Current capabilities

• Four specialized microbiology labs, for low and high risk BS2 pathogens
  • One for bacteria, low risk, with automation for reading and incubation
  • One for bacteria, low to high risk, with Anaerobic chamber
  • One for bacteria, high risk, manual workflows (BSL-3 built, but not certified)
  • One for fungal, low risk, with automation for reading
• Microbe Collection, with +450 isolates with tested growth conditions
  • 219 Gram-negative
  • 232 Gram-positive, including mycrobacteria
  • 17 Fungi

• Optimized screening workflow, for consistency and throughput
• Panel of standard organisms
  • Escherichia coli ATCC 25922, FDA control strain
  • Klebsiella pneumoniae ATCC 700603, MDR
  • Acinetobacter baumannii ATCC 19606, Type strain
  • Pseudomonas aeruginosa ATCC 27853, Quality control strain
  • Staphylococcus aureus ATCC 43300, MRSA
  • Candida albicans ATCC 90028, CLSI reference
  • Cryptococcus neoformans ATCC 208821, H99 - Type strain
• Panel of membrane deficient organisms
  • Escherichia coli MB4902, ∆(lpxC) (Merck)
  • Escherichia coli MB5747, ∆(tolC) (Merck)
  • Pseudomonas aeruginosa PAO397, ∆(mexAB-oprM) ∆(mexCD-oprJ) ∆(mexEF-oprN) ∆(mexJKL) ∆(mexXY) ∆(opmH) ∆(pscC). (H. Schweizer U. Florida)
• Standard assay conditions
  • All plate handling and dispensing under sterile conditions, with handling of only 1 organism at a time, with separate dispensing units for each organisms, and/or usage of single disposable tips
  • 384 well plate format, with 2 columns for controls
  • Negative control: media only
  • Positive control: bacteria only, without compound
  • Standard control: 4 different concentration of vancomycin (Gram-positive), colistin (Gram-negative) and fluconazole (fungi)
  • Non-binding surface (NBS) plates (Corning #3640)
  • Media: 50 uL of Cation adjusted Mueller Hinton Broth (CaMHB) for bacteria
    Media: 50 uL of Yeast Nitrogen Base broth (YNB) for fungi
  • Incubation with 5x10⁵ CFU/mL, for 18 h at 37C, without shaking, for bacteria
    Incubation with 2.5x10⁵ CFU/mL, for 24 h at 37C, without shaking, for fungi
    Dispensing of bacterial/fungal media with bulk dispensers.
  • Readout by Absorbance at 600nm (OD₆₀₀) for bacteria, absorbance at 530nm (OD₅₃₀) for candida spp., absorbance at 600nm-570nm (OD_600-570) for cryptococcus spp., after addition of Resazurin. Readouts are done without plate-lids, using Tecan M1000 Pro and BioTek HTX multimode readers.
  • Automated analysis of plates using inhouse developed workflow with Pipeline Pilot (Biovia), and integrated into sample tracking software, with the following characteristics:
    • Percentage inhibition, based on median values of negative and positive control wells
    • Calculation of modified Z-score (samples), using median and MAD values (instead of mean and standard deviation) : Mi= (0.6745 (x_i-x ̃))/MAD
    • QC criteria for accepting plate is, Z’Factor (controls) > 0.45 and standard controls display at least 2 actives and 2 inactive wells.
    • Selection for hit-confirmation: Actives = Inhibition ≥ 80% and Z-Score > 3.5
      Inhibition ≥ 50% and Z-Score > 2.5 are classified partial actives and included in hit-confirmation for Gram-negative bacteria only.
    • Automatic MIC calculation using inhouse script with Pipeline Pilot (Biovia), following the CSLI guidelines for selecting an MIC value, i.e. requirement of at least two inactive or active wells. Wells are classified as active by Inhibition ≥ 80%.
• Throughput of assays is dependent on incubation space and reading time.
  • Bacteria : 210,000 data points (wells) per week
    200 plates per screening day, with max 3 screening days per week
  • Fungi: 56,000 data points (wells) per week
    80 plates per screening day, with max 2 screening days per week
• Current Primary Screen includes 5 bacteria, 2 fungi and 3 Gram-negative strains with membrane deficiency, and in duplicates (n=2):
  • 15,000 compounds/week – full panel
• Current Hit-Confirmation includes 8 concentrations, 5 bacteria, 2 fungi and 1 Gram-negative strains with membrane deficiency, and in duplicates (n=2):
  • 3,500 compounds/week– full panel, without cytotoxicity

CO-ADD’s Hit-to-Lead facilities consists of:

• Fully functional Medicinal/Organic Chemistry labs
  • 14 Fume hoods
  • Inventory of chemicals and solvent
  • Preparative HPLC for purification
  • Analytical LS/MS for chemical analysis
  • Access to NMR (500, 600 and 900 MHz)
  • Access to HiRes MS/MS and TOF spectrometers
  • Access to Peptide synthesizer
  • Chemical Database (Abase IDBS)

The organic chemistry lab has synthesized and purified 2500 compounds since 2010, with an average of around 350 per year. The range of compounds include mainly small molecules (<600 Da), peptides, peptidomimetics and glycopeptides.

• Fully functional Biological labs, with a range of established assays for Hit development and Hit-to-Lead optimization:
  • Bacterial growth inhibition in Broth, as high-throughput screen (384well)
  • Bacterial growth inhibition on Agar, as medium-throughput (24 well)
  • Time-kill evaluation by CFU
  • Resistance frequency determination
  • Resistant induction studies, with 20 day passaging and genetic analysis
    Access to Whole Genome Sequencing, transcriptome, gene expression profiling, whole human exome. Illumina NextSeq 500 and MiSeq, Agilent 2100 Bioanalyzer, PE LabChip GX Oxford Nanopore MinION.
  • Microscopy with fluoro-probes, for localization, morphology and mode-of-action studies
    using inhouse probes for Vancomycin, linezolid, trimethoprim, polymyxin, octapeptin, and b-hairpin AMPs
    Access to 25 high end microscopes, including confocal (upright & inverted), STED, SR-SIM, spinning disc confocal, deltavision, lattive light sheet microscope, brightfield/darkfield, fluoro, FLIM/FRET. Cryo-TEM facility.
  • FACS with fluorescent probes
  • Membrane penetration and depolarization assay, using fluorescent probes like NPN, DisC3-5, PI, SYTOX-Green
  • Structural Target investigations, using XRay crystallography or NMR
    including Target production and purification inhouse or using UQ Protein Expression Facilities (expression with a wide range of host cells, including insect cells)
    Access to High-Performance Computer facilities and software for molecular dynamics, docking, machine/deep learning, for virtual ligand-based and structure-based screening.
  • Binding studies with protein targets, using:
    • label-free technologies, including Surface Plasmon Resonance (SPR), isothermic Titration Calorimetry (ITC), or xCELLigance, NMR or MS;
    • Labeled technologies, including FRET/BRET, AlphaScreen, Fluorescent Polarisation, or fluoro-probes.
  • Chemical property studies, including solubility and stability in serum and solvent.
  • Liver microsome stability assays, in-house and by CRO.
  • Toxicity analysis, by cytotoxicity and haemolytic activity
  • In vivo murine thigh infection model, including with bioluminescent strains (MRSA), for efficacy and PK studies.
  • Off-target screening, extended PK, in vivo toxicity using CRO, with good relationship to frequently used CRO, like WuXi Apptech and Cerep/Eurofin.