Scientific Rationale

Group A streptococcus (GAS) is among the most common human pathogens affecting millions of people globally each year. Even though treatment with antibiotics has greatly reduced mortality in the developed world, complications of GAS infection are sometimes still lethal. Further, emergence of epidemic invasive GAS diseases poses another critical medical challenge, but little is known about the mechanism of invasiveness (3). Knowledge of the underlying molecular mechanisms required for infection by this important pathogen will be crucial for the development of novel therapies for GAS infection.

Streptokinase (SK) is a key virulence factor for GAS infection through interacting with host plasminogen (2,8,9). This project aims to take advantage of this observation and discover novel antimicrobial agents for the treatment of GAS infection. One key design of the project is to target specifically the bacterial virulence without impact on bacterial growth. Such agents provide a promising alternative approach for treatment of infectious diseases by reducing the risks of antibiotic resistance. Antibiotic resistance represents an increasing concern as a major global public health threat contributed by widespread use of antibiotics that cause death or growth arrest in the target bacteria. This screening campaign has identified a probe (CID-1612038/134; 4-methoxy-2-(4-propoxyphenyl)quinazoline) with an IC₅₀ of 4.2 μM, which is inactive in a bacterial viability test within a range of testing concentrations up to 50 μM.

Materials and Reagents

All reagents and solvents were purchased from commercial vendors and used as received. Phosphate-buffered saline (PBS) was acquired from the Broad Institute Supply and Quality Management (SQM; Cambridge, MA). SK assay solution was acquired from Innov Research (Novi, Michigan), and S2403 was acquired from Diapharma (West Chester, OH). BacTiter-Glo was purchased from Promega (catalog no. G8233; Madison, WI). White assay plates were purchased from Corning (catalog no. 3570; Corning, NY), and clear plates were purchased from Nunc (catalog no. 242757; Rochester, NY).

2.1. Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A. Refer to Appendix B for the detailed assay protocols.

2.1.3. Secondary Screen for SK Expression in UMAA 2616 Strain (AID nos. 1914, 2137, 504351)

On Day 1, UMAA 2616 was streaked out on THY/S medium (30g/L THB/0.2% yeast extract with streptomycin 100 μg/mL), and incubated at 37°C overnight. On Day 2, a single colony was picked and grown overnight in THY/S medium at 37°C. On Day 3, the overnight culture was diluted 20-fold into fresh THY/S medium in a flask and grown until it reached OD600 of 0.6–0.8. The culture was again diluted into cold THY/S medium to result in OD600 value of 0.038, and the diluted culture was kept at 4°C overnight. On Day 4, the diluted culture was warmed to room temperature, and 20 μl per well of culture was dispensed with a Combi (Thermo Fisher Scientific Inc., Waltham, MA) into assay plates that contained 30 μl per well of THY/S medium. Next, 100 nL per well of compound was pinned with Cybi-Well (CyBio; Woburn, MA), and the plates were incubated at 37°C for 6 hours in a Liconic incubator. The cells were pelleted at 3000 rpm in a Sorvall centrifuge. Next, 10 μl per well of the supernatant was transferred twice but into separate clear plates with Cybi-Well Vario and stored at −20°C. The culture plates were warmed to room temperature for 30 minutes. Then, 50 μl per well of half strength BacTiter-Glo was added with a Combi multidrop. The plates were incubated at room temperature for 10 minutes, and luminescence was detected on an EnVision (Perkin Elmer; Waltham, MA) multimode reader for indications of bacterial cell viability.

SK expression was measured by adding 50 μl/well of SK assay solution (5 μl per well of human plasma, 1.2 μl to 2.4 μl per well of 4.2 mg/ml S2403, and 43.8 μl per well of PBS) to the 10 μl per well supernatant that had been thawed and warmed up to room temperature in clear SK assay plates. Compound CID-657963 was used as a positive control in the SK expression assay (see Figure 1). OD405 was taken at 0 h and after 1.5 h to 2.5 h of incubation at 37°C depending on the lot of the S2403.

Figure 1

Prior Art Compound CID-657963 for SK Expression. Compound CID-657963 (positive control) in the SK expression assay. This compound did not significantly inhibit bacterial cell viability up to approximately 100 μM.

