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N-(pyridin-4-yl)benzo[d]thiazole-6-carboxamide inhibits E. coli UT189 bacterial capsule biogenesis

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Author Information

Received: ; Last Update: April 5, 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 (AP) 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, the team 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 were further characterized for structure-activity relationships, mechanism of action, and selectivity. The probe compound, N-(pyridin-4-yl)benzo[d]thiazole-6-carboxamide, was identified as a small molecule inhibitor of K1 capsule formation with an IC50 value of 1.04 ± 0.13 μM and a >200-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 and Characteristics

ML333.

ML333

SID/CID/ML#Target NameIC50 (μM) [AID]Anti-target Name(s)CC50 (μM) [SID, AID]Fold SelectiveaSecondary Assay(s) Name: IC50 (μM) [AID]
SID 103147597CID 18109210/ML333K1 Capsule1.04 ± 0.13; [AID 488970]Mammalian cell toxicityCC50 = 239 ± 89; [SID 10314760], AID 488970, AID 493020, AID 504769, AID 504831, AID 588399230T7 phage assay <0.39 μM; AID 488970
Orcinol assay 24% of control value; AID 488970, AID 624060
a

Calculated as CC50/IC50

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). 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. 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 the consequences of antibiotic resistance are already being realized.

This probe is designed to specifically inhibit UPEC during infection. Dissemination of UPEC from the lower urinary tract is associated with morbidity and mortality through infection of the kidneys, bloodstream, and central nervous system. A probe resulting from this work would be used to specifically 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. 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, commensal 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 (™P-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 ™P-SMX exceed 10% to 20%, ™P-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 results 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 to candidates for combination therapy with standard care models.

1. Introduction

Probe Project Purpose

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 (14). UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. Antimicrobial resistance among UPEC is on the rise (10, 1518), driving efforts to discover vulnerable targets in the molecular pathogenesis of infection.

During UTI, UPEC lives in intracellular and extracellular locales. UPEC adheres to the apical bladder epithelium and invades into it (1921). Within the bladder epithelium, UPEC typically reproduces in a biofilm-like state called intracellular bacterial communities (IBC; (5)). 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 reproduction phases again. Past studies have revealed bacteria encased in the IBC within a complex matrix of fibrous protein assemblies and polysaccharides (5). Our prior studies have also shown that disruption of the IBC pathway aborts experimental UTI, highlighting the importance of this intracellular lifecycle (2225). A detailed study of urine samples from women with acute UTI demonstrated IBC in shed bladder epithelial cell, showing that the pathway is conserved in humans (26).

Investigators have found that bacterial encapsulation is an important UPEC virulence factor (46), and experiments show that the K capsule contributes to multiple aspects of pathogenesis, including IBC formation. K capsules, also called K antigens, are enveloping structures composed of high-molecular-weight polysaccharides. Among UPEC, the K antigens K1, K2, K5, K30, and K92 are thought to be most prevalent (27). Capsules are well-established virulence factors for a variety of pathogens that are thought to protect the cell from opsonophagocytosis and complement-mediated killing (reviewed in (28, 29)). While they did not study the effects of K antigen from UPEC, Llobet et al. recently demonstrated that the highly acidic polysaccharide capsules of diverse organisms including Klebsiella pneumoniae, Pseudomonas aeruginosa, and Streptococcus pneumoniae interact strongly with APs, acting as “sponges” to sequester and neutralize the APs (30).

Of the different K types, the Group 2 and Group 3 capsules are most prevalent among UPEC isolates, with K1 and K5 being leading types. Although the capsules have different compositions, they are regulated, synthesized, assembled, and exported by functionally homologous factors, leading us to hypothesize that we can develop small molecular inhibitors of K-type encapsulation that target the most medically important K capsule types. Furthermore, the medically important infectious agents Campylobacter jejuni, Haemophilus influenzae, Neisseria meningitides, and Salmonella typhimurium among others, use homologous components in the biogenesis of their capsules. The K1 capsule type is closely associated with pathogenic isolates; not only is it the leading type in UTI, but it also accounts for much of the extra-urinary tract complications. Animals studies of E. coli K1 sepsis demonstrated that injection of a K1 capsule degrading enzyme abrogates infection (7). However, the enzyme treatment is immunogenic; accordingly, chemical inhibition may prove to be a superior approach.

There are currently no therapeutics that specifically inhibit the formation of any bacterial capsule, and this is a novel strategy for preventing or decreasing the prevalence of chronic or re-occurring urinary tract infections. New insights into the roles of K1 capsules in UPEC virulence during UTI make capsules an attractive target for therapeutic intervention. Antimicrobial resistance among UPEC is on the rise (11, 12, 15, 31, 32), and the discovery of novel small molecules that can act as probes or lead compounds for the investigation and treatment of UTI will add to the arsenal of compounds available for single or combination therapies.

