Identification of small molecules that selectively inhibit fluconazole-resistant Candida albicans in the presence of fluconazole but not in its absence

Hartland CL, Youngsaye W, Morgan B, et al.

Publication Details

The effectiveness of the potent antifungal drug fluconazole has been compromised by the rise of drug-resistant fungal pathogens. It has been observed that inhibition of heat shock protein 90 (Hsp90) can reverse drug resistance in Candida; however, it is challenging to find fungal-specific inhibitors of Hsp90 that do not also impair the human host protein. The Molecular Libraries Probe Production Centers Network (MLPCN) library was screened in duplicate dosings to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline (CID 1251584) was identified as the initial hit, and after considerable SAR studies, was selected as the new probe compound (CID 1251584/ML189).

Assigned Assay Grant No: 1 R03 MH086456-01

Screening Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber, PhD

Chemistry Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber, PhD

Assay Submitter & Institution: Susan Lindquist, PhD. Whitehead Institute, Cambridge, MA

PubChem Summary Bioassay Identifier (AID): 2007

Probe Structure & Characteristics

ML 189.

ML 189

Recommendations for the scientific use of this probe

Acquired drug resistance by medically relevant microorganisms poses a grave threat to human health and has enormous economic consequences. Fungal pathogens present a particular challenge because they are eukaryotes and share many of the same mechanisms that support the growth and survival of the human host cells they infect. The number of drug classes that have unique targets in fungi is very limited, and the usefulness of current antifungal drugs is compromised by either dose-limiting host toxicity or the frequent emergence of high-grade resistance.

The objective of this project is to discover compounds capable of reversing fungal drug resistance, thereby making currently available antifungal drugs more effective and reducing dosage-related side effects. Inhibition of the molecular chaperone Hsp90 is one approach that can abrogate drug resistance in diverse fungi including species of both Candida and Aspergillus (1). At concentrations that demonstrate no antifungal activity on their own, classical Hsp90 inhibitors dramatically reduce azole antibiotic resistance of clinical isolates and transform conventional fungistatic azole compounds into fungicidal drug combinations. This probe could greatly improve the ability to control otherwise lethal fungal infections in severely immunocompromised hosts, especially cancer patients undergoing high-dose chemotherapy and/or bone marrow transplantation. This high-throughput screen (HTS) project seeks to identify novel small molecules that can prevent fungal growth in combination with a sublethal dose of fluconazole.

1. Introduction

Scientific Rationale

Modulators of the fungal stress response could greatly improve the ability to control otherwise lethal fungal infections in immunocompromised hosts, especially cancer patients undergoing high-dose chemotherapy and/or bone marrow transplant procedures. Although Hsp90 and calcineurin inhibitors under current development appear to be well-tolerated in early phase cancer clinical trials, compromise of host chaperone protein function could have very deleterious effects in the context of active infection and the associated stresses of fever and cardiovascular instability. For example, host immune response is compromised by treatment with nonselective calcineurin inhibitors such as cyclosporin A and FK506. An obvious way to avoid this problem would be the identification of fungal-specific inhibitors that do not impair the host chaperone protein. To address these concerns, this project seeks to identify new fungal-selective chemosensitizers. In particular, the design of the screening cascade will also allow capture of compounds targeting still other, as yet unknown, components of fungal stress response pathways that enable the emergence and maintenance of resistance to current antifungal drugs.

Several compounds have been previously identified as chemosensitizers, increasing the susceptibility of various C. albicans strains to fluconazole treatment (2,3,4,5). Cernicka et al. previously reported that the compound 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT, Figure 1) was capable of chemosensitizing C. albicans strains to fluconazole (3). Against fluconazole-susceptible C. albicans strain 90028 and fluconazole-resistant C. albicans strain Gu5, CTBT was effective with an MIC value of 2.4 μM when combined with fluconazole. In the absence of fluconazole, CTBT demonstrated no activity against C. albicans strain 90028, but did inhibit growth of C. albicans strain Gu5 at concentrations greater than 2.4 μM. The antiarrhythmic drug amiodarone was recently demonstrated to act synergistically with fluconazole in C. albicans with MIC values ranging between 1.6 μM to 18.8 μM (4). Plagiochin E, a natural product isolated from liverwort, increased yeast susceptibility to fluconazole at 2.4 μM (5). These agents were not considered for the current project because of their documented single-agent antifungal activity (amiodarone, an MIC50 value of 3.1 μM [6]; plagiochin E, an IC50 value of 3.8 μM [5]).

Figure 1. Chemosensitizing Agents for Reversing Fluconazole Resistance.

Figure 1

Chemosensitizing Agents for Reversing Fluconazole Resistance.

The most potent compounds currently known are several HDAC inhibitors previously reported by Mai et al. (7). As depicted in Figure 2, compounds 4 and 5 are uracil-derived hydroxamic acids that exhibited MIC values ranging from 1.2 μM to 1.4 μM when combined with fluconazole. When tested independently, neither compound demonstrated activity against C. albicans at concentrations up to 368 μM. When compounds 4 and 5 were evaluated in a biochemical binding assay with murine HDAC1, their IC50 values were measured at 37 and 51 nM, respectively (7). This finding suggests that these HDAC inhibitors would not be particularly selective for fungal protein targets and diminishes their potential as fungal-selective chemosensitizers. At the present time, neither compound 4 nor 5 have been registered with MLSMR and were not available for evaluation in the current investigation.

Figure 2. Uracil-derived HDAC Inhibitors Capable of Reversing Antifungal Drug Resistance.

Figure 2

Uracil-derived HDAC Inhibitors Capable of Reversing Antifungal Drug Resistance.

Ideally, this endeavor will identify small molecule probes possessing novel modes of action to complement existing Hsp90, calcinuerin, and HDAC inhibitors. If necessary, however, an alternative approach to identify chemosensitizers entails investigation of fungal selective HDAC inhibitors.

2. Materials and Methods

Compounds that can successfully inhibit the growth of fluconazole resistant Candida string CaCi-2 in the presence of sublethal doses of fluconazole as measured in a fluorescence reporter assay are further tested against a more highly-resistant Candida CaCi-8 strain in the presence of fluconazole. Those that inhibit growth in this more resistant strain are also tested for toxicity against Candida in the absence of fluconazole, and for toxicity against mammalian cells using a luminescence reporter assay. The positive control, acting in concert with fluconazole, for screening in the Candida and mammalian cell assays is the Hsp90 inhibitor geldanamycin (GA, Figure 3). Compounds that pass these four hurdles are binned through use of a Saccharomyces assay to determine whether the mechanism of action might be through an Hsp90 pathway, a calcineurin pathway, or an uncategorized mechanism.

Figure 3. Positive Controls for Biological Assays.

Figure 3

Positive Controls for Biological Assays.

2.1. Assays

Table 1. Summary of Assays Completed.

Table 1

Summary of Assays Completed.

Protocols

Refer to Appendix A for all detailed protocols.

Assay Materials

Clear flat bottom, black 384-well plates (Cat# 3712BC Lot# 35808016) and white 384-well plates (Cat# 8867BC, Lot 22609019) were obtained from Corning. Geldanamycin was obtained from AG Scientific (Cat# G-1047) and prepared as a 15 mM stock solution in DMSO. Pen/Strep was obtained from Gibco (Cat#10378-016 Lot 21040170) and added to a final concentration of 1% to all media. Fluconazole was obtained from Sigma (F829-100MG Lot 098K4715) and prepared as a 2 mg/mL stock solution in PBS. Alamar Blue was obtained from AG Scientific (DAL1100, Lot 151016SA). Phosphate buffered saline (PBS) without calcium and magnesium was obtained from Cellgro (Cat #21-040-CV). Radicicol (Cat# F4679) and FK506 (Cat#R2146) were obtained from Sigma. Tropix Gal-Screen was obtained from Applied Biosystems (Cat#T2359, Lot 0903044).

