Scientific Rationale

Bacteria utilize quorum sensing (QS) to assess the population density and species complexity of their environment and adapt their physiological behavior to the prevailing conditions. A large number of human pathogens utilize QS as a means to produce virulence factors. Therefore, the ability to prevent virulence factor production could have a huge impact on human health. An example of a QS bacterium is the human pathogen Vibrio cholerae. The World Health Organization estimates that reported cases represent only 5–10% of the actual number of cholera incidents. There are an estimated 3 to 5 million cases that occur every year and approximately 120,000 deaths [6]. These high case numbers persist despite the development of a vaccine and a simple treatment regimen. In addition to V. cholerae, there are at least six other Vibrio species that are human pathogens. It has been determined that V. cholerae, as well as other bacteria, rely on a two-component system to relay exogenous information to the cell interior. A transmembrane histidine kinase (i.e. sensor) and an internal response regulator are the primary constituents of these two-component systems [7]. V. cholerae possess two parallel QS pathways. One pathway is responsible for interspecies communication wherein LuxPQ is the transmembrane receptor. The second, genus-specific pathway is controlled by the CqsS sensor [8]. Both pathways converge upon a common intermediary, LuxO [9].

The endogenous ligand of CqsS is the α-hydroxy ketone 1 (Figure 1), also known as cholera autoinducer 1 (CAI-1) [9-10]. Through careful studies of mutant CqsS receptors, it has been determined that CAI-1 inhibits His194 auto-phosphorylation of CqsS [11]. The auto-phosphorylation of His194 is believed to initiate a phosphorelay sequence involving the intermediary transfer protein LuxU and terminates at the response regulator LuxO (Figure 2). LuxO is a member of the NtrC family of response regulators that require interaction with a σ-54 factor to activate transcription [12]. LuxO binds DNA upstream of RNA polymerase and contains an inherent ATPase activity that is needed for changes to the transcriptional complex which involves DNA looping and subsequent formation of an open promoter complex [13]. LuxO integrates the CqsS and AI-2 signaling pathways and a homologue has been found in all Vibrio species examined. There are species-specific differences on the number of pathways that feed into LuxO.

Figure 1

The Endogenous Ligand of the Vibrio cholerae Sensor Histidine Kinase CqsS, Cholera autoinducer-1 (CAI-1).

Figure 2

The V cholerae CqsS/CAI-1 Quorum Sensing Phosphorelay system. At low cell density, there are insufficient amounts of CAI-1 to adequately bind CqsS. This leads to the production of AphA and the adoption of individual cell behaviors. At high cell density, (more...)

When CAI-1 is present in minute quantities, the phosphorelay system favors the phosphorylation of LuxO at Asp47 (Figure 2) [11]. Phosphorylated LuxO up-regulates transcription of the small regulatory RNAs qrr1-4, which subsequently promote translation of AphA while simultaneously suppressing translation of the HapR regulator. AphA regulates genes that are beneficial to individual behaviors, while HapR regulates genes that promote group (quorum) behaviors. Another level of regulating the RNA is the RNA chaperone Hfq that base pairs with and destabilizes the hapR mRNA [12]. When dense populations of V. cholerae synthesize and secrete sufficient quantities of CAI-1, this ligand will bind to CqsS and promote the production of HapR. The induction of HapR expression then allows the colonizing V. cholerae population to detach from the gut epithelium and vacate its host. Among the genes regulated by HapR is the expression of haemagglutinin protease (HapA) that facilitates the expulsion of V. cholerae from its human host [13]. In most QS bacteria, QS induces virulence factor production but Vibrio cholerae is distinct from non-vibrio bacterial species because QS results in repression of virulence factor and biofilm production, [2]. From a therapeutic perspective, the artificial activation of CqsS or inhibition of LuxO or Hfq to promote HapR expression could lead to a new generation of anti-cholera treatments. A potential combinatorial therapeutic approach targeting multiple targets in the QS pathway could ensure effective and genus-specific antimicrobial response.

Antagonism of LuxO has not been well-studied in the literature thus far. The only known small molecule inhibitors of LuxO were reported after the initiation of this project [2]. Derived from an aza-uracil scaffold, these compounds show moderate inhibition of LuxO activity (IC₅₀'s range from 5 to 20 µM) in cellular assays. Further investigation of the most potent compound, (Figure 3, Aza-U), determined that these LuxO antagonists block ATPase hydrolysis and prevent subsequent transcriptional activation. Aza-U was selected as the positive control for all cell-based SAR assays in the post-HTS medicinal chemistry phase of this project.

Figure 3

Aza-uracil-derived antagonists of LuxO ATPase activity. Aza-U (4) and its analogs have been shown to inhibit the ATPase activity of LuxO. Quorum-sensing modulation was measured in V. cholera strains BH1578 and BH1651 (see Section 2) and their respective (more...)

It is hoped that the identification of additional small molecule modulators of LuxO could elucidate the mechanisms of the latter steps underpinning the V. cholerae QS phosphorelay system. The additional chemical matter will also assist in the structural characterization of potential binding pockets and, in combination with a clearer understanding of how the transcriptional activation occurs, could guide development of new QS agonists as potential anti-cholera agents. Since all vibrios possess a LuxO homologue, LuxO inhibitors should be efficacious against all pathogenic vibrios. Aza-U compounds were found to inhibit LuxO activity in V. harveyi and V. parahaemolyticus [2].

