Tut 4: How to improve drugs (Peptide mimetics)

Question 0

What is peptidomimetics?

Answer 0

Peptidomimetics = peptide mimetics = designed compounds that contain non-peptide structural elements but are capable of mimicking the biological action of a natural parent peptide. The drug mimetics can include backbone-modified peptides and small non-peptide molecules.
Peptide mimetics no longer contain any classical peptidic bonds, instead they contain unnatural amino acids and/or unusual compounds that integrated into the backbone.

Question 1

Why do we want to design peptidomimetics?

Answer 1

Various experimental techniques yield particles that have significant and wide spread biological activity, but have undesirable pharmacological properties such as unstable, easily excreted, can’t cross membranes etc. Therefore, there is a need to design structurally similar organic compounds - peptidomimetics.
A peptide is an easy lead for biology but has poor drug properties, making is a poor drug candidate.
Mimetics take advantage of the peptide date, and produced a drug candidate with good properties, stable and bioavailability.

Question 2

What is the different between modified peptides and pseudopeptides?

Answer 2

Modified peptides - Peptide derivatives with relatively small modifications that don’t alter the peptide bond but make the peptide more resistant to proteases and are therefore more stable.
Pseudopeptides - peptide derivatives containing modifications to the peptide bond

Question 3

Describe the nature of peptide mimetics

Answer 3

Peptidomimetics are usually small compounds that consist of structural elements that mimic a peptide binding element in 3D and mimics the function (i.e. same job, same receptor, same agonist/antagonist activity)
Peptide modifications are used to stabilise structure, prevent degradation and alter biological activity.

Question 4

What is the difference between agonists and antagonists?

Answer 4

Agonist - An agonist drug has an active site of similar shape to the endogenous ligand so it binds to the receptor and produces the same effect.
Antagonist - An antagonist drug is close enough in shape to bind to the receptor, but isn’t close enough to produce an effect. It take up all the receptor space, hence inhibiting the endogenous ligand from binding.

Question 5

List and describe some of the modifications that are made by chemists.

Answer 5

• Peptide N-end protection: Eg. N-terminal end acetylation. (Alkylation is widely used to improve stability and lipophilicity)
• Replacing an amino acid with a non-natural amino acid.
• Enantiomers: Replacing L-enantiomers to D-enantiomers confers resistance to proteolytic degradation.
• Peptide cyclisation: Used to mimic natural peptide structures of to synthesise more stable peptide analogues therefore enhancing conformational stability and resistance to proteolytic hydrolysis.
• Minor side chain modifications of amino acids including amide bond surrogates (Amide bonds replaced with other chemical groups to prevent protease degradation of the amide bond), peptoids (the moving of the alpha-carbon side chain by one position to the amide nitrogen to increase flexibility), azapeptides (the alpha-carbon is replaced with a nitrogen to increase stability)

Question 6

What are the steps for designing peptidomimetics?

Answer 6

1) Identify a peptide sequence within protein that shows activity to a relevant assay
2) develop structure-activity relationships
3) define minimal active sequence and identify the key residues and portions of peptide backbone responsible for the biological effect
4) consider 3D conformation - Modify/add constraints to modify the key residues regions to create mimetics.

Alternatively, large libraries of compounds can be synthesised and screened for activity. Screening mimetics is more effective than random screening, however agonists and antagonists of receptors have been found through random screening.

Question 7

What are some of the issues associated to the detection of erythropoiesis-stimulating agents?

Answer 7

• Technically challenging; Affinity purification, IEF, SDS-PAGE, immunoassays, mass spectrometry
• Distinguishing between ESA’s and native EPO
• Are they detectable in blood and or urine?
• When should the sample be taken?
• Speed of urine/blood collection and sample analysis (I.e. half-life, stability etc).
• Exclusion of underlying conditions that can lead to an increase in EPO and hence and increase in red blood cells.
• The results combine the measure of major haematopoietic pharmacodynamic parameters in blood.

Question 8

What are some of the methods of doping?

Answer 8

• High altitude training
• Blood transfusion
• Human growth hormone (injectable): Used for patients with a deficiency in growth hormones, many healthy athletes use this as they think it bulks up muscle and improves physical performance, however studies have shown that it may increase the ability to sprint, but there are no effects on the fitness/jumping/lifting ability of an individual.
• Gene doping (emerging method): Recombinant protein is directly produced within a human cell (Not introduced). It is developed from gene therapy trials to treat genetic diseases. When artificial genes are introduced by a viral vector, there are several health risks associated. so gene doping reduces this risk. In this method there are issues with detecting because proteins are not shown in blood or urine analysis so a muscle biopsy would be required.

Question 9

Identify methods to optimise EPO mimetics

Answer 9

• Xray crystal structure: Information on they key residues involved in binding of EMP1 to the EPO receptor.
• Key amino acids binding group identified by an EPO-receptor binding screen modified chemically: The creation of libraries of compound derivatives of EMP1 designed to bind to the EPO receptor.
• Compounds are screened for EPO receptor binding: Evaluated in vitro for the ability to compete with EPO in the receptor binding assay
• Compound are screen for EPO biological activity: Evaluated in vitro for the ability to support cell proliferation in EPO-responsive cell lines.
• Identification of peptides that possess mimetic properties and contain a minimal agonist epitope: Less potent than EPO, but can be improved.
• Optimisation: Screening for dimeric, trimeric and tetrameric versions of candidate ligand (If it contains multiple copies of identical binding subunits there is an increase in the chance to interact with a second receptor molecule.)