Growth Media

For the synthetic defined growth medium, RPMI 1640 medium (powder without sodium bicarbonate) was obtained from Invitrogen (Cat#31800-089, Lot 648072). Uridine (8 mg/mL in water; Cat#U3750, Lot 028K0760), glucose 40% (w/v) in water (Cat# G-5400), and MOPS buffer (Cat#M-1254, Lot 098K0033) were obtained from Sigma. Optimem medium was obtained from Invitrogen. Fetal Bovine Serum 2.5% (v/v) was obtained from Hyclone. SD Growth Media was obtained from MP Biomedical (Cat#4027-012, Lot 119458), and Complete supplement minus adenine was obtained from Sunrise Science (Cat #1029-100, Lot 070409). DOC steroid (Cat#D7000) and calcium chloride were obtained from Sigma (Cat#C3306, Lot 058K0664).

Cells Strains

The following test strains were used in this study:

  • Fluconazole resistant C. albicans CaCi-2 or more highly resistant strain CaCi-8 NIH-3T3
  • NIH 3T3 mammalian fibroblasts
  • ATCC 201238 Saccharomyces cerevisiae p2A/GRGZ reporter strain
  • ATCC 201238 Saccharomyces cerevisiae 4XCDRE-lacZ strain

2.1.1. Primary CaCi-2 and CaCi-2 Dose-Response Retest

Refer to Appendix A for all detailed protocols.

RPMI medium (1X) was prepared by dissolving 10.4 g powdered medium in 800 mL water. A buffer of 34.52 g MOPS was added. While stirring, pH was adjusted to 7.0 with 10N NaOH. Next, 10 mL uridine solution and 50 mL glucose solution were added. The final volume was adjusted to 1000 mL and filter sterilized.

The following yeast strain was used in this study: C. albicans CaCi-2 (9). Fungal inoculum was prepared as follows: 500 μL of strain was inoculated from cryopreserved stock into a 250-mL shaker flask containing 30 mL growth medium and shaken overnight at 30 °C. The optical density (OD 600) of 1 mL fungal culture in a cuvette was read using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank. The desired volume of fungal inoculum was diluted according to the formula as specified in the protocol. Fluconazole stock solution was added to fungal inoculum to achieve 8 μg/mL. Pen/Strep was added to a final concentration of 1% (v/v). A Thermo Combi was used to dispense 20 μL/well of assay media into all wells. Next, 25 nL test compound was pinned from compound plates into assay plates using CyBi-Well pin tool. A further 20 μL/well of culture was dispensed into the assay media in all wells. Plates were incubated in a humidified (90% humidity) Liconic incubator at 37 °C without agitation for 48 h. Alamar Blue Reagent was diluted 1:40 in Ca/Mg-free PBS. To all plates, 5 μL/well of the diluted Alamar Blue was added to the plates for a final dilution factor of 1:200. Plates were incubated a further 2 h. Relative Fluorescence Intensity (RFU) of wells was read on a standard plate reader as a measure of relative fungal growth.

2.1.2. Orthogonal Resistant Strain Dose-Response (AID 488807)

Refer to Appendix A for all detailed protocols.

RPMI medium (1X) was prepared by dissolving 10.4 g powdered medium in 800 mL water. A buffer of 34.52 g MOPS was added. While stirring, pH was adjusted to 7.0 with 10N NaOH. Next, 10 mL uridine solution and 50 mL glucose solution were added. The final volume was adjusted to 1000 mL and filter sterilized.

The following yeast strain was used in this study: C. albicans CaCi-8 (9). Fungal inoculum was prepared as follows: 500 μL of strain was inoculated from cryopreserved stock into a 250-mL shaker flask containing 30 mL growth medium and shaken overnight at 30 °C (16 h). The optical density (OD 600) of 1 mL fungal culture in a cuvette was read using a standard optical density reader with growth medium as a background blank. The desired volume of fungal inoculum was diluted according to the formula as specified in the protocol. Fluconazole stock solution was added to fungal inoculum to achieve 8 μg/mL. Pen/Strep was added to media to 1% concentration. A Thermo Combi was used to dispense 20 μL/well of assay media into all wells. Geldanamycin was dispensed in positive control wells using Thermo Combi nL for a final concentration of 3 μM. Then, 100 nL of test compound was pinned from compound plates into assay plates using a CyBi-Well pin tool. A further 20 μL/well of culture was dispensed into the assay media in all wells. Plates were incubated in a humidified (90% humidity) Liconic incubator at 37 °C without agitation for 48 h. Alamar Blue was diluted 1:40 in Ca/Mg-free PBS. To all plates, 5 μL/well of the diluted Alamar Blue was added to plates (final dilution factor 1:200). Plates were incubated a further 2 h. The (RFU) of wells was read on a standard plate reader as a measure of relative fungal growth.

2.1.3. Counterscreen Mammalian Cell Toxicity Dose Response (AID 488809)

Refer to Appendix A for all detailed protocols.

The following cells were used in this study: NIH-3T3 mammalian fibroblasts. Cells were plated in 384-well plates at 6,000/well in 20 μL assay medium. The plates were incubated overnight at 37 °C under 5% CO2. After overnight culture, compounds were pinned into wells at 100 nL/well using the CyBio CyBi-Well pinning instrument. After compounds were pinned, an additional 20 μL of assay medium supplemented with fluconazole was added to each well. The final nominal concentration in the well was 8 μg/mL fluconazole. The plates were returned to the tissue culture incubator and culture continued for 48 h at 37 °C under 5% CO2. At the completion of this incubation, Alamar Blue solution diluted 1:40 in PBS was added to each well (10 μL/well) to achieve a final dilution of 1:200. Plates were incubated for a further 2 h at 37 °C under 5% CO2. The RFU of wells was read on a standard plate reader as measure of relative cell growth.

2.1.4. Secondary No Fluconazole Dose Response (AID 488802)

Refer to Appendix A for all detailed protocols.

RPMI medium (1X) was prepared by dissolving 10.4 g powdered medium in 800 mL water. A buffer of 34.52 g MOPS was added. While stirring, pH was adjusted to 7.0 with 10N NaOH. Next, 10 mL uridine solution and 50 mL glucose solution were added. The final volume was adjusted to 1000 mL and filter sterilized.

The following yeast strain was used in this study: C. albicans CaCi-2 (9). Fungal inoculum was prepared as follows: 500 μL of strain was inoculated from cryopreserved stock into a 250 mL shaker flask containing 30 mL growth medium and shaken overnight (16 h) at 30 °C. The OD 600 of 1 mL of fungal culture in a cuvette was read using a standard optical density reader with growth medium as a background blank. The desired volume of fungal inoculum was diluted according to the formula as specified in the protocol. Pen/Strep was added to the media to 1% concentration. A Thermo Combi was used to dispense 20 μL/well of assay media into all wells. Geldanamycin and fluconazole were mixed for positive control. Positive control solution was dispensed in positive control wells using Thermo Combi nL for a final concentration of 3 μM Geldanamycin and 8 μg/ml Fluconazole. Then, 100 nL of test compound was pinned from compound plates into assay plates using a CyBi-Well pin tool. A further 20 μL/well of culture was dispensed into the assay media in all wells. Plates were incubated in a humidified (90% humidity) Liconic incubator at 37 °C without agitation for 48 h. Alamar Blue was diluted 1:40 in Ca/Mg-free PBS. To all plates, 5 μL/well of the diluted Alamar Blue was added to the plates (final dilution factor 1:200). Plates were incubated a further 2 h. The RFU of wells was read on a standard plate reader as measure of relative fungal growth.