Prior Art

Through the efforts of the Seed lab toward developing assay and screening techniques for inhibitors of bacterial capsule biogenesis, a library of 2,195 compounds obtained from the Developmental Therapeutics Program at the National Cancer Institute was tested. In the K1F phage lysis 96-well plate format assay, 35 (1.59%) of the compounds were found to have inhibitory activity, of which only nine compounds gave reproducible phage lysis inhibition activity in shaken tube format. These nine compounds were taken on to a secondary screening process from which two capsule biogenesis inhibitors emerged. Malachite green oxalate (NCS5550), a compound not known to inhibit capsule biogenesis, was found to produce metabolites with previously reported toxicities to mammalian systems and was thus discarded. The second inhibitor, 2-(4-phenylphenyl)-benzo[g]quinoline-4-carboxylic acid (NSC136469), or C7, was employed as a prototype small molecule inhibitor of capsule biogenesis since it inhibited K1F phage lysis of UPEC K1 strain UTI80 reproducibly in the tests following HTS. Furthermore, this inhibition was found to behave in a concentration-dependent manner with the inhibitory effect reaching saturation at approximately 25 μM C7, producing approximately 50% inhibition of K1F phage lysis of UPEC at 12.5–25 μM (33).

Image ml333fu2

This team previously reported a probe ML317 that arose from the same high-throughput screen that led to the probe ML333 reported herein. In short, the probe ML317 was found to have an IC50 of 1.89 μM in a bacterial viability assay in the presence of K1 phage, a TC50 (toxicity) of 51.6 μM, a selectivity of 27, a T7 phage bacterial viability assay IC50 of <0.39 μM, and to reduce the bacterial capsule-dependent orcinol stain to 21% of the control sample. Additional biological characterization for probe ML317 is provided in Section 4.1 of this report.

Image ml333fu3

Intellectual Property Landscape for the Probe Chemotype

On September 30th, 2012, a search was performed using SciFinder to explore the intellectual property landscape around the probe compound ML333 and analogues from the probe chemotype. A substructure search using the structure shown revealed one patent and zero publications related to the preparation and use of N-(pyridin-4-yl)benzo[d]thiazole-6-carboxamide derivatives for use as tryptase inhibitors. No publications or patents reporting the use of ML333 derivatives as antibacterial agents were found.

ML333.

ML333

  1. “Preparation of (hetero)arylmethlamines as tryptase inhibitors” Lively, Sarah Elizabeth; Waszkowycz, Bohdan; Harrison, Martin James; Clase, Juha Andrew; Naylor, Neil Jason PCT Int. Appl. (2001), WO 2001027096 A1 20010419.

An exact structure search on the probe ML333 using SciFinder revealed that the compound was commercially available, and no references to publications or patents were found. An exact structure search for some of the most active compounds from this chemotype (i.e., CID 53484233, CID 53484226, CID 53484228, and CID 53484225) revealed no publications or patents. Overall, these search results suggest that compounds derived from the ML333 chemotype could be claimed for use as anti-bacterial agents, specifically for the inhibition of bacterial capsule biogenesis for the treatment of bacterial infection.

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 alamarBlue 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 counterscreen (same concentration range) to determine compound 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 (secondary assays 1 and 2) and specificity (secondary assays 3 and 4). Confirmed actives with a selectivity index (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 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 alamarBlue 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 assay 4).

2.1. Assays

A. Primary Assay: Screening for Inhibitors of Bacterial Capsule Biogenesis

Purpose: The primary inhibition assay using E. coli UT189 and bacteriophage K1F was conducted to screen the MLSMR 300K compound library, to confirm 1,767 hits from the primary screen, and to verify the activity for purchased/synthesized compounds.

Summary AID: AID 488970

Assigned AID: AID 488966

B. 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

C. Secondary Assay 1: Screening for Inhibitors of Bacterial Capsule Biogenesis - E.coli strain UT189 with C7 control

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 alamarBlue reagent.

Summary AID: AID 488970

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

D. Secondary Assay 2: Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition

Purpose: This secondary assay measures compound mechanistic specificity for inhibition of bacterial capsule formation and excludes inhibitors of phage replication using a different bacteriophage (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

E. Secondary Assay 3: 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. The ability of the test compounds to inhibit the K1 capsule formation was measured by a reduction in absorbance due to 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 Delta Region II capsule mutant bacterial strains.

Summary AID: AID 488970

Assigned AID: AID 504733, AID 624060

F. Secondary Assay 4: K5 Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis

Purpose: 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 using a method identical to the T7 assay test with modification of only the test strain and bacteriophage 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 and promoted lysis in the T7 phage assay were analyzed using this assay. The positive control drug C7 was used in this assay.

Summary AID: AID 488970

Assigned AID: AID 624061

2.2. Probe Chemical Characterization

Probe Chemical Structure and Properties

Figure 1. Structure Verification and Purity.

Figure 1Structure Verification and Purity

1H NMR, 13C NMR, RP HPLC/UV/HRMS Data

Proton and carbon NMR data for ML333/MLS004555969/SID 103147597/CID 18109210: Detailed analytical methods used and the 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. The associated spectra are included in Appendix B.

