 37°C, at which point Bocillin FL (Life Technologies, Grand Island, NY) was added to a final concentration of 20 μM and incubated for an additional 10 min. The reaction mixture was quenched by addition of SDS-sample buffer and boiling before analysis by SDS-PAGE. The fluorescence signal was quantified and plotted against the compound 2 concentration, and the data were fit by nonlinear regression using a previously reported equation (31).

Intrinsic fluorescence assay.Allosteric-site binding affinity was determined using an intrinsic fluorescence assay, as previously reported (23). Briefly, purified PBP 2a was allowed to react with 500 μM oxacillin for 45 min at room temperature in order to irreversibly (and covalently) block the active site, followed by removal of excess oxacillin using a protein-desalting column. The acylated protein was quantified by intrinsic fluorescence using a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA) monitoring the excitation at 280 nm and scanning the emission from 300 to 425 nm. The decrease in the maximum fluorescence was quantified relative to the buffer control and plotted versus the compound 2 concentration. Each experiment was performed in triplicate, and the averaged data were fit by nonlinear regression. Assays were performed in 25 mM HEPES buffer, pH 7.0, with 1 M NaCl.

Animals.Female ICR mice (6 to 8 weeks old and with a body weight of 20 to 25 g; Harlan Laboratories, Inc., Indianapolis, IN) were housed in polycarbonate shoeboxes with bedding consisting of ¼-in. corncob (The Andersons Ind., Maumee, OH) and Alpha-Dri (Shepherd Specialty Papers, Inc., Richland, MI). Mice were maintained on a 12-h light/dark cycle at 72°F and were given Teklad 2918 irradiated extruded rodent diet and water ad libitum. All procedures were performed in accordance with and with approval by the University of Notre Dame Institutional Animal Care and Use Committee.

Neutropenic thigh infection study.The efficacy of the triple combination of compound 2 with TZP compared to those of the individual compounds was evaluated in a mouse neutropenic thigh infection model (32). Briefly, mice (n = 8 per group) were rendered neutropenic by intraperitoneal treatment with 100 μl of a 50-mg/ml solution of cyclophosphamide (Alfa Aesar, Haverhill, MA; corresponding to 200 mg/kg), on days 4 and 1 prior to the infection. An inoculum of 0.1 ml of MRSA strain NRS70 at a final concentration of approximately 106 CFU/ml in brain heart infusion broth was injected intramuscularly into the right thighs of all the animals. The infected animals were treated with either a vehicle (5% dimethyl sulfoxide [DMSO], 25% Tween 80, 70% water), 40 mg/kg of compound 2, or 24 mg/kg of PIP and 3 mg/kg of TZB or a combination of compound 2 and TZP. The drugs were administered subcutaneously (three doses) every 8 h starting 1 h after the infection. The animals were euthanized 24 h later (8 h after the last dose), and the infected thighs were harvested aseptically for bacterial counts. The uninfected thigh and terminal blood were also collected to measure drug levels.

Statistical analysis.Statistical analysis was done using the Mann-Whitney U test on GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA).

Dose preparation.Compounds were dissolved in 5% DMSO–25% Tween 80–70% water (vehicle) at the required concentrations. For subcutaneous injections, all the compounds were filter sterilized using nylon membranes (0.2 μm; Pall Life Sciences, Port Washington, NY).

Drug levels in plasma and thigh.A 50-μl aliquot of plasma was quenched with 100 μl of internal standard in acetonitrile to precipitate protein. Control mouse plasma (50 μl) was spiked with standards of each compound (compound 2, PIP, and TZB) ranging from 0.05 μg/ml to 40 μg/ml and quenched with 100 μl of internal standard in acetonitrile. Samples and standards were then centrifuged for 10 min at 12,000 × g. The uninfected thighs were cut into small pieces and homogenized in an equivalent volume of acetonitrile containing internal standard and were centrifuged at 12,000 × g for 20 min. Plasma and thigh supernatants were analyzed using a Waters Acquity ultraperformance liquid chromatograph (UPLC; Waters Corp., Milford, MA) coupled with a triple quadrupole mass spectrometer (TQD; Waters, Milford, MA) with multiple-reaction monitoring (MRM).