2.1.5. Hsp90 Binning

Refer to Appendix A for all detailed protocols.

To prepare the assay media, 100 mL 20% dextrose and 780 mg Complete Supplement were added to 100 mL SD Growth Media. Water was added to a final volume of 1L and the solution was filter sterilized. For DOC media, 1 mL DOC was added to 100 mL SD-ADE media.

The following test strain was used in this study: Saccharomyces cerevisiae p2A/GRGZ reporter strain. Cell inoculum was prepared as follows: Reporter Saccharomyces strain was inoculated from cryopreserved stock into a 250 mL shaker flask containing 20 mL SD-ADE media. The flask was incubated overnight (16 h) at 37 °C and 150 RPM. The OD 600 of 1 mL of culture in a cuvette was read using a standard optical density reader with growth medium as a background blank. Cells were diluted to OD = 0.04 in SD-ADE media. To each well of 384 white plates was added 20 μL of diluted culture using a Thermo Combi. Then, 100 nL of test compounds were pinned into plates with a CyBi-Well pin tool. Next, 5 μM Radicicol was added as a positive control in control wells, dispensing with Thermo Combi nL. With Combi, 20 μL of 20 μM DOC (steroid) in SD-ADE media was dispensed in pinned plates. Plates were incubated at 30°C for 75 minutes with agitation. Using Combi, 40 μL Gal-Screen reagent was dispensed. Plates were incubated at 30°C for 25 minutes. Luminescence of wells was read on a standard plate reader as a measure of relative fungal growth.

2.1.6. Calcineurin Binning

Refer to Appendix A for all detailed protocols.

To prepare the assay media, 100 mL 20% dextrose and 770 mG Complete Supplement was added to 100 mL SD Growth Media. Water was added to a final volume of 1L. Filter sterilize. For SD-URA plus 1.25 M calcium chloride, 3.68 g calcium chloride was added to 100 mL SD-ADE media. The media was filter sterilized through a 0.2 μm filter.

The following yeast strain was used in this study: Saccharomyces cerevisiae 4XCDRE-lacZ strain (Stathopoulos, A.M., and Cyert, M.S. 1997. Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes & Dev. 11:3432-44.) Cell Inoculum was prepared as follows: Reporter strain Saccharomyces cerevisiae 4XCDRE-lacZ was inoculated from cryopreserved stock into 250 mL shaker flask containing 20 mL SD-URA media. The flask was incubated overnight (16 h) at 37 °C and 150 RPM. The optical density (OD 600) of 1 mL of culture in a cuvette was read using a standard optical density reader with growth medium as a background blank. Cells were diluted to OD = 0.01 in SD-URA media. To each well of 384-well white plates was added 35 μL of diluted culture using a Thermo Combi. Then, 100 nL of test compounds were pinned into plates with a CyBi-Well pin tool. Next, 5 μM FK506 was added as positive control in control wells, dispensing with Thermo Combi nL. Plates were incubated at 23°C for 75 minutes with agitation. Then, 5 μL of SD-URA were added to each well with 1.25 M calcium chloride and incubated at 30°C for 90 minutes, with agitation. Using Combi, 40 μL Gal-Screen reagent was dispensed. The plates were incubated at 30°C for 25 minutes. Luminescence of wells was read on a standard plate reader as measure of relative fungal growth.

2.2. Probe Chemical Characterization

After preparation as described below, the probe (CID 1251584, SID 99376514, ML 189) was analyzed by HPLC, 1H and 13C NMR, and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy were consistent with the structure of the probe, and HPLC indicated an isolated purity of greater than 99%.

The observed solubility of the probe was calculated to be less than 1 μM in PBS. Aqueous stability in PBS was monitored over a 48-hour period, and the data is provided below (Figure 4). Precipitation of the poorly soluble probe was believed responsible for the apparent loss of material in the PBS stability assay (Figure 4, blue line). To address this possibility, DMSO was added to each well after the initial UPLC injection, and the resulting PBS/DMSO mixture was analyzed to determine the presence of ML189. The results of the DMSO recovery are provided in Figure 4 (red line) and indicate that the probe is still intact after 48 hours.

Figure 4. Probe Stability in PBS solution at 23 °C.

Figure 4

Probe Stability in PBS solution at 23 °C.

Plasma protein binding (PPB) was determined to be greater than 99% bound in human plasma and 98% bound in murine plasma. The probe is stable in human and murine plasma with greater than 99% remaining after a 5-hour incubation period. The solubility, PPB, and plasma stability results are summarized in Section 3.4 (Table 3, entry 1). The probe and five additional analogs were submitted to the SMR collection [MLS003179195 (probe), MLS003179198, MLS003179199, MLS003179197, MLS003179196, MLS003179194].

2.3. Probe Preparation

Figure 5. Scheme 1: Synthesis of Probe.

Figure 5Scheme 1: Synthesis of Probe

The probe compound 10 (CID 1251584, SID 99376514, ML 189) was prepared in a two-step sequence beginning with commercially available 4,7-dichloroquinoline. 4,7-Dichloroquinoline was combined with excess piperazine and subsequently heated in triethylamine to facilitate nucleophilic substitution. The resulting product was coupled with 4-chlorobenzoic acid to afford the probe. Full experimental details are provided below.

General details. All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz or Varian UNITY INOVA 500 MHz spectrometer as indicated. Proton and carbon chemical shifts are reported in ppm (δ) relative to tetramethylsilane or CDCl3 solvent (1H δ 7.24, 13C δ 77.0). NMR data are reported as follows: chemical shifts, multiplicity (obs. = obscured, br = broad, s = singlet, d = doublet, t = triplet, m = multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated NMR data were collected at 25 °C. Flash chromatography was performed using 40–60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash Rf system. Tandem Liquid Chromatography/Mass Spectrometry (LCMS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with ultraviolet (UV) light and aqueous potassium permanganate (KMnO4) stain followed by heating. High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility with a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer.

Image ml189fu3

Step 1. Preparation of 7-chloro-4-(piperazin-1-yl)quinoline (8): 4,7-Dichloroquinoline (2.00 g, 10.1 mmol) was combined with piperazine (4.35 g, 50.5 mmol) and neat triethylamine (2.81 mL, 20.2 mmol). The reaction flask was sealed under nitrogen and heated to 130 °C whereupon the solid material gradually melted to give a clear orange-yellow solution. After a period of time, a yellow precipitate was observed. The reaction was completed in approximately 4 hours as determined by TLC (5% v/v methanol in dichloromethane was used as the solvent system). At this point, the reaction was cooled to room temperature before being diluted with dichloromethane (25 mL) and water (25 mL). The layers were separated and the organic layer was washed with water (3 × 25 mL). The combined aqueous washes were then back-extracted with dichloromethane (50 mL). The combined organic layers were washed with brine (50 mL) and dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give a yellow solid (2.60 g). The crude material was purified by column chromatography over silica gel (dichloromethane/methanol: 100/0 to 98/2) to afford 2.44 g (9.85 mmol, 98% yield) of the title compound as a yellow solid. The 1H and 13C spectra were consistent with published data (8). 1H NMR (300 MHz, CDCl3): δ 8.71 (d, J = 4.9 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 2.0, 9.0 Hz, 1H), 6.82 (d, J = 5.0 Hz, 1H), 3.17 (obs. br. s, 4H), 3.16 (obs. br. s, 4H), 1.82 (s, 1H); 13C NMR (75 MHz, CDCl3): δ 157.28, 151.89, 150.13, 134.73, 128.80, 125.97, 125.16, 121.87, 108.87, 77.42, 77.00, 76.58, 53.49, 46.01; ESI-MS:m/z 248.