2.2. Probe Chemical Characterization

After preparation as described in Section 2.3, the probe (CID-1612038/ML134, SID-99432327) was analyzed by UPLC, ¹H NMR, ¹³C NMR, and LC-MS. The data obtained from NMR spectroscopy were consistent with the structure of the probe (CID-1612038/ML134), and UPLC indicated an isolated purity of 100%. The relevant data are provided in Appendix C.

The observed solubility of the probe (CID-1612038/ML134) was calculated to be less than 1 μM in PBS solution. The stability of the probe (CID-1612038/ML134) in PBS (0.1% DMSO) was measured over 48 hours, and the data are shown in Figure 2 (blue line). We suspected that poor solubility (and not instability) as the reason behind the dramatic drop in the amount of sample over time. To test this, we added acetonitrile to each well (final concentration 50%) and measured the amount of the probe. Amounts detected after addition of acetonitrile are shown in Figure 2 (red line). Since addition of acetonitrile leads to increased detection of the probe, it is more likely that the compound is stable in PBS. The apparent instability as suggested by Figure 2 (blue line) is an artifact resulting from poor solubility of the probe in PBS.

Figure 2

Stability of the Probe (CID-1612038/ML134) in PBS.

Plasma protein binding (PPB) was determined to be 97.7% bound in human plasma. The probe (CID-1612038/ML134) is stable in both human and murine plasma with approximately 63.6% and 90% remaining respectively after a 5-hour incubation period. The solubility, PPB, and plasma stability results are summarized in Section 3.4 (entry 1, Table 1).

Table 1

SAR Analysis and Properties of 4-substituted Quinazoline Analogues.

The probe (CID-1612038/ML134) and four additional analogs were submitted to the SMR collection MLS002699829 (probe), MLS002699827 (CID-914635), MLS002699835 (CID-946943), MLS002699830 (CID-946273), and MLS002699828 (CID-1634732).

2.3. Probe Preparation

Chemistry Experimental Methods

The probe (CID-1612038/ML134;SID-99432327) was prepared in three steps outlined in Scheme 1.

Scheme 1

Synthesis of the Probe (CID-1612038/ML134).

General details. All reagents and solvents were purchased from commercial vendors and used as received. Unless otherwise mentioned, all of the reactions were run in over-dried, round-bottom flasks or in regular vials under nitrogen. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker 300 MHz spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) relative to tetramethylsilane (1H δ 0.00) or residual chloroform in CDCl3 solvent (¹H δ 7.24, ¹³C δ 77.0). NMR data are reported as follows: chemical shifts, multiplicity (s = singlet, m = multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25°C. Flash chromatography was performed using 40 μm to 60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash Rf system. Tandem Liquid Chromatography/Mass Spectrometry (LC-MS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO₄) stain followed by heating.

4-Hydroxy-2-(4-propoxyphenyl)quinazoline. To 50 mL of dichloromethane was added 5.0 g (28 mmol) of 4-propoxybenzoic acid followed by 2.6 mL (30 mmol) of oxalyl chloride and 2 to 3 drops of dimethylformamide (DMF), and the mixture was stirred overnight. After concentrating, the crude product was dissolved in 50 mL of tetrahydrofuran (THF) and 3.8 g (28 mmol) of 2-aminobenzamide and 5 mL of triethylamine were added. The mixture was stirred for 30 minutes. After concentration, 250 mL of methyl alcohol (MeOH) and a solution of 16 g of sodium hydroxide (NaOH) in 125 mL of water were added, and the reaction was heated at 90°C for 1 hour. After cooling to room temperature, 40 mL of concentrated hydrogen chloride (HCl) was added, and the white precipitate was filtered off. More solid was obtained by concentrating the mother liquors and filtering off precipitate; the combined solids were rinsed with ethanol and pumped on to give 5.66 g white solid (73%).

¹H NMR (300 MHz, DMSO-d₆) δ 12.41 (s, 1H), 8.19 (d, J = 8.81, 2H), 8.14 (d, J = 7.88, 1H), 7.78 (t, J = 7.61, 1H), 7.71 (d, J = 8.07, 1H), 7.49 (t, J = 7.47, 1H), 7.08 (d, J = 8.83, 2H), 4.03 (t, J = 6.53, 2H), 1.77 (sext, J = 7.00, 2H), 1.00 (t, J = 7.39, 3H).

