antibiotics - drug discovery examples Flashcards
(28 cards)
What was the target enzyme in the Mur Ligase inhibitors study?
MurA
Which compound was used to validate the target enzyme MurA?
fosfomycin
Where was the high-throughput screening (HTS) performed?
Novartis, Cambridge, Massachusetts
What was the filter criterion for activity against any prokaryote?
≤ 32 μg/ml
What was the required inhibition percentage at 1 mM for the screening?
≥ 50% inhibition
What was the maximum allowed inhibition percentage at 100 μM?
≤ 10% inhibition
What was the requirement for complete solubility?
Complete solubility at 1 mM
In which organisms was in vitro activity tested against MurA?
E. coli
What type of assays were performed for specificity?
Chymotrypsin and MDH
How many compounds entered the screen?
~650K compounds
How many compounds passed the 1 mM activity screen?
~2000 compounds
What was the filter criterion for inhibition at 50 μM?
≥ 50% inhibition
How many compounds passed the selectivity screens?
26 compounds
What assay was used to exclude membrane damaging agents?
BacLight assay
What was the maximum allowed membrane damage at 4 X MIC?
≤ 10% membrane damage
How many compounds passed the Baclight screen?
10 compounds
What was the focus of the low throughput academic investigation?
Purification of an antimicrobial peptide from the venom of Bungarus fasciatus
What chromatography technique was used for the initial purification?
Cation Exchange Chromatography (IEX)
What was the elution gradient used in IEX?
0-1M NaCl
Which pool showed activity against E. coli?
Pool 2
What was used for further purification and desalting after IEX?
Solid Phase Extraction
What was the elution solution used in Solid Phase Extraction?
80% Acetonitrile (ACN) + 0.05% TFA
What chromatography method was used for the final stage of purification?
C18 Reverse Phase HPLC
What was the gradient used in C18 Reverse Phase HPLC?
0-100% 80% ACN + 0.05% TFA