Image ml189fu4

Step 2. Preparation of (4-chlorophenyl)(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methanone (10): 7-chloro-4-(piperazin-1-yl)quinoline (0.100 g, 0.404 mmol) was dissolved in dichloromethane (2.0 mL) and then treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.116 g, 0.606 mmol) and 4-(N,N-dimethylamino)pyridine (0.005 g, 0.04 mmol). 4-Chlorobenzoic acid (0.0695 g, 0.444 mmol) was added and the resulting clear, colorless solution was stirred overnight at room temperature. The reaction was quenched with water (5 mL) and stirred until both layers were clear. The layers were separated, and the aqueous layer was further extracted with dichloromethane (3 × 5 mL). The combined organic portions were then washed sequentially with water (10 mL), saturated sodium bicarbonate (aqueous, 10 mL), and brine (10 mL). The organic layer was shaken over magnesium sulfate, filtered, and concentrated under reduced pressure to give a white solid. The crude material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 0/100) to afford 0.132 g (0.342 mmol, 85% yield) of the probe as a white solid. 1H NMR (500 MHz, CDCl3): δ 8.76 (d, J = 4.9 Hz, 1H), 8.07 (s, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 9.3 Hz, 1H), 7.43 (s, 4H), 6.86 (d, J = 4.6 Hz, 1H), 4.06 (br. s, 2H), 3.75 (br. s, 2H), 3.22 (br. s, 4H); 13C NMR (125 MHz, CDCl3): δ 169.51, 156.21, 151.95, 150.16, 136.18, 135.19, 133.59, 129.12, 128.92, 128.68, 126.67, 124.61, 121.77, 109.37, 77.25, 77.00, 76.75, 52.27. HRMS (ESI): calculated mass for C20H17Cl2N3O [M+H] 386.0821, found 386.0815.

Figure 6. Proton NMR spectroscopy of probe compound (CID 1251584, SID 99376514, ML 189).

Figure 6Proton NMR spectroscopy of probe compound (CID 1251584, SID 99376514, ML 189)

Figure 7. Carbon NMR spectroscopy of probe compound (CID 1251584, SID 99376514, ML 189).

Figure 7Carbon NMR spectroscopy of probe compound (CID 1251584, SID 99376514, ML 189)

Figure 8. High resolution mass spectra of probe compound (CID 1251584, SID 99376514, ML 189).

Figure 8High resolution mass spectra of probe compound (CID 1251584, SID 99376514, ML 189)

3. Results

Probe attributes

  • Compounds that inhibit yeast growth in the presence, but not in the absence of 8 μg/ml fluconazole.
  • Compounds that show at least 10-fold selectivity between the primary Candida test strain and mammalian cells.
  • Compounds that show activity toward resistant clinical isolates at an IC50 < 50 μM.
  • IC50 ≤ 1 μM in primary or resistant screen cell line.
Compound Summary in PubChem.

Table

Compound Summary in PubChem.

3.1. Summary of Screening Results

A high throughput screen of 302,509 compounds (PubChem AID 1979) was performed in duplicate in the fluconazole-resistant Candida albicans strain CaCi-2 in the presence of a sublethal concentration of fluconazole. Using a screening active cutoff of ≥75% inhibition at a screening concentration of 9.5 μM, 1,893 hits were identified as Candida CaCi-2 growth inhibitors in the presence of fluconazole. 1,654 hits were available as cherry picks. These picked compounds were retested in dose against the C. albicans strain CaCi-2 to confirm their inhibitory activity and determine an IC50. There were 622 compounds which met the criterion of inhibitory activity of less than or equal to 1 μM.

An orthogonal screen of these 1,654 cherry picks against CaCi-8, a more highly-resistant C. albicans strain, in the presence of a sub-lethal concentration of fluconazole (AID 2423), selected for compounds that were active to at least 10-fold greater inhibition difference from the original strain (>50 M). There were 836 compounds that met this criterion, and 403 of these compounds were included in the 622 CaCi-2 retest actives.

Murine 3T3 fibroblasts provided an assay for overt compound toxicity to mammalian cells. Of the 1,654 cherry picks, 1,012 compounds were inactive in this assay, indicating fungal selectivity, of which 350 also met the criteria in the prior CaCi-2 and CaCi-8 assays.

To eliminate cherry pick compounds that intrinsically inhibit Candida growth, an additional secondary screen of the 1,654 cherry picks in the absence of fluconazole was included, with a 10 μM IC50 cutoff. Of the 350 compounds of interest, 296 met this criterion.

Figure 9. Screening Assay Antifungal Campaign in CaCi-2, CaCi-8, and Murine 3T3 Fibroblasts in the Presence and Absence of Fluconazole.

Figure 9Screening Assay Antifungal Campaign in CaCi-2, CaCi-8, and Murine 3T3 Fibroblasts in the Presence and Absence of Fluconazole

Two ancillary secondary assays were run to bin the remaining 296 compounds into one of three categories: 1) Hsp90 inhibitors, 2) calcineurin inhibitors, or 3) other mechanisms. The Hsp90 test used a Saccharomyces cerevisiae strain engineered to express beta-galactosidase driven by the glucocorticoid response element. The glucocorticoid hormone receptor depends heavily on Hsp90 for function. Of the 296 compounds of interest, 17 were active as defined by a 10 μM upper threshold of inhibition.

The second binning assay for calcineurin inhibition was evaluated in a yeast carrying a construct encoding calcineurin-dependent response elements (CDRE) driving expression of beta-galactosidase. Reporter activity with or without the prior addition of test compounds was measured following challenge with the stressor CaCl2. Of the 296 compounds of interest, two compounds were active as defined by a 10 μM upper threshold of inhibition.

The remaining 277 compounds were binned as “Other”.

Thirty compounds were chosen for initial dry powder confirmation studies from the 296 identified above by first clustering into small groups of related analogs, and then picking representative analogs from each of those families. After re-testing these dry powders in the test cascade, three compounds were chosen as potential probe candidates, and a first round of 31 analogs (plus three probe candidates) were obtained and assayed. Using the results from these assays as guidance, a second round of 26 compounds (plus probe candidate) was prepared for SAR. Figure 10 illustrates the progression of the project toward the probe compound.

Figure 10. Critical Path.

Figure 10

Critical Path.

3.2. Dose Response Curves for Probe

Figure 11. Dose-dependent activity of probe (CID 1251584) against various cell lines.

Figure 11Dose-dependent activity of probe (CID 1251584) against various cell lines

(A) C. albicans CaCi-2 in the presence of fluconazole (IC50 = 306 nM, AID 488943); (B) C. albicans CaCi-8 in the presence of fluconazole (IC50 = 179 nM, AID 488950); (C) Murine 3T3 fibroblasts in the absence of fluconazole (inactive, AID 488933); (D) C. albicans CaCi-2 in the absence of fluconazole (inactive, AID 488932).

3.3. Scaffold/Moiety Chemical Liabilities

A search of PubChem for the probe compound (CID 1251584) indicated that the probe has been previously evaluated in 256 assays and identified as active in nine assays. Three of those assays are the primary HTS campaign (AID 1979), primary screen in dose (AID 2467), and the preliminary CaCi-8 orthogonal assay (AID 2423) performed during the course of this project. ML189 was identified as a weak inhibitor of ROR gamma transcriptional activity (IC50 = 25 μM, AID 2546). In addition, ML189 was implicated in STAT1 inhibition (AID 920, 1263) and growth inhibition of Leishmania major promastigote (AID 1063) during their respective primary screening, but subsequent assays determined ML189 was not active in these projects (STAT1 confirmatory assay AID 1396; L.major re-test assay AID 1258). While screening for small molecule inhibitors of T. cruzi replication, the Broad Institute reported an IC50 value of 1.3 μM for ML189 [AID 1885 (primary HTS assay), 2044 (confirmatory)]. This last result, coupled with ML189’s structural resemblance to known anti-malarial chloroquine, underscores the utility of this scaffold in the infectious disease arena.