4-Chloro-2-(4-propoxyphenyl)quinazoline. To 25 mL phosphorus oxychloride (POCl₃) was added 2.4 g of 4-hydroxy-2-(4-propoxyphenyl)quinazoline (8.6 mmol), and the mixture was heated at 110°C for 1 hour before cooling and concentration. Ice water was added. The contents were transferred to a separatory funnel and rinsed several times with dichloromethane. The combined dichloromethane rinses were dried (MgSO₄), filtered, concentrated, and chromatographed with 10% ethyl acetate (EtOAc) to yield 1.6 g product (49%).

¹H NMR (300 MHz, CDCl₃) δ 8.53 (d, J = 8.9, 2H), 8.22 (d, J = 8.4, 1H), 8.04 (d, J = 8.5, 1H), 7.90 (t, J = 7.7, 1H), 7.61 (t, J = 7.6, 1H), 7.02 (d, J = 8.9, 2H), 4.02 (t, J = 6.6, 2H), 1.85 (dt, J = 6.9, 13.9, 2H), 1.07 (t, J = 7.4, 3H).

4-Methoxy-2-(4-propoxyphenyl)quinazoline. To 50 mL of anhydrous methyl alcohol (MeOH) was added 25 mg of sodium (Na;1 mmol) with stirring. Once gas evolution ceased, 300 mg (1.0 mmol) of 4-chloro-2-(4-propoxyphenyl)quinazoline was added, and the mixture stirred overnight. The mixture was concentrated and partitioned between water and dichloromethane, the water was rinsed twice with dichloromethane and the combined organic layers were dried (MgSO₄), filtered, and concentrated to 250 mg of probe (CID-1612038/ML134) as white solid (85%), which was clean by LC and NMR analysis.

¹H NMR (500 MHz, CDCl₃) δ 8.54 (d, J = 8.7, 2H), 8.13 (d, J = 8.1, 1H), 7.94 (d, J = 8.3, 1H), 7.78 (t, J = 7.7, 1H), 7.47 (t, J = 7.5, 1H), 7.01 (d, J = 8.8, 2H), 4.27 (s, 3H), 4.01 (t, J = 6.6, 2H), 1.96 – 1.77 (m, 2H), 1.07 (t, J = 7.4, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.84, 161.30, 159.87, 151.90, 133.34, 130.57, 130.02, 127.65, 125.87, 123.39, 114.98, 114.23, 69.54, 53.96, 22.57, 10.53.

The ¹H NMR spectra, ¹³C NMR spectra, and UPLC chromatograms of the probe (CID-1612038/ML134) and analogs are provided in Appendix C.

2.4. Additional Analytical Analysis

Plasma Protein Binding. Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology, Rockford, IL) for both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 μL) with PBS (350 μL; pH 7.4) added to the other side. The compounds were incubated at 37°C for 5 hours in an orbital shake at 250 rpm. Following the incubation, the samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Plasma Stability. Plasma stability was determined at 37°C for 5 hours in both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). The compounds were incubated at 37°C for 5 hours in an orbital shake at 250 rpm with time points taken at 0 hours and 5 hours. The samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Probe Attributes

• IC₅₀ ≤ 10 μM
• 30- to 100-fold selectivity as defined by: IC₅₀ ratios of the inhibition of SK expression and the inhibition of cell growth where both IC₅₀ ratios are measurable.
• Compounds that are active in SK expression with IC₅₀ ≤ 10 μM but don’t result in cell growth inhibition at test concentrations no less than 50 μM.

The project included a primary high-throughput screen of the entire MLSMR collection (greater than 300,000 substances). Approximately 1% of the compounds tested were selected for dose retest and selectivity. Of these, approximately 60 compounds were identified as having the desired potency and selectivity. Resupply of these compounds was ordered from commercial sources for subsequent verification. In addition to these commercially available compounds, multiple rounds of chemistry were performed to generate new compounds for determination of the SAR of on the scaffolds. Compounds with the best combination of potency and selectivity and chemical properties were designated as the probes. This report concerns one structural series, Probe 2 (CID-1612038/ML134).

3.1. Summary of Screening Results

Figure 3 displays the critical path for probe development.

