Lecture 2

Question 1

Describe the typical drug development pipeline.

Answer 1

Lab-based target discovery
In vitro validation (eg. cell lines)
In vivo validation (eg xenografts)
In vivo PD and PK profiling

Question 2

What is a xenograft?

Answer 2

Human cells grown in immunocompromised animals.

Question 3

What precedes drug development?

Answer 3

Comprehensive validation of a target.

Question 4

What must be considered for development of a new therapy?

Answer 4

The chosen target has an activity that can be druggable
Inactivation of the target has a negative effect on the disease cell growth and may also drive cell apoptosis.
The target is expressed, potential aberrantly, in the disease.
If these criteria are satisfied then can go ahead with a drug development programme.

Question 5

What does X-Ray crystallography do in regards to drug development?

Answer 5

Gives globular structure and secondary helices structure.
Used for refining and adapting chemistry’s of drugs so they bind better to the target protein.

Question 6

What does high-throughput sequencing do in regards to drug development?

Answer 6

Identifies several drugs that could target the protein of interest.
Knowing the structure of the protein from x-ray crystallography allows the list of drugs to be refined and then the best candidates are generated for further testing.

Question 7

How could you identify a suitable cell line that expresses the protein of interest in drug development?

Answer 7

Need to select a cell line that expresses the protein of interest.

Question 8

What is a clonogenic assay (colony-forming)?

Answer 8

Most immortal cancer cells can grow in 2 dimensions without the help form surrounding tissue.
When seeded out at single-ce;; densities on flasks/dishes they will form colonies.
Trying to understand if you take a cell and treat it with a drug does it have an impact on the viability of the cell.
If there is reduced colony forming ability then it suggests the compound is cytotoxic to the cell.

Question 9

How do you calculate plating efficiency of a clonogenic assay?

Answer 9

Count the colonies - 1 colony = 1 viable cell
Divide the colonies by the number of initial seeded cells then multiply by 100.

Question 10

What do adherent cells grow as in vitro?

Answer 10

Mono-layers on specific plastic ware in growth medium that contains foetal-calf serum.

Question 11

Why do cells being grown in vitro need to be routinely passaged (split)?

Answer 11

Cells in culture grow indefinitely and need to be split to help keep them healthy.

Question 12

What must be done once cells have been split when growing them in culture?

Answer 12

They must be counted to allow accurate repeating of a desired cell number - imperative for clonogenic assays.

Question 13

How can we count cells?

Answer 13

Using a haemocytometer.
Inject 10um of the cell suspension onto the coverslip, capillary flow goes through chamber and then can look down microscope and count cells.

Question 14

How would you count cells in a haemocytometer that is 1mmx1mm with a 0.1mm gap?

Answer 14

Vol of liquid over the 1mm square = 0.1cm x 0.1cm x 0.01cm = 10^-4cm3 so 10^-4ml.
Number of cells over the 1mm square = n so n is the number of cells in 10^-4ml.
So 1ml = n x 10^4 so the suspension contains n x 10^4 cells per ml.
Count the cells that are within the confines of the 1mm^2 grid then multiply by 10000.

Question 15

Why should a cell line be counted multiple times?

Answer 15

To aid accuracy.

Question 16

Why aren’t all replicates the same?

Answer 16

Errors in pipetting
Errors in timing
Variation in cell cultures
Variation in state of cells

Question 17

What must else be done to validate a new drug as a novel cancer treatment?

Answer 17

1. plot graphs of mean number of colonies expressed at % of control
2. determine the concentrations of the drugs that are necessary to kill 50% of the cells (IC50)
3. Determine the ratio between the IC50 of the 2 drugs
4. Determine if the compound is more cytotoxic than the other compound

Question 18

How would you calculate the IC50 of compounds?

Answer 18

% survival plotted on y-axis
Where lines cross at 50% survival and read the concentration of the drug off the x-axis
This is the IC50 value and represents the concentration of drug required to kill 50% of cells

Question 19

Why might a compound be less cytotoxic than another compound even though it was initially found to be 10-fold more effective in inhibiting the target protein in the in vitro assays?

Answer 19

Compound is less able than the other to enter cells
Compound is more readily exported from the cells than the other
The recombinant enzyme used in the initial tests does not reflect the properties of the protein in the cell
The protein is not the key target for cytotoxic effects of these compounds