External ID: HMS937_MLP

Protocol: 10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to each well of 384-well plates (Corning 3820), except for column 1, which was used for positive controls.

100 nL of each experimental compound were transferred to the assay plates by pin transfer, and then incubated for 30 minutes at room temperature.

10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells of the assay plates, and then incubated for 25 minutes at room temperature. The fluorescence intensity of each well was then read using a Perkin Elmer EnVision plate reader at 355 nm/460 nm (excitation/emission).

For positive controls, 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 1 on each assay plate, and incubated for 25 minutes at room temperature before fluorescence reading.

For negative controls, 10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 2 on each assay plate, and then 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) was added. After incubation at room temperature for 25 minutes, fluorescence intensity was read.

Comment: Activity scores were calculated based on percent (%) inhibition, which was in turn calculated from fluorescence intensity (FI) readings as follows: % Inhibition = (1-((Vi-Vp)/(Vn-Vp)))*100, where Vi is the FI of the individual well, Vp the average FI of the positive controls on that plate, and Vn the average FI of the negative controls on that plate. A final % inhibition value was calculated as the average of replicate results for each individual compound and used to determine the Activity Score. If the % inhibition was less than zero, the Activity Score was set to Zero. Compounds showing >20% inhibition were considered active.

External ID: HMS1158_MLP

Protocol: A 5 mL overnight pre-culture of E. coli is started by inoculating a tube of lysogeny broth with a single colony from an agar plate. The tube is incubated at 37 degrees C overnight with shaking. The resulting saturated culture is removed from the shaker and diluted into fresh lysogeny broth at 1:100 proportion and incubated at 37 degrees C with shaking for two hours. The culture is removed from the shaker and absorbance is measured at 600 nm. The culture is diluted into fresh lysogeny broth to an estimated absorbance of 0.0002. For each library plate to be screened, two 384-well assay plates are filled with 30 microL per well of fresh lysogeny broth, except for the positive control columns, which are left empty. The multiple of two is necessary to expose the strain in duplicate to the same compounds from a given library plate.

300 nL of experimental compounds are transferred into the assay plates from library plates using a robotically manipulated stainless steel pin array, exposing the strain to the same compounds at the same concentration. The positive control columns are filled with 30 microL of the positive control compound in LB. Thirty microL of E. coli culture at absorbance of 0.0002 are then added to the assay plates, producing a total volume of 60 microL in each well. Plates are incubated at 37 degrees C without shaking and with humidification for 23 hours. The plates are removed from the incubator, and absorbance of the cultures is measured at 600 nm using a PerkinElmer EnVision plate reader. The absorbance is used as the readout.

Positive control: (R)-4-(4-(2-fluoro-4-methoxyphenyl)-2-oxopyridin-1(2H)-yl)-N-hydroxy-2-methyl-2-(methylsulfonyl)butanamide was present at 0.0754 microM in all wells of column 24.

Negative control: No treatment in remaining empty wells of the assay plate (minimally, wells in column 23).

Comment: A threshold constant was calculated by subtracting 3.6 X experimental well standard deviation for absorbance from the experimental well average absorbance, combining all plates in a screening run. Well absorbance was then subtracted from this threshold value. Wells were considered active if the resulting value after subtracting the threshold constant > 0 for at least one replicate. Activity scores were calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: HMS1364-MLP_WT

Protocol: The day before screening, RN4220 was grown overnight (16-18 h) in sterile-filtered tryptic soy broth (TSB) at 30 degrees C with shaking.

On the day of screening, columns 1-24 of assay plates (Corning 3710, clear) were prefilled with 30 uL of TSB using the Combi. Next, 100 nL of each compound was pin-transferred to each plate. For every compound library plate, there were two daughter plates (A & B). Overnight cultures were diluted 625x into TSB. 50 uL of the diluted cultures were added to columns 1-24 of the corresponding assay plates. Wells in column 2 contained only medium (with culture) and served as the negative controls. Positive control (0.8 uL of erythromycin, 10 ug/mL) was added to column 1 using the Hewlett Packard D300. Final assay well volume was 80 uL. Plates were spun down and stacked 5 high, covered with lids (Corning 3009), and incubated at 30 degrees C overnight (~16 h).

The following day, assay plates were removed from the incubator and cell density quantitated using a PerkinElmer EnVision (600 nm filter).

Comment: Z-scores were calculated for every well based on plate average and standard deviation of experimental well absorbance (OD600). Wells were considered active for reduced WT survival if Z-score for both replicates <= -3 and absorbance for both replicates <= 0.18. Percent inhibition was calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. The replicate values were then averaged to determine activity scores. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: HMS1364-MLP_ugtP

Protocol: The day before screening, RN4220 ugtP was grown overnight (16-18 h) in sterile-filtered tryptic soy broth (TSB) at 30 degrees C with shaking.