Materials and Reagents

• CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega (Catalog No. G7573; Madison, WI)

Bacterial strains & Cell Lines

The following cell lines were used in this study:

• Vibrio cholerae BH1578; a genetically modified strain lacking LuxS and CqsA autoinducer synthases that was provided by the Bassler lab. This strain was used in the primary assay.
• Vibrio cholerae BH1651; a genetically modified strain with constitutively active LuxO that was provided by the Bassler lab.
• Vibrio cholerae DH231; a genetically modified strain lacking the CqsS receptor that was provided by the Bassler lab.
• Vibrio cholerae WN1103; a genetically modified strain lacking the LuxQ receptor that was provided by the Bassler lab.
• HeLa obtained from ATCC (Catalog Number CCL-2; Manassas, VA) is a human epithelial adenocarcinoma cell line used for mammalian cytotoxicity profiling

Note: All of the V. cholerae strains also carry the heterologous V. harveyi luxCDABE luciferase operon

2.1. Assays

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

2.2. Probe Chemical Characterization

Probe ML370 was prepared as described in Section 2.3. Data from ¹H NMR, ¹³C NMR and high resolution mass spectrometry are all consistent with the proposed structure. The purity of the compound is 96.1 area% at 214 nm by UPLC. Synthetic procedures for the preparation of the probe from commercially available m-anisidine are found in Appendix C and spectral data for the probe are in Appendix E.

The solubility of ML370 was experimentally determined to be 33.5 µM in phosphate buffered saline (PBS) with 1% (v/v) DMSO. Solubility was also measured to be 49.4 µM in the Luria Broth (LB) media used in the primary assay. Table 2 summarizes the solubility of ML370 and several analogs in both PBS and LB media. In general, LB media is a better solvent than PBS for these compounds.

Table 2

Solubility of ML370 and Analogs in PBS and LB Media.

The probe ML370 is stable in 1:1 acetonitrile:PBS solution (>99% remaining after 48-hour incubation at 23 °C). No significant reaction with DTT was observed for ML370 (>99% remaining after 8-hour incubation). The data from the PBS stability and DTT stability assays is provided in Figure 4. The probe is also stable to human and murine plasma with greater than 98% and 85% remaining respectively after incubation at 37 °C for 5 hours. Table 3 summarizes the results for the various stability assays performed. Experimental procedures for all analytical assays are provided in Appendix D.

Figure 4

Stability of Probe ML370 in 1:1 Acetonitrile:PBS (pH 7.4, 23 °C). MLS004820389 (CID 70680248, SID 160655705) was tested over a time course in aqueous stability assay and thiol stability assay. (A) Stability of the probe over 8 hours in 1:1 acetonitrile:PBS; (more...)

Table 3

Thiol Stability (DTT) and Plasma Stability for ML370.

The physical properties of probe ML370 are summarized in Table 4

Table 4

Summary of Probe Properties Computed for ML370.

2.3. Probe Preparation

Probe ML370 was synthesized in five steps and 7.5% overall yield as shown in Scheme 1. m-Anisidine was acetylated using neat acetic acid under microwave irradiation to afford acetanilide KU2. A Meth-Cohn quinolone synthesis using this material gave aldehyde KU3, which was converted to the nitrile (KU4) in one step using hydroxylamine and propylphosphonic anhydride [17]. Reaction of nitrile KU4 with methylhydrazine afforded the 1H-pyrazolo[3,4-b]quinolone core (penultimate compound KU5), which was acylated using furoyl chloride to give probe ML370.

Scheme 1

Synthesis of Probe ML370.

Full experimental details for the preparation of ML370 are provided in Appendix C.

3.1. Summary of Screening Results

As described above, the Molecular Libraries and Small Molecules Repository (MLSMR) collection of 352,083 unique compounds were tested at a single concentration (20 µM) for their ability to induce the production of luciferase by BH1578, thus indicating successfully activation of the QS signaling pathway. 553 compounds were active at 20 µM, representing a nominal hit rate of 0.15%.

The active compounds were then re-tested in a dose-response format, wherein only 2 compounds actually met or exceeded the project requirement of AC₅₀ ≤ 1.0 µM. However, the low success rate of the primary assay prompted us to expand the primary assay cut-off to include substances with AC₅₀ ≤ 10 µM.

Subsequently, dry powders for 26 compounds were procured for re-testing. After purity analysis and structural confirmation by NMR spectroscopy, these substances were screened according to the workflow outlined below in Figure 6.

Figure 6

Critical Path for Probe Development.

The assay progression outlined in Figure 6 assessed the activation of QS pathways in several V. cholerae strains: BH1578, BH1651, and SLS353. CID 665390 emerged as a promising LuxO inhibitor and was subsequently prioritized for development. A number of analogs were prepared and assayed for QS activation. The results of the SAR investigation are presented in detail in Section 3.4.