Proton NMR Data for ML333/SID 103147597/CID 18109210:1H NMR (400 MHz, DMSO) δ 10.76 (s, 1H), 9.60 (s, 1H), 8.12 (d, J = 1.2 Hz, 1H), 8.51 (dd, J = 1.2, 5.2 Hz, 2H), 8.25 (d, J = 8.8 Hz, 1H), 8.12 (dd, J = 1.6, 6.8 Hz, 1H), 7.81 (dd, J = 1.2, 3.6 Hz, 2H) ppm.

Carbon NMR Data for ML333/SID 103147597/CID 18109210:13C NMR (100 MHz, DMSO) δ 165.9, 159.6, 155.1, 150.3, 145.8, 133.7, 131.3, 125.9, 122.9, 113.9 ppm.

RP HPLC/UV/HRMS Data for ML333/SID 103147597/CID 18109210: Detailed analytical methods used and the 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 103147597 (CID 18109210) revealed purity of 100.0 % (retention time = 2.287 minutes). The experimental RP HPLC/UV/HRMS spectra are included in Appendix B. HRMS (m/z) calcd. for C13H9N3OS [M+H+] 256.0539, found 256.0543.

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 (34). Probe ML333 (SID 103147597/CID 18109210) was found to have a solubility of 104 μg/mL, or 407 μM, under these conditions. Solubility was also assessed in primary assay media (Luria-Bertani Broth). Probe ML333 was determined to have an assay media solubility of >126 μg/mL or >662 μM. The solubility in PBS buffer is good, and only slightly less than in assay media, and, in any case, the solubility for the probe is well above its activity in the K1 and 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 the percent compound remaining after 48 hours. ML333 was found to be quite stable (>75% remaining) in both solvent systems under these experimental conditions over the 48 hour study (Figure 2).

Figure 2. Graph depicting the stability for ML333/KUC107756 after 48 hours in two separate solvent systems.

Figure 2

Graph depicting the stability for ML333/KUC107756 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 (35).

Figure 3. Graph depicting stability of the probe to thiol.

Figure 3

Graph depicting stability of the probe to thiol.

ML333 was found to be completely stable to the presence of five times its concentration of both glutathione and DTT across all time points in our analysis (Figure 3). Statistically, there is no significant drop in the % remaining concentration of ML333 during the course of these experiments. This data is easily contrasted to the data for the ethacrynic acid positive 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 ML333 and numerous analogues were synthesized using the reaction sequence shown in Figure 4.

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 (Table 1). The structures for the five supporting analogues are shown in Figure 5.

Table 1. Five probe analogues submitted to the NIH MLSMR, and their associated screening data.

Table 1

Five probe analogues submitted to the NIH MLSMR, and their associated screening data.

Figure 5. Five analogues chosen to support probe ML333.

Figure 5

Five analogues chosen to support probe ML333.

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. Fraction collection was 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 flow rate pH 9.8 NH4OH aqueous mobile phase.

The probe was prepared using the following protocols

Preparation of N-(pyridin-4-yl)benzo[d]thiazole-6-carboxamide (ML333)
Step 1

Benzo[d]thiazole-6-carboxylic acid (0.181 g, 1.012mmol) was dissolved in 10 mL of dichloromethane in a round bottomed flask containing a stir bar. N,N-Dimethylformamide (7.40 mg, 0.101 mmol) was added to this stirring mixture, and then the mixture was cooled to 0 °C. A solution of oxalyl chloride (0.193 g, 0.132 mL, 1.518 mmol) in 3 mL of dichloromethane was added dropwise. The reaction was allowed to slowly warm to room temperature and then to further mix for one hour. The reaction was concentrated in vacuo to remove solvent and excess oxalyl chloride, while not heating over 30 °C. This crude mixture was then redissolved in 2 mL of dichloromethane and added dropwise to a solution of pyridin-4-amine (0.095 g, 1.012 mmol), triethylamine (0.358 g, 0.494 mL, 3.54 mmol) in dichloromethane (2 mL) that had been previously placed in Mettler-Toledo Bohdan MiniBlock™ reaction tube (Mettler-Toledo Autochem Reaction tubes 10.0 mi Part # 1352118) (Note: 6 × 4 MiniBlock™ setups were used to generate 24 different products per block in parallel). After the addition, the septum layer and cover plate were secured onto the MiniBlock™ with spring clamps. The block was then secured onto a Bohdan MiniBlock™ Compact Shaking and Washing Station, in which the shaker was set at 600 rpm for 16 hours. The MiniBlock™ was then removed from the shaker, followed by a subsequent draining of the reaction mixture into a second MiniBlock™ containing a Biotage ISOLUTE ® SPE Accessories Phase Separator Tube (Part # 120-1905-CG), containing water (3 mL). A cover plate was placed on the second MiniBlock™ containing the reaction mixture, and then the MiniBlock™ was placed on the shaker and was allowed to shake for five minutes at 600 rpm. After removal of the MiniBlock™ from the shaker, the organic phase was allowed to drain into a sample collection tube. Sample was concentrated in vacuo in a GeneVac HT-4X centrifugal evaporator and then purified via automated preparative reverse-phase HPLC purification (method listed in Section 3) to give N-(pyridin-4-yl)benzo[d]thiazole-6-carboxamide (0.228 g, 88 % yield).

