Theme 2 Lecture 4 Brief History of Drug Discovery

Question 1

Describe the evolution of drug screening and lead discovery

Answer 1

The beginning was the discovery of penicillin, then receptor binding assays, recombinant DNA technology, HTS and MTS

Question 2

Explain the evolution of classical in vitro and in vivo pharmacology.

Answer 2

In vitro tissues and in vivo measurements (blood pressure/HR). Was limited by lack of gene sequences and protein structures. However had quantitative agonism and antagonism.

Question 3

Identify key blockbuster drugs

Answer 3

Beta blockers
Histamine h2 antagonists
Beta 2 adrenoreceptors
5-HT receptors
Dopamine D2 receptor antagonists
CCBS

Question 4

Explain molecular target-based screens

Answer 4

Chemical libraries
HTS-using automated systems to test thousands to millions of compounds rapidly.
High content screening
Low successes as drugs usually fail in clinical trials

Question 5

List examples of Screening Technologies

Answer 5

FLIPR-Ca2+
NanoBRET
Reporter Genes
Label Free

Question 6

Intracellular Calcium

Answer 6

Fluo4AM detects changes in calcium levels in response to the GPCR activation

Question 7

GPCR activation pathways calcium via subunits

Answer 7

Gq and Gi/o which modulates via Gβγ subunits.

Question 8

What does BRET stand for?

Answer 8

Bioluminescence Resonance Energy Transfer

Question 9

Explain how BRET works?

Answer 9

Uses N-terminal Nluc tag on the (GPCR)
Measures the energy transfer between NanoLuc and the fluorescent ligand.

Thus showing Ligand Binding

Question 10

How do reporter genes work for cell signalling?

Answer 10

Reporter genes are luciferase and GFP.
These measure the transcriptional activity resulting from GPCR signalling.

Question 11

What is DMR

Answer 11

Dynamic mass redistribution

Question 12

Explain label free technologies

Answer 12

Monitors changes in the cell monolayer which is caused by GPCR activation without the need for fluorescent or luminescent labels

Question 13

Target identificiation

Answer 13

Find a disease phenotype
Find drugs that interfere with the phenotype
Then find its specific protein targets

Question 14

Target based vs phenotypic screening

Answer 14

Target based: Known molecular targets, high throughput, clear mechanism, biophysical lead confirmation and disease relevance risk
Molecular based; No knowledge of targets, lower throughput, unknown/multiple mechanisms, lead confirmation by cell assays and in vivo and disease relevant.

Question 15

Examples of phenotypic screens

Answer 15

Cell=Thyrocytes Assay=TSH responsive proteins
Tumour cells or assay would be Cell viability (ATP content)

Question 16

what is pluripotent cells

Answer 16

They can be differentiated into any cell type

Question 17

Chlorcyclizine

Answer 17

Repurposed for treating HCV infection.
CAG promoter has high levels of gene expression.
Therefore allows luciferase expression.

Question 18

MOA for chlorcyclizine

Answer 18

Targets E1
Prevents viral fusion of the lipid envelope at a low pH environment
Thus no release of the viral RNA into the cytosol.