External ID: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

External ID: HMS979

Protocol: Cation-adjusted Mueller Hinton II broth supplemented with 0.005% Tween-80 was inoculated with a single colony of S. aureus RN4220. Following overnight incubation, the culture was diluted 1:100 into fresh medium, and incubation was continued until the bacterial suspension reached an optical density at 600 nm (OD600) of 0.6, corresponding to a bacterial density of 5 x 10E8 colony forming units (CFU)/mL. An aliquot of this culture was diluted 1:400 with fresh medium and stored on ice until use. Assay plates were prepared for compound transfer in quadruplicate to enable screening of compounds at two temperatures in duplicate. The plate layout was as follows: wells in columns 1-22 were loaded with culture medium at 25 microL/well; wells in column 23 contained the screening negative control (medium only); wells in column 24 contained the screening positive control (medium containing 25 microg/mL chloramphenicol). 300 nL of experimental compounds were transferred by stainless steel pin array from library plate to assay plates.

25 microL of the bacterial suspension was added to each assay well. Assay plates were incubated at 42 degrees C in humidified chambers (humidity > 85%). After 20 h, the OD600 was measured using a plate reader in absorbance mode.

Comment: Data analysis description:

Absorbance values at 42 degrees C and 30 degrees C for each replicate were normalized to positive and negative control plate average absorbance, and replicate normalized values were averaged to calculate an average relative absorbance at both temperatures. A substance was considered a temperature-dependent positive at 42 degrees C if average relative absorbance <= 50 and the difference between relative absorbance at 30 degrees C and 42 degrees C >= 20 (relative absorbance 30 degrees C - relative absorbance 42 degrees C >= 20). A substance was considered a temperature-independent positive if relative absorbance at both 30 degrees C and 42 degrees C < 10.

Activity scores were calculated using average relative absorbance at both temperatures. Average relative absorbance <= 0 was scored as 100 for activity; average relative absorbance >= 100 was scored as 0 for activityy. Average relative absorbance between 0 and 100 was subtracted from 100 to generate activity scores for intermediate values (i.e. average relative absorbance = 40 corresponds to an activity score of 60).

Note that since this is treated as a panel assay, the query for active compounds includes many that were considered active at both temperatures. The final Pubchem activity score is the activity score at 42 degrees C, and compounds scored as active (PUBCHEM_ACTIVITY_OUTCOME = 2) include both temperature-dependent and temperature-independent active substances.

External ID: HMS750

Protocol: The stalled elongation complex used as a substrate in the screening assay was generated by pre-incubating 600 nM 3Dpol with 12 nM 5' fluorescein labeled 8-6 PETE RNA and 240 nM ATP for 30 minutes at room temperature in 50 mM HEPES, pH 7.0, 40 mM NaCl, 1.5 mM magnesium acetate, 60 microM ZnCl2, 4 mM DTT, and 0.1% Igepal detergent. This solution was dispensed into 384-well microplates (Corning 3710) at a volume of 25 microL/well. Experimental compounds were added by robotic pin transfer at 100 nL/well.

After an incubation time of ~60 min, the total fluorescence and fluorescence polarization signals from the labeled RNA were measured with Perkin Elmer EnVision microplate readers. This first reading provided data indicating whether compounds interfered with RNA binding to the stalled elongation complex. Polymerase elongation activity was then tested by adding 5 microL of a solution containing GTP, CTP, and UTP at 1.2 microM each, resulting in a final concentration of 200 nM for each of the four NTPs and 16.7 microg/ml for the compounds. This allowed for elongation to the end of the RNA template, and assay data were obtained with a second reading done after an incubation period of ~60 min, which is four times longer than the ~15 min needed for elongation under non-inhibited conditions.

Comment: Potential inhibitors were identified using a correlation plot combining standard Z-scores with an elongation efficiency measurement. A Z-score for each compound was calculated by the normal method of Z=(FPcompound-FPplate_mean)/StdDevplate_mean. An elongation efficiency value (FP %Elongation) was then calculated as E=(FPcompound-FPpositive)/(FPnegative-FPpositive), which reflects how the FP signal obtained in the presence of a compound compared to those of the unelongated positive controls (FPpositive) and fully elongated negative controls (FPnegative). Compounds that gave a FP signal greater than the starting material but less than the fully elongated controls thus had E values between 0% and 100%, while compounds that caused the RNA to dissociate from 3Dpol had negative E values due to the FP value associated with free RNA being lower than the unelongated FPpositive value. Finally, the elongation reaction also results in an increase in the total fluorescence intensity due to deprotonation of the fluorescein as it approaches the positively charged polymerase surface, providing an additional assessment of elongation. Compounds that resulted in total fluorescence %Elongation higher than that of the fully elongated control samples were rejected from the analysis. Positives were defined as having experimental FP Z-scores < -0.5 and FP %Elongation < 90%. Activity scores were calculated based on FP %Elongation. FP %Elongation <= 0 was scored as 100 for activity; FP %Elongation >= 100 was scored as 0 for activity. FP %Elongation values between 0 and 100 were subtracted from 100 to generate activity scores.