3.2. Representative Dose Response Curves for Probe ML370

Figure 7ML370 Induces Quorum Sensing via LuxO ATPase Inhibition without Toxicity

ML370 was tested across a range of concentrations up to 35 µM in the primary BH1578 assay, the SLS353 qrr4 reporter assay, the in vitro LuxO ATPase assay and a HeLa cytotoxicity assay. Concentration response curves were generated with Genedata Screener Condeseo and show normalized percent activity for the individual doses. ML370 DH231 assay (AID 686931), IC₅₀ = 1.8 µM (A); ML370 and ML366 SLS353 assay, (B); ML370 in vitro LuxO ATPase assay (C); ML370 HeLa cytotoxicity (AID 686928), IC₅₀ ≥ 35 µM (D). ○ = replicate 1, △ = replicate 2.

3.3. Scaffold/Moiety Chemical Liabilities

A search of PubChem for ML370 returns no biological assay results (as of April 8, 2013). A structure-based search (80% similarity to ML370) of the Chemical Abstracts database using the SciFinder software returned 12 analogs of ML370 with biological studies reported (as of April 8, 2013). All of the compound analogs were reported in the same patent application, “Method for altering the lifespan of eukaryotic organisms” [18]. An exact structure search of the Chemical Abstracts database using the SciFinder software for ML370 did not return a hit.

A search of PubChem for CID 665390 on August 06, 2013 (the original hit compound, entry 1 in Table 6) shows that this compound has been evaluated in a total of 732 assays. There are 16 assays unrelated to this project in which CID 665390 showed dose-dependent activity with a reported AC₅₀ below 20 µM. Table 5 lists these assays and the measured activity of CID 665390.

Table 6

Structural Modifications of the Core Region.

Table 5

PubChem Assays Reporting Dose-Dependent Activity of CID 665390 (the hit compound).

ML370 (CID 70680248) possesses no obvious chemical liabilities, and a 1:1 acetronitrile:phosphate-buffered saline solution of the probe showed no significant decomposition over a 48-hour period (see Figure 4B). In addition DTT did not react with the probe appreciably over 8 hours (see Figure 4C). See Appendix D for the experimental procedures for the solubility, PBS stability, GSH/DTT stability and plasma stability measurements.

3.4. SAR Tables

The 1H-pyrazolo[3,4-b]quinolone core was one of a small number of cores chosen after analysis of the high throughput data (AID 588436) and subsequent confirmatory analyses (data not shown). One derivative of this structure, N-(1,7-dimethyl-1H-pyrazolo[3,4-b]quinolin-3-yl)furan-2-carboxamide (CID 665390), was the ninth most active compound in the high throughput screen. Confirmatory and related screens were run on this compound (Figure 5A & B) and, given the acceptable results and drug-likeness of the compound, this compound was chosen as a lead structure for a new chemotype.

A search of AID 588436 (350,317 substances tested) for the 1H-pyrazolo[3,4-b]quinoline substructure (SMILES C12=CC=CC=C1C=C3C(NN=C3)=N2) showed that 543 substances with this substructure were tested, of which two were active (CID 665390 and N-(1-ethyl-1H-pyrazolo[3,4-b]quinolin-3-yl)furan-2-carboxamide (CID 663294)). A broader search using the simpler substructure 1H-pyrazolo[3,4-b]pyridine (SMILES C12=NC=CC=C1C=NN2) showed 2,894 substances with this substructure, of which only the same two compounds noted above were active. (The overall hit rate for AID 588436 was 0.16%.) Given the structural similarities between CIDs 665390 and 663294, and the data for CID 665390, we proceeded with CID 665390 as a lead structure.

In order to investigate the activity of the hit compound CID 665390, 20 structurally related analogs were synthesized and evaluated for their ability to initiate the V. cholera QS pathway in the BH1578 V. cholera strain.

The biological assay data of these analogs are presented in Table 6-7. Characterization data (¹H and ¹³C NMR spectra and RP HPLC chromatograms and HRMS data) for these analogs are provided in Appendix F.

Table 7

Structural Modifications of the Acyl Group.

With respect to structural modifications to the pyrazoloquinoline core, while holding the 2-furoyl amide constant, only entries 5 and 6 (Table 6) gave desirable activity. Truncating the quinoline ring, as in entries 2 through 4 (Table 6) significantly diminished activity. Di-substitution on the far-left portion of the pyrazoloquinoline core uniformly led to diminished activity, as shown for entries 10 through 13 (Table 6). Depending on the nature of the mono-substitution on the far-left portion of the pyrazoloquinoline core, activity was increased or decreased, as seen for entries 6 and 7 through 9 (Table 6), respectively. With the limited number of analogs tested, in this regard, inferring causation is difficult and unwarranted.

In Table 7, structural modifications to the 2-furoyl moiety, while holding the pyrazoloquinoline core constant, uniformly decreased activity for the compounds tested.

Given the above results, entry 6, Table 6, was nominated as the probe compound.

3.5. Cellular Activity

The primary assay and several secondary assays were performed with intact Vibrio cholerae bacteria and a few compounds were observed to be capable of inhibiting an intracellular kinase, LuxO. In addition, compounds were tested in a cytotoxicity screen utilizing mammalian HeLa cells without apparent cytotoxicity. An overview of these assays is provided above in Section 2.1, and full experimental details can be found in Appendix B. The probe ML370 satisfies the cellular activity criteria specified for this project (refer to Section 4, Table 8).

Table 8

Comparison of New & Existing Cholera QS Probes to Project Criteria.

