Open Source Antibiotics: Simple Diarylimidazoles Are Potent against Methicillin-Resistant Staphylococcus aureus

Antimicrobial resistance (AMR) is widely acknowledged as one of the most serious public health threats facing the world, yet the private sector finds it challenging to generate much-needed medicines. As an alternative discovery approach, a small array of diarylimidazoles was screened against the ESKAPE pathogens, and the results were made publicly available through the Open Source Antibiotics (OSA) consortium (https://github.com/opensourceantibiotics). Of the 18 compounds tested (at 32 μg/mL), 15 showed >90% growth inhibition activity against methicillin-resistant Staphylococcus aureus (MRSA) alone. In the subsequent hit-to-lead optimization of this chemotype, 147 new heterocyclic compounds containing the diarylimidazole and other core motifs were synthesized and tested against MRSA, and their structure–activity relationships were identified. While potent, these compounds have moderate to high intrinsic clearance and some associated toxicity. The best overall balance of parameters was found with OSA_975, a compound with good potency, good solubility, and reduced intrinsic clearance in rat hepatocytes. We have progressed toward the knowledge of the molecular target of these phenotypically active compounds, with proteomic techniques suggesting TGFBR1 is potentially involved in the mechanism of action. Further development of these compounds toward antimicrobial medicines is available to anyone under the licensing terms of the project.


Summary 1.1 Study
Hit Confirmation of active compounds by whole cell growth inhibition assays was conducted as an 8-point dose response to determine the Minimum Inhibitory Concentration (MIC), in duplicate (n=2).The inhibition of growth is measured against those microorganisms that showed susceptibility to the compounds tested in the Primary Screen.
Included in the Hit Confirmation were 5 bacteria: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus, and 2 fungi Candida albicans and Cryptococcus neoformans.
In addition to determining MIC, active compounds were counter screened for cytotoxicity against a human embryonic kidney cell line, HEK293, by determining their CC 50 value.The compounds were also screened for haemolysis of human red blood cells.As per the T&C's of CO-ADD, structures for all submitted compounds for antimicrobial screening should be disclosed to CO-ADD following Primary Screening.Without structures for all submitted compounds, Hit Confirmation assays will not be triggered.

Assay
If you have not already done so, please provide CO-ADD with the chemical structure of the full sample set in this study (both for compounds showing activity and those that do not), which will allow CO-ADD to filter out future samples with the same, or highly similar structure.
In addition, please notify CO-ADD if you agree to publish the data (i.e.structures and activity) in the public bioactive database ChEMBL (www.ebi.ac.uk/chembl/).CO-ADD aims to increase the public knowledge of antimicrobial research, including data about non-active compounds.
All confirmed hits, without cytotoxicity or haemolytic activity, will be considered for further Hit-Validation, after a detailed analysis of structure-activity relationship and antimicrobial novelty, within CO-ADD samples, as well as, within public antimicrobial activity databases, like ChEMBL (www.ebi.ac.uk/chembl/).

Publishing CO-ADD Data
If you wish to publish data provided by CO-ADD, we kindly ask that you acknowledge CO-ADD appropriately with the following reference: Please advise CO-ADD at your earliest convenience that you have used provided data for publication purposes.This information is extremely helpful in keeping track of the outputs from the CO-ADD initiative and supports the program in renewed funding possibilities to continue CO-ADD as a free screening service available to the academic community.
CO-ADD also asks, that where possible you publish your data in an Open Access journals.

Sample Preparation
Samples were provided by the collaborator and stored frozen at -20 °C.Samples were prepared in DMSO and water to a final testing concentration of 32 µg/mL or 20 µM (unless otherwise indicated in the data sheet) and serially diluted 1:2 fold for 8 times.Each sample concentration was prepared in 384-well plates, non-binding surface plate (NBS; Corning 3640) for each bacterial/fungal strain, tissue-culture treated (TC-treated; Corning 3712/3764) black for mammalian cell types and polypropylene 384-well (PP; Corning 3657) for haemolysis assays, all in duplicate (n=2), and keeping the final DMSO concentration to a maximum of 0.5%.All the sample preparation was done using liquid handling robots.
Compounds that showed notable solubility issues during stock solution preparation are detailed in the Data sheet for the individual Project.

Procedure
All bacteria were cultured in Cation-adjusted Mueller Hinton broth (CAMHB) at 37 °C overnight.A sample of each culture was then diluted 40-fold in fresh broth and incubated at 37 °C for 1.5-3 h.The resultant mid-log phase cultures were diluted (CFU/mL measured by OD 600 ), then added to each well of the compound containing plates, giving a cell density of 510 5 CFU/mL and a total volume of 50 µL.All the plates were covered and incubated at 37 °C for 18 h without shaking.

Analysis
Inhibition of bacterial growth was determined measuring absorbance at 600 nm (OD 600 ), using a Tecan M1000 Pro monochromator plate reader.The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references.
The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate.The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by an inhibition ≥ 80%.In addition, the maximal percentage of growth inhibition is reported as D Max , indicating any compounds with partial activity.

Procedure
Fungi strains were cultured for 3 days on Yeast Extract-Peptone Dextrose (YPD) agar at 30 °C.A yeast suspension of 1 x 10 6 to 5 x 10 6 CFU/mL (as determined by OD 530 ) was prepared from five colonies.The suspension was subsequently diluted and added to each well of the compound-containing plates giving a final cell density of fungi suspension of 2.5 10 3 CFU/mL and a total volume of 50 µL.All plates were covered and incubated at 35 °C for 36 h without shaking.