Figure 3

Critical Path for Probe Development.

A high-throughput screen of 303,546 compounds (AID-1662) was performed in duplicate in the engineered strain SKKanGAS, in which the kanamycin resistance gene was placed under control of the SK promoter to identify compounds that can inhibit kanamycin resistance. Using a cutoff of 50% inhibition at a screening concentration of 7.5 μM average, 3962 hits were identified as SK expression inhibitors; among these, 3268 were available as cherry picks. These picked compounds were retested in dose in the SKKanGAs strain to confirm their inhibitory activity as well as in SK(-)GAS strain where the kanamycin gene is driven by an SK-independent promoter to investigate promoter dependence.

The next set of secondary tests provided a more physiologically relevant analysis of the compounds specificity and potency. Interference with SK expression was measured in the non-engineered UMAA 2616 strain by measuring SK activity in culture supernatant via activation of plasminogen enzymatic activity (AID nos.1914, 2137, 504351). Bacterial viability was also measured via ATP content from the sample assay sample (AID nos.1915, 2138, 504396). From this secondary panel, 95 compounds were prioritized, and 61 dry powders were obtained and tested multiple times in the secondary assays.

From this set of dry powder compounds, a quinozoline series was prioritized, and additional analogs were synthesized and tested in the secondary assays. This quinozoline series became probe series 2 with a group of 33 compounds (AID nos. 2137, 2138; 504351, 504396) (see Table 1, Section 3.4).

3.2. Dose Response Curves for Probe

The probe (CID-1612038/ML134) met the defined probe criteria and was not active in the bacterial cell viability assay. Approximately 100% inhibition of endogenous streptokinase in the non-engineered strain UMAA2616 is obtained. Figure 4 displays dose response curves for the probe (CID-1612038/ML134).

Figure 4

Concentration-dependent Activities (%) of Probe (CID-1612038/ML134) in the Target Assay and Counterscreen Assays. Probe CID-1612038/ML134 concentration-dependent activities: SK expression (IC₅₀=4.2 μM; AID-504351) (A); Bacterial viability (inactive; (more...)

3.3. Scaffold/Moiety Chemical Liabilities

The scaffold does not have obvious chemical liabilities. The probe (CID-1612038/ML134) has acceptable solubility and stability in aqueous conditions. It is stable over 48 hours in PBS buffer and shows good stability in human and murine plasma. A PubChem activity survey performed on February 24, 2011 revealed no other confirmatory nonstreptokinase assay (out of 442 reported in PubChem) in which the probe (CID-1612038/ML134) was active. A structure-based search in SciFinder and Reaxys did not identify any publications or patents in which the probe compound appeared. Therefore, the probe is considered not to be a promiscuous binder.

3.4. SAR Tables

The initial hit from the HTS campaign was 4-methoxy-2-(4-propoxyphenyl)quinazoline (entry 1, CID-1612038/ML134,Table 1). A collection of 28 structurally related analogs were synthesized and evaluated for their ability to inhibit SK expression in the primary assay and array of secondary assays. Two diversification points selected for chemical modification are depicted in Figure 5. The biological assay data and physical properties of the HTS hit and the analogs are presented in Table 1 and Table 2.

Figure 5

Summary of SAR Performed on the Quinazoline Scaffold. Two diversity points (highlighted in red and blue) were explored and the number of analogs screened for each site is specified.

Table 2

SAR Analysis and Properties of Substituted Phenyl Side chain Analogues.

Table 1 presents the initial hit and several derivatives of it wherein the C-4 position of the quinazoline scaffold was substituted with various alkoxy, phenoxy, and amino groups to probe the SAR of this region. The inactivity of the hydroxy quinazoline (entry 2; Table 1) highlights the importance of the presence of an alkoxy group at C-4 position. Increasing the size or length of the alkoxy group on the quinazoline ring profoundly decreased potency (entry 3–7; Table 1) suggesting a small binding pocket in this region. Similar to the effect of sterically hindered alkoxy groups, aryloxy groups at C-4 position led to inactive analogs (entries 10–13; Table 1). Loss of potency was also observed for the amino substituted quinazoline analogs (entries 8–9; Table 1).

