2.1. Kinase Inhibitors Increase Sensitivity of MRSA to β-Lactam Antibiotics, Such as Nafcillin
We hypothesized that inhibition of Stk1 by kinase inhibitors should increase the sensitivity or minimal inhibitory concentration (MIC) of MRSA to β-lactam antibiotics. To this end, using methods described [
8], we first derived Δ
stk1 mutants from CA-MRSA strains linked to sequential and overlapping epidemics in the United States (USA300 LAC and USA400 MW2 [
9]). Antibiotic susceptibility testing was performed using standards established by the Clinical and Laboratory Standards Institute (CLSI, [
10]). Consistent with previous findings [
6,
7], we observed that ∆
stk1 mutants derived from MRSA strains MW2 and LAC showed a dramatic increase in susceptibility to β-lactams (lower MIC, see
Table 1). The above results further confirmed that MRSA ∆
stk1 mutants are more sensitive to β-lactam antibiotics and provided support for the identification of kinase inhibitors.
Table 1.
Role of Stk1 in β-lactam resistance of methicillin-resistant S. aureus (MW2 and LAC).
Table 1.
Role of Stk1 in β-lactam resistance of methicillin-resistant S. aureus (MW2 and LAC).
MIC (µg/mL) | PEN | AMP | NAF | CXM | CAZ | FEP | IPM |
---|
Newman (MSSA) | 0.094 | 0.25 | 0.25 | 1.5 | 16 | 4 | 0.032 |
MW2 | 48 | 24 | 32 | >256 | >256 | >256 | 1 |
MW2∆stk1 | 12 | 12 | 2 | 6 | 32 | 8 | 0.125 |
LAC | 48 | 32 | 16 | >256 | >256 | >256 | 0.75 |
LAC∆stk1 | 24 | 24 | 4 | 6 | 12 | 4 | 0.06 |
We next screened a library consisting of 280 drug-like, low molecular weight compounds that can inhibit protein kinases (
http://www.timtec.net/kinase-modulators-actitarg-k-library.html) for their ability to increase the sensitivity of wild type (WT) MRSA to β-lactams antibiotics. Briefly, WT MRSA (LAC) was grown overnight in the presence of sub-lethal concentrations of NAF (4 µg/mL) in the presence of 40 µg/mL of the respective kinase inhibitor. Controls included MRSA (LAC) grown in the presence of the kinase inhibitor alone, NAF alone, or media alone. As controls, LACΔ
stk1 was also grown in the presence of the antibiotic, the kinase inhibitor, or media only. The data are shown as % inhibition compared to growth of WT MRSA LAC in the absence of the inhibitor (
Figure 1A). These studies revealed that four inhibitors namely ST085384 ([N-(1benzylpiperidin-4-yl)-1-(naphthalen-1ylsulfonyl)piperidine-3carboxamide], ST085399 (N-(4-methylphenyl)-2-oxo-1H-benzo[cd]indole-6-sulfonamide), ST085404 (2-oxo-N-(2-oxonaphtho[2,1-d][1,3]oxathiol-5-yl)-1,2-dihydrobenzo[cd]indole-6-sulfonamide), ST085405 (N-(4-methoxyphenyl)-2-oxo-2H-naphtho[1,8-bc]thiophene-6-sulfonamide), increased sensitivity of WT MRSA to β-lactams (>66% inhibition
Figure 1A, see
Figure 1B for structures). These kinase inhibitors also did not inhibit growth of the MRSAΔ
stk1 mutant (
i.e., ≤0% growth inhibition).
Figure 1.
Identification of sulfonamides that increase the sensitivity of MRSA to Nafcillin (NAF). (A) Cultures of MRSA WT LAC were grown in the presence of sub-lethal concentration of NAF with various sulfonamides to identify putative kinase inhibitors. Four compounds (ST085384, ST085399, ST085404, and ST085405) caused growth inhibition greater than two standard deviations of the data set; and (B) structures of the four identified sulfonamides.
Figure 1.
Identification of sulfonamides that increase the sensitivity of MRSA to Nafcillin (NAF). (A) Cultures of MRSA WT LAC were grown in the presence of sub-lethal concentration of NAF with various sulfonamides to identify putative kinase inhibitors. Four compounds (ST085384, ST085399, ST085404, and ST085405) caused growth inhibition greater than two standard deviations of the data set; and (B) structures of the four identified sulfonamides.
Using ST085384 (
Figure 2A) and ST085399 (
Figure 2B), we confirmed that growth inhibition of MRSA LAC was observed only in the presence of the β-lactam (
Figure 2A,B). At 12.7 µM, ST085384 inhibited growth of MRSA by 52% (
Figure 2A), which is comparable to the 58% inhibition observed with staurosporine (a general and potent kinase inhibitor) at a similar concentration (
Figure 2C, 13.4 µM). Similar results were observed with MRSA MW2.
