External ID: Cornell_MTB_2017

Protocol: 1#Two weeks prior to the assay start ~50 ml culture of WT M. tuberculosis in 7H9 OADC + kanamycin medium by using 1 freezer stock tube. After a week, transfer the culture into 7H12 + acetate + kanamycin medium at an OD600 of 0.1.
2#Day of screen make 10 microM rifampin solution in 7H12 + cholesterol + 25 microg/ml kanamycin + 100 microg/ml cyclohedamide medium. (Use 2.5 mM stock. 250-fold dilution will give 10 microM rifampin at 0.4% DMSO. Subsequent 2-fold dilution will give 5 microM Rif, 0.2% DMSO final).
3#Also make up similar volume of medium with DMSO instead of Rifampin. For that, repeat step 2 but replace Rifampin by 100% DMSO.
4#Add 25 ul rifampin medium to column 24 of the compound plates. This is the positive control. Add 25 ul DMSO medium to column 23 of the compound plates. This is the negative control.
5#On the day 0 of assay, first turn on the HVAC system, robot head, plate stack, pump and computer.
6#Measure the OD600 of the culture. Calculate the total cell mass required to inoculate all the plates at 25 microl/well at a final OD600 of 0.1. Account for dead volume and prepare 20 ml of extra inoculum. Then spin down the culture by centrifugation at 3K rpm for 10 minutes. Decant the medium. Resuspend the WT cell pellet in 7H12 + 200 microM cholesterol + 25 microg/ml Kanamycin + 100 microg/ml cyclohexamide. The inoculum will be added to 25 microl of 2X compounds in the well to give a final OD600 of 0.05.
7#Prepare the Janus for liquid handling. On the desktop screen, click on WinPrep icon to use the robot head. Go to Utilities > Instrument > Initialize. Then load "Cholesterol screen VanderVen" protocol. This protocol is set to dispense 25 microl of inoculum to each well of the 384-well plate.
8#In the Biosafety cabinet, replace the lids of the 384-well plates with stacker lids (Use 1 lid per 5 plates. Then discard). Stack the plates in the down stacker on the right.
9#Carefully remove the adhesive tape from the 384 tip box so as to neither open the lid nor get the tape to stick on your gloves. Set the tip box on the left top corner. Use the lid of the tip box as a reservoir for bacterial culture. Make sure the lid is not broken before adding M. tuberculosis.
10#Click on "Execute test". When prompted for plate number, select the number of plates that will be inoculated. If inoculating more than 20 plates, it is advisable to replace the pipet tips after filling 20 plates.
11#When done inoculating, re-lid the plates and store the plates in a Ziplock bag with an OPEN 50 ml conical tube containing wet kim wipes to prevent evaporation. Incubate at 37oC.
12#At day 7 add 10 microl of 0.16 mg/ml Resazurin. Turn on the HVAC system, robot head, plate stack, pump and computer 1. Prepare the Janus for liquid handling, on the desktop screen, click on WinPrep icon to use the robot head. Go to Utilities > Instrument > Initialize. Then load "10 ul Alamarblue addition" protocol.
13#Carefully remove the adhesive tape from the 384 tip box. Set the tip box on the left top corner. Use the lid of the tip box as a reservoir for Resazurin. Make sure the lid is not broken before adding Resazurin. Click on "Execute test". When prompted for plate number, select the number of plates that will be inoculated. If inoculating more than 20 plates, it is advisable to replace the pipet tips after filling 20 plates.
14#Read the plates after 24 hours with Envision using the "384 well Alamarblue" protocol.

Comment: The compounds that reduced growth of Mtb by more than two standard deviations as compared to DMSO control were taken as hits. In the current submission, hits are marked as active and non-hits are marked as inactive under the PUBCHEM_ACTIVITY_OUTCOME column.

To see the structural details of the compounds please search for this substance upload ID- 73810