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3-(2,6-difluorobenzamido)-5-(4-ethoxyphenyl) thiophene-2-carboxylic acid inhibits E.coli UT189 bacterial capsule biogenesis

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Author Information

Received: ; Last Update: March 14, 2013.

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. During UTI, UPEC exists in both intracellular and extracellular spaces. Infection is initiated by adherence to the apical bladder epithelium and then invading this layer of cells. Within the bladder epithelium, UPEC typically reproduces in a biofilm-like state composed of intracellular bacterial communities (IBC). After maturation of IBCs, UPEC disperses away from the IBC and exits the infected cells. Extracellular UPEC must then re-adhere, initiating the invasion and intracellular propagation phases again. Bacterial-epithelial interactions incite a strong inflammatory response through which the UPEC must persist. One persistence factor is the K type polysaccharide capsule. Capsule protects against phagocytosis, complement action, and antimicrobial peptide killing. Recent studies have also revealed that capsule along with fibrous protein assemblies is a key part of the IBC formation. Antimicrobial resistance among UPEC is increasing, driving efforts to identify therapeutic targets in the molecular pathogenesis of infection. Capsules are an attractive target because of new insights into the roles of bacterial K capsules in UPEC virulence during UTI. Specific investigations have shown that K capsule contributes to multiple aspects of pathogenesis, including IBC formation. In this program we used a cell-based assay to screen 335,740 compounds from the MLSMR library and identified 1,767 hits that inhibited K1 bacterial capsule formation. Of those hits, 59 were confirmed as active in a dose-responsive manner, and eight compounds were shown in secondary assays to specifically inhibit capsule formation. Of those eight compounds, three of those were further characterized for structure-activity relationships, mechanism of action, and therapeutic index. The probe compound, 3-[(2,6-difluorobenzoyl)amino]-5-(4-ethoxyphenyl)thiophene-2-carboxylic acid, was identified as a small molecule inhibitor of K1 capsule formation with an IC50 value of 4.5 ± 2.4 μM and a >10-fold selectivity index (SI) in BC5637 bladder cells. The probe has been broadly profiled for off-target liabilities and assessed for aqueous solubility, parallel artificial membrane permeability, and hepatocyte microsome and plasma stability. It is suitable for use as a lead compound for inhibition of K1 capsule formation.

Assigned Assay Grant #: 1 R03 MH090791

Screening Center Name & PI: Southern Research Specialized Biocontainment Screening Center, E. Lucile White

Chemistry Center Name & PI: Kansas University Specialized Chemistry Center, Jeffrey Aubé

Assay Submitter & Institution: Patrick Seed, Duke University

PubChem Summary Bioassay Identifier (AID): 488970

Resulting Publications

  1. Goller CC, Arshad M, Noah JW, Ananthan S, Evans CW, et al. Lifting the Mask: Identification of New Small Molecule Inhibitors of Uropathogenic Escherichia coli Group 2 Capsule Biogenesis. PLoS ONE. 2014;9(7):e96054. [PMC free article: PMC4077706] [PubMed: 24983234] [Cross Ref]

Probe Structure & Characteristics

ML317.

ML317

SID/CID/ML#Target NameIC50 (μM) [AID]Anti-target Name(s)CC50 (μM) [SID, AID]Fold SelectiveaSecondary Assay(s) Name: IC50 (μM) [AID]
SID 103147605/CID 23602075/ML317

(commercial sample)
K1 Capsule4.5 ± 2.4; [AID 488970]Mammalian cell toxicityCC50 = 47.2 ± 7.2; [SID 10314760, AID 488970, AID 493020, AID 504769, AID 504831, AID 588399]10.5T7 phage assay <0.39 μM; AID 488970
Orcinol assay 24% of control value; AID 488970, AID 624060
SID 126497311/CID 23602075/ML317

(synthesized sample)
K1 Capsule1.89 ±1.56; [AID 488970]Mammalian cell toxicityCC50 = 51.6 ±13.69; [SID 126497311, AID 488970, AID 493020, AID 504769, AID 504831, AID 588399]27.3T7 phage assay <0.625 μM; AID 488970
Orcinol assay 21% of control value; AID 488970, AID 624060
a

Calculated as CC50/IC50

1. Recommendations for Scientific Use of the Probe

There is currently a mounting crisis in clinical infectious diseases. The vast majority of infectious problems are treated in community clinics. However, many of the bacteria that produce common community-acquired infections are steadily gaining multi-drug resistance, compromising the ability to treat the infections in the outpatient setting due to a limited number of orally available antibiotics (1). A loss of effective antibiotics for use against community-acquired infections will result in delays in treatment, increased rates of hospitalization, sizable increases in healthcare costs, and higher morbidity and mortality. Indeed, some of these consequences of antibiotic resistance are already being realized.

Urinary tract infections (UTI) are the second most common infection of humans. There are over 10 million UTI per year in the USA alone, and over 80% of the community-acquired infections are caused by uropathogenic Escherichia coli (UPEC) (2). Furthermore, UPEC produces recurrent UTI whereby ~25–40% of individuals experience at least one recurrence within the 6 months following the first infection. There are effectively 5 types of antibiotics routinely used to treat community-acquired UTI in outpatient clinics, and resistance to 4 of these antibiotics among UPEC ranges from 15% to over 50% in many communities (2). The inability to effectively treat UTI in outpatient clinics with oral antibiotics will result in major health care costs and increased morbidity. Dissemination of UPEC from the lower urinary tract is associated with morbidity and mortality through infection of the kidneys, bloodstream, and central nervous system. The probe is specifically in development to address this rising crisis and has major potential advantages relative to conventional antibiotic technology, as highlighted below. In addition, chemical-assisted molecular studies of the pathogenesis of UTI, as facilitated by this probe, will reveal further novel targets for advanced therapeutics.

