L4: Case study in drug development

Question 1

describe how azacitidine was first discovered.

Answer 1

eg. DNA Methylation
1. we know that DNA methylation leads to gene silencing/ inactive chromatin -> wanted to see if DNA methylation is an active driver of cancer
2. identified target: DNMT1 in breast cancer by comparing normal vs disease state
3. target validation: loss of function of DMNT1 assay showed that tumour shrinks
4. azacitidine is a methyltransferase inhibitor –> prevents gene silencing of tumour suppressor genes

Question 2

how did the first azacitidine perform?

Answer 2

drug response was highly variable between diff cell lines

Question 3

how was azacitidine improved? [2]

Answer 3

create new versions of the drug, either ‘me toos’ or ‘next gen’ drugs

1. researchers first did structure based screening [rational drug design] with diff libraries –> identified hit/lead
2. lead optimisation: created a lead series via bioisosteric replacement aka making modifications to the side groups

Question 4

how did researchers validate azacitidine HIT/leads?

Answer 4

1. they did primary assays on the HITS identified [cell free assay: prepare enzyme on plate and add substrate then add capture anibody] ==> compare with parent compound and choose those that have lower DNA methylation than parent compound [become LEADS]
2. did another round of primary assay on 2 LEADS identified from ^, and chose the one more effective at lower dose.

Question 5

what were the steps taken after LEAD validation?

Answer 5

1. LEAD optimisation using SAR analysis
2. Pre-clinical drug development: find out CMC of drug

Question 6

why did direct matrix metalloproteinases (MMPs) fail in clinical trials?

Answer 6

they were too toxic

Question 7

how did researchers eventually find a drug to target MMPs?

Answer 7

1. used high-throughput screening and identified a small molecule repressive for MMP-9
2. did a cell based primary assay using luciferase assay [new*]. assay targets a specific chromatin region. if MMP-9 expressed, will have luciferase read-out
3. also evaluated robustness of luciferase assay by finding Z’.
4. underdo high throughput luciferase screening –> identified 4 HITs
5. HIT validation via secondary assays
   - MTT assay
   - zymography
   - endogenous cellular efficacy test
   - migration and invasion test
6. identify LEADS using SAR analysis and optimise LEADs

Question 8

MOA of MMP-9 inhibition

Answer 8

bind to activator protein (AP-1) and inhibits endogenous MMP-9