There are no obvious chemical liabilities associated with the probe compound.

3.4. SAR Tables

Figure 12. Probe substructures for SAR investigation.

Figure 12Probe substructures for SAR investigation

To investigate the activity of the probe compound, an additional 49 structurally related analogs were synthesized and evaluated for their ability to reverse fluconazole resistance in the C. albicans test strains. The biological assay data and physical properties of these analogs are presented below in Tables 2 through 9. The associated AID numbers for the biological assays are 488943 (CaCi-2 growth inhibition in the presence of fluconazole); 488932 (CaCi-2 growth inhibition in the absence of fluconazole); 488950 (CaCi-8 growth inhibition in the presence of fluconazole); and 488933 (murine 3T3 fibroblast toxicity in the absence of fluconazole). Spectroscopic data of these analogs may be found in Appendix B.

The first site of diversification evaluated was the 4-chlorobenzamide substructure (Table 2 and Table 3). Replacing the para-chloro substitutent with hydrogen, methyl, methoxy or fluoro derivatives (Table 2, entries 2, 3, 6, 7) attenuates probe activity, implicating the chloride as a necessary component. While a para-fluoro substitution still afforded nanomolar potency in the CaCi-2 strain, it experienced a 10-fold reduction against the more resistant CaCi-8 cells (Table 2, entry 7). Migration of the chloride around the phenyl ring was not tolerated (Table 2, entries 4 and 5), although the 3,4-dichloro variant displayed comparable levels of activity to the parent probe (Table 2, entry 10). In order to increase the solubility of the probe, chloropyridines were introduced in place of the benzamide. As predicted, solubility in PBS was increased (Table 3, entries 1, 8, and 9), but this was accompanied by a significant loss in activity (Table 2, entries 8 and 9).

Table 2. Summary of SAR Performed Upon the 4-chlorobenzamide.

Table 2

Summary of SAR Performed Upon the 4-chlorobenzamide.

Table 3. Physical Properties of 4-chlorobenzamide Derivatives.

Table 3

Physical Properties of 4-chlorobenzamide Derivatives.

The necessity of the benzamide itself was evaluated by preparing a series of alkyl or heteroaromatic amides (Table 4 and Table 5). As a general trend, the introduction of an alkyl amide greatly increased PBS solubility of the compounds, but negated all significant activity against CaCi-2 (Table 4, entries 1–8). Minimal inhibition of CaCi-8 could still be observed however. One exception is cyclohexane carboxamide (Table 4, entry 8); this analog retained considerable potency against both fungal strains, but also benefited from slightly increased solubility when compared to the original probe (Table 5, entry 8). Attempts to replace the amide linkage with urea or sulfonamide moieties were counterproductive; most activity was lost (Table 4, entries 9–12) with no discernable solubility gains (Table 5, entries 10–12). Alternative aryl groups were considered (Table 4, entries 13 and 14), and it appeared the larger ring system yielded a better result, suggesting there may be a steric requirement associated with the benzamide position. If the entire amide functionality was eliminated, the remaining N-aryl piperazine was completely inactive (Table 4, entry 15).

Table 4. Evaluation of Additional Amides and Amide Bond Surrogates.

Table 4

Evaluation of Additional Amides and Amide Bond Surrogates.

Table 5. Physical Properties of Additional Amides and Amide Bond Surrogates.

Table 5

Physical Properties of Additional Amides and Amide Bond Surrogates.

Following SAR evaluation of the benzamide, a focused collection of analogs bearing modified piperazines was prepared to evaluate the tolerance of ring substitution and ring expansion (Table 6 and Table 7). A 2-methylpiperazine core was introduced (as a racemate) with only minor loss in potency (Table 6, entry 1). Ring expansion to the seven-membered homopiperazine, however, was not beneficial (Table 6, entry 2), although removal of the amide nitrogen did not significantly alter the probe’s activity as indicated by a piperidine derivative (Table 6, entry 3).

Table 6. Biological Assay of Piperazine Derivatives.

Table 6

Biological Assay of Piperazine Derivatives.

Table 7. Physical Properties of Piperazine Derivatives.

Table 7

Physical Properties of Piperazine Derivatives.

The final site for diversification was the chloroquinoline moiety, and the envisioned SAR analysis was divided into two families. The first cluster of analogs comprised substituted quinolines (Table 8 and Table 9); the second family evaluated alternative aromatic rings in lieu of the 7-chloroquinoline (Table 10 and Table 11). The chloride substituent at the 7-position is critical to potency as its deletion led to almost complete loss of activity (Table 8, entry 1). Similarly, a methyl ether situated at the 7-position was nearly inactive (Table 8, entry 3). Interestingly, relocating the chlorine to the 6-position only increased the IC50 values slightly (Table 8, entry 2). Further substitutions at the 2- or 8-positions were generally ineffective (Table 8, entries 4–7).

Table 8. Summary of SAR Performed with Substituted Quinolines.

Table 8

Summary of SAR Performed with Substituted Quinolines.

Table 9. Physical Properties of Substituted Quinoline Analogs.

Table 9

Physical Properties of Substituted Quinoline Analogs.

Table 10. Heteroaromatic Substitution of the 7-chloroquinoline.

Table 10

Heteroaromatic Substitution of the 7-chloroquinoline.

Table 11. Physical Properties of the Heteroaromatic Surrogates.

Table 11

Physical Properties of the Heteroaromatic Surrogates.

A second library of analogs was designed to substitute the entire quinoline moiety with aromatic surrogates. Replacement of the 7-chloroquinoline with alternative heterocycles was generally detrimental to probe activity (Table 10 and Table 11). In several isolated cases, the observed IC50 values were low micromolar, but removal of the chloroquinoline often eliminated the probe’s chemosensitizing properties.

Of all the analogs prepared, the original HTS hit proved the most potent in the cellular assays with IC50 values of 0.31 μM and 0.18 μM against CaCi-2 and CaCi-8, respectively. Aqueous solubility of ML189 remains a liability; numerous attempts to incorporate solubilizing moieties produced inactive compounds.

3.5. Cellular Activity

All assays were performed with whole cells. A murine 3T3 fibroblast mammalian cell toxicity assay was included as a secondary screen. Experimental details are provided above in Section 2.1.2. The probe (CID 1251584/ML189) clearly met the established probe criteria specified for this project (Table 12).

Table 12. Comparison of the Probe to Project Criteria.

Table 12

Comparison of the Probe to Project Criteria.

3.6. Profiling Assays

The probe compound was evaluated for inhibitory activity against calcineurin and Hsp90. Assay results indicated the probe compound did not display any significant inhibition of either target. Additional details are provided below in Section 4.2.

4. Discussion

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

Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO), PatFT, AppFT, and World Intellectual Property Organization (WIPO). The search terms applied and hit statistics are provided in Table 13. The searches were performed on and are current as of February 7, 2011.

Table 13. Search Strings and Databases Employed in Prior Art Search.

Table 13

Search Strings and Databases Employed in Prior Art Search.