3.6. Profiling Assays

ML370 was submitted to Eurofins Panlabs for evaluation in their LeadProfilingScreen (Catalog No. 68) to determine possible off-target effects against a broad panel of receptors, ion channels, and transporters. The probe compound was determined to be inactive against most of the 68 biological targets, and showed significant activity against three targets, the Adenosine A1 (85% Inh at 10 µM), Adenosine A2A (68% Inh at 10 µM), and Adenosine A3 (86% Inh at 10 µM) receptors. This potential for activity at the adenosine receptors should be kept in mind when contemplating using ML370 in animal studies. The complete listing of the results for the Eurofins Panlabs LeadProfilingScreen for ML370 can be found in Appendix H.

Because its tentative mode of action is ATPase inhibition, ML370 was also tested in Merck Millipore's KinaseProfiler screen for potential cross-reactivity towards a panel of 59 human kinase targets. The probe compound was determined to be inactive against all of the 59 kinase targets. The complete listing of the results for the Merck Millipore KinaseProfiler panel assay for ML370 can be found in Appendix I.

4.1. Comparison to existing art and how the new probe is an improvement

A series of aza-uracil-containing compounds are the only known, reported inhibitors of V. cholerae LuxO [2]. The most potent member of this family, designated Aza-U (cf. Figure 3), was synthetically prepared and used as the positive control for all cell-based assays. ML370 is more potent than Aza-U in the primary assay when compared directly (EC₅₀ = 2.5 µM and > 35 µM, respectively). Consistently across all of the assays, ML370 performed better than Aza-U (Figure 5C, Figure 5D, Table 9). In BH1578, BH1651, and DH231, only the positive control dose of 40 µM induces light production and the lower doses do not generate a consistent dose-dependent response (see Figures 5C & 5D). In the WN1103 assay, ML370 is more potent compared to Aza-U (1.7 to 21 µM, respectively, Table 9).In addition to Aza-U, ML370 was compared to the natural QS ligand, CAI-1, as well as two other small molecule CqsS agonists and the results of this comparison are summarized in Table 9. The discovery and optimization of the CqsS agonist probes, ML343 and ML344, were previously reported by the Broad in late 2012. It is anticipated that the current LuxO probes, ML366 and ML370, will provide valuable information regarding available binding pockets of V. cholera LuxO and guide the development of the next generation of quorum sensing focused therapeutics.

Table 9

Probe Activation of Quorum Sensing in Vibrio cholerae Mutant Strains. Table 9. ML370, positive controls and the previously submitted probes were evaluated in alternative V. cholerae mutants to establish possible modes of actions. The reported EC₅₀ values (more...)

4.2. Mechanism of Action Studies

ML370 was evaluated for its ability to activate the quorum sensing pathways of additional V. cholerae strains (Table 9). Strain BH1651 expresses a constitutively active LuxO^D47E mutant (Pubchem AID 651847) as described above in Section 2.1.3. For this particular bacterial strain, LuxO is locked into an active conformation and thus the signaling processes downstream of LuxO are actively repressed. The production of luciferase is repressed independent of CqsS or LuxPQ signaling. The ability of ML370 to promote light production in BH1651 suggests the compound acts upon or downstream of LuxO, consistent with the putative activity of LuxO inhibitors. V. cholerae DH231 and WN1103 are mutant strains lacking CqsS and LuxQ membrane receptors, respectively. If ML370 is working downstream of either CqsS or LuxPQ, the DH231 and WN1103 cells should activate light production with compound treatment. This predicted phenotype is precisely what is observed (Table 9, entries 1 & 4). This activity profile appears to be conserved across both LuxO inhibitor scaffolds (ML370 and ML366); QS agonists like CAI-1, ML343 and ML344 activate V. cholerae QS only if the cells express the CqsS receptor.

Compared to DMSO, ML370 significantly decreases LuxO ATPase activity in vitro (Figure 6C). This suggests that ML370 inhibits LuxO in a similar manner to azaU by directly targeting LuxO itself [2].

4.3. Planned Future Studies

In addition to V. cholerae, there are at least six Vibrio species that cause disease in humans that are contracted through contaminated water or ingested during consumption of seafood [19]. Aza-U inhibits LuxO in multiple Vibrio species, including V. harveyi and V. haemolyticus [2]. ML370 will be tested against these and multiple vibrios for the ability to shutdown virulence factor production. Combinatorial therapies have proven efficacious in a number of infectious disease treatment regimens and could also be applicable in treating cholera. ML370 will be tested in combination with several CqsS agonists and the natural CAI-1 ligand. ML370 and the other probes will also be tested in animal models of cholera. Identification and characterization of the binding site(s) occupied by ML370 will be undertaken. These studies will include analysis with several LuxO mutants in cells and using in vitro kinase and ATPase assays. Other experiments could include structural efforts. This structural information will undoubtedly be beneficial in the development of additional LuxO inhibitors as potential anti-cholera therapeutics. Co-crystallization of LuxO with the probe is the most attractive possibility to obtain structural information, but protein NMR binding studies are also an option. Other methods to determine probe-protein interaction, albeit with less precision, are biophysical experiments to determine direct binding of the compound to the LuxO protein. Differential scanning fluorimetry (DSF) or thermal shift is a means of detecting binding of a ligand to purified protein. This technique is routinely used in our laboratories and could be applied to LuxO.