Mass spectrometry.Acquisition parameters were as follows: Kinetex C18 column (2.6 μm; 75 mm by 2.1 mm), electrospray ionization positive mode (ESI+), flow rate of 0.4 ml/min, capillary voltage of 4 kV, cone voltage of 30 V, and collision voltage of 25 V. The solvent program was as follows: 90% solvent A and 10% solvent B for 2 min, 6-min linear gradient to 10% solvent A and 90% solvent B, and hold for 2 min, where solvent A is 0.1% formic acid-water and solvent B is 0.1% formic acid-acetonitrile. The methods were linear between 0.05 and 40 μg/ml (R2 = 0.99) for compound 2. Multiple-reaction monitoring transitions were 393→274 for compound 2, 518→143 for PIP, 301.0→168.0 for TZB, and 300→93 for the internal standard. Concentrations in the unknown plasma and thighs were determined using standard curve regression parameters relative to the internal standard.

Crystallization.Native PBP 2a crystals were obtained by the sitting-drop vapor diffusion method at 4°C by mixing 1 μl of protein solution and 1 μl of precipitant solution containing 20% (wt/vol) polyethylene glycol 1000 (PEG 1000), 880 mM NaCl, 100 mM HEPES buffer at pH 7.0, and 16 mM CdCl2. Drops were equilibrated against 150 μl of precipitant in the reservoir chamber. The complex of PBP 2a with compound 2 and PIP, in the allosteric and the active sites, respectively (PBP 2a-compound 2-PIP [complex 1]), was obtained by the soaking of PBP 2a crystals in the precipitant solution and 1 mM compound 2 for 8 h; then crystals were transferred into a solution containing the precipitant solution, 1 mM compound 2, and 4 mM PIP for 3.5 h. Complex 2 (PBP 2a-PIP obtained in the presence of compound 2) was obtained by soaking PBP 2a crystals in the precipitant solution containing 2 mM compound 2 for 10 min, followed by soaking into a solution with mother liquor, 2 mM compound 2, and 4 mM PIP for 15 min.

Data collection, structure determination, and structure refinement.All crystals were cryoprotected in a 70:30 (vol/vol) mixture of Paratone-paraffin oil before flash cooling at 100°K. Data sets were collected using synchrotron radiation at beamline XALOC of the ALBA synchrotron (Barcelona, Spain) using a Pilatus 6 M detector and were indexed and integrated using XDS (33) and AIMLESS (34).

X-ray diffraction data for complex 1 were collected using a wavelength of 0.979257 Å and diffracted up to a 2.50-Å resolution. Crystals belonged to the orthorhombic space group P212121, with the following cell dimensions: a = 81.17 Å, b = 102.88 Å, and c = 187.37 Å. The crystals present two molecules in the asymmetric unit. The complex 2 data were collected using a wavelength of 0.979260 Å and diffracted up to a 2.82-Å resolution. Crystals belonged to the orthorhombic space group P212121, with the following cell dimensions: a = 80.71 Å, b = 105.01 Å, and c = 185.62 Å. The crystals present two molecules in the asymmetric unit.

The structures of both complexes were solved by the molecular-replacement method using the coordinates of the PBP 2a native structure (PDB code 1VQQ) as the search model. The rotational and translational searches were performed using PHASER (35) followed by manual modeling using COOT (36) and refined with PHENIX (37). All the statistics for data processing and refinement are shown in Table S1. In complex 1, the electron density map allowed modeling of complete PBP 2a molecules, a molecule of PIP at the active site, and a molecule of compound 2 at the allosteric site. However, the electron density map for compound 2 does not allow unambiguous orientation of the molecule at the allosteric site, so it was removed from the model. In complex 2, the electron density map allowed modeling of the complete PBP 2a molecule in one chain, while the other presented disorder in the α2-α3 loop; two molecules of PIP were found bound to the active sites of both chains. No electron density was found for compound 2 at the allosteric site in complex 2.

ACKNOWLEDGMENTS

We thank Tatyana Orlova for assistance with SEM imaging and the staff from the ALBA Synchrotron Facility for help during crystallographic data collection.

This work was supported by grant AI116548 (to M.C.) and AI104987 (to S.M.) from the National Institutes of Health and by grant BFU2017-90030-P from the Spanish Ministry of Science, Innovation and Universities (to J.A.H.). R.B. was supported by training grant T32GM075772 and by individual Ruth L. Kirschstein National Research Service Award F31AI115851 from the National Institutes of Health. The SEM work was partially supported by the University of Notre Dame Integrated Imaging Facility.

FOOTNOTES

    • Received 18 December 2018.
    • Returned for modification 7 January 2019.
    • Accepted 5 March 2019.
    • Accepted manuscript posted online 11 March 2019.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02637-18.

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KEYWORDS

MRSA
allosterism
piperacillin
quinazolinone
synergy
tazobactam

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