Andy Wattes - Basic principles of drug design 4 - Biological Therapeutics

Question 1

How is technology impacting the drug discovery process?

Answer by listing the new steps involved…

Answer 1

1) Identify disease
2) Isolate protein (genomics, proteomics, biopharm)
- Identify more targets, personalise drugs
3) High throughput screening of existing
- Screen up to 100,000 molecules a day for activity against target
- Test large number of compounds faster
4) Virtual screening
- Use PC to predict activity (no need to make compound)
5) Combinatorial chemistry
- Rapidly produce vast numbers of compounds
6) Molecular modelling
- Computer graphics and models to help improve activity
- Predict better compounds
7) In vitro and in silico screening
- Computer and tissue models to begin to replace animal testing
- Faster, more ethical, cheaper

Question 2

What are Biological Therapeutics?

Answer 2

• Wide range of medicinal products such as:
  1) Vaccines
  2) Gene therapy
  3) Recombinant therapeutic proteins and peptides

Question 3

Where can Biological Therapeutics be isolated from?

Answer 3

• Humans
• Animals
• Micro-organisms

Question 4

When might Gene-based and cellular biologics be particularly useful?

Give 3 examples…

Answer 4

• To treat a variety of medical conditions for which no other treatments are available
• To treat medical conditions where existing therapies were inadequate
• e.g Rheumatology, oncology, cardiology

Question 5

What was the earliest example of protein and peptide based therapeutics?

Answer 5

• Insulin

Question 6

What type of inhibitor is Fuzeon (Enfuviritide)?

How does it work?

Answer 6

• Fusion inhibitor polypeptide - made synthetically
• Blocks the entry of HIV into cells (CD4 or T cells)
• Does this by mimicking components of GP41
  (GP41 anchors virus to cell membrane of host and pulls the membranes together to permit fusion)
• Enfuviritide displaces the GP41 and prevents this normal fusion

Question 7

What structure is Enfuviritide (Fuzeon) related to?

Answer 7

• Related to viral protein GP41 which anchors virus to cell membrane of host cell and pulls membranes together to permit fusion

Question 8

What is the mechanism of action of HIV fusion?

Answer 8

1) HIV approaches host CD4 T cell. Viral membrane has glycoprotein spikes with gp41 and gp120 subunit
2) Fusion begins with gp120 binding to CD4 and chemokine receptors on cell membrane
3) Induces a conformational change, exposing gp41
4) Fusion mediated by gp41 which has two repeat domains HR1 and HR2
5) gp41 exposed and hydrophobic terminus embeds itself into cell membrane. HR2 domain coils into grooves on HR1 domain of gp41 - ZIPPING
6) Cell and viral membrane = destabilised and viral membrane punches a hole (fusion pore) through both membranes
7) HIV capsid passes through cell membrane and infection occurs

Question 9

What is the mechanism of action of Fusion (Enfuvirtide)?

Answer 9

1) Fuzeon is a peptide mimic of HR2 region of gp41
2) Fuzeon binds to HR1 region
3) Zipping cannot take place
4) Infection is blocked

Question 10

What are some advantages of Fuzeon?

What are some disadvantages of Fuzeon?

Answer 10

• Shows antiviral activity in vitro against wild type virus as well as virus resistant to all three classes of current antiretroviral
• Has a unique mode of action so no cross resistance with other ARV classes (GP41 domain is therapeutic target exclusive to Fuzeon)
• Expensive
• Has BD administration by SC injection

Question 11

What type of drug design was used in the discovery of Fuzeon?

Answer 11

• Rational drug design

- Identified druggable target and went from there

Question 12

What are some general issues with biological therapeutics?