Analysis
Growth inhibition of C. albicans was determined measuring absorbance at 630 nm (OD 630 ), while the growth inhibition of C. neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD 600-570 ), after the addition of resazurin (0.001% final concentration) and incubation at 35 °C for 2 h.The absorbance was measured using a Biotek Multiflo Synergy HTX plate reader.
In both cases, the percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (fungi without inhibitors) on the same plate.The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by an inhibition ≥ 80% for C. albicans and an inhibition ≥ 70% for C. neoformans.Due to a higher variance in growth and inhibition, a lower threshold was applied to the data for C. neoformans.In addition, the maximal percentage of growth inhibition is reported as D Max , indicating any compounds with marginal activity.
Hits were classified by MIC ≤ 16 µg/mL or MIC ≤ 10 µM in either replicate (n=2 on different plates).HEK293 cells were counted manually in a Neubauer haemocytometer and then plated in the 384-well plates containing the compounds to give a density of 5000 cells/well in a final volume of 50 µL.DMEM supplemented with 10% FBS was used as growth media and the cells were incubated together with the compounds for 20 h at 37 °C in 5% CO 2 .
CC 50 (concentration at 50% cytotoxicity) were calculated by curve fitting the inhibition values vs. log(concentration) using a sigmoidal dose-response function, with variable fitting values for bottom, top and slope.In addition, the maximal percentage of cytotoxicity is reported as D Max , indicating any compounds with partial cytotoxicity.
The curve fitting was implemented using Pipeline Pilot's dose-response component, resulting in similar values to curve fitting tools such as GraphPad's Prism and IDBS's XlFit.Any value with > indicate sample with no activity (low D Max value) or samples with CC 50 values above the maximum tested concentration (higher D Max value).
Cytotoxic samples were classified by CC 50 ≤ 32 µg/mL or CC 50 ≤ 10 µM in either replicate (n=2 on different plates).In addition, samples were flagged as partial cytotoxic if D Max ≥ 50%, even with CC 50 > the maximum tested concentration.

Procedure
Human whole blood was washed three times with 3 volumes of 0.9% NaCl and then resuspended in same to a concentration of 0.5 x 10 8 cells/mL, as determined by manual cell count in a Neubauer haemocytometer.The washed cells were then added to the 384-well compound-containing plates for a final volume of 50 µL.After a 10 min shake on a plate shaker the plates were then incubated for 1 h at 37 °C.After incubation, the plates were centrifuged at 1000g for 10 min to pellet cells and debris, 25 µL of the supernatant was then transferred to a polystyrene 384-well assay plate.

Analysis
Haemolysis was determined by measuring the supernatant absorbance at 405 mm (OD 405 ).
The absorbance was measured using a Tecan M1000 Pro monochromator plate reader.
HC 10 and HC 50 (concentration at 10% and 50% haemolysis, respectively) were calculated by curve fitting the inhibition values vs. log(concentration) using a sigmoidal dose-response function with variable fitting values for top, bottom and slope.In addition, the maximal percentage of haemolysis is reported as D Max , indicating any compounds with partial haemolysis.
The curve fitting was implemented using Pipeline Pilot's dose-response component, resulting in similar values to curve fitting tools such as GraphPad's Prism and IDBS's XlFit.Any value with > indicate sample with no activity (low D Max value) or samples with HC 10 values above the maximum tested concentration (higher D Max value).
Haemolysis samples were classified by HC 10 ≤ 32 µg/mL or HC 10 ≤ 10 µM in either replicate (n=2 on different plates).In addition, samples were flagged as partial haemolytic if D Max ≥ 50%, even with HC 10 > the maximum tested concentration.

Antibiotic, Cytotoxic and Haemolytic Standards Preparation and Quality Control
Colistin and Vancomycin were used as positive bacterial inhibitor standards for Gramnegative and Gram-positive bacteria, respectively.Fluconazole was used as a positive fungal inhibitor standard for C. albicans and C. neoformans.Tamoxifen was used as a positive cytotoxicity standard.Melittin was used as a positive haemolytic standard.
Each antibiotic standard was provided in 4 concentrations, with 2 above and 2 below its MIC or CC 50 value, and plated into the first 8 wells of column 23 of the 384-well NBS plates.
Tamoxifen and melittin was used in 8 concentrations in 2 fold serial dilutions with 50 µg/mL highest concentration.
The quality control (QC) of the assays was determined by Z'-Factor, calculated from the Negative (media only) and Positive Controls (bacterial, fungal or cell culture without inhibitor), and the Standards.Plates with a Z'-Factor of ≥ 0.4 and Standards active at the highest and inactive at the lowest concentration, were accepted for further data analysis.

Outcome
All standard compound controls displayed inhibitory values within the expected range for each assay type and each organism tested.For further information please contact the CO-ADD team at support@co-add.org.

Microbial strains and cell lines
ID: HC-Procedures & Materials Date: Last updated June 2018 Confidential Page 10 of 11

4.0 Controls 4.1 Antimicrobial susceptibility of tested strains
Values are the average of ≥ 6 independent biological replicates.All values are within the expected range as per CLSI guidelines.

3 Susceptibility profile of human washed red cells
Values are the average of > 6 independent biological replicates.HC10 and HC50 are the concentrations at 10% and 50% haemolysis, respectively.