This probe is designed to specifically inhibit UPEC during infection. While most conventional antibacterials inhibit central metabolic and structural targets such as protein and DNA synthesis and the cell wall, they are indiscriminant, targeting not only the bacteria responsible for an infection but also benign bacteria in commensal reservoirs. Conventional antibiotics induce stress that results in mutational changes and horizontal exchange of antibiotic resistance among bacteria in the commensal flora (3). Antibiotic resistance primarily arises in the commensal reservoirs with an outgrowth of resistant mutants in the presence of antibiotic stress.

A novel strategy to overcome many of the drawbacks of conventional antibiotics is to target bacterial virulence factors that are non-essential for viability and unnecessary for commensalism, but critical for an infectious process. Thus, the organisms may be impeded during infection, but the stress that drives the emergence of antibiotic resistance may be reduced and depletion of commensal bacteria avoided. One candidate UPEC factor is the ubiquitous polysaccharide encapsulation. Investigators, including those of our collaborative team, have found that polysaccharide encapsulation is an important UPEC virulence factor (46). Although several capsule types are prevalent among UPEC, such as K1 and K5, there are up to 70 different types in total, limiting the practicality of designing vaccines. Similarly, some investigators have proposed capsule-specific degrading enzymes or capsule-specific lytic phage as therapeutics (7). However, the variety of capsule type, antigenicity of proteinaceous therapeutics, and limitations in orally delivering, such biologicals to the urinary tract, raises serious questions about the utility of these approaches.

Despite the variety of composition and thus antigenicity among the capsules of UPEC, some mechanisms of capsule regulation and biogenesis are common to these strains and may be targeted to inhibit capsule production across a wide variety of UPEC. Furthermore, commenal E. coli do not use similar mechanisms for capsule biogenesis, making inhibitors of UPEC capsule biogenesis sparing of commensal organisms. The probe in this report inhibits capsule biogenesis in UPEC with 2 distinct major capsule types, K1 and K5, indicating that it has the important characteristic of acting on a common target shared between UPEC with different capsules.

This probe addresses the problem of multidrug resistance. Multidrug resistant UPEC has quickly emerged and has complicated clinical management. While ampicillin resistance has been established for over a decade (>50% in most communities), resistance to trimethoprim-sulfamethoxazole (TMP-SMX) has recently emerged with rates in excess of 20% in some areas (e.g., (8)). The Infectious Diseases Society of America recommends that in regions where resistance rates to TMP-SMX exceed 10% to 20%, TMP-SMX should not be used for empirical therapy (9). Fluoroquinolones, in particular ciprofloxacin, are used increasingly, but resistance to ciprofloxacin is also on the rise (e.g., (10)), and fluoroquinolone-resistant isolates of E. coli are often multidrug resistant (11). Nitrofurantoin is a common choice for empiric therapy of acute uncomplicated UTI with rising resistance rates (12). Prior exposure to antibiotics significantly increases the risk of drug-resistant UPEC on subsequent infections, as is common for a recurrent infection such as UTI (13). Further complicating the development of antibiotic resistance is the multiple uses for most antibiotics, including infections of the respiratory tract, skin-soft tissues, and urinary tract. This probe targets a process independent from conventional antibiotics.

The probe will be used to further study the biogenesis pathways that result in K capsule formation. Small molecule inhibitors of capsule biogenesis may provide opportunities to further dissect the molecular processes involved in capsule biogenesis. The genetic identity of the target for small molecule inhibitors of capsule biogenesis would provide a better understanding to the research community of the capsule biosynthesis process and could greatly enhance the search for chemotherapeutics that target highly conserved components of the capsule assembly machinery. This could lead to the attenuation of a broad range of encapsulated bacteria with commonalities in capsule assembly. Lastly extended study of the probe could lead to compounds with potential as single antibiotics with novel inhibitory mechanisms or candidates for combination therapy with standard care model.

2. Materials and Methods

Overall Assay Strategy

The ability of the T1 bacteriophage to bind to bacterial capsule, infect, and lyse E. coli UT189 was used as the primary assay to identify the effects of screened compounds on bacterial capsule formation. The phenotypic end-point assay measured the fluorescence generated by cellular processing of alamar Blue as a direct indicator of cell viability. A total of 335,740 compounds were screened using the primary assay. Following this, a concentration-dependent confirmatory assay (in a compound concentration range of 50 – 0.19 μM) was used in parallel with a eukaryotic cytotoxicity counter-screen (same concentration range) to determine hit IC50s and CC50s (and selective indices). Hits were further evaluated using three secondary assays for alternative measurement of compound-induced reduction in bacterial capsule formation and specificity. Confirmed actives with an SI >5 were further investigated and subjected to chemical optimization, followed by secondary assay evaluation. Secondary assays more closely characterized the ability of the compounds to reduce bacterial capsule formation and can be used to examine the mechanism of action of the compounds. The combination of primary assay (to measure capsule reduction), counter assay (for general eukaryotic cell toxicity), and secondary assay (to measure reduction in capsule formation and specificity) were combined to allow a determination of probe efficacy, selectivity, and specificity.

