Drug discovery Flashcards
2 approaches to drug discovery
- Structure-based drug
2. Ligand-based
Drug discovery vs development
Discovery: identification of small synthetic molecule or large biomolecule for comprehensive evaluation as potential drug candidate
Development: candidate continues through several stages to make safe/effective medicine
Process of drug discovery
Target identification > development of high-throughput assays > high-throughput screening > hit compounds > refinement (structure based durg design) > lead compounds
Fluo-4 acetoxymethyl ester assay
Detection of intracellular Ca2+ levels.
Precursor of calcium-sensitive dye diffuses into cell > precursor gets converted to fluorescent dye, GPCR (Gq) receptor activation leads to calcium-release, calcium forms complex with dye > fluorescent
HIV therapies
Entry inhibitors, RT inhibitors, integrase inhibitors, protease inhibito (eg Amprenavir: structure-based drug design!)
How can 3D (protein) structures be obtained?
X-ray crystallography
Cryogenic electron microscopy
Nuclear magnetic resonance spectroscopy
Homology (comparative modelling(
Modelling based on amino acid sequences and 3D structures of related protein
Computational docking
Docking of ligands into putative binding site based on binding affinity predictions.
Differences between high-throughput and virtual screening
HTS: automated testing of large number of chemicals/biological compounds (appropriate assay required)
Virtual: searching of large databases of 3D structures of small molecules t find those binding pocket or receptor using fast approximate docking programs
Databases
Protein DataBase (PDB): open access digital data resource providing 3D structures of proteins/nucleic acids and complexes
UniProt: freely accessible resource of protein sequence and functional information
ZINC: database for virtual screening
Steps in drug discovery
Target identification and validation >hit identificaiton and validation >hit to lead > lead optimisation
Cystic fibrosis
Mutations in CFTR gene (F508del > degradation, G542X > no synthesis) > viscous mucus in lung and other secretory organs
CFTR
Membrane protein, chloride channel.
2 TM domains, 1 nucleotide binding domain NBD each, 1 regulatory domain connecting the NBDs.
The ion channel only opens when its R-domain has been phosphorylated by PKA and ATP is bound at the NBDs. CL- efflux + osmotic water efflux
Potentiator vs corrector (CF)
Corrector drugs: help CFTR protein to form right shape, traffic to cell surface, stay there longer.
Potentiators: increase proteins ability to hold gate open longer
Lipinskis rule of five
Rules for choosing drugs for assay: compounds with five or less H-bond donors, ten or less H-bond acceptors, log P of or lower than 5, less than 500 Da
Most common approach of drug discovery nowadays
HTS (finding drug) and structure-based approach (optimization)
Protein-/Structure-based drug design/discovery
Structure of biological target is known.
Based on databases and structure candidate is searched that binds to structure with high affinity/selectivity.
» find known molecules / design new ligands / optimize known molecules
Ligand-/Fragment-based drug design/discovery
Molecules that bind to target are known. Pharmacore model is derived (minimum necessary structural characteristics for binding).
»modification of pharmacore model to create new molecules with new/improved properties
FRET assay
Emitting of light at adequate wavelength > plasma membrane localized fluorescent dye > excites other, voltage-sensitive membrane solvable dye > voltage change by CI-efflux