3. Results

3.1. Summary of Screening Results

Primary assay: An end-point assay to measure the amount of K1 phage-induced bacterial cell lysis was employed to determine test compound effect on capsule biogenesis. K1 bacteriophage specifically binds the bacterial capsule during the initial stages of infection. Bacteria without capsule cannot be infected and lysed by the bacteriophage. The ability of the test compounds to inhibit the K1 capsule formation was measured by an increase in the fluorescence of alamarBlue, which correlated with the amount of intact bacterial cells compared to control reaction wells. Inhibition of phage-induced lysis indicated an active compound in the primary screen. A total of 338,740 compounds were screened at 100 μM in the primary screen. Inhibition of phage lysis was calculated relative to the mean of the bacterial (positive) control on each microtiter plate. Primary screen average Z values = 0.75; average signal to background (S/B) = 11; and average coefficient of variance = 4.6%. For calculation of S/B/Z value, and CV, the following formulae (in which SD stands for standard deviation) were used: S/B = mean signal/mean background; Z = 1 − 3SD of sample + 3SD of control mean of sample - mean of control; % CV = (SD mean signal/mean signal) × 100.

Confirmatory efficacy: The background cutoff (15% inhibition) for the primary screen was calculated using the mean of all compound results plus 3× SD. Compounds that inhibited more than 30% (1,767) were considered for evaluation by confirmatory dose response and cytotoxicity counter screening assays. Only 1,219 compounds were available for confirmatory assays. The confirmatory efficacy assay was performed as described for the primary screen except that each compound was tested at 10 concentration points starting from 300 μM and continuing to lower concentrations by serial 2-fold dilutions to 0.69 μM. Twenty-six compounds were confirmed as effective at inhibiting the formation of capsule at compound concentrations below 50 μM. Confirmatory screen average Z values = 0.79; average signal to background (S/B) = 32; and average coefficient of variance = 6.4%. IC50 values were calculated using a 4 parameter Levenburg-Marquardt algorithm, with the maximum and minimum locked at 0 and 100 respectively.

Cytotoxicity assay: The cytotoxicity assay was performed as described in Appendix A. Hit cytotoxicity and the 50% toxic concentration (CC50) were determined and compared to the IC50 to calculate the selectivity index. Twenty-nine compounds were tested, and 11 were inactive (CC50 > 50 μM).

Figure 6. HTS to compound hit flowchart.

Figure 6HTS to compound hit flowchart

Outcome

3.2. 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 ML333 potency in the primary phase lysis inhibition assay was determined to be IC50 = 1.04 ± 0.13 μM, and the CC50 239 ± 89 μM. The calculated selectivity was 230 (CC50/IC50). The ML333 concentration response curves for efficacy and cytotoxicity are graphed (Figure 7).

Figure 7. Efficacy and cytotoxicity analysis of ML333.

Figure 7

Efficacy and cytotoxicity analysis of ML333. The graph on the left (green line) shows the efficacy of the probe in the primary bacterial phage lysis inhibition assay (n = 8). The small gray inset magnifies the corresponding region in the larger graph. (more...)

3.3. Scaffold/Moiety Chemical Liabilities

The probe compound and analogues appears to be stable based on our observations from day-to-day handling related to their synthesis, analysis, dissolution-transfer, lyophilization, and storage. The probe compound and analogues do not contain moieties that are known or expected to be reactive. The probe was found to be stable in aqueous solution and in the presence of thiol (please see section 2.2).

3.4. SAR Tables

The high throughput screen of 338,740 compounds at 100 μM concentration in the primary screen (AID 488966) resulted in a number of validated compound hits. One of the compound hit chemotypes the team chose to explore further resided in a cluster of nine compounds, in which one, Table 2, entry 1, exhibited 53% inhibition at 100 μM concentration. Related compounds in this chemotype, Table 2, entries 2–9 were found to be inactive at 100 μM.

Table 2. HTS data for compounds in the benzothiazole chemotype.

Table 2

HTS data for compounds in the benzothiazole chemotype.

Considering the limited SAR information available for this chemotype, for the initial SAR exploration, the team chose to focus on varying the benzothiazole, amide, and pyridyl sub-structural components of the chemotype, as shown schematically in Figure 8.

Figure 8. Regions of the compound hit identified for initial SAR exploration.

Figure 8

Regions of the compound hit identified for initial SAR exploration.