Figure 2.
Kinase inhibitors induce a dose-dependent increase in the sensitivity of MRSA to NAF in vitro. Cultures of MRSA WT LAC were grown in the presence or absence of NAF (4 µg/mL) with various concentrations of (A) ST085384; (B) ST085399; or (C) staurosporine. The concentration required for 50% growth inhibition for ST085384 (12.7 µM) was comparable to that of staurosporine (13.4 µM). At the highest concentration used, the kinase inhibitors were at ~25 µg/mL. * p < 0.05; ** p < 0.01; Bonferroni Multiple Comparisons following Two-way ANOVA; n = 2–3).
Figure 2.
Kinase inhibitors induce a dose-dependent increase in the sensitivity of MRSA to NAF in vitro. Cultures of MRSA WT LAC were grown in the presence or absence of NAF (4 µg/mL) with various concentrations of (A) ST085384; (B) ST085399; or (C) staurosporine. The concentration required for 50% growth inhibition for ST085384 (12.7 µM) was comparable to that of staurosporine (13.4 µM). At the highest concentration used, the kinase inhibitors were at ~25 µg/mL. * p < 0.05; ** p < 0.01; Bonferroni Multiple Comparisons following Two-way ANOVA; n = 2–3).
To confirm that the decrease in optical density observed on treatment of MRSA with kinase inhibitor and NAF correlated with decreased bacterial CFU, we performed a time-to-kill analysis, as described [
11,
12]. Briefly, 10
4 CFU of WT MRSA (LAC) was grown for 24 h in either media alone, antibiotic NAF alone (4 µg/mL), kinase inhibitor alone (40 µg/mL), or kinase inhibitor containing NAF. For enumeration of viable bacterial CFU, aliquots were serially diluted and plated at 0, 2, 4, 8, and 24 h post-inoculation. As a control, approximately 10
4 CFU/mL of LACΔ
stk1 (which is sensitive to NAF) was added to either media alone or media containing NAF (4 µg/mL). The results shown in
Figure 3A confirm that treatment of MRSA LAC with the kinase inhibitor and antibiotic NAF was bactericidal, whereas treatment with either only the kinase inhibitor or only the antibiotic NAF did not confer bactericidal activity. Additionally, the bactericidal activity of NAF to MRSA LAC treated with ST085384 is similar to that observed with the β-lactam sensitive MRSA LACΔ
stk1 treated with NAF.
The MRSA strain LAC was grown overnight from single colony. The next morning, approximately 104 CFU/mL of LAC was added to 500 µL of either TSB (shown below as LAC), TSB containing NAF (4 µg/mL, shown below as LAC + NAF), TSB containing kinase inhibitor ST085384 (40 µg/mL, shown below as LAC + ST085384), or TSB containing both NAF (4 µg/mL) and ST085384 (40 µg/mL); shown below as LAC + ST085384 + NAF, see green dashed line. As a control, approximately 104 CFU/mL of LACΔstk1 (which is sensitive to NAF) was added to either 500 µL of TSB (see LACΔstk1) or 500 µL of TSB containing NAF (4 µg/mL, see LACΔstk1 + NAF, red dashed line). All cultures were grown in a 37 °C shaker at 220 rpm. Aliquots of the media were removed immediately after subculture (T0) and at 2, 4, 8, and 24 h post-inoculation and were serially diluted and plated on TSA. Plates were incubated for 24–48 h at 37 °C and viable CFU were counted. The experiment was repeated three times. Note that NAF is bactericidal to MRSA LAC only in the presence of the kinase inhibitor ST085384 and is similar to that observed with MRSA LACΔstk1 treated with NAF (see red and green dashed lines).
Figure 3.
The kinase inhibitor ST085384 increases sensitivity of MRSA LAC to the bactericidal activity of Nafcillin (NAF).
Figure 3.
The kinase inhibitor ST085384 increases sensitivity of MRSA LAC to the bactericidal activity of Nafcillin (NAF).