Answer 12

• Proteins and peptides are not good ‘drug-like’ molecules
• Need to improve PK properties of biological therapeutics

e. g Increasing serum half life of protein to…
- Reduce cost of therapy
- Improve quality of life (less freq injections)

Question 13

List 4 ways that Biological Therapeutics can be produced…

Answer 13

1) Synthetically (peptides such as Fuzeon)
2) Human cell lines (retains post-translations modifications, but gives low yields)
3) Yeast cells (humanised) - greater production and retains post-translational modifications
4) Bacterial cells - much greater yields but lacks post-translational modifications

Question 14

What are 4 ways that proteins are modified after translation?

Answer 14

• Phosphorylation
• Sulfation
• Glycosylation (add carbohydrates)
• Acylation

Question 15

What are the two major types of glycosylation? (post translational modification)

Answer 15

1) O-linked glycosylation
- Important for protein targeting to specific receptors

2) N-linked glycosylation
- Important role in protein regulation and serum half-life
- Important role in PK properties of Biological Therapeutics

Question 16

What pharmacokinetic parameter can be influenced by N-linked glycosylation?

Answer 16

• Half life

Question 17

Which drug is a good example of how PK properties of biologics can be improved?
- What type of drug is this?

Answer 17

• Filgrastim
• Human granulocyte colony-stimulating factor (G-CSF)
• AA protein made by recombinant DNA tech
• High molecular weight

Question 18

How does Filgrastim (human granulocyte colony stimulating factor) work?

Answer 18

• Colony stimulating factors = glycoproteins that act on hematopoietic cells by binding to specific cell surface receptors and stimulating proliferation, differentiation and end-cell functional activation
• Used to treat neutropenia (low no of neutrophils)
• Stimulates bone marrow to inc production of neutrophils
• Used to treat variety of cancers

Question 19

What was an issue with Filgrastim?

Answer 19

• Had a very short T1/2
• 3.5 hours
• Produced by E.coli that had been inserted with human granulocyte colony stimulating factor gene
• AA sequence identical to natural sequence in human DNA BUT addition of N-terminal methionine for expression in E coli
• Produced in E.coli so product = NONGLYCOSYLATED
• Differs from G-CSF isolated from human cells
• Reason for short T 1/2?

Question 20

Name a way of improving PK properties of Biologics…

How does this work?

Answer 20

PEGylation

• attach strands of PEG (polyethylene glycol) polymer to molecules - peptides, proteins, antibody fragments
• do this by incubating reactive derivative of PEG with target macromolecule
• improves safety and efficiency of many therapeutics
• covalent attachment of PEG to therapeutic protein can ‘mask’ agent from host’s immune system (reduced immunogenicity and antigenicity)
• increase hydrodynamic size (size in solution) of agent thus prolonging its circulatory time by reducing renal Cl

Question 21

List some ways in which PEGylation of a molecule can impart pharmacological advantages over the unmodified form…

Answer 21

• Increases the MW of a molecule
• Improved solubility
• Reduced dosage freq, without diminished efficacy with potentially reduced toxicity
• Extended circulating life - Inc MW so less renal Cl (not filtered)
• Increased drug stability
• Enhanced protection from proteolytic degradation - hydrophilic, polymeric shield (protects and disguises)
• Extend patent life of previously approved drugs
• New delivery formats and dosing regimens

Question 22

What was done to Filgrastim to improve activity and PK?

Answer 22

• Made into Pegfilgrastim
• Covalent conjugate of recombinant methanol human granulocyte colony stimulating factor and PEG (monomethoxypolyethylene glycol)
• PEG bound to N-terminal methanol residue of Filgrastim
• HIGH MW
• Studies on cellular proliferation, receptor binding and neutrophil function show that Filgrastim and PEGfilgrastim have same mech of action
• Colony stimulating factors that act on haematopoeitic cells, stim proliferation, differentiation, end cell functional activity

Question 23

What are the differences between Filgrastim and Pegfilgrastim?

Answer 23

• Pegfilgrastim:
• Reduced renal Cl due to increased MW meaning it is not filtered into the kidney
• Prolonged persistance in vivo
• Improved potency
• Sub cut injection but only ONCE A FORTNIGHT compared to daily for Filgrastim