A chemical probe for this project was defined as a small molecule that

  • Has an IC50 of < 10 μM in the primary and confirmatory Alamar Blue Screen of E. coli strain UT189 lysis (primary assay)
  • Has a therapeutic index of > 5 relative to the cytotoxicity in the bladder cell line Hu5637 (counterscreen)
  • Has an IC50 of < 10 μM in the confirmatory Bacterial Growth Screen of E. coli strain UT189 lysis (secondary assay 1)
  • Has an IC50 of < 10 μM in the T7 lysis inhibition assay, indicating the desired target specificity (secondary assay 2)
  • Will yield low orcinol levels that are 50% of the levels for capsule export control strain (secondary assay 3)
  • Will yield K5 phage sensitivity in E. coli strain DS17 (secondary assay4).

2.1. Assays

Screening for Inhibitors of Bacterial Capsule Biogenesis

Purpose: The primary screening was conducted to screen the MLSMR 300K compound library for selection of 1,767 hit compounds.

Summary AID: AID 488966

Assigned AID: AID 488970

Assay Description: Screening for Inhibitors of Bacterial Capsule Biogenesis

Primary assay: The primary assay was conducted in the 1,536 well plate format. Bacterial cultures of E. coli K1 strain UTI89 (cystitis isolate) and isogenic capsule mutant strains (as controls for phage infection) were grown and prepared at the screening center immediately prior to use. Overnight starting cultures of UTI89 were grown at 37 °C, and inoculated into 1 liter of LB (starting OD600 ~0.03), which was sufficient for screening 40 plates (including ~10% waste) and ~40,000 compounds as single points.

Control wells contained UTI89 (wt K1) with 0.5% DMSO as a simulated positive control; UTI89 with 0.5% DMSO and tetracycline for a negative growth control; and media plus vehicle control. 50 mL of LB Broth was inoculated with stock 150 μL E. coli UT189 and grown overnight at 37 °C. The next day, cultures were diluted 1:75 in 1 L of LB Broth containing 0.5% DMSO. 3 μL of this culture was added to each plate well, and plates were incubated, inverted, at 37 °C for 2 hr. The K1F bacteriophage stock was diluted 1:8 in LB Broth containing 0.5% DMSO, and 1.5 μL of diluted phage (or media only) was added to the pre-plated test compound wells and appropriate control wells. The plates were centrifuged briefly, and then were incubated, inverted, at 37 °C for an additional 2 hr. Afterward, alamar Blue reagent (Invitrogen, #DAL1100) was diluted 1:2 with LB broth and 1 μL was added to each plate well. The plates were again centrifuged briefly, then were further incubated, inverted, at 37 °C for 30 min.

Single Dose Compound Preparation: For single dose screening, compounds or carrier control (DMSO) were diluted to a final well concentration of 1:200 in assay media. Compounds (45 nL in 100% DMSO) were dispensed to assay plates using an Echo non-contact dispenser. Compounds from the libraries were added to the plates at a final concentration of 100 μM, before the addition of bacteria or phage. Each compound was tested as a single point, and ~1200 compounds were tested per plate. The entire primary screening campaign was divided into eight batches, each screened in this manner.

Control Drug: The positive control drug C7 in 1% DMSO (2-(4-phenylphenyl)benzo[g]quinoline-4-carboxylic acid) that was previously identified in a pilot screen was not used in the primary screen due to aqueous insolubility. Tetracycline (50 μM) was used as a negative growth control drug.

Endpoint Read: The plates were read at ambient temperature from the top for fluorescence intensity in an Envision plate reader (Perkin Elmer) by excitation at 560 nm and emission at 590 nm, and the degree of phage-mediated lysis was determined based on the metabolic processing of alamar Blue by live bacterial cells. A positive hit was defined by the compound producing greater than a 50% inhibition of phage-induced lysis.

Dose Response Compound Preparation

Concentration dependent confirmatory and cytotoxicity assays. Dose response testing (dose range = 300-0.58 μM) was used to confirm and characterize the primary screen hits, which was necessary to determine the number of compounds advanced to secondary screens.

Efficacy: Compounds were plated in 1536-well microplates, and the dose response efficacy assay was performed as described for the primary screen, with the exception that each compound was tested in duplicate at 10 concentration points starting from 300 μM and continuing to lower concentrations by 2-fold serial dilutions. The strain UTI89 delta-kpsM, a K1 capsule export mutant, was evaluated with the wt strain as a phage insensitive control (mimicking 100% capsule inhibition).

Counterscreen: Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis

Purpose: This cell-based assay measures the cytotoxicity of compounds in bladder carcinoma 5637 cells using luminescent cell viability assay readout.

Summary AID: AID 488970

Assigned AID: AID 493020, AID 504769, AID 504831, AID 588399

Assay Description:Cytotoxicity: Dose response testing (dose range = 300-0.58 μM) established the hit cytotoxicity data. Compounds were plated in 384-well microplates in a stacked dose response format using the same doses used in the efficacy dose response. Bladder carcinoma 5637 cells were added to the compounds, and 72 hr later cell viability was measured using CellTiter Glo (14). Hit cytotoxicity and the 50% toxic concentration (TC50) was determined and compared to the IC50 to calculate the therapeutic index. Test compounds are serially diluted in a plate-to-plate matrix or stacked-plate matrix. All 320 compounds in a source plate are diluted together resulting in a 10 point dose response dilution series. It is visualized as a serial dilution series proceeding vertically through a stack of plates with the high dose plate on top and the low dose plate on the bottom.

