Drug discovery

Question 1

2 approaches to drug discovery

Answer 1

1. Structure-based drug

2. Ligand-based

Question 2

Drug discovery vs development

Answer 2

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

Question 3

Process of drug discovery

Answer 3

Target identification > development of high-throughput assays > high-throughput screening > hit compounds > refinement (structure based durg design) > lead compounds

Question 4

Fluo-4 acetoxymethyl ester assay

Answer 4

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

Question 5

HIV therapies

Answer 5

Entry inhibitors, RT inhibitors, integrase inhibitors, protease inhibito (eg Amprenavir: structure-based drug design!)

Question 6

How can 3D (protein) structures be obtained?

Answer 6

X-ray crystallography
Cryogenic electron microscopy
Nuclear magnetic resonance spectroscopy

Question 7

Homology (comparative modelling(

Answer 7

Modelling based on amino acid sequences and 3D structures of related protein

Question 8

Computational docking

Answer 8

Docking of ligands into putative binding site based on binding affinity predictions.

Question 9

Differences between high-throughput and virtual screening

Answer 9

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

Question 10

Databases

Answer 10

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

Question 11

Steps in drug discovery

Answer 11

Target identification and validation >hit identificaiton and validation >hit to lead > lead optimisation

Question 12

Cystic fibrosis

Answer 12

Mutations in CFTR gene (F508del > degradation, G542X > no synthesis) > viscous mucus in lung and other secretory organs

Question 13

CFTR

Answer 13

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

Question 14

Potentiator vs corrector (CF)

Answer 14

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

Question 15

Lipinskis rule of five

Answer 15

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

Question 16

Most common approach of drug discovery nowadays

Answer 16

HTS (finding drug) and structure-based approach (optimization)

Question 17

Protein-/Structure-based drug design/discovery

Answer 17

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

Question 18

Ligand-/Fragment-based drug design/discovery

Answer 18

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

Question 19

FRET assay

Answer 19

Emitting of light at adequate wavelength > plasma membrane localized fluorescent dye > excites other, voltage-sensitive membrane solvable dye > voltage change by CI-efflux