In particular, in a combinatorial fashion, the benzothiazole moiety was replaced using the quinolone, indole, and benzimidazole ring systems, while the 4-pyridyl moiety was replaced using p-methylphenyl, p-trifluoromethyl-phenyl, p-methoxyphenyl, 2-pyridyl, 3-pyridyl, o-fluorophenyl, m-fluorophenyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl, p-acetylphenyl, and furan-2-yl-methyl moieties. Each compound was screened to determine IC50 values in the K1 phage, human bladder cell cytotoxicity, and T7 phage assays. To our surprise, only one of the 49 analogues synthesized (Table 3, entry 42) showed any hint of activity.

Table 3. First round of SAR study on synthesized analogues.

Table 3

First round of SAR study on synthesized analogues.

Since none of the more significant structural modifications studied in the first round of SAR exploration resulted in active compounds, the team decided that the second round of compounds for SAR study should consist of only slightly modified analogues (Table 4). Specifically, the pyridyl moiety was substituted to give 2-bromopyridyl, 2,6-dichloropyridyl, 2-methoxypyridyl, 2-chloropyridyl, 2-hydroxypyridyl, 2-fluoropyridyl, and 3-fluoropyridyl analogues. To explore the SAR around the amide bond, we included the N-methyl analogue (entry 1), the reverse amide (entry 7), as well as amide-cyclized oxazolo[4,5-b]pyridyl and oxazolo[5,4-c]pyridine analogues (entries 8 and 9). Each compound was screened to determine IC50 values in the K1 phage, human bladder cell cytotoxicity, and T7 phage assays. Entries 2, 5, 6, and 12 of Table 4 all exhibited activity within the range of our desired probe criteria, showing that mono-halogenation and methoxy substitutions on the pyridyl moiety were tolerated. However, N-methylation of the amide bond, reversal of the amide bond, di-substitution on the pyridyl ring, hydroxyl substitution of the pyridyl ring or cyclization through the amide bond onto the pyridyl ring were not tolerated. Replacing the pyridyl group in the molecule with a phenyl group (entry 16) reduced activity significantly.

Table 4. Second round of SAR study on synthesized analogues.

Table 4

Second round of SAR study on synthesized analogues.

The third round of SAR study focused on very specific modifications to the pyridyl portion of the hit compound (Table 5). Saturated, homologated, and dihomologated analogues were prepared. The parent carboxylic acid, benzothiazole-6-carboxylic acid, was also tested. Each compound was screened to determine IC50 values in the K1 phage, human bladder cell cytotoxicity, and T7 phage assays. None of these analogues showed biological activity.

Table 5. Third round of SAR study on synthesized analogues.

Table 5

Third round of SAR study on synthesized analogues.

For the final round of SAR study, the team explored combining the most promising substitutions discovered from our studies on the pyridyl ring with substitutions on the, as yet, unexplored 2-position of the benzothiazole ring (Table 6, entries 1–32). At this stage of the project, compounds were screened for activity in the T7 phage assay (and were not screened using the K1 phage assay) and for cytotoxicity against human bladder cell carcinoma 5637 cells. This final round of SAR resulted in some of the most potent and selective analogues prepared, to date, entries 13 and 17. In the case of entry 13, methyl-group substitution adjacent to the nitrogen atom of the pyridine ring and introduction of a Boc-protected-amine at the 2-position of the benzothiazole provided a compound with an IC50 of 490 nM in the T7 phage assay and a selectivity index of nearly 200. In addition, methyl-group (entries 10 and 11) and amine-group (entry 23) substitution was shown to be tolerated at the 2-position of the benzothiazole.

Table 6. Fourth round of synthetic analogues.

Table 6

Fourth round of synthetic analogues.

Probe Selection

Several compounds generated during these studies met the criteria established at the outset of the project for a useful probe compound (see Section 2). Early in the project, however, ML333 was found to have good activity across the suite of assays used to define the probe criteria and in a suite of in vitro PK assays. In addition, in PubChem, the probe ML333 is reported to have shown activity in only 3 (distinct) of 379 bioassays in which it was tested (<1% hit rate), which suggests that ML333 is not a promiscuous hitter. In as much, this compound was nominated as the probe candidate and used for advanced, preliminary studies such as those described in sections 4.2 and 4.3. In any case, as was mentioned previously, quite a number of useful compounds were identified across the ML333 chemotype, and, depending on the specific intended use for these compounds, many of them could have been chosen as probes (for example, CID 53484225, entry 2, Table 4; CID 53484232, entry 5, Table 4; CID 53484228, entry 6, Table 4; CID 126587005, entry 12, Table 4; CID 57412031, entry 13, Table 6; and CID 57412040, entry 17, Table 6).

3.5. Cellular Activity

ML333 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 (300 μM), and the eukaryotic 50% cytotoxic concentration (CC50) was 239 ± 89 μM, which afforded a calculated selectivity index of 230. 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 analog were routinely tested using the cytotoxicity counterassay.

3.6. Profiling Assays

In vitro pharmacokinetics profiling

The in vitro pharmacokinetic (PK) properties of the probe (ML333) were profiled using a standard panel of assays (Table 7) across which the probe displayed encouraging results.

