Tutorial 4: How to improve drugs: peptide mimetics Flashcards
How is a peptidomimetic designed?
- Identify a peptide sequence within protein (eg a hot spot) that shows activity in a relevant assay.
- develop structure-activity relationships.
- Define minimal active sequence and identify key residues and portions of peptide backbone responsible for biological effect
- consider 3D conformation - modify/add constraints to modify key residues/regions to create mimetic. Need to consider optimal fit of the binding site, stability, degradation, whether transport across biological membrane…
Alternatively, or in parallel to rational design of mimetics, large libraries of compounds can be synthesised and screened for activity. Screening mimetics is more effective than random screens.
What are peptide mimetics?
AKA peptidomimetics. Designed compounds that contain non-peptidic structural elements, but are capable of mimicking the biological action of a natural parent peptide.
Drug mimetics include backbone-modified peptides, and small non-peptide molecules.
Typically, peptide mimetics no longer contain any classical peptide bonds. They have unnatural AAs and/or unusual compounds integrated within the backbone.
Peptide mimetics are especially useful in drug development as they may antagonise, stimulate, or otherwise modulate the physiological activity of natural ligands. peptide modifications used to stabilise structure, prevent degradation, and alter biological activity.
Describe how binding of the ligand to the EPO receptor is critical for its biological activity?
The EPO receptor is activated by ligand-induced homodimerisation, which triggers a signalling cascade that is responsible for EPO-mediated erythropoeisis.
Describe how the EPO mimetic promotes active EPO receptor dimerisation.
The dimerisation of the EPO receptor is mediated by non-covalent association of 2 peptides that is promoted by EPO.
Agonist ligand EMP1 (EPO mimetic) favours formation of active dimeric receptor conformation by bringing together the Intracellular domains of the receptor into correct proximity and orientation to allow the phosphorylation events that innitiate the signal cascade.
How was the EPO mimetic EMP1 identified?
First, there was a need to obtain information about the functional importance of minimal mimetic peptide sequence, and amino acids required for effective EPO mimetic action.
Some strategies employed to find this information included:
- series of truncation peptides was generated to find minimum sequence necessary for mimetic action
- conserved residues of a known peptide mimetic of EPO were subjected to an alanine replacement strategy. This allowed researchers to see whether a specific amino acid was required for biological activity, without imposing too much steric constraints.
These strategies led to the identification of EMP1 sequence.
Describe the optimisation of EPO mimetics.
X-Ray crystal structure: gives information on key residues involved in binding of EMP1 to EPO receptor
The binding group of key AAs were identified and chemically modified to create libraries of compound derivatives of EMP1 designed to bind to EPO receptor.
These compounds were then screened for EPO receptor binding and evaluated in vitro for ability to compete with EPO in receptor binding assay.
Compounds were also screened for EPO biological activity, and evaluated in vitro for ability to support cell proliferation in EPO-responsive cell lines.
This led to the identification of peptides that possess mimetic properties and contain a minimal agonist epitope (less potent than EPO but can be improved).
Optimisation: screening of dimeric, trimeric and tetrameric versions of candidate ligand. Contains multiple copies of identical binding subunits, leading to increased chances to interact with second receptor molecule (active EPO receptor is a dimer) –> dimers sufficient for biological activity.
Describe the development of synthetic erythropoeisis-stimulating agents (ESAs).
rHuEPO is widely used to treat anaemia caused by chronic renal disease.
First recombinant haematopoeitic growth factor produced - available commercially as recombinant protein drug since 1989.
Several types of rHuEPO commercially available for some time: Epoetin alpha/beta (first gen) and darbepoetin alpha (second generation)
Loss of patents combined with important therapeutic use led to the recent development of new EPOs (rhEPOs: CERA, and EPO mimetics)
Describe the use of ESAs for doping and the associated risks.
ESAs are widely used for doping in sports. Since rhEPO was introduced, approximately 3-7% of elite endurance athletes use doping with the drug.
Associated risks include increased blood viscosity, which leads to increased risk of thrombosis and CVD.
Discuss the many issues related to detection of ESAs.
Technically challenging: affinity purification, immunoassays, mass spec
Must be able to distinguish ESAs from native EPO
Must consider when sample is taken, at length of intervals.
Speed of urine and blood collection, and sample analysis (ESA half life and stability should be considered)
Must exclude underlying conditions that can lead to increased levels of native EPO and RBCs (eg tissue hypoxia, defect in erythroid progenitors, impaired oxygen delivery)
What are some other methods of doping?
High altitude training and blood transfusion used to increase the amount of red blood cells and increase oxygen delivery to tissues.
Human growth hormone: many healthy athletes use rGH with the belief that it will increase muscle mass and improve physical performance. It may increase ability to sprint on a bicycle, but no effects on fitness or ability to pull weight or jump.
Gene doping is an emerging technology.
Describe gene doping.
Recombinant protein is directly produced within the human cells (not introduced).
Developed from legitimate gene therapy trials in animals aimed at treating genetic diseases by introducing and expressing a gene that is deficient, or by modulating activity of an existing gene.
Artificial gene introduced by viral vector-mediated delivery or engineered, genetically modified cells introduced to host. many potential health risks.
Problems with detection: protein does not show up in blood or urine - need muscle biopsy
New sophisticated methods currently being developed (proteomic profiling, immune response to viral vectors, analysis of specific sequences in transgene, structural differences in proteins encoded by transgene)