To determine if ST085384 had potential off-target effects that also increased sensitivity of MRSA LACΔ
stk1 to NAF, we compared growth of LACΔ
stk1 in the presence of sub-MIC NAF with and without the kinase inhibitor ST085384. To this end, we first determined the MIC and sub-MIC of NAF for LACΔ
stk1 under the time to kill assay conditions. To this end, approximately 10
4 CFU/mL of LACΔ
stk1 was added to 500 μL of TSB containing two-fold serial dilutions of Nafcillin. Under these conditions, we determined that the MIC of NAF for LACΔ
stk1 to be 0.5 μg/mL, see
Figure 4A). The MIC of NAF for LAC∆
stk1 under the time to kill assay conditions was lower than that shown in
Table 1, likely due to different inoculum densities. Subsequently, ~10
4 CFU/mL of LACΔ
stk1 was added to 500 μL of either TSB or TSB containing MIC NAF (0.5 µg/mL), TSB containing sub-MIC NAF (0.25 µg/mL) and TSB containing sub-MIC NAF (0.25 µg/mL + kinase inhibitor ST085384 (40 µg/mL)). Aliquots of the media were removed immediately after subculture (T
0) and at 2, 4, 8, and 24 h post-inoculation and were serially diluted and plated in duplicate on TSA. Note that ST085384 did not increase the sensitivity of LACΔ
stk1 to NAF. Collectively, these data confirm that the kinase inhibitor used in this study increases sensitivity of WT MRSA to ß-lactam antibiotics.
Figure 4.
The kinase inhibitor ST085384 does not increase sensitivity of MRSA LAC∆stk1 to the bactericidal activity of Nafcillin (NAF). (A) The MRSA strain LAC∆stk1 was grown overnight from a single colony. The next morning, approximately 104 CFU/mL of LAC∆stk1 was added to 1 mL TSB without antibiotics or 500 µL of TSB containing two-fold serial dilutions of Nafcillin from 0.0625 µg/mL to 4 µg/mL and was incubated O/N at 37 °C with shaking. The MIC of NAF for LAC∆stk1 under these conditions was determined to be 0.5 µg/mL (see loss of turbidity or lack of growth in Panel A); (B) approximately 104 CFU/mL of LAC∆stk1 was added to 500 µL of either TSB or TSB containing MIC NAF (0.5 µg/mL, indicated as LAC∆stk1 + MIC NAF), TSB containing sub-MIC NAF (0.25 µg/mL, indicated as LAC∆stk1 + sub-MIC NAF), and TSB containing sub-MIC NAF (0.25 µg/mL + kinase inhibitor ST085384 (40 µg/mL), indicated as LAC∆stk1 + sub-MIC NAF + ST085384). All cultures were grown in a 37 °C shaker at 220 rpm. Aliquots of the media were removed immediately after subculture (T0) and at 2, 4, 8, and 24 h post-inoculation, and were serially diluted and plated on TSA. Plates were incubated for 24–48 h at 37 °C and viable CFU were counted. The experiment was repeated twice. Note that ST085384 did not increase sensitivity of LACΔstk1 to NAF.
Figure 4.
The kinase inhibitor ST085384 does not increase sensitivity of MRSA LAC∆stk1 to the bactericidal activity of Nafcillin (NAF). (A) The MRSA strain LAC∆stk1 was grown overnight from a single colony. The next morning, approximately 104 CFU/mL of LAC∆stk1 was added to 1 mL TSB without antibiotics or 500 µL of TSB containing two-fold serial dilutions of Nafcillin from 0.0625 µg/mL to 4 µg/mL and was incubated O/N at 37 °C with shaking. The MIC of NAF for LAC∆stk1 under these conditions was determined to be 0.5 µg/mL (see loss of turbidity or lack of growth in Panel A); (B) approximately 104 CFU/mL of LAC∆stk1 was added to 500 µL of either TSB or TSB containing MIC NAF (0.5 µg/mL, indicated as LAC∆stk1 + MIC NAF), TSB containing sub-MIC NAF (0.25 µg/mL, indicated as LAC∆stk1 + sub-MIC NAF), and TSB containing sub-MIC NAF (0.25 µg/mL + kinase inhibitor ST085384 (40 µg/mL), indicated as LAC∆stk1 + sub-MIC NAF + ST085384). All cultures were grown in a 37 °C shaker at 220 rpm. Aliquots of the media were removed immediately after subculture (T0) and at 2, 4, 8, and 24 h post-inoculation, and were serially diluted and plated on TSA. Plates were incubated for 24–48 h at 37 °C and viable CFU were counted. The experiment was repeated twice. Note that ST085384 did not increase sensitivity of LACΔstk1 to NAF.


