in silico screening strategy for discovering antibacterial compounds

28 Pages·2016·3.44 MB·English

INTEGRATED IN VITRO – IN SILICO
SCREENING STRATEGY FOR
DISCOVERING ANTIBACTERIAL
COMPOUNDS
Leo Ghemtio & Päivi Tammela
Faculty of Pharmacy
BACKGROUND
Cell-based antimicrobial HTS assay using
recombinant luminescent bacterial strain (E. coli K-
12/pTetLux1)

Aiming to improve antimicrobial screening
• Antimicrobial assay time reduction from 24 to 2 hours
• Higher-throughput: miniaturization feasible to 384-
format using automated liquid handling

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ASSAY PRINCIPLE
E. coli K12 (pTetLux1)
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HTS ASSAY
DEVELOPMENT

• Optimize conditions for sensor bacteria storage
Preparations
• Determination of CFU count in overnight cultures
Assay optimization
• Optimize the amount of bacteria and assay temp.
1 • Determine luminescence time point measurements
Assay optimization
• DMSO tolerability, pre-incubation time testing
2 • Creating SOP
Assay
• Miniaturization from 96 to 384-well plate format
miniaturization • Optimising automated liquid handling, HTS SOP
• Known antibiotics
Assay validation
• Screen of known bioactives, 2000 compounds
ASSAY OPTIMIZATION &
VALIDATION
Plate uniformity test
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PILOT SCREEN: SPECTRUM BIOACTIVES
LIBRARY (TOTAL 2000 COMPOUNDS)
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DOSE-RESPONSE
RESULTS FOR SELECTED
HITS
Azithromycin Chloramphenicol Doxycycline
Meclocycline
Minocycline Rifaximin
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WORKFLOW SUMMARY
Cell-based
primary HTS
Growth inhibition
Compound
against standard
solubility in
bacterial ATCC
Hit identification assay medium
strains
Preliminary Novelty
human cell assessment,
cytotoxicity promiscuity
Hit expansion by
virtual screening
Secondary testing
Compounds
confirmed active
PRIMARY HTS
• 10 240 compounds from the ChemBridge DIVERset collection
• Compounds pre-plated into 384-well plates (30 nL volumes)
using Echo® liquid transfer system
• Screened in singles (320 compounds per plate) at a final
concentration of 10 µM (0.1% DMSO). Positive controls:
erytromycin and rifampicin
• Bacterial culturing & HTS protocol:

Automated liquid handling using
Biomek FX:
Luminescense
• 30 µl total well volume
measurement
• Bacteria: 5×104 CFU/well
• Pre-incubation 30 min 37 °C
• Luminescence induction by
addition of tetracycline
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HTS RESULTS
• Assay performance quality (32 plates):
Z’ = 0.7±0.2, S/B =122±11 S/N = 14±12

Number of

Inhibition
• 8 hit compounds above 50%
compounds
inhibition
50-100% 8

40 - 50 % 22 • Hit rate (50% cutoff) : 0.08%
3 0 - 40 % 189
• Hit inhibitions ranged
20 – 30 % 907
between 52% and 87%

10 – 20 % 734

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