Several compounds have been previously identified as chemosensitizers, increasing the susceptibility of various C. albicans strains to fluconazole treatment (27). Of these, the most potent belonged to a series of HDAC inhibitors reported by Mai et al. (7). Depicted in Figure 13, these compounds are uracil-derived hydroxamic acids and exhibited MIC values ranging from 1.2 μM to 2.8 μM when combined with fluconazole. When tested in the absence of fluconazole, neither compound demonstrated activity against C. albicans at concentrations up to 368 μM. The potent activity of these compounds against murine HDAC1 (IC50 <51 nM) suggests they would not likely possess the fungal selectivity required for the probe.

Figure 13. Best-in-class Chemosensitizers for Reversing Fluconazole Resistance (MIC=1.2–2.8 μM).

Figure 13

Best-in-class Chemosensitizers for Reversing Fluconazole Resistance (MIC=1.2–2.8 μM).

The current probe (CID 1251584) has a potency of 306 nM against the C. albicans strain CaCi-2, and does not exhibit any effect in the absence of fluconazole or against mammalian fibroblasts. Remarkably, this probe is very potent (179 nM) against a highly drug resistant Candida strain CaCi-8. This probe is the result of synthesizing about 50 analogs, comprehensively exploring many different substituents around the benzamide group, the chloroquinoline group, and even the piperazine linker. It should prove extremely useful to researchers investigating novel pathways to overcome resistance mechanisms in pathogenic fungi.

4.2. Mechanism of Action Studies

All compounds were binned into one of three categories: 1) Hsp90-inhibitors, 2) calcineurin inhibitors, or 3) other mechanism.

Potential inhibition of the Hsp90-based chaperone machinery was evaluated using a yeast reporter assay involving the glucocorticoid hormone receptor. This well-established client protein depends heavily on Hsp90 for function. Refer to Section 2.1.5 for method details.

Potential inhibition of calcineurin function was evaluated in yeast carrying a construct encoding calcineurin-dependent response elements (CDRE) driving expression of a reporter enzyme. Reporter activity with or without the prior addition of test compounds was measured following challenge with the stressor CaCl2.

The probe (CID 1251584/ML189) was classified into the “Other” categorization, showing no inhibition of calcineurin or Hsp90 pathways.

4.3. Planned Future Studies

The goal of probe optimization is to generate potent, fungal-specific agents for future mechanistic studies and target identification projects. The activity of probes against drug-resistant yeast will make possible the use of the powerful genetic approaches available including genome-wide, overexpression, and deletion libraries, both in parallel arrayed format and pooled bar-coded format. To complement genetic approaches, affinity precipitation and proteomic approaches based on Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technology, which are already being employed in other projects, may be used. The drug metabolism/pharmacokinetic (DMPK) properties of the probe can be further optimized, and the probe (CID 1251584/ML189) could be studied in well-established mouse models of invasive, drug-resistant Candida fungemia, which are available and have been employed in the Whitehead Institute in conjunction with the laboratory of Gerald Fink. There is currently an IACUC-approved protocol in place to enable such experimentation in future work and to evaluate the therapeutic potential of probes generated by this screen.

5. References

1.
Cowen LE, Lindquist S. Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science. 2005 Sep 30;309(5744):2185–89. [PubMed: 16195452]
2.
DiGirolamo JA, Li X.-C, Jacob MR, Clark AM, Ferreira D. Reversal of fluconazole resistance by sulfated sterols from the marine sponge Topsentia sp. J Nat Prod. 2009 Aug;72(8):1524–28. [PMC free article: PMC3697152] [PubMed: 19653640]
3.
Cernicka J, Kozovska Z, Hnatova M, Valachovic M, Hapala I, Riedl Z, Hajós G, Subik J. Chemosensitisation of drug-resistant and drug-sensitive yeast cells to antifungals. Int J Antimicrobial Agents. 2007 Feb;29(2):170–8. Epub 2007 Jan 3. [PubMed: 17204400] [Cross Ref]
4.
Gamarra S, Rocha EM, Zhang Y.-Q, Park S, Rao R, Perlin DS. Mechanism of the synergistic effect of amiodarone and fluconazole in Candida albicans. Antimicrob Agents Chemother. 2010 May;54(5):1753–61. Epub 2010 Mar 1. [PMC free article: PMC2863688] [PubMed: 20194694]
5.
Guo X.-L, Leng P, Yang Y, Yu L.-G, Lou H.-X. Plagiochin E, a botanic-derived phenolic compound, reverses fungal resistance to fluconazole relating to the efflux pump. J Appl Microbio. 2008 Mar;104(3):831–38. Epub 2008 Jan 9. [PubMed: 18194250] [Cross Ref]
6.
Courchesne WE. Characterization of a novel, broad-based fungicidal activity for the antiarrhythmic drug amiodarone. J Pharmacol Exp Ther. 2002 Jan 1;300:195–99. Epub. [PubMed: 11752116] [Cross Ref]
7.
Mai A, Rotili D, Massa S, Brosch G, Simonetti G, Passariello C, Palamara AT. Discovery of uracil-based histone deacetylase inhibitors able to reduce acquired antifungal resistance and trailing growth in Candida albicans. Bioorg Med Chem Lett. 2007 Mar 1;17(5):1221–25. Epub 2006 Dec 12. [PubMed: 17196388]
8.
Solomon VR, Hu C, Lee H. Design and synthesis of anti-Breast cancer agents from 4-Piperazinylquinoline: a Hybrid pharmacophore approach. Bioorg Med Chem. 2010;18:1563–1572. [PubMed: 20106668]
9.
Redding S, Smith J, Farinacci G, Rinaldi M, Fothergill A, Rhine-Chalberg J, Pfaller M. Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS: documentation by in vitro susceptibility testing and DNA subtype analysis. Clin Infect Dis. 1994 Feb 1;18(2):240–42. [PubMed: 8161633]

Appendix A. Detailed Assay Protocols

Primary CaCi-2 (AID no. 1979) and CaCi-2 Dose-Response Retest (AID nos. 2467, 488836, 488930, 488907, 488943)

Materials and Reagents

Clear, flat bottom, black, 384-well plates (Corning Catalog no. 3712BC; Lot no. 35808016);Geldanamycin (AG Scientific, Catalog no. G-1047) 15 mM stock solution in DMSO; Fluconazole (Sequoia Research Products Ltd) 2 mg/ml stock solution in PBS; Pen/Strep (Gibco Catalog no.10378-016; Lot no. 21040170) 100X in PBS; Alamar Blue (AG Scientific Catalog no. DAL 1100; Lot no.151016SA); PBS without Calcium and Magnesium (Cellgro Catalog no. 21-040-CV)

Synthetic Defined Growth Medium

RPMI 1640 medium, (powder without sodium bicarbonate; Invitrogen Catalog no. 31800-089; Lot no. 648072); Uridine 8 mg/ml in water (Sigma Catalog no. U3750; Lot no. 028KO760); Glucose 40% (w/v) in water (Sigma Catalog no. G-5400); MOPS Buffer (Sigma Catalog no. M-1254; Lot no. 098K0033)

  1. Prepare 1X RPMI medium by dissolving 10.4 grams powdered medium in 800 ml water.
  2. Add 34.52 g MOPS. While stirring, adjust pH to 7.0 with 10 N NaOH.
  3. Add 10 ml uridine solution, 50 ml glucose solution, adjust final volume to 1000 ml. Filter sterilize.
Fungal Inoculum

Test Strain: C. albicans CaCi-2

  1. Inoculate 500 μl of strain from cryopreserved stock into a 250 ml shaker flask containing 30 ml growth medium. Shake at 30 °C overnight.
  2. Read OD 600 of 1 ml fungal culture in a cuvette using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank.
  3. Dilute to desired volume of fungal inoculums according to the following formula: (1/OD measured) × (Desired Final Volume of Inoculum) × 0.3 = Volume of fungal culture (μl) to add to desired volume of growth medium. When added to media in wells, this yields a calculated starting OD of the fungal inoculum of 0.00015.

