Cells, Vectors, Rational Drug Design

Question 1

What are the differences between gram positive and gram negative bacterial cell walls?

Answer 1

Gram-positive bacteria = Thick peptidoglycan layer
Gram-negative bacteria = Double membrane; thin peptidoglycan layer; LPS (drugs must also attack this when targeting gram negative bacteria)

Question 2

What are the minimal requirements for media?

Answer 2

Ions and a carbon source (glucose, glycerol, lactate)

Question 3

What is a prototroph?

Answer 3

Bacterium that can grow in minimal medium (i.e., can synthesize all necessary organic substances)

Question 4

What is an auxotroph?

Answer 4

Designated an auxotroph is any other organic substance other than a carbon source must be added to media

Question 5

What are yeast? How do they multiply?

Answer 5

Free-living, unicellular organisms (eukaryotes)

Multiply by budding, not division

Question 6

What is a bacteriophage? What are the three types (based on appearance)?

Answer 6

Virus that infects bacteria

Icosahedral tailless; Icosahedral tailed; Filamentous

Question 7

Describe the life cycle of the bacteriophage

Answer 7

• Approach
• Attach
• Inject DNA
• Transcription of phage DNA in bacteria
• Phage proteins made; replication of phage DNA; conversion of bacterium to phage factory
• “Factory” bacterium produces phage structures
  DNA packaged into phage; assembly of phage particle
• Lysis
• Cycle continues

Question 8

What are plasmids?

Answer 8

Double-stranded, circular DNA that can replicate

Can encode antibiotic resistance genes

Question 9

What are the steps of plasmid replication?

Answer 9

• Nicking of supercoiled plasma DNA and binding of protein to 5’ terminus
• Looped rolling-circle replication and transfer of displaced strand
• Completion of transfer of displaced strand (to recipient)
• Copy of transferred strand in recipient and supercoiling

Question 10

What are the four phases in batch culture growth?

Answer 10

Lag
Exponential
Stationary
Death

Question 11

How do you isolate cells from broth?

Answer 11

Centrifugation

Filtration

Question 12

In drug discovery, what is a hit and what is a lead?

Answer 12

Hit = Compound passes a screen (relative to locus)
Lead = Maintains an effect after further testing

Question 13

What are the two established approaches to drug discovery?

Answer 13

Compound-centred (compound identified, explore biological profile)
Target-centred (drug target is identified (e.g., receptor, enzyme, molecule in pathway), search for compounds that interact with it)

Question 14

What is structure-based drug design based on?

Answer 14

Drug is based on the 3D structure of target which is determined by X-ray crystallography or NMR

Question 15

What are the steps to rational drug design?

Answer 15

• Identify “active site” within the structure
• Design a drug to fit that space (stimulatory or inactive “analogue” (inhibitor))
  OR
• Identify target (disease locus or functional molecule - ion channel, proteases, kinases, nuclear hormone receptors)
• Obtain accurate structural information (crystallography, NMR, homology modelling)
• Identify target site (co-crystallize ligand with receptor)

Question 16

What are two drug design methods?

Answer 16

Computer aided (inspection, virtual screening, de-novo generation)
Experimental methods (high-throughput screening)

Question 17

Describe the inspection component of computer-aided drug design

Answer 17

Inspection (known molecules that bind to site are modified)

Question 18

Describe the virtual screening component of computer-aided drug design

Answer 18

Databases of known molecules are “docked” and scored based on predicted interactions

Question 19

Describe the de-novo generation component of computer-aided drug design

Answer 19

Small molecular fragments are positioned within the site, scored, and linked “in-silico”

Question 20

What are three critical questions to ask when designing a drug?

Answer 20

1) Are molecules available for modification as inhibitors?
2) Can we synthesize the new molecule?
3) Is the molecule accurately designed with respect to its true structure (i.e., protein and ligand flexibility)?

Question 21

What is peptidomimetics?

Answer 21

• Active fragments of larger proteins
• Increase oral bioavailability
• Parenteral drug-delivery mechanisms