ML370 will be assessed for pharmacokinetic properties in anticipation of evaluating the therapeutic value of QS agonists in animal models of cholera infection [3-5]. One attribute of a potential cholera therapeutic is that it should be well distributed within the host gut, but systemic exposure is not required. In addition, ML370 could be tested in combination with CqsS agonists as a possible therapeutic regiment. Such a combination therapy would be potent and species selective.

ML370 might benefit from additional chemical optimization investigating substitution on the pyrazolo moiety and bioisosteric replacement of the amide linker (Figure 9).

Figure 9

Possible Directions for Future SAR Investigation of ML370.

Our recommendations for future SAR expansion around the probe ML370 involve: (1) exploring additional electron-donating and electron-withdrawing groups on the far-left of the pyazoloquinoline core, (2) replacing the amide with the sulfonamide group, or reducing the amide, or N-alkylating the amide or sulfonamide, (3) replacing the furan using bioisosteric groups that may be less prone to metabolism, and (4) exploring a variety of replacements for the N-methyl group. The regions shaded in mauve and green, especially, could benefit from SAR exploration.

V. cholerae BH1578 primary bioluminescence assay (2132-01) (AID 588346, AID 602243, AID 624270, AID 651854, AID 651809, AID 651816, AID 652239, AID 686929)

Materials and Reagents

• Bacterial strain: BH1578 (V. cholerae ΔcqsA ΔluxS carrying pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
• LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
• Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 °C, protect from light.
• LB/tet: add 1 mL tetracycline (10 mg/mL) to 1 L of LB medium. Final concentration of tetracycline is 10 µg/mL. Make it fresh for every use.
• CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 °C

Procedure

1. Grow up BH1578 reporter strain in 50 mL LB/Tet at 30 °C for >16 hours with shaking (200 rpm). The final OD₆₀₀ of each culture should be > 3.0.
2. Adjust culture to OD₆₀₀ = 0.3, mix well. (Note: a low speed centrifugation (200 rpm for 1 min) removed most biofilm aggregates).
3. Add 20 µL LB/tet per well with Thermo Combi MultiDrop fluid dispenser into a 384 well plate. (Greiner black, clear bottom plates were used for HTS while Corning 8867BC white opaque 384 plates were used for subsequent assays).
4. Pin transfer 100 nL compound or 1 µM CAI-1 as positive control per well from compound source plate to assay plate (pinning volume was 150 nL for the primary HTS).
5. Dispense 10 µL of diluted culture into each well of a 384 well plate.
6. Incubate the plates at 30 °C without shaking for 6 hours.
7. Measure bioluminescence (Lum(384)) and OD₆₀₀ in a Perkin-Elmer Envision Multilabel Reader

For dose retests, compounds were arrayed so that 2 rows of DMSO were located between each test compound. This minimized any signal from a neighboring well yielding a false positive due to an extremely high bioluminescence signal.

V. cholerae BH1651 secondary LuxO inhibitor bioluminescence assay (2132-03) (AID 624254, AID 624269, AID 651847, AID 652289)

Materials and Reagents

• Bacterial strain: BH1651 (V. cholerae luxO^D47E carrying pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
• LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
• Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 °C, protect from light.
• LB/tet: add 1 mL tetracycline (10 mg/mL) to 1 L of LB medium. Final concentration of tetracycline is 10 µg/mL. Make it fresh for every use.

Procedure

1. Grow up BH1651 reporter strain in 50 mL LB/Tet at 30 °C for >16 hours with shaking (200 rpm). The final OD₆₀₀ of each culture should be > 3.0.
2. Adjust culture to OD₆₀₀ = 0.3, mix well. (Note: a low speed centrifugation (200 rpm for 1 min) removed most biofilm aggregates).
3. Add 20 µL LB/tet per well with Thermo Combi MultiDrop fluid dispenser into a 384 well plate. (Greiner black, clear bottom plates were used for HTS while Corning 8867BC white opaque 384 plates were used for subsequent assays).
4. Pin transfer 100 nL compound per well from compound source plate to assay plate.
5. Dispense 10 µL of diluted culture into each well of a 384 well plate.
6. Incubate the plates at 30 °C without shaking for 6 hours.
7. Measure bioluminescence (Lum(384)) and OD₆₀₀ in a Perkin-Elmer Envision Multilabel Reader

For dose retests, compounds were arrayed so that 2 rows of DMSO were located between each test compound. This minimized any signal from a neighboring well yielding a false positive due to an extremely high bioluminescence signal.