Control Drug: Hyamine was used as a positive cytotoxic control. All wells contained 0.5% DMSO.

Preparation of Bladder carcinoma 5637 cells: Cells are harvested and resuspended to 80,000 cells per ml in Complete DMEM/F12®.

Endpoint Read: Following the three day incubation period, the assay plates were equilibrated to room temperature for 30 min and an equal volume (30 μL) of Cell Titer-Glo® reagent (Promega Inc.) is added to each well using a WellMate™ (Matrix, Hudson, NH) and plates are incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence is measured using a Perkin Elmer Envision™ multi-label reader (PerkinElmer, Wellesley, MA) with an integration time of 0.1 s.

Secondary Assay: Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189

Purpose: This confirmatory cell-based assay provides an alternative measurement of inhibitory activity on phage-induced lysis. It measures reduction in bacterial capsule formation using an absorbance readout at A600 instead of the alamar Blue reagent.

Summary AID: AID 488970

Assigned AID: AID 504358, AID 504543, AID 504675, AID 504768, AID 588321, AID 588386, AID 588395

Assay Set-up: This secondary assay was conducted in the 96 well plate format. Bacterial cultures of E. coli K1 strain UTI89 (cystitis isolate) and isogenic capsule mutant strains (as controls for phage infection) were grown and prepared at the screening center immediately prior to use. Overnight starting cultures of UTI89 were grown at 37 °C, and diluted 1:100 into LB. Compounds were added to plates in a concentration dependent manner in the range of 100-0.39 μM, followed by addition of 100 μL of bacterial culture. Each concentration was tested in quadruplicate. 1% DMSO (final well concentration) was included. The plates were tape sealed and shaken vigorously for 1.5 hr. An initial OD600 reading at the time of infection was measured to identify compounds that cause growth retardation or bacterial killing in the absence of phage. Next, K1F phage (5 μL) was added to all of the test wells. The plates were resealed and shaken vigorously at 37 °C, and measurements of OD600 for phage-mediated lysis were taken after 3 hrs.

Endpoint Read: The plates were read at ambient temperature from the bottom for absorbance at A600 in an Envision plate reader (Perkin Elmer) and the degree of phage-mediated lysis was determined based on the absorbance.

Secondary Assay: Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition

Purpose: This secondary assay measures the compound mechanistic specificity for inhibition of bacterial capsule formation using a different bacterial phage (T7). In this assay, an increase in phage-induced lysis correlates to a decrease in capsule formation.

Summary AID: AID 488970

Assigned AID: AID 504349, AID 504538, AID 504676, AID 504767, AID 588322

Assay Description: T7 phage-mediated lysis assay: This assay determined if the mechanism of action of the compound is inhibition of phage infectivity or replication. The T7 phage has a nearly identical genome to K1F, without encoding an endosialidase. Its cycle of replication is similar to that of K1F as well. However, T7 entry into E. coli is inhibited by K capsules. This secondary assay was conducted in the 96 well plate format. Bacterial cultures of E. coli K1 strain UTI89 (cystitis isolate) and isogenic capsule mutant strains (as controls for phage infection) were grown and prepared immediately prior to use. Overnight starting cultures of UTI89 were grown at 37 °C, and diluted 1:100 into LB. Compounds were added in quadruplicate to plates in a concentration dependent manner in the range of 100-0.39 μM, followed by addition of 100 μL of bacterial culture. 1% DMSO (final well concentration) was included. The plates were tape sealed and shaken vigorously for 1.5 hr. An initial OD600 reading was measured to identify compounds that cause growth retardation or bacterial killing in the absence of phage. Next, T7 phage (5 μL) was added to all of the test wells. The plates were resealed and shaken vigorously at 37 °C, and measurements of OD600 for phage-mediated lysis were taken after 3 hr. True inhibitors of capsule yielded bacteria that were susceptible to T7 phage and lysed within 2 hr of the addition of phage. However, compounds inhibiting phage replication did not promote bacterial lysis. The positive control drug C7 (100 μM final well concentration) was used in this screen.

Endpoint Read: The plates were read at ambient temperature from the bottom for absorbance at A600 in a BioTek Quantplate reader and the degree of phage-mediated lysis was determined based on the absorbance.

Secondary Assay: Orcinol Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis

Purpose: This assay was performed only on the probe candidate. This is an end-point assay to measure the amount of K1 bacterial cell capsule formation in the presence of a test compound concentration range. The ability of the test compounds to inhibit the K1 capsule formation was measured by a reduction of absorbance from the complex formed by orcinol and the capsule polysaccharide. The biochemical determination of cell-surface associated capsule was performed using UTI89 or the delta Region I and Region II capsule mutant bacterial strains.

Summary AID: AID 488970

Assigned AID: AID 504733, AID 624060

Assay Description: The biochemical measurement of cell-surface associated capsule was performed. UTI89 or capsule mutants were grown in culture tubes with and without the test compound (50 μM). The cultures were centrifuged, and the cell pellets were washed in PBS and resuspended in Tris buffer, pH5. We have found that low pH releases surface polysaccharide without lysing the bacteria. Released polysaccharide was harvested by separation from whole bacteria by centrifugation followed by deproteination with phenol/chloroform and precipitation with ethanol. The precipitated material was subjected to acid hydrolysis (pH 2 at 80°C for 1 hr) and incubated with orcinol, which reacts with periodic intermediates to produce a violet color quantified at OD 570 (3). Inhibitors reducing or abrogating surface encapsulation yielded low orcinol levels similar to the capsule export and synthesis mutants (Region I and II). The positive control drug C7 (100 μM final well concentration) was used in this screen.

