ML212: A small-molecule probe for investigating fluconazole resistance mechanisms in Candida albicans

The National Institutes of Health Molecular Libraries and Probe Production Centers Network (NIH-MLPCN) screened >300,000 compounds to evaluate their ability to restore fluconazole susceptibility in resistant Candida albicans isolates. Additional counter screens were incorporated to remove substances inherently toxic to either mammalian or fungal cells. A substituted indazole possessing the desired bioactivity profile was selected for further development, and initial investigation of structure–activity relationships led to the discovery of ML212.


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.

S3
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.
3) Use a Thermo Combi nL to dispense 20 µL/well of assay media into all wells.  2) Add 34.52 g MOPS. While stirring, adjust pH to 7.0 with 10 N NaOH.
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.
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 concentration of 3 µM.
S5 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.  S6 2) Add 34.52 g MOPS. While stirring, adjust pH to 7.0 with 10 N NaOH.
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.

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% CO 2 .
S8 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% CO 2 . 6) Read the relative fluorescence intensity (RFU) of wells on a standard plate reader as a measure of relative cell growth. EnVision (Perkin Elmer) plate reader setup: Ex 544 nm, Em 590 m, bandwidth12 nm, top read.

Compound synthesis
General details. All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz spectrometer.

Representative preparation of methyl 3-alkylindazolyl acetates:
Cyclohexyl ( The reaction was quenched with a saturated solution of ammonium chloride (aqueous, 2 mL) and further diluted with water (2 mL). The resulting mixture was stirred at room temperature until both layers were clear. The lower aqueous layer was separated and extracted with diethyl ether (2 × 3 mL). The combined organic layers were shaken over magnesium sulfate, filtered, and concentrated under reduced pressure to give a clear, colorless oil, which was used without further purification.
The reaction was slowly warmed to room temperature and stirred until complete as

NMR and MS data for reported compounds
General details: NMR spectra were recorded on a Bruker 300 MHz spectrometer.