V. cholerae DH231 secondary sensor mechanism bioluminescence assay (2132-04) (AID 624281, AID 651808, AID 686931)

Materials and Reagents

• Bacterial strain: DH231 (V. cholerae ΔcqsS ΔluxS carrying pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
• LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
• Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 °C, protect from light.
• LB/tet: add 1 mL tetracycline (10 mg/mL) to 1 L of LB medium. Final concentration of tetracycline is 10 µg/mL. Make it fresh for every use.
• CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 °C

Procedure

1. Grow up reporter strain in 50 mL LB/Tet at 30 °C for >16 hours with shaking (200 rpm). The final OD₆₀₀ of each culture should be > 3.0.
2. Adjust culture to OD₆₀₀ = 0.3, mix well. (Note: a low speed centrifugation (200 rpm for 1 min) removed most biofilm aggregates).
3. Add 20 µL LB/tet per well with Thermo Combi MultiDrop fluid dispenser into a 384 well plate. (Corning 8867BC white opaque 384 plates were used).
4. Pin transfer 100 nL compound per well or 1 µM CAI-1 as positive control from compound source plate to assay plate.
5. Dispense 10 µL of diluted culture into each well of a 384 well plate.
6. Incubate the plates at 30 °C without shaking for 6 hours.
7. Measure bioluminescence (Lum(384)) and OD₆₀₀ in a Perkin-Elmer Envision Multilabel Reader

For dose retests, compounds were arrayed so that 2 rows of DMSO were located between each test compound. This minimized any signal from a neighboring well yielding a false positive due to an extremely high bioluminescence signal.

V. cholerae WN1103 Secondary sensor mechanism bioluminescence assay (2132-05) (AID 624275, AID 651841, AID 686930)

Materials and Reagents

• Bacterial strain: WN1103 (V. cholerae ΔcqsA ΔluxQ carrying pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
• LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
• Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 °C, protect from light.
• LB/tet: add 1 mL tetracycline (10 mg/mL) to 1 L of LB medium. Final concentration of tetracycline is 10 µg/mL. Make it fresh for every use.
• CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 °C

Procedure

1. Grow up reporter strain in 50 mL LB/Tet at 30 °C for >16 hours with shaking (200 rpm). The final OD₆₀₀ of each culture should be > 3.0.
2. Adjust culture to OD₆₀₀ = 0.3, mix well. (Note: a low speed centrifugation (200 rpm for 1 min) removed most biofilm aggregates).
3. Add 20 µL LB/tet per well with Thermo Combi MultiDrop fluid dispenser into a 384 well plate. (Corning white opaque 8867BC 384 plates were used).
4. Pin transfer 100 nL compound or 1 µM CAI-1 as positive control per well from compound source plate to assay plate.
5. Dispense 10 µL of diluted culture into each well of a 384 well plate.
6. Incubate the plates at 30 °C without shaking for 6 hours.
7. Measure bioluminescence (Lum(384)) and OD₆₀₀ in a Perkin-Elmer Envision Multilabel Reader

For dose retests, compounds were arrayed so that 2 rows of DMSO were located between each test compound. This minimized any signal from a neighboring well yielding a false positive due to an extremely high bioluminescence signal.

V. cholerae SLS353 secondary qrr4:GFP fluorescence reporter assay (2132-06) (AID 652219, AID 686942)

Materials and Reagents

• Bacterial strain: SLS353 (V. cholerae luxO^D47E carrying qrr4:GFP plasmid)
• LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
• Kanamycin (100 mg/mL): Dissolve 100 mg kanamycin in 1 mL water, store at -20 °C
• LB/Kan: add 1 mL Kanamycin (100 mg/mL) to 1 L of LB medium. Final concentration of Kanamycin is 100 µg/mL. Make it fresh for every use.

Procedure

8.

Grow up strain in 50 mL LB/Kan at 30 °C for >16 hours with shaking (200 rpm). The final OD₆₀₀ of each culture should be > 3.0.

9.

Adjust culture to OD₆₀₀ = 0.3, mix well. (Note: a low speed centrifugation (200 rpm for 1 min) removed most biofilm aggregates).

10.

Add 20 µL LB/Kan per well with Thermo Combi MultiDrop fluid dispenser into a 384 well plate. (Corning black opaque 384 plates were used).

11.

Pin transfer 100 nL compound per well from compound source plate to assay plate.

12.

Dispense 10 µL of diluted SLS353 culture into each well of a 384 well plate.

13.

Incubate the plates at 30 °C without shaking for 16 hours.

14.

Measure GFP fluorescence in a Perkin-Elmer Envision Multilabel Reader

LuxO in vitro ATPase assay (AID 686936)

A modified coupled-enzyme assay was used to measure the rate of ATP hydrolysis by LuxO^D47E. Briefly, ADP released from ATP by LuxO D47E is reacted with phosphoenolpyruvate (PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is reacted with NADH to form NAD and lactate using lactate dehydrogenase (LDH). The rate of NAD production is followed at 340 nm using a spectrophotometer. The rates of ATP hydrolysis by LuxO^D47E were measured in reactions containing 100 mM sodium phosphate buffer pH 7.4, 5 mM MgCl₂, 0.2 mM NADH, 1 mM PEP, 5–20 units of PK/LDH mix (Sigma), and 10 µM LuxO^D47E. ATP was added at a final concentration of 2.5 mM. The rate of ATP hydrolysis was monitored for 5 minutes. The values reported are ΔOD₃₄₀/min.