Several derivatives were synthesized next wherein the phenyl ring at the C-2 position of the qunizoline scaffold was substituted with various electron-withdrawing and electron-donating groups on the meta- and para-positions to probe the SAR of this region. As expected, all the C-4 hydroxy analogs of the corresponding substituted phenyl quinazolines turned out to be inactive (entries 11–16, Table 2). Any modification on the meta-position led to a decrease in potency (entries 1–5; Table 2) as well as in case of unsubstituted phenyl analog (entry 6; Table 2). Presence of an electron-withdrawing group at the para position adversely affects the potency (entries 7–9; Table 2), although it is interesting to see from Table 2 how potency is affected dramatically by shortening the alkoxy side chain from propoxy to methoxy (entry 10, Table 2) and then to hydrogen (entry 6, Table 2) suggesting that there may be a deep hydrophobic pocket in the biological target of this scaffold.

In conclusion, several analogs of the original hit were synthesized and assayed to better understand the structure-activity relationships. The previously reported probe (entry 1, CID-1612038/ML134,Table 1),4-methoxy-2-(4-propoxyphenyl)quinazoline, for the quinazoline series remains as the most active analog and will retain its’ status as the declared probe.

3.5. Cellular Activity

The SK expression assay and the bacterial cell viability assay are bacterial cell-based phenotypic assays. Therefore, active compounds possess reasonable permeability across the bacterial membrane. No mammalian cell permeability was measured.

3.6. Profiling Assays

A PubChem activity survey performed on February 24, 2011 using Broad’s internal algorithm revealed no other confirmatory nonstreptokinase assay in which the probe (CID-1612038/ML134) was active (out of 442 reported in PubChem). Other profiling screens (e.g., CEREP) have not been performed with this compound to date.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

The quinazoline probe (CID-1612038/ML134) has demonstrated selective inhibition of SK expression in a GAS strain without any effect on bacterial growth. The probe (CID-1612038/ML134) fulfilled all the probe criteria (Table 3) and will serve quite well in further cell-based investigations concerning SK expression.

Table 3

Probe Criteria.

It is interesting to see from Table 1 and Table 2 how potency is affected by the various alkoxy groups and modification of phenyl ring substituents. The range of potencies provides some confidence that the carbazole analogs are binding to one target specifically (i.e., a target that must control SK gene expression). The probe (CID-1612038/ML134) and the structural analogs shown are fairly hydrophobic molecules, possibly reflecting the hydrophobic character of the site to which they bind.

To support the novelty of the probe (CID-1612038/ML134) and its’ phenotypic effects, an investigation into the relevant prior art was performed by searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO), PatFT, AppFT, and World Intellectual Property Organization (WIPO). Specifically, we were looking for reports of chemical matters that demonstrated inhibition of SK expression. The search terms applied and hit statistics are provided in Table 4. Abstracts were obtained for all references returned including journal articles, patents, and other form of public disclosures and were analyzed for relevance to the current project. The searches were performed on, and are current as of, January 28, 2011.

Table 4

Search Strings and Databases Employed in the Prior Art Search.

The literature and patent searches in Table 4 uncovered no known small molecule that is capable of inhibiting SK expression. The only compound (CID-657963) that is known to inhibit SK inhibition is shown in Figure 6. This compound was identified by the Assay Provider, Dr. Hongmin Sun, for this probe development project (Note: Initial results associated with the discovery of CID-657963 are not yet published). The moderate potency (≥ 10.0 μM) and the presence of a reactive alkylthioacetonitrile moiety in CID-657963 have motivated us to discover a superior scaffold with higher inhibitory potency from the MLPCN compound collection. The improved potency and no obvious chemical liabilities will make the quinazoline probe (CID-1612038/ML134) a very useful tool molecule to better understand the biology of SK expression in group A streptococci.

Figure 6

Compound (CID-657963) Inhibitor of SK Inhibition.