Endpoint Read: The samples were read at ambient temperature from the bottom for absorbance at A570 in a BioTek Quantplate reader and the degree of orcinol-reactive material was determined based on the absorbance compared to a wild-type encapsulated strain (UTI89) and a standard curve using purified sialic acid.

Secondary Assay: K5 Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis

Purpose: This assay was performed only on the probe, and determined if compounds considered active in the T7 and orcinol secondary assays were able to inhibit also inhibit K5 capsule biogenesis.

Summary AID: AID 488970

Assigned AID: AID 624061

Assay Description: This assay was performed only on the probe candidate, and determined if compounds considered active in the T7 and orcinol secondary assays were able to also inhibit K5 capsule biogenesis. This assay was performed in a method identical to the T7 assay test, but a different bacterial test strain and bacteriophage are used. In this validation test, we used E. coli strain DS17, a pyelonephritis clinical isolate expressing a K5 capsule. DS17 is highly susceptible to K5 phage-mediated lysis. Thus, compounds that were active in the K1F phage assay, but did not inhibit phage in the T7 phage assay, were analyzed using this assay. This secondary assay was conducted in the 96 well plate format. Bacterial cultures of E. coli strain DS17 were grown and prepared immediately prior to use. Overnight starting cultures of DS17 were grown at 37 °C and diluted 1:100 in LB. Compounds were added to plates in quadruplicate at 50 and 100 μM, followed by addition of 100 μL of bacterial culture. 1% DMSO (final well concentration) was included. The plates were tape sealed and shaken vigorously for 1.5 hr. An initial OD600 reading was measured to identify compounds that cause growth retardation or bacterial killing in the absence of phage. Next, K5 bacteriophage (5 μL) was added to all of the test wells. The plates were resealed and shaken vigorously at 37 °C, and measurements of OD600 for phage-mediated lysis were taken after 3 hr. True inhibitors of capsule yielded bacteria that were not susceptible to K5 bacteriophage and did not show lysis within 2 hr of the addition of phage. The positive control drug C7 (100 μM final well concentration) was used in this screen.

Endpoint Read: The plates were read at ambient temperature from the bottom for absorbance at A600 in a BioTek Quantplate reader and the degree of inhibition of phage-mediated lysis was determined based on the absorbance.

2.2. Probe Chemical Characterization

Figure 1. Probe Chemical Structure and Properties.

Figure 1Probe Chemical Structure and Properties

Structure Verification and Purity: 1H NMR, 13C NMR, RP HPLC/UV/HRMS Data

Proton and carbon NMR data for ML317/MLS004256629/SID 126497311/CID 23602075: Detailed analytical methods and associated instrumentation are described in section 2.3, entitled “Probe Preparation”, under general experimental and analytical details. The numerical experimental proton and carbon NMR data are presented below.

Proton NMR Data for ML317/SID 126497311/CID 23602075:1H NMR (400 MHz, DMSO) δ 13.62 (s, 1H), 8.24 (s, 1H), 7.68 (d, J = 6.8 Hz, 2H), 7.64 (m, 1H), 7.31 (t, J = 6.8 Hz, 2H), 7.03 (d, J = 7.2 Hz, 2H), 4.09 (q, J = 5.6 Hz, 2H), 1.36 (t, J = 5.6 Hz, 3H) ppm.

Carbon NMR Data for ML317/SID 126497311/CID 23602075:13C NMR (100 MHz, DMSO) δ 164.6, 160.2, 159.6, 158.2, 158.1, 157.0, 148.4, 142.6, 133.5, 133.3, 127.4, 124.7, 116.7, 115.2, 113.5, 112.7, 112.5, 111.1, 63.3, 14.5 ppm.

RP HPLC/UV/HRMS Data for ML317/SID 126497311/CID 23602075: Detailed analytical methods and associated instrumentation are described in section 2.3, entitled “Probe Preparation”, under general experimental and analytical details. Purity assessment by RP HPLC/UV/HRMS at 214 nm for SID 126497311 (CID 23602075) revealed purity of 97.6% (retention time = 2.423 minutes), HRMS (m/z) calcd for C20H15F2NO4S [M+H+] 404.0763, found 404.0770.

Aqueous Solubility

Solubility was measured in phosphate buffered saline (PBS) at room temperature (23 °C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4 (15). Probe ML317 (SID 103147597/CID 18109210) was found to have a solubility measurement of 92.6 μg/mL, or 230 μM, under these conditions. Solubility was also assessed in primary assay media (Luria-Bertani Broth). Probe ML317 was determined to have an assay media solubility of 68 μg/mL or 169 μM. The solubility in PBS buffer is good and only slightly less than in assay media, and the solubility for the probe is well above its activity in the K1and T7 assays.

Aqueous Stability

Aqueous stability for the probe was assessed using two solvent systems (100% aqueous PBS, and 50:50 aqueous PBS:acetonitrile). The probe stability was measured in aqueous PBS (no antioxidants or other protectants, DMSO concentration below 0.1%, room temperature) and the results are reported as circles in the graph in Figure 2. The probe stability was also measured in 50:50 aqueous PBS and acetonitrile and the results are reported as squares in the graph in Figure 2. Stability data in each case is depicted as the loss of compound with time over 48 hours with a minimum of six time points and providing the percent compound remaining after 48 hours. ML317 was found to be stable in both solvent systems under these experimental conditions over the entire 48 hour study (Figure 2).