Table 7. Summary of in vitro ADME properties of ML333.

Table 7

Summary of in vitro ADME properties of ML333.

Broad-spectrum target profiling

The probe compound ML333 was submitted to Ricerca for LeadProfiling to assess off-target pharmacology. The probe was tested in duplicate at 10 μM concentration and significant activity was noted for only one target across the panel of 67 targets (i.e., norepinephrine transporter, 58% inhibition, see Appendix D for the complete list of results). In addition, in PubChem, the probe ML333 is reported to have shown activity in only 3 (distinct) of 379 (<1%) bioassays in which it was tested. These results suggest that ML333 is not a promiscuous compound with respect to off-target effects.

4. Discussion

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.

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

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 (33) and the previous MLPCN probe compound ML317. 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, along with the MLPCN probe ML317, serves as the only prior art for this probe development project. Relative to C7, the probe discovered during this 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 9). Furthermore, we have demonstrated the synthetic tractability for the ML333 chemotype, and prepared an array of highly active analogues, whereas C7 does not share either of positive these attributes. To further demonstrate the usefulness of ML333 as a probe, we have shown ML333 to have acceptable in vitro pharmacokinetic properties. Aqueous stability in 1× 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 acceptable. In contrast, while C7 has not been screened in such in vitro PK assays, one might predict poor results based on its chemical structure and calculated physiochemical properties. 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 ML333. Compared to the previous MLPCN probe ML317, the current probe compound ML333, has a better selectivity index (BC5637 bladder cells TC50/K1 phage IC50), comparable aqueous solubility, permeability, and plasma stability (human and mouse), whereas the hepatic microsome stability is observed to be slightly less promising.

Figure 9. Structures and select properties for C7, ML317, and ML333.

Figure 9

Structures and select properties for C7, ML317, and ML333.

4.2. Mechanism of Action Studies

Moving forward, genetic and biochemical approaches are being employed to identify the mechanism of action for the probe. Currently, overexpression of a whole genome open reading frame library of E. coli is being used to identify gene products that enhance or reduce susceptibility to the probe. A number of plasmids expressing open reading frames have been selected in the screen and the identity and function of gene products are being analyzed. The target of the probe may be within a signal transduction pathway to capsule regulation but distinct from the actual transcription factor affecting capsule expression. Cellular localization of probe-interacting factors and biochemical studies of probe-target interactions will be explored.

Recent studies have also employed chemical mutagenesis to derive strains resistant to the action of the probe. Whole genome sequencing using Illumina Hiseq technology has been completed on five independent isolates. An analysis of polymorphisms shared among independent resistant bacterial clones will be used to localize putative factors that interact with the probes. These may be transporters or the actual target of the probe. The genes from the mutant and wild type strains will be cloned and expressed in the isogenic bacterial backgrounds to ascertain their roles in sensitivity to the probe.

4.3. Planned Future Studies

The probe will be optimized further for potency, selectivity, physiochemical properties, and in vitro pharmacokinetics (using the K1 phage, T7 phage, human bladder cell cytotoxicity, aqueous solubility, aqueous stability, plasma stability, plasma protein binding, logD, cell permeability, and microsomal stability assays). Based on the current SAR story, substitution at the positions R1, R2, and R3 will be explored, along with the introduction of nitrogen atoms at the positions X, as shown in Figure 10.

Figure 10. Future SAR studies.

Figure 10

Future SAR studies.

Subsequently, in vivo pharmacokinetic measurements will be performed in a preclinical murine model with additional optimization of the probe for bioavailability and renal excretion. After completion of these studies and refinements to the probe, the refined probe will be employed in prophylaxis and treatment trials using preclinical murine infection models of E. coli urinary tract infection and bloodstream infection.

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Appendix A. Detailed Assay Descriptions

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: 488966

Assigned 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, alamarBlue 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, and 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 alamarBlue 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: 488970

Assigned AID: 493020, 504769, 504831, 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 (38). 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: 488970

Assigned AID: 504358, 504543, 504675, 504768, 588321, 588386, 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 hr.

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: 488970

Assigned AID: 504349, 504538, 504676, 504767, 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: 488970

Assigned AID: 504733, 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: 488970

Assigned 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.

Appendix B. Structure Verification and Purity: 1H NMR, 13C NMR, RP HPLC/UV/HRMS Spectra for the Probe Compound

Figure B1. 1H NMR Spectra of ML333.

Figure B11H NMR Spectra of ML333

Figure B2. 13C NMR Spectra of ML333.

Figure B213C NMR Spectra of ML333

Figure B3. RP HPLC/UV/HRMS Spectra for ML333.