4.2. Mechanism of Action Studies

The experimental scope of this project did not include target identification due to the complexity of and considerably much more resource commitment to such tasks. Researchers can carry out mechanism of action (MOA) studies through the following approaches. First, determine if the decreased level of SK expression is due to transcriptional control. Second, if transcription is a control mechanism, quantitative gene expression measurement can be used to test the activities of known pathways and/or expression level of known pathway components that control SK expression. Third, based on the knowledge gained from the above experiments, one can generate specific hypotheses of possible protein targets and test activity of these targets biochemically. Alternatively, researchers can employ several affinity-based techniques using the probe (CID-1612038/ML134) (or its’ active but modified version) as “bait’ to fish out proteins that specifically interact with the probe (CID-1612038/ML134). Inactive structurally similar analogs provided in this report are excellent control compounds for this purpose. The biological insight gained from the initial MOA studies should help narrow down the likelihood of the real targets if multiple specific binding proteins are identified.

4.3. Planned Future Studies

In addition to the MOA studies proposed in Section 4.2, researchers can further evaluate the probe (CID-1612038/ML134) or activity of its’ active analogs in animal models if the pharmacokinetic parameters are favorable. An animal model successfully used in the Assay Provider’s laboratory to evaluate compound activity is provided in Sun et al. (2).

Primary Screen Protocol in SKKanGAS Strain (AID no. 1662)

1. Day 1: Streak out SKKanGAS for colonies on THY/S/S medium plate (30 g/L THB/0.2% yeast extract with streptomycin 100 μg/ml, spectinomycin 100 μg/ml) agar. Incubate at 37°C overnight.
2. Day 2: Grow an overnight culture in THY/S/S medium from a single colony. Incubate at 37°C overnight.
3. Day 3: Measure OD600 of the overnight culture, and then dilute the culture into fresh THY/S/S medium (1:20) in a flask. Monitor OD600 until it reaches 0.6–0.8 (between 3–5 hours).
4. Dispense assay plates at 30 μl/well of THY/kanamycin (THY plus 40 μg/ml kanamycin) with Combi. Load plates into incubators.
5. Dilute the 0.8 OD culture down to OD 0.038 in cold THY/kanamycin; keep the diluted culture at 4°C overnight.
6. Day 4: Warm the diluted culture to room temperature. Dispense 20 μl/well of diluted culture with a Combi onto the pinned assay plates containing 30 μl/well of THY/S medium.
7. Pin approximately100 nL/well of compounds with Cybi-Well, and incubate the plates at 37°C for 6 hours.
8. Cool plates to room temperature for 30 minutes.
9. Add 30 μl/well solution (25 μl/well BacTiter-Glo and 5 μl/well PBS) to the culture, and incubate at room temperature for 10 minutes.
10. Read on an EnVision plate reader.

Secondary Assay Protocol (SK Expression and Bacterial Cell Viability) (AID nos. 1914,1915, 2137, 2138)

1. Day 1: Streak out UMAA 2616 for colonies on THY/S medium plate (30 g/L THB/0.2% yeast extract with streptomycin 100 μg/mL) agar. Incubate at 37°C overnight.
2. Day 2: Grow an overnight culture in THY/S medium from a single colony. Incubate at 37°C overnight.
3. Day 3: Dilute the overnight culture into fresh THY/S medium (1:20) in a flask. Monitor OD600 until it reaches 0.6–0.8 (between 3–5 hours).
4. Dilute the 0.8 OD culture down to OD 0.038 in cold THY/S medium; keep the diluted culture at 4 °C overnight.
5. Day 4: Warm the diluted culture to room temperature. Dispense 20 μl/well of diluted culture with a Combi onto the pinned assay plates containing 30 μl/well of THY/S medium.
6. Pin 100 nL/well of compounds with Cybi-Well, and incubate the plates at 37°C for 6 hours in a Liconic incubator.
7. Pellet the cells at 3000 rpm in a Sorvall centrifuge. Transfer 10 μl/well of supernatant twice into separate, clear plates for assay with Cybi-Well Vario. Store at −20°C.
8. Warm the culture plates to room temperature for 30 minutes, then add 50 μl/well of BacTiter-Glo (1/2x).
9. Incubate at room temperature for 10 minutes. Read on an EnVision plate reader.
10. Add 50 μl/well of SK assay solution (5 μl/well of human plasma, 1.2 μl/well of 4.2 mg/ml S2403, 43.8 μl/well of PBS) to the 10 μl/well supernatant (thawed and warmed to room temperature) in clear SK assay plates with a Combi.
11. Read OD405 at 0 hours and after incubation at 37°C for 1.5–2.5 hours depending on the specific lot of substrate.
12. Read OD405.