Figure 2. Graph depicting the stability for ML317 after 48 hours in two separate solvent systems.

Figure 2

Graph depicting the stability for ML317 after 48 hours in two separate solvent systems.

Thiol Stability

Compounds were dissolved at 10 μM in PBS at pH 7.4 (1% DMSO) and incubated at room temperature with either no thiol source as a negative control, 50 μM glutathione (GSH), or 50 μM dithiothreitol (DTT). The mixtures were sampled every hour for eight hours and analyzed by RP HPLC/UV/HRMS. The analytical RP HPLCUV/HRMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 × 50mm, 1.8um) and elution with a linear gradient of 1% water to 100% CH3CN at 0.6 mL/min flow rate. Peaks on the 214 nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent compound remaining. The masses of potential adducts were searched for in the final samples to determine if any detectable adduct formed. All samples were prepared in duplicate (Figure 3). Ethacrynic acid, a known Michael acceptor, was used as a positive control. If solubility of the compound was an issue at 10 μM, PBS was substituted with PBS with 50% acetonitrile (16).

Figure 3. Graph depicting thiol stability of probe.

Figure 3

Graph depicting thiol stability of probe.

ML317 was found to be completely stable to the presence of five times its concentration of both glutathione and DTT thiol sources across all time points in our analysis (Figure 3). Statistically, there is no drop in the % remaining concentration of ML317 in the course of these experiments. This data is easily contrasted to the data for the ethacrynic acid control compound, which is shown to react rapidly with five times its concentration of glutathione and even more rapidly with DTT.

Synthesis Route

The probe compound ML317 and numerous analogues were synthesized using the reaction sequence shown in Figure 4. Briefly, a series of aryl ketones was converted to (Z)-3-chloro-3-arylacrylonitriles through treatment with POCl3, DMF and hydroxylamine. These nitriles were then converted via Fisselman reaction to tert-butyl 3-amino-5-(aryl)thiophene-2-carboxylates through a reaction with sodium methoxide and tert-butyl 2-mercaptoacetate. Lastly, these amines were coupled with various acid chlorides in the presence of triethylamine in dichloromethane followed by a removal of the tert-butyl group with trifluoroacetic acid in dichloromethane.

Figure 4. Synthetic route for probe and analogue generation.

Figure 4

Synthetic route for probe and analogue generation.

Submission of Probe and Five Analogues to the MLSMR

Samples of the probe and five analogues were prepared, analytically characterized, and shipped to the MLSMR (see Table 1). The structures for the five supporting analogues are shown in Figure 5.

Table 1. Five probe analogues with screening data.

Table 1

Five probe analogues with screening data.

Figure 5. Five thiophene analogues chosen to support of probe ML317.

Figure 5

Five thiophene analogues chosen to support of probe ML317.

2.3. Probe Preparation

General experimental and analytical details

All reagents were used as received from commercial suppliers. The 1H and 13C spectra were recorded on a Bruker Avance 400 MHz or 500 MHz spectrometer. Chemical shifts are reported in parts per million and were referenced to residual proton solvent signals. Flash column chromatography separations were performed using the Teledyne Isco CombiFlash RF using RediSep RF silica gel columns. TLC was performed on Analtech UNIPLATE silica gel GHLF plates (gypsum inorganic hard layer with fluorescence). TLC plates were developed using iodine vapor. RP HPLC/UV/HRMS analysis was carried out with gradient elution (5% CH3CN to 100% CH3CN) on an Agilent 1200 RRLC with a photodiode array UV detector and an Agilent 6224 TOF mass spectrometer (also used to produce high resolution mass spectra). Purification was carried out by mass directed fractionation with gradient elution (a narrow CH3CN gradient was chosen based on the retention time of the target from LCMS analysis of the crude sample) on an Agilent 1200 instrument with photodiode array detector, an Agilent 6120 quadrupole mass spectrometer, and a HTPAL LEAP autosampler. Fractions were triggered using a MS and UV threshold determined by RP HPLC/UVHRMS analysis of the crude sample. The conditions for RP HPLC analysis included the following: Waters BEH C-18, 1.7 μm, 2.1 × 50 mm column; 0.6 ml/min flow rate; and pH 9.8 NH4OH aqueous mobile phase. The conditions for purification included: Waters XBridge C18 5μm, 19 × 150mm column; 20 ml/min flowrate pH 9.8 NH4OH aqueous mobile phase.