Data Analysis

For the primary screen and other assays, negative-control (NC) wells and positive-control (PC) wells were included on every plate. The raw signals of the plate wells were normalized using the ‘Stimulators Minus Neutral Controls’ or the ‘Neutral Controls’ method (when no positive control was available) in GeneData Screener Assay Analyzer (v7.0.3 & v10.0.2). The median raw signal of the intra-plate NC wells was set to a normalized activity value of 0, while the median raw signal of the intra-plate PC wells was set to a normalized activity value of 100. Experimental wells were scaled to this range, resulting in an activity score representing the percent change in signal relative to the intra-plate controls. The mean of the replicate percent activities were presented as the final 'PubChem Activity Score'. The 'PubChem Activity Outcome' class was assigned as described below, based on an activity threshold of 70%:

• Activity_Outcome = 1 (inactive), less than half of the replicates fell outside the threshold.
• Activity_Outcome = 2 (active), all of the replicates fell outside the threshold, OR at least half of the replicates fell outside the threshold AND the 'PubChem Activity Score' fell outside the threshold.
• Activity_Outcome = 3 (inconclusive), at least half of the replicates fell outside the threshold AND the ‘PubChem Activity Score did not fall outside the threshold.

Probe CID 70680248; SID 160655705

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃)

UPLC-MS Chromatogram

HRMS calcd for [C₁₇H₁₄N₄O₃ + H]⁺: 323.1139, found: 323.1170.

Analog CID 665390; SID 160655702

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.34, 150.76, 149.19, 147.08, 144.98, 141.84, 137.97, 135.12, 129.66, 126.39, 126.11, 122.46, 116.12, 112.81, 109.17, 33.62, 22.25.

UPLC-MS Chromatogram

HRMS calcd for [C₁₇H₁₄N₄O₂ + H]⁺: 307.1190, found: 307.1234.

Analog CID 70680249; SID 160655703

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.83, 155.39, 150.78, 149.18, 145.38, 141.82, 138.29, 135.38, 129.70, 126.35, 126.04, 122.44, 117.43, 109.31, 109.17, 33.60, 22.25, 13.94.

UPLC-MS Chromatogram

HRMS calcd for [C₁₈H₁₆N₄O₂ + H]⁺: 321.1346, found: 321.1357.

Analog CID 1385812; SID 160655704

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 159.30, 150.75, 149.22, 141.89, 138.40, 137.93, 135.13, 131.74, 129.66, 129.43, 128.15, 126.38, 126.15, 122.48, 109.18, 77.27, 77.01, 76.76, 33.59, 22.25.

UPLC-MS Chromatogram

HRMS calcd for [C₁₇H₁₄N₄OS + H]⁺: 323.0961, found: 323.0984.

Analog CID 70680252; SID 160655706

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.37, 148.82, 147.03, 145.02, 138.00, 135.61, 131.19, 130.11, 127.74, 124.13, 123.41, 116.20, 112.84, 109.76, 77.26, 77.22, 77.01, 76.76, 33.69.

UPLC-MS Chromatogram

HRMS calcd for [C₁₆H₁₂N₄O₂ + H]⁺: 293.1033, found: 293.1048.

Analog CID 70680251; SID 160655707

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.35, 150.75, 149.31, 147.97, 147.08, 144.98, 137.99, 135.17, 129.76, 125.23, 124.90, 122.67, 116.13, 112.82, 109.20, 77.27, 77.01, 33.64, 29.38, 14.84.

UPLC-MS Chromatogram

HRMS calcd for [C₁₈H₁₆N₄O₂ + H]⁺: 321.1346, found: 321.1360.

Analog CID 70680254; SID 160655708

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.72, 147.44, 144.60, 141.39, 138.14, 127.07, 123.36, 120.23, 116.55, 115.67, 112.58, 108.75, 35.34.

UPLC-MS Chromatogram

HRMS calcd for [C₁₃H₁₁N₃O₂ + H]⁺: 242.0924, found: 242.0942.

Analog CID 19405046; SID 160655709

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.27, 147.50, 146.21, 144.36, 130.97, 115.20, 112.47, 97.40, 77.28, 77.22, 77.02, 76.77, 38.82.

UPLC-MS Chromatogram

HRMS calcd for [C₉H₉N₃O₂ + H]⁺: 192.0768, found: 192.0766.

Analog CID 70680250; SID 160655710

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.35, 150.70, 149.65, 147.10, 144.85, 137.48, 134.13, 116.32, 115.97, 112.72, 108.22, 77.27, 77.22, 77.02, 76.76, 33.58.

UPLC-MS Chromatogram

HRMS calcd for [C₁₂H₁₀N₄O₂ + H]⁺: 243.0877, found: 243.0914.

Analog CID 70680253; SID 160655711

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 159.94, 150.77, 149.23, 145.80, 144.43, 141.91, 138.35, 135.07, 129.65, 126.39, 126.16, 122.47, 121.97, 109.24, 108.43, 77.26, 77.21, 77.01, 76.75, 33.60, 22.25.

UPLC-MS Chromatogram

HRMS calcd for [C₁₇H₁₄N₄O₂ + H]⁺: 307.1190, found: 307.1198.

Analog CID 70680247; SID 160655712

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 174.17, 150.74, 149.15, 141.76, 138.86, 135.18, 129.65, 126.33, 125.98, 122.34, 109.25, 45.98, 33.51, 30.58, 26.09, 22.23.

UPLC-MS Chromatogram

HRMS calcd for [C₁₈H₂₀N₄O + H]⁺: 309.1710, found: 309.1717.

Analog CID 1400696; SID 160655713

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 161.77, 150.78, 149.14, 149.00, 148.34, 141.73, 138.35, 137.72, 135.03, 129.67, 126.84, 126.42, 126.02, 122.46, 122.38, 109.31, 33.65, 22.24.