2.2. Kinase Inhibitors Decrease Stk1 Autophosphorylation
To confirm that kinase inhibitors such as ST085384 can inhibit Stk1 from
S. aureus, we performed autophosphorylation assays. To this end, we cloned the coding sequence of the
stk1 gene from
S. aureus into the expression vector pET32ck [
13] to generate a C-terminal His-tagged fusion. Although we used genomic DNA from
S. aureus RN4420 as the template for
stk1 cloning, we confirmed that the coding sequence of the
stk1 gene in
S. aureus RN4220 [
14] is 100% identical to that found in MRSA USA300 [
15]. Stk1 protein was then purified and an aliquot was analyzed on 10% SDS-PAGE followed by Coommasie staining (
Figure 5A). Then autophosphorylation asssays were performed in buffer containing 10 µCi [γ
32P]-ATP and either DMSO (control) or Staurosporine (2 µM) or ST085384 (2 µM) for 15 min. Subsequently, all samples were separated by SDS-PAGE followed by autoradiography.
Figure 5B shows that autophosphorylation of Stk1 is decreased in the presence of Staurosporine or ST085384 (compare lanes 2 and 3 to 1). These data indicate that the kinase inhibitor ST085384 and Staurosporine can inhibit the activity of
S. aureus Stk1.
Figure 5.
Kinase inhibitor ST085384 decreases Stk1 autophosphorylation in vitro. (A) S. aureus Stk1-His6 protein was purified as described in the methods, and an aliquot of the purified protein was resolved on 10% SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1 represents pre-stained protein standard (BioRad) and Lane 2 presents Stk1-His6 (~1 µg); and (B) autophosphorylation assays of Stk1 was performed using S. aureus Stk1-His6(~1 µg) in kinase buffer containing 10 µCi [γ 32P]-ATP and either DMSO control (lane 1) or Staurosporine (2 µM, lane 2) or ST085384 (2 µM, lane 3). Samples were resolved on 12% SDS-PAGE followed by autoradiography. Note that autophosphorylation of Stk1 is decreased in the presence of staurosporine (lane 2) and ST085384 (lane 3).
Figure 5.
Kinase inhibitor ST085384 decreases Stk1 autophosphorylation in vitro. (A) S. aureus Stk1-His6 protein was purified as described in the methods, and an aliquot of the purified protein was resolved on 10% SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1 represents pre-stained protein standard (BioRad) and Lane 2 presents Stk1-His6 (~1 µg); and (B) autophosphorylation assays of Stk1 was performed using S. aureus Stk1-His6(~1 µg) in kinase buffer containing 10 µCi [γ 32P]-ATP and either DMSO control (lane 1) or Staurosporine (2 µM, lane 2) or ST085384 (2 µM, lane 3). Samples were resolved on 12% SDS-PAGE followed by autoradiography. Note that autophosphorylation of Stk1 is decreased in the presence of staurosporine (lane 2) and ST085384 (lane 3).
2.3. The Kinase Inhibitor ST085384 Is Tolerated in Mice
Bioinformatic analysis using the Mobyle FAF-drugs2 software [
16] indicated that “alerting structures” commonly associated with toxic effects were not present in ST085384, ST085404, ST085405, or ST085399. Additionally, ST085384, ST085404, ST085405, ST085399 did not exhibit any violations to Lipinski’s rule of five that are important for solubility and permeability of compounds in drug discovery and development [
17]. Therefore, we were next interested to determine if the kinase inhibitor/s is tolerated by mice. We chose to test this with the kinase inhibitors ST085384 and ST085405. To this end, we administrated a weight-adjusted dose of ST085384 or ST085405 in Cremaphor EL intraperitoneally to six-week old female C57BL/6J mice (
n = 14
/group, weight range 15–20 g). Inhibitor doses included 0 (Cremaphor EL, vehicle control), 10 and 100 mg/kg. All groups of mice were continuously monitored for signs of morbidity for a period of nine days. Collectively, these results shown in
Figure 6 indicates that at a 100 mg/kg dose, ST085384 and ST085405 are non-toxic to mice, and are better tolerated than the general and potent kinase inhibitor, staurosporine (LD
50 = 6.5 mg/kg [
18]). These studies provide the foundation for future work to determine if kinase inhibitors that enhance MRSA susceptibility to β-lactams
in vivo.
Figure 6.
The kinase inhibitors ST085384 and ST085405 are tolerated in mice. Survival of mice injected with 100 mg/kg (black circle and solid line), 10 mg/kg (hollow square and dashed line), or vehicle only (0 mg/kg; black triangle and dotted line) of (A) ST085384 or (B) ST085405 was monitored for nine days post-injection. Note that all mice survived.
Figure 6.
The kinase inhibitors ST085384 and ST085405 are tolerated in mice. Survival of mice injected with 100 mg/kg (black circle and solid line), 10 mg/kg (hollow square and dashed line), or vehicle only (0 mg/kg; black triangle and dotted line) of (A) ST085384 or (B) ST085405 was monitored for nine days post-injection. Note that all mice survived.