Figure B3RP HPLC/UV/HRMS Spectra for ML333

Appendix C. Analytical Characterization Data for the Five Supporting Analogues

Image ml333fu51

N-(3-Fluoropyridin-4-yl)benzo[d]thiazole-6-carboxamide (CID-53484233): 1H NMR (400 MHz, DMSO) δ 10.62 (s, 1H), 9.59 (d, J = 1.6 Hz, 1H), 8.83 (d, J = 1.2 Hz, 1H), 8.61 (d, J = 2.4 Hz, 1H), 8.41 (d, J = 4.0 Hz, 1H), 8.24 (d, J = 7.2 Hz, 1H), 8.12 (dd, J = 1.6, 5.6 Hz, 1H), 7.99 (t, J = 4.4 Hz, 1H) ppm; 13C NMR (100 MHz, DMSO) δ 165.6, 159.7, 155.2, 152.0, 150.0, 146.3, 146.2, 138.2, 133.6, 133.4, 133.3, 130.5, 126.1, 123.3, 122.8, 118.2 ppm; IR νmax (cm−1) 2961, 1604, 1516, 1408, 1033; HRMS calcd for C13H8FN3OS [M+H+] 274.0372, found 274.0433.

Image ml333fu52

N-(2-Fluoropyridin-4-yl)benzo[d]thiazole-6-carboxamide (CID- 53484226): 1H NMR (400 MHz, DMSO) δ 11.02 (s, 1H), 9.61 (s, 1H), 8.81 (d, J = 1.6 Hz, 1H), 8.25 (d, J = 6.8 Hz, 1H), 8.17 (d, J = 4.8 Hz, 1H), 8.13 (dd, J = 1.6, 5.2 Hz, 1H), 7.66 (m, 1H), 7.27 (d, J = 1.6 Hz, 1H) ppm; 13C NMR (100 MHz, DMSO) δ 166.2, 164.8, 162.9, 159.8, 155.2, 150.1, 150.4, 147.9, 133.8, 130.9, 125.9, 123.1, 122.9, 112.5, 98.5, 98.2 ppm; IR νmax (cm−1) 1607, 1512, 1055, 1033; HRMS calcd for C13H9N3OS [M+H+] 274.0372, found 274.0359.

Image ml333fu53

N-(2-Chloropyridin-4-yl)benzo[d]thiazole-6-carboxamide (CID-53484228):1H NMR (400 MHz, DMSO) δ 10.94 (s, 1H), 9.61 (s, 1H), 8.81 (d, J = 1.6 Hz, 1H), 8.33 (d, J = 4.4 Hz, 1H), 8.25 (d, J = 6.8 Hz, 1H), 8.11 (dd, J = 1.2, 5.6 Hz, 1H), 7.86 (d, J = 1.6 Hz, 1H), 7.76 (dd, J = 1.6, 3.2 Hz, 1H) ppm; 13C NMR (100 MHz, DMSO) δ 166.1, 159.8, 155.2, 150.9, 150.3, 148.5, 133.8, 130.9, 125.9, 123.1, 122.9, 113.3, 113.2 ppm; IR νmax (cm−1) 3253, 3040, 1662, 1613, 1583, 1307 ; HRMS calcd for C13H9N3OS [M+H+] 290.0766, found 290.0118.

Image ml333fu54

N-(Pyridin-4-yl)benzo[d]thiazole-6-carboxamide (CID-53484229): 1H NMR (400 MHz, DMSO) δ 10.77 (s, 1H), 9.33 (s, 1H), 8.12 (dd, J = 1.2, 2.4 Hz, 2H), 8.7 (d, J = 1.2 Hz, 1H), 8.09 (d, J = 7.2 Hz, 1H), 7.90 (dd, J = 1.2, 2.4 Hz, 2H), 7.84 (2.0, 5.2 Hz, 1H) ppm; 13C NMR (100 MHz, DMSO) δ 164.2, 155.3, 150.3, 149.7, 141.8, 136.2, 134.1, 122.9, 121.6, 119.9, 113.2 ppm; IR νmax (cm−1) 3307, 2944, 2833, 1654, 1409, 1130, 1023; HRMS calcd for C13H9N3OS [M+H+] 256.0466, found 256.0541.

Image ml333fu55

N-(2-Bromopyridin-4-yl)benzo[d]thiazole-6-carboxamide (CID-53484225): 1H NMR (400 MHz, DMSO) δ 10.92 (s, 1H), 9.60 (s, 1H), 8.01 (s, 1H), 8.31 (d, J = 6.0 Hz, 2H), 8.11 (s, 1H), 7.79 (s, 1H) ppm; 13C NMR (100 MHz, DMSO) δ 166.1, 159.8, 155.2, 150.8, 148.1, 141.9, 133.8, 130.8, 125.9, 123.1, 122.9, 116.9, 113.6 ppm; IR νmax (cm−1) 2922, 1680, 1578, 1493, 1463, 1274, 868; HRMS calcd for C13H8BrN3OS [M+H+] 333.9571, found 333.9632.

Appendix D. Ricerca LeadProfiling Report for ML333

The following text was provided along with the Ricerca LeadProfiling Report for ML333.

Study Objective

To evaluate, in radioligand binding assays, the activity of probe compound ML333 across a panel of 67 receptors.