UPLC-MS Chromatogram

HRMS calcd for [C₁₈H₁₅N₅O + H]⁺: 318.1349, found: 318.1250.

Analog CID 70698449; SID 160778928

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 160.18, 158.14, 155.34, 146.94, 145.03, 138.61, 122.04, 116.36, 113.93, 113.79, 112.88, 109.00, 108.85, 33.86, 22.52, 22.51.

UPLC-MS Chromatogram

HRMS calcd for [C₁₇H₁₃FN₄O₂ + H]⁺: 325.1095, found: 325.1093.

Analog CID 70698444; SID 160778929

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.36, 147.01, 146.11, 145.02, 137.52, 134.11, 134.06, 129.27, 129.22, 116.20, 112.85, 111.24, 111.06, 109.52, 33.63, 16.08, 16.05.

UPLC-MS Chromatogram

HRMS calcd for [C₁₇H₁₃FN₄O₂ + H]⁺: 325.1095, found: 325.1131.

Analog CID 70698450; SID 160778930

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.87, 155.07, 150.76, 149.23, 147.70, 141.91, 137.82, 135.13, 129.68, 127.66, 127.57, 126.43, 126.19, 124.08, 122.96, 122.50, 112.15, 111.98, 109.07, 33.68, 22.26.

UPLC-MS Chromatogram

HRMS calcd for [C₂₁H₁₆N₄O₂ + H]⁺: 357.1346, found: 357.1347.

Analog CID 70698445; SID 160778931

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.32, 150.53, 148.23, 147.14, 144.93, 142.20, 137.79, 133.97, 133.34, 128.66, 126.78, 123.09, 116.04, 112.78, 109.17, 33.62, 20.82, 19.94.

UPLC-MS Chromatogram

HRMS calcd for [C₁₈H₁₆N₄O₂ + H]⁺: 321.1346, found: 321.1351.

Analog CID 70698443; SID 160778932

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 164.98, 162.98, 155.32, 150.75, 150.51, 150.40, 147.00, 145.01, 139.96, 139.87, 138.38, 132.31, 121.12, 116.18, 115.32, 115.11, 112.83, 108.89, 108.73, 108.70, 33.59, 19.65, 19.64.

UPLC-MS Chromatogram

HRMS calcd for [C₁₇H₁₃FN₄O₂ + H]⁺: 325.1095, found: 325.1134.

Analog CID 70698446; SID 160778933

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 166.35, 149.15, 147.63, 143.14, 141.84, 138.08, 134.79, 129.61, 126.38, 126.12, 122.39, 111.05, 109.47, 109.17, 36.66, 33.53, 22.24.

UPLC-MS Chromatogram

HRMS calcd for [C₁₈H₁₆N₄O₂ + H]⁺: 321.1346, found: 321.1379.

Analog CID 70698448; SID 160778934

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.34, 152.52, 150.77, 149.34, 147.08, 144.98, 138.00, 135.16, 129.81, 124.12, 123.44, 122.80, 116.12, 112.82, 109.24, 34.56, 33.65, 23.54.

UPLC-MS Chromatogram

HRMS calcd for [C₁₉H₁₈N₄O₂ + H]⁺: 335.1503, found: 335.1519.

Analog CID 70698447; SID 160778935

¹H NMR Spectrum (500 MHz, CDCl₃)

¹³C NMR Spectrum (126 MHz, CDCl₃) δ 155.33, 154.71, 150.81, 149.21, 147.08, 144.99, 137.99, 134.95, 129.47, 122.88, 122.55, 122.38, 116.13, 112.82, 109.34, 35.43, 33.69, 30.96.

UPLC-MS Chromatogram

HRMS calcd for [C₂₀H₂₀N₄O₂ + H]⁺: 349.1659, found: 349.1675.

Study Objective

To evaluate, in radioligand binding assays, the activity of probe compound ML370 (KUC112612N) across a panel of 68 receptors.

Methods

Methods employed in this study performed at Eurofins Panlabs [20] have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assay results are presented as the percent inhibition of specific binding or activity (for n = 2 replicates) for the probe compound tested at a concentration of 10 µM.

Table A3Results for ML370 for the Eurofins Panlabs LeadProfilingScreen

Assay Formats

Protein kinases are tested in a radiometric assay format and the raw data are measured by scintillation counting (in cpm). Lipid kinases are measured in an HTRF® format, with fluorescence measured at two wavelengths and raw data subsequently expressed as HTRF ratio.

Controls and Blanks

Positive control wells contain all components of the reaction except the compound of interest; however, DMSO is included in these wells to control for solvent effects. For protein kinase assays, blank wells contain all components of the reaction with a reference inhibitor replacing the compound of interest. This abolishes kinase activity and establishes the base line (0% kinase activity remaining). For lipid kinase assays, the 0% activity base

% Activity Values

In the graph, results are expressed as a percentage of the mean kinase activity in the positive control samples. The positive control value is considered to be 100%, and all test samples are measured in relation to this value. For example, a result of 42% means that, in comparison to the positive control, 42% kinase activity remains in the presence of the test compound. Expressed another way, the test compound inhibits the kinase activity by 58%.

Table A4Merck Millipore Kinase Report for ML370