External ID: HMS1126

Protocol: 384-well assay plates (Corning 3820) were filled with Mix 1 at 10 microL per well. Mix 1 contained 133.3 microM potassium phosphate pH 7.5, 133.3 microM manganese sulfate, 5% Cayman enzyme mix, and 2 microM BmaI1. 300 nL of each experimental compound were pin-transferred to assay wells. 3.33 microL of 40 mM acetovanillone in DMSO was added to positive control wells before the reaction was started.

The primary assay was initiated with 3.33 microL Mix 2. Mix 2 contained 100 microM octanoyl-ACP, 800 microM 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), and 140 microM S-adenosylmethionine (SAM). The secondary assay was initiated by addition of 3.33 microL of Mix 4. Mix 4 contained 133.3 microM potassium phosphate pH 7.5, 133.3 microM manganese sulfate, and 4% Cayman enzyme mix. The assay was allowed to proceed for 40 to 60 minutes, at which time it was stopped by the addition of 3.33 microL of 40 mM acetovanillone to all wells except the positive control wells.

Plates were read on a PerkinElmer EnVision (525/25 excitation filter, 590/20 emission filter, 555 mirror. Gain 165, excitation light 5%, 10 flashes).

Library plates were screened using a primary assay and a counterscreen assay, with all assay plates for a given library plate prepared on the same day. For every compound library plate, there were four daughter plates. Primary plates A & B were prepared as described; counterscreen plates CNTL-A and CNTL-B contained the same components except for 5'-Deoxy-5'-(methylthio)adenosine instead of BmaI1, acyl-ACP, and SAM. Counterscreen plates also contained 3% enzyme mixture instead of 3.5%.

Positive control: All wells of column 24 contained the final enzymatic reaction mix, with acetovanillone added before the assay was started, and no experimental compound.
Negative control: All wells in column 23 contained only the final enzymatic reaction mix (no experimental compound).

Comment: For each non-excluded experimental well, a robust Z-score was calculated based on the fluorescence intensity median and normalized MAD for each replicate in the primary assay and the counterscreen assay. The absolute value of each experimental screen well Z-score was subtracted from the absolute value of plate average positive control Z-score. Wells with a robust Z-score < 2 units from the plate average positive control Z-score for both primary screen replicates, and a robust Z-score < 3 for at least one counterscreen replicate, were considered positive.

Activity scores were calculated for both the primary screen and the counterscreen using normalized percent of control values. For each well (both replicates), the positive control plate average fluorescence intensity was subtracted from the well fluorescence intensity, divided by the difference between plate average negative and positive control fluorescence intensity, subtracted from 1, and multiplied by 100. Some plates lacked a positive control; the positive control average of all plates was used for these plates. Replicate normalized percent of control values were averaged for the primary screen and counterscreen separately to determine activity scores for each. Values less than 0 were set to 0 and values greater than 100 were set to 100.

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.