Methods

Methods employed in this study 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.

Where presented, IC50 values were determined by a non-linear, least squares regression analysis using MathIQ™ (ID Business Solutions Ltd., UK). Where inhibition constant (Ki) are presented, the Ki values were calculated using the equation of Cheng and Prusoff (Cheng. Y., Prusoff, W.H., Biochem. Pharmacol. 22:3099–3108, 1973) using the observed IC50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values of the KD of the ligand (obtained experimentally at Ricerca Biosciences, LC). Where presented, the Hill coefficient (nH), defining the slope of the competitive binding curve, was calculated using MathIQ™. Hill coefficients significantly different than 1.0, may suggest that the binding displacement does not follow the laws of mass action with a single binding site. Where IC50, Ki, and/or nH data are presented without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (Ki, IC50, nH) should be interpreted with caution.

Cat #Assay NameBatch*Spec.Rep.Conc.% Inh.
Compound: KUC107756N-04, PT #: 1160397
200510Adenosine A1314096hum210 μM−17
200610Adenosine A2A314151hum210 μM−6
200720Adenosine A3314213hum210 μM−5
203100Adrenergic α1A314153rat210 μM−3
203200Adrenergic α1B314154rat210 μM14
203400Adrenergic α1D314156hum210 μM7
203620Adrenergic α2A314100hum210 μM−14
204010Adrenergic β1314161hum210 μM3
204110Adrenergic β2314163hum210 μM8
285010Androgen (Testosterone) AR314097rat210 μM5
212510Bradykinin B1314221hum210 μM12
212620Bradykinin B2314223hum210 μM4
214510Calcium Channel L-Type, Benzothiazepine314226rat210 μM−1
214600Calcium Channel L-Type, Dihydropyridine314169rat210 μM18
216000Calcium Channel N-Type314227rat210 μM−18
217030Cannabinoid CB1314171hum210 μM20
219500Dopamine D1314173hum210 μM4
219700Dopamine D2S314174hum210 μM−6
219800Dopamine D3314297hum210 μM6
219900Dopamine D4,2314238hum210 μM0
224010Endothelin ETA314240hum210 μM−4
224110Endothelin ETB314241hum210 μM3
225510Epidermal Growth Factor (EGF)314243hum210 μM8
226010Estrogen ERα314245hum210 μM13
226600GABAA, Flunitrazepam. Central314115rat210 μM12
226500GABAA, Muscimol. Central314175rat210 μM4
228610GABAB1A314248hum210 μM7
232030Glucocorticoid314321hum210 μM16
232700Glutamate, Kainate314298rat210 μM3
232810Glutamate, NMDA, Agonism314451rat210 μM−8
232910Glutamate, NMDA, Glycine314253rat210 μM6
233000Glutamate, NMDA, Phencyclidine314178rat210 μM2
239610Histamine H1314180hum210 μM−16
239710Histamine H2314258hum210 μM2
239820Histamine H3314302hum210 μM13
241000Imidazoline I2, Central314181rat210 μM−23
243520Interleukin IL-1314186muose210 μM4
250460Leukotriene, Cysteinyl CysLT1314262hum210 μM19
251600Melatonin MT1314266hum210 μM7
252610Muscarinic M1314320hum210 μM10
252710Muscarinic M2314183hum210 μM5
252810Muscarinic M3314185hum210 μM−3
257010Neuropeptide Y Y1314271hum210 μM2
257110Neuropeptide Y Y2314303hum210 μM−6
258590Nicotinic Acetylcholine314188hum210 μM22
258700Nicotinic Acetylcholine α1, Bungarotoxin314189hum210 μM8
260130Opiate δ (OP1, DOP)314191hum210 μM10
260210Opiate κ(OP2, KOP)314304hum210 μM10
260410Opiate μ(OP3, MOP)314194hum210 μM1
264500Phorbol Ester314195mouse210 μM1
265010Platelet Activating Factor (PAF)314197hum210 μM−1
265600Potassium Channel [KATP]314201ham210 μM12
265900Potassium Channel hERG314202hum210 μM9
268420Prostanoid EP4314208hum210 μM−4
268700Purinergic P2X314300rabbit210 μM6
268810Purinergic P2Y314301rat210 μM1
270000Rolipram314203rat210 μM3
271110Serotonin (5-Hydroxytryptamine) 5-HT1A314275hum210 μM11
271700Serotonin (5-Hydroxytryptamine) 5-HT2B314205hum210 μM19
271910Serotonin (5-Hydroxytryptamine) 5-HT3314281hum210 μM−7
278110Sigma σ1314095hum210 μM9
255520Tachykinin NK1314269hum210 μM7
285900Thyroid Hormone314309rat210 μM20
220320Transporter, Dopamine (DAT)314094hum210 μM−13
226400Transporter, GABA314299rat210 μM−4
204410Transporter, Norepinephrine (NET)314086hum210 μM58
274030Transporter, Serotonin (5-Hydroxytryptamine)314284hum210 μM5

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