Probe preparation and corresponding protocols

Image ml317fu2

(Z)-3-Chloro-3-(4-ethoxyphenyl)acrylonitrile: Prepared according to an experiment outlined by Romagnoli et al. (17). To an ice cold solution of dry dimethyl formamide (8.90 g, 122 mmol) was added phosphorus oxychloride (5.57 ml, 60.9 mmol) dropwise while stirring for 15 minutes. To this cold mixture, 1-(4-ethoxyphenyl)ethanone (5.00 g, 30.5 mmol) was added dropwise while maintaining the temperature between 45–55 °C via external heat source for ten minutes. The reaction mixture was then cooled to room temperature and allowed to stir for 30 minutes. Then, a solution of hydroxylamine hydrochloride (8.46 g, 122 mmol) in dry DMF was added slowly (7 mL of 33 mL for 10 g reaction). This mixture was allowed to stir at 80 °C (an ice bath was briefly employed to control a rapid exotherm) for five minutes. Then, the remaining hydroxylamine solution was added so that the reaction mixture heated exothermically to no greater than 150 °C. After the completion of addition, the reaction mixture was allowed to cool to RT and stir for an additional 30 minutes. The reaction mixture was cooled to 0 °C. Cold water was then added and the reaction mixture was extracted with chloroform. The crude product was purified via normal phase, silica gel chromatography (98:2 ethylacetate/hexane) (59.1%, 6.32 g). 1H NMR (400 MHz, DMSO) δ 7.62 (d, J = 9.2, 2H), 6.93 (d, J = 9.2 Hz, 2H), 5.89 (s, 1H), 4.09 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 6.8 Hz, 3H); ppm; 13C NMR (100 MHz, DMSO) δ 162.1, 152.9, 128.7, 126.3, 115.9, 114.7, 93.4, 63.9, 14.7 ppm; IR (ATR) 2983, 2217, 1593, 1506, 1238, 1039 cm−1.

Image ml317fu3

tert-Butyl 3-amino-5-(4-ethoxyphenyl)thiophene-2-carboxylate: Following the protocol outlined by Gokaraju et al. (18), to a solution of tert-butyl 2-mercaptoacetate (1.1 g, 7.42 mmol) in methanol (5mL) was added a solution of sodium methoxide (0.401 g, 7.42 mmol) in methanol (5mL). The reaction mixture was then stirred for 30 minutes. A solution of (Z)-3-chloro-3-(4-ethoxyphenyl)acrylonitrile (1.23 g, 5.94 mmol) in DMF was added dropwise for 10 minutes to the reaction mixture and then stirred at 60 °C for an additional two hours. Then, a solution of sodium methoxide (0.802 g, 14.84 mmol) in methanol (10 mL) was added dropwise to the reaction mixture which was then stirred for another two hours at 60 °C. Cold water was added to the reaction mixture and the mixture was allowed to stir for 15 minutes. The aqueous solution was extracted three times with chloroform. The combined organic phases were washed with water, brine, dried over sodium sulfate and then concentrated in vacuo. The crude residue was purified with a DCM/MeOH eluent on a normal phase, silica gel column, giving the product (60.5%, 1.67 g). 1H NMR (400 MHz, DMSO) δ 7.51 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.64 (s, 1H), 5.37 (s, 2H), 4.06 (q, J = 6.8 Hz, 2H), 1.57 (s, 9H), 1.41 (t, J = 6.8 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO) δ 164.3, 159.6, 148.4, 127.2, 126.2, 114.8, 114.5, 80.6, 63.6, 28.6, 14.8 ppm; IR (ATR) 3459, 3352, 2978, 1664, 1602, 1548, 1460 cm−1; HRMS calcd for C17H21NO3S [M+H+] 320.1242, found 320.1320.

Image ml317fu4

3-(2,6-Difluorobenz amido)-5-(4-ethoxyphenyl) thiophene-2-carboxylic acid (ML317, SID 126497311, CID 23602075): To a solution of tert-butyl 3-amino-5-(4-ethoxyphenyl)thiophene-2-carboxylate (0.1866 g, 0.584 mmol) and triethylamine (0.163 mL, 1.168 mmol) in dichloromethane was added 2,6-difluorobenzoyl chloride (0.103 g, 0.584 mmol) dropwise. The reaction mixture was allowed to stir overnight. Then, 1N HCl was added to the reaction mixture. The organic phase was then washed with brine, concentrated en vacuo. A 1:3 solution of trifluoroacetic acid:dichloromethane was added slowly to the residue. The reaction was allowed to stir for two hours. The reaction was then concentrated and the residue was triturated with diethyl ether, resulting in a white solid (94%, 0.221 g). 1H NMR (400 MHz, DMSO) δ 13.62 (s, 1H), 8.24 (s, 1H), 7.68 (d, J = 6.8 Hz, 2H), 7.64 (m, 1H), 7.31 (t, J = 6.8 Hz, 2H), 7.03 (d, J = 7.2 Hz, 2H), 4.09 (q, J = 5.6 Hz, 2H), 1.36 (t, J = 5.6 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO) δ 164.6, 160.2, 159.6, 158.2, 158.1, 157.0, 148.4, 142.6, 133.5, 133.3, 127.4, 124.7, 116.7, 115.2, 113.5, 112.7, 112.5, 111.1, 63.3, 14.5 ppm; IR (ATR) 3319, 2942, 2531, 1693, 1651, 1607, 1565, 1449, 1254, 1024 cm−1; HRMS calcd for C20H15F2NO4S [M+H+] 404.0763, found 404.0770.

3. Results

3.1. Dose Response Curves for Probe

The primary assay (Summary AID 488970) and counterscreen methods (AID 493020); were used to measure both probe efficacy and cytotoxicity. The probe ML317 potency in the primary phase lysis inhibition assay was determined: IC50 = 4.5 ± 2.4 μM, and the CC50 47.2 ± 7.2 μM. The calculated selectivity was determined as (CC50/IC50) = 10.5. The ML317 dose response profiles for efficacy and cytotoxicity curves are graphed (Figure 6).

Figure 6. Efficacy and cytotoxicity analysis of ML317.

Figure 6

Efficacy and cytotoxicity analysis of ML317. The graph on the left (green line) shows the efficacy of the probe in the primary bacterial phage lysis inhibition assay (n = 3). The small gray inset magnifies the corresponding region in the larger graph. (more...)