Procedures

  1. Add fluconazole stock solution to fungal inoculum to achieve a final concentration of 8 μg/ml.
  2. Add Pen/Strep at 0.1 ml per 10 ml media (1% v/v).
  3. Use a Thermo Combi nL to dispense 20 μl/well of assay media into all wells.
  4. Pin 25 nl test compound from compound plates into assay plates using CyBi-Well pin tool.
  5. Dispense 20 μl/well of culture into the assay media in all wells.
  6. Incubate plates in a humidified (90% humidity) Liconic incubator at 37 °C without agitation for 48 hours.
  7. Dilute Alamar Blue Reagent 1:40 in Ca/Mg-free PBS.
  8. To all plates, add 5 μl/well of the diluted Alamar to a final dilution factor of 1:200.
  9. Incubate the plates for an additional 2 hours.
  10. Read the Relative Fluorescence Intensity (RFU) of wells on a standard plate reader as a measure of relative fungal growth. EnVision (Perkin Elmer) plate reader set-up: Ex 544 nm, Em 590 nm, Bandwidth 12 nm, Top read.

Orthogonal Resistant Strain Dose Response (AID nos. 2423, 488807, 488909, 488905, 488950)

Materials and Reagents

Clear, flat-bottom, black, 384-well plates (Corning Catalog no. 3712BC; Lot no. 35808016); Geldanamycin (AG Scientific Catalog no. G-1047) 15 mM stock solution in DMSO; Pen/Strep (Gibco Catalog no.10378-016;Lot no.21040170) 100X in PBS; Fluconazole (Sigma Catalog no.F829-100MG;Lot no. 098K4715) 2 mg/ml stock solution in PBS; Alamar Blue (AG Scientific Catalog no. DAL1100; Lot no.151016SA); PBS w/o Calcium and Magnesium (Cellgro Catalog no. 21-040-CV)

Synthetic Defined Growth Medium

RPMI 1640 medium, (powder without sodium bicarbonate; Invitrogen Catalog no. 31800-089, Lot no.648072); Uridine 8 mg/ml in water (Sigma Catalog no. U3750; Lot no. 028K0760); Glucose 40% (w/v) in water (Sigma Catalog no. G-5400); MOPS Buffer (Sigma Catalog no. M-1254; Lot no. 098K0033)

  1. Prepare 1X RPMI medium by dissolving 10.4 g powdered medium in 800 ml water.
  2. Add 34.52 g MOPS. While stirring, adjust pH to 7.0 with 10N NaOH.
  3. Add 10 ml uridine solution, 50 ml glucose solution, adjust final volume to 1000 ml. Filter sterilize
Fungal Inoculum

Test Strain: C. albicans CaCi8 (9)

  1. Inoculate 500 μl of strain from cryopreserved stock into a 250 ml shaker flask containing 30 ml growth medium. Shake at 30 °C overnight (16 hours).
  2. Read OD 600 of 1 ml of fungal culture in a cuvette using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank.
  3. Dilute to a desired volume of fungal inoculum according to following formula: (1/OD measured) × (Desired Final Volume of Inoculum) × 0.3 = Volume of fungal culture (μl) to add to desired volume of growth medium. When added to media in wells, this yields a calculated starting OD of the fungal inoculum of 0.00015.

Procedures

  1. Add fluconazole stock solution to fungal inoculum to achieve 8 μg/ml.
  2. Add Pen/Strep to media to 1% concentration.
  3. Use a Thermo Combi nL to dispense 20 μl/well of assay media into all wells.
  4. Dispense geldanamycin in positive control wells using Thermo Combi nL for a final cocentration of 3 μM.
  5. Then, pin 100 nl of test compound from compound plates into assay plates using a CyBi-Well pin tool.
  6. Dispense 20 μl/well of culture into the assay media in all wells.
  7. Incubate the plates were incubated in a humidified (90% humidity) Liconic incubator at 37 °C without agitation for 48 hours.
  8. Dilute Alamar Blue 1:40 in Ca/Mg-free PBS.
  9. To all plates, add 5 μl/well of the diluted Alamar Blue to plates to a final dilution factor of 1:200.
  10. Incubate the plates for 2 hours.
  11. Read the Relative Fluorescence Intensity (RFU) of wells on a standard plate reader as a measure of relative fungal growth. EnVision (Perkin Elmer) plate reader set-up: Ex 544 nm, Em 590 nm, Bandwidth 12 nm, Top read.

Counterscreen Mammalian Cell Toxicity Dose Response (AID Nos. 2327, 488809, 488911, 488933)

Materials and Reagents

Clear, flat-bottom, black, 384-well plates (Corning Catalog no. 3712BC Lot no. 35808016); Geldanamycin (AG Scientific Catalog no. G-1047) 15 mM stock solution in DMSO; Fluconazole (Sequoia Research Ltd.) 2 mg/ml stock solution in PBS; Alamar Blue (AG Scientific Catalog no. DAL1100, Lot no. 151016SA); PBS w/o Calcium and Magnesium (Cellgro Catalog no. 21-040-CV)

Assay Medium

Optimem medium (Invitrogen Catalog no. 31985-070; Lot no. 548536); 2.5% (v/v) Fetal Bovine Serum (Hyclone Catalog no.30071.03; Lot no. ARF26748); 1% (v/v) Pen/Strep solution (Invitrogen Catalog no.15140-122; Lot no. 529891)

Cell Inoculum

Test Strain: NIH-3T3 mammalian fibroblasts (ATCC CRL No. 1658)

  1. Plate cells in 384-well plates at 6,000 cells/well in 20 μl assay medium.
  2. Incubate plates overnight at 37 °C under 5% CO2.

Procedures

  1. After overnight culture, pin compounds into wells at 100 nl/well using the CyBio CyBi-Well pinning instrument.
  2. After pinning compounds, add 20 μl of assay medium supplemented with fluconazole to each well. To a final nominal concentration of 8 μg/ml fluconazole.
  3. Return the plates to the tissue culture incubator and incubate the culture for an additional 48 hours at 37 °C under 5% CO2.
  4. At the completion of this incubation, add Alamar Blue solution diluted 1:40 in PBS to each well (10 μl/well) to achieve a final dilution of 1:200.
  5. Incubate the plates for an additional 2–3 hours at 37 °C under 5% CO2.
  6. Read the Relative Fluorescence Intensity (RFU) of wells was read on a standard plate reader as a measure of relative cell growth. EnVision (Perkin Elmer) plate reader set-up: Ex 544 nm, Em 590 m, Bandwidth12 nm, Top read.