On the day of screening, columns 1-24 of assay plates (Corning 3710, clear) were prefilled with 30 uL of TSB using the Combi. Next, 100 nL of each compound was pin-transferred to each plate. For every compound library plate, there were two daughter plates (A & B). Overnight cultures were diluted 625x into TSB. 50 uL of the diluted cultures were added to columns 1-24 of the corresponding assay plates. Wells in column 2 contained only medium (with culture) and served as the negative controls. Positive control (0.8 uL of erythromycin, 10 ug/mL) was added to column 1 using the D300. Final assay well volume was 80 uL. Plates were spun down and stacked 5 high, covered with lids (Corning 3009), and incubated at 30 degrees C overnight (~16 h).

The following day, assay plates were removed from the incubator and cell density quantitated using a PerkinElmer EnVision (600 nm filter). Library plates were screened in duplicate, with both assay plates in a given set prepared on the same day.

Comment: Z-scores were calculated for every well based on plate average and standard deviation of experimental well absorbance (OD600). Wells were considered active for reduced ugtP survival if Z-score for both replicates < -3 and absorbance for both replicates <= 0.15. Percent inhibition was calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. The replicate values were then averaged to determine activity scores. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: HMS1149-MLP

Protocol: A 558 bp region upstream of hspX was PCR amplified and cloned upstream of the GFP-mut2 (Cormack et al., 1996). This construct was cloned into a modified version of the replicating plasmid pSE100 and transformed into CDC1551 to generate CDC1551(hspX'::GFP).

The M. tuberculosis CDC 1551 (hspX'::GFP) fluorescent biosensor was grown in pH 7.0 Middlebrook 7H9 medium to mid- to late-log phase. Black-walled, clear-bottom, tissue-culture treated 384-well microtiter plates (Corning #3712) were filled with 25 uL of buffered pH 7.0 7H9 medium using a Matrix Combi. 100 nL of compound was then transferred to each well via stainless steel pin array. Plates were manually sealed with DMSO-resistant aluminum seals and frozen at -80C until they were ready to be screened. The cultures were re-suspended in pH 7.0 7H9 and 25 uL was dispensed into each well of the 384-well assay plates already containing the perturbator utilizing a Cy-Bio Selma liquid handler robot to an OD595 of 0.05. The plates were then placed in a Ziploc bag with a moistened paper towel (to limit evaporation) and incubated for 6 days at 37C. Fluorescence and optical density (OD) readings were made on an EnSpire plate reader (Perkin Elmer, Inc.) at excitation and emission wavelengths of 488 and 509 nm as a top read, with the OD being taken at 595 nm as a bottom read.

The ~328,633 compound library was arrayed into black-walled, clear-bottom tissue-culture treated 384-well microtiter plates (Corning #3712) by pin transfer (100 nL), containing 25 uL of buffered pH 7.0 7H9 medium. In a previous screen utilizing the same compound library and screening method, four random compound plates were screened in duplicate to observe variation in hit robustness across plates and method. The result was that the duplicate plates did not vary in a statistically significant manner and due to the high volume of compounds to be screened (~328,633), replicates were not utilized.

Negative control: DMSO, 0.1% final concentration in buffered pH 7.0 7H9 in column 2 or column 23 of each plate

Positive control: 0.3 uM Rifampicin final concentration in buffered pH 7.0 7H9 in column 1 or column 24 of each plate

Comment: Analysis methods:
To calculate normalized fluorescence percent inhibition, plate average negative control fluorescence intensity (based on all plates within a screening run) was subtracted from well fluorescence intensity, divided by the difference between plate negative and positive control average fluorescence intensity (based on all plates within a screening run), and multiplied by 100. To calculate normalized growth inhibition, plate average negative control absorbance (based on all plates within a screening run) was subtracted from well absorbance, divided by the difference between plate negative and positive control average absorbance (based on all plates within a screening run), and multiplied by 100. Normalized fluorescence percent inhibition was divided by normalized growth inhibition to calculate a ratio. Wells were considered active if growth was not greatly decreased and showed either a 3-fold selectivity fluorescence to growth inhibition ratio (with 35-45% fluorescence inhibition) or a 1.5-fold selectivity fluorescence to growth inhibition ratio (with > 45% fluorescence inhibition). These compounds may be directly or indirectly inhibiting DosRS signaling. Wells were considered general inhibitors of M. tuberculosis growth if both fluorescence and growth inhibition > 50%. Activity scores were scaled from 100 to 0 and derived from normalized fluorescence percent inhibition, with 100 (or > 100) = 100% inhibition and 0 (or < 0) = no inhibition.