3.2. Cellular Activity

ML317 was identified using a phenotypic bacterial cell-based assay that determined the amount of bacterial capsule formation in the presence of the probe compound. No biochemical assays were used for determination of compound biological activity. Additional cell-based assays were performed to determine probe specificity. Also, a counterassay was performed to determine eukaryotic cytoxicity using human bladder cell carcinoma 5637 cells, which are considered the physiologically-relevant target cell type. The compound was shown to have no effect on bacterial cell viability in the highest tested concentration (100 μM), and the cytotoxic concentration was 47.2 ± 7.2 μM, which determined a calculated selective index of 10.5. Although the primary and secondary assays were performed using bacterial cultures, it is recognized that probe will be used in in vitro eukaryotic and in vivo assay systems. Because of this, each synthesized probe and analogs were routinely tested using the cytotoxicity counterassay.

3.3. Profiling Assays

In vitro pharmacokinetics profiling

The in vitro pharmacokinetic (PK) properties of the probe (ML317) were profiled using a standard panel of assays (Table 2) across which the probe displayed encouraging results.

Table 2. Summary of in vitro ADME properties of ML317.

Table 2

Summary of in vitro ADME properties of ML317.

Broad-spectrum target profiling

The probe compound ML317 was submitted to Ricerca for LeadProfiling to assess off-target pharmacology. The probe was tested in duplicate at 10 μM concentration and no significant activity was noted across the panel of 67 targets (i.e., <50% inhibition). In addition, in PubChem, the probe ML317 is reported to have shown activity in only 16 of 341 (4.7%) bioassays in which it was tested. These results suggest that ML317 is not a promiscuous compound with respect to off-target effects.

4. Discussion

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

Traditional anti-infectives for the treatment of UTI are almost exclusively directed toward inhibiting central metabolic and structural targets such as folate metabolism, protein synthesis, DNA replication machinery, and cell wall assembly. Traditional targets of inhibition are attractive because of the potential broad spectrum of inhibition of a variety of organisms. However, the lack of specificity then also means that microbes not involved in an infectious process and not intended as targets of the therapeutic, are indiscriminately targeted. The major consequences are two-fold: 1) Normal flora are eliminated, resulting in side effects such as antibiotic-associated diarrhea and the emergence of pathogens such as Clostridium difficile, and 2) stress within microbial reservoirs such as the enteric tract drives the acquisition and emergence of antibiotic resistance. Furthermore, more traditional anti-infectives are not disease specific, and thus the multiplicity of their use for a variety of infection prevention and treatment increases the amount of human and agricultural exposure to these agents, further driving antibiotic resistance.

Targeting factors required by microbial pathogens almost exclusively during infection, so called anti-virulence therapeutics, is predicted to dramatically reduce chemical stress on commensal microbes and thus lessen the emergence of resistance. Furthermore, many infections may be cleared by inhibiting microbial factors that subvert the host immune response, thus allowing natural clearance of the infection and possibly enhancing immune memory of the infectious agent to allow the immune system to better recognize and clear subsequent infections. Since the vast majority of UTI occur in the community in other healthy individuals with competent immune systems, this strategy for novel anti-virulence agents is rationale and practical.

The probe described herein is entirely novel with the only precedent being the structurally dissimilar molecule 2-(4-phenylphenyl)benzo[g]quinoline-4-carboxylic acid that we previously described in proof-of-concept studies (19). The probe described herein is uniquely poised for development as an infection-specific prevention and treatment therapeutic that enhances natural immune clearance of an infection, namely UTI. The highly soluble probe abrogates capsule development in several K type UPEC as demonstrated through capsule-specific phage assays and orcinol biochemical tests. However, the probe has no effect on in vitro growth and viability of UPEC in the absence of immune factors. Unlike with many traditional antibiotics, exposure of UPEC to a range of concentrations of the probe has not resulted in the emergence of spontaneous resistance in the laboratory, consistent with the concept that this probe does not induce stress and adaptive changes that confer resistance. Furthermore, this probe would be expected to act upon traditional antibiotic resistant strains of E. coli.

As previously outlined, 2-(4-phenylphenyl)-benzo[g]quinoline-4-carboxylic acid C7, was used as the prototype small-molecule inhibitor of capsule biogenesis and serves as the only prior art for this probe development project. The probe discovered during this Molecular Libraries Probe Production Centers Network (MLPCN) project demonstrates improved potency in the K1 phage assay, a better selectivity index, similar performance in the orcinol screen, and greater than an order-of-magnitude lower IC50 value in the T7 phage assay (Figure 7). Furthermore, we have demonstrated the synthetic tractability for the ML317 chemotype, and prepared an array of highly active analogues, whereas C7 does not share either of these positive attributes. We feel these factors to be of utmost importance, since these compounds are believed to have a unified mode of action. To further demonstrate the usefulness of ML317 as a probe, we have shown ML317 to have good in vitro pharmacokinetic properties. Aqueous stability in 1X PBS at pH 7.4, aqueous stability in LB, both human and mouse hepatic microsomal stability, PAMPA permeability and both human and mouse stability were all found to be good. In contrast, while C7 has not been screened in such in vitro PK assays, one might predict poor results. For example, one would not imagine a compound such as C7 to have good solubility characteristics. Lastly, the many aryl rings of the C7 structure pose liabilities to oxidative metabolism that might render this compound less attractive compared to ML317.

Figure 7. Structures of C7 and ML317.

Figure 7

Structures of C7 and ML317.

5. References

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