Secondary Single Agent (No Fluconazole) Activity Assay Protocol (AID Nos. 2387, 488802, 488893, 488937, 488932)

Materials and Reagents

Clear, flat-bottom, black 384-well plates (Corning Catalog no. 3712BC; Lot no. 35808016);Geldanamycin (AG Scientific Catalog no. G-1047) 15 mM stock solution in DMSO; Pen/Strep (Gibco Catalog no. 10378-016; Lot no21040170) 100X in PBS; Fluconazole (Sigma Catalog no. F829-100MG; Lot no. 098K4715) 2 mg/ml stock solution in PBS; Alamar Blue (AG Scientific Catalog no. DAL1100; Lot no.151016SA);PBS w/o Calcium and Magnesium (Cellgro Catalog no. 21-040-CV)

Synthetic Defined Growth Medium

RPMI 1640 medium, (powder without sodium bicarbonate; Invitrogen 31800-089, Lot 648072); Uridine 8 mg/ml in water (Sigma Catgalog no. U3750; Lot no. 028K0760); Glucose 40% (w/v) in water (Sigma Catalog no. G-5400); MOPS Buffer (Sigma Catalog no. M-1254; Lot no. 098K0033)

  1. Prepare 1X RPMI medium by dissolving 10.4 g powdered medium in 800 ml water.
  2. Add 34.52 g MOPS. While stirring, adjust pH to 7.0 with 10N NaOH.
  3. Add 10 ml uridine solution, 50 ml glucose solution, adjust final volume to 1000 ml. Filter sterilize.
Fungal Inoculum

Test Strain: C. albicans CaCi-2 (9)

  1. Inoculate 500 μl of yeast from cryopreserved stock into a 250 ml shaker flask containing 30 ml growth medium. Shake at 30 °C overnight (16 hours).
  2. Read OD 600 of 1 ml of fungal culture in a cuvette using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank.
  3. Dilute to desired volume of fungal inoculum according to following formula: (1/OD measured) × (Desired Final Volume of Inoculum) × 0.3 = Volume of fungal culture (μl) to add to desired volume of growth medium. When added to media in wells, this yields a calculated starting OD of the fungal inoculum of 0.00015.

Procedures

  1. Add Pen/Strep to the media to a final 1% concentration.
  2. Use a Thermo Combi nL to dispense 20 μl/well of assay media into all wells.
  3. Mix geldanamycin and fluconazole for positive control.
  4. Dispense positive control solution into the positive control wells using Thermo Combi nL for a final concentration of 3 μM geldanamycin, and 8 μg/ml fluconazole.
  5. Then, pin 100 nl of test compound from compound plates into assay plates using a CyBi-Well pin tool.
  6. Dispense 20 μl/well of culture into the assay media in all wells.
  7. Incubate the plates in a humidified (90% humidity) Liconic incubator at 37 °C without agitation for 48 hours.
  8. Dilute Alamar Blue 1:40 in Ca/Mg-free PBS.
  9. To all plates, add 5 μl/well of the diluted Alamar Blue to the plates to a final dilution factor 1:200.
  10. Incubate the plates for 2 hours.
  11. Read Relative Fluorescence Intensity (RFU) of wells was read on a standard plate reader as measure of relative fungal growth. EnVision (Perkin Elmer) plate reader settings: Ex 544 nm, Em 590 nm, Bandwidth 12 nm, Top read.

Hsp90 Binning (AID No. 2400)

Materials and Reagents

White, 384-well plate (Corning Catalog no. 8867BC, Lot no. 22609019); Radicicol (Sigma Catalog no. R2146);Tropix Gal-Screen (Applied Biosystems Catalog no. T2359, Lot no. 0903044)

Assay media
SD-ADE Yeast nitrogen base w/o ammonium sulfate, minus adenine

SD Growth Media (MP Biomedical Catalog no. 4027-012; Lot no. 119458); Dextrose 20%; Complete supplement minus adenine (Sunrise Science Catalog no. 1029-100; Lot no. 070409)

  1. To 100 ml SD Growth Media, add 100 ml 20% dextrose and 780 mg Complete Supplement.
  2. Add water to 1 liter. Filter and sterilize.
DOC media

SD-ADE (see above); DOC steroid (Sigma Catalog no. D7000)

  1. To 100 ml SD-ADE media, add 1 ml DOC.
Cell Inoculum

Test Strain: ATCC 201238 Saccharomyces cerevisiae W303 reporter strain

  1. Inoculate reporter Saccharomyces strain (ATCC Catalog no. 201238) from cryopreserved stock into a 250 ml shaker flask containing 20 ml SD-ADE media.
  2. Incubate the flask overnight (16 hours) at 37 °C and 150 RPM.

Procedures

  1. Read OD 600 of 1 ml of culture in a cuvette using a standard optical density reader (Eppendorf BioPhotometer Plus), with growth medium as a background blank.
  2. Dilute cells to OD = 0.04 in SD-ADE media.
  3. To each 384-well white plate, add 20 μl of diluted culture using a Thermo Combi.
  4. Then, pin 100 nl compounds into plates with a CyBi-Well pin tool.
  5. Next, add 5 μM radicicol as a positive control in control wells, dispensing with Thermo Combi NL.
  6. With Combi, dispense 20 μl of 20 μM DOC (steroid) in SD-ADE media in pinned plates.
  7. Incubate the plates at 30 °C for 75 minutes with agitation.
  8. Using Combi, dispense 40 μl Gal-Screen reagent.
  9. Incubate the plates at 30 °C for 25 minutes.
  10. Read the luminescence of wells on a standard plate reader as measure of relative fungal growth. EnVision (Perkin Elmer) plate reader set-up: Top read; Luminescence filter (560 nm) at 0.1 second

Appendix B. Spectroscopic data for SAR analogs reported in Section 3.4

Figure 14. Spectroscopic data for SID 99245537

Figure 15. Spectroscopic data for SID 99245531

Figure 16. Spectroscopic data for SID 99376520

Figure 17. Spectroscopic data for SID 99376521

Figure 18. Spectroscopic data for SID 99376522

Figure 19. Spectroscopic data for SID 99376499

Figure 20. Spectroscopic data for SID 99376501

Figure 21. Spectroscopic data for SID 99376507

Figure 22. Spectroscopic data for SID 99245567

Figure 23. Spectroscopic data for SID 99376531

Figure 24. Spectroscopic data for SID 99376500

Figure 25. Spectroscopic data for SID 99376504

Figure 26. Spectroscopic data for SID 99376527

Figure 27. Spectroscopic data for SID 99245535

Figure 28. Spectroscopic data for SID 99376509

Figure 29. Spectroscopic data for SID 99376510

Figure 30. Spectroscopic data for SID 99376511

Figure 31. Spectroscopic data for SID 99245553

Figure 32. Spectroscopic data for SID 99376523

Figure 33. Spectroscopic data for SID 99245557

Figure 34. Spectroscopic data for SID 99245539

Figure 35. Spectroscopic data for SID 99245543

Figure 36. Spectroscopic data for SID 99245548

Figure 37. Spectroscopic data for SID 99376512

Figure 38. Spectroscopic data for SID 99376525

Figure 39. Spectroscopic data for SID 99376508

Figure 40. Spectroscopic data for SID 99376517

Figure 41. Spectroscopic data for SID 99376498

Figure 42. Spectroscopic data for SID 99376530

Figure 43. Spectroscopic data for SID 99376518

Figure 44. Spectroscopic data for SID 99376526

Figure 45. Spectroscopic data for SID 99245559

Figure 46. Spectroscopic data for SID 99376513

Figure 47. Spectroscopic data for SID 99376519

Figure 48. Spectroscopic data for SID 99376502

Figure 49. Spectroscopic data for SID 99376538

Figure 50. Spectroscopic data for SID 99376557

Figure 51. Spectroscopic data for SID 99245561

Figure 52. Spectroscopic data for SID 99245569

Figure 53. Spectroscopic data for SID 99376524

Figure 54. Spectroscopic data for SID 99376503

Figure 55. Spectroscopic data for SID 99376554

Figure 56. Spectroscopic data for SID 99376537

Figure 57. Spectroscopic data for SID 99376553

Figure 58. Spectroscopic data for SID 99376545

Figure 59. Spectroscopic data for SID 99376552

Figure 60. Spectroscopic data for SID 99245570

Figure 61. Spectroscopic data for SID 99376515

Figure 62. Spectroscopic data for SID 99376533

Appendix C. Compounds submitted to BioFocus

Table 14. Compounds submitted to BioFocus.

Table 14

Compounds submitted to BioFocus.