Structure Affinity Relationship Study of Macrocyclic Peptide Scaffold Binding to PSD-95

Abstract

Acute ischemic stroke is one of the major causes of death and disability worldwide. New
neuroprotective strategies have emerged, with the postsynaptic density protein 95 (PSD-95)
identified as a promising target. PSD-95 plays a crucial role in controlling downstream NMDA
receptors and neuronal death. Consequently, therapies targeting PSD-95 could mitigate stroke
damage while promoting brain protection and recovery.
This project focused on investigating cyclic peptides for the inhibition of the PDZ2 domain of
PSD-95. The study was based on a lead peptide binding to the PDZ2 domain with a Ki value of
0.3 µM. To further understand its interaction with PDZ2, an alanine scan was conducted,
synthesizing alanine variants of the parent 17-mer cyclic peptide. These peptides were evaluated
for their binding to PDZ2 using a fluorescence polarization assay to assess the contribution of each
residue. Additionally, the plasmin stability of the parent 17-mer peptide was investigated.
Due to synthesis challenges, a complete alanine scan was not possible; only nine out of the planned
14 peptides were synthesized and tested for PDZ2 binding. Results indicated that residues E3, E12,
and T13 are crucial for binding to PDZ2, while I11 and F15 are also highly important. In contrast,
I2, T5, S14, and T16 were found to be less significant for binding. Stability assays demonstrated
that the parent 17-mer peptide appears to be metabolically stable when tested in a plasmin stability
assay.
Future work should aim to complete the alanine scan and include additional positional scans, such
as N-methylated amino acid and D-amino acid scans, to further evaluate each residue's function.
Moreover, efforts should be made to miniaturize the 17-mer lead peptide by removing less
important residues, potentially enhancing cell permeability—a highly desirable characteristic
when targeting PSD-95.

Popular Abstract

Cyclic Peptides: A Novel Approach to Stroke Protection
Acute ischemic stroke is a leading cause of death and disability worldwide. Despite advances in
medical science, effective treatments to reduce stroke-induced damage remain limited. However,
new research into neuroprotective strategies is showing promise. One such strategy targets a
protein inside postsynaptic nerve cells called postsynaptic density protein 95 (PSD-95), which is
crucial in regulating brain cell survival after a stroke.
During an ischemic stroke, blood vessels to the brain are blocked, preventing oxygen from
reaching brain tissue. This blockage leads to the formation of toxic compounds that harm the brain
and cause neuronal cell death. By blocking PSD-95 protein, we hope to prevent neuronal cell death
and enhance brain recovery following a stroke. This master thesis research focuses on developing
cyclic peptides that inhibit a specific part of the PSD-95 protein to stop the formation of these toxic
compounds during a stroke. To achieve this, the project was based on a lead peptide known to bind
to PSD-95 with high affinity. An "alanine scan" was conducted to understand how each part of
this peptide interacts with PDZ2, a domain of PSD-95. This involved creating variants of the
peptide where specific amino acids were replaced with alanine, thereby losing the unique
properties of those side chains. These variants were tested to assess their binding affinity to the
PSD-95 protein. Additionally, the stability of the original peptide was assessed to ensure it could
withstand enzymatic breakdown in the body.
The study found that certain amino acids within the peptide are crucial for binding to PSD-95.
Specifically, three amino acids were essential for binding, while two more played significant roles.
In contrast, three amino acids were less important for binding. Moreover, stability tests
demonstrated that the original peptide is stable against the tested enzyme, indicating it is likely to
remain intact and effective in the body. These findings represent a significant step towards
developing a new treatment for stroke. By understanding which parts of the peptide are most
important for binding to PSD-95, the peptide designs can be refined and improved. Future work
will focus on completing the alanine scan and exploring additional modifications to further
enhance the peptide’s effectiveness and cell permeability.
In conclusion, targeting PSD-95 with specially designed cyclic peptides offers a promising new
approach to reducing stroke damage and promoting brain recovery. This master thesis research
provides valuable insights into how these peptides interact with PSD-95 and lays the groundwork
for developing more effective neuroprotective therapies.

• author: Berg, Frida
• supervisor: Ulf Ellervik; Kristian Strømgaard
• organization: Centre for Analysis and Synthesis
• course: KASM05 20241
• year: 2024
• type: H1 - Master's Degree (One Year)
• subject: Technology and Engineering
• keywords: Cyclic peptides, PSD-95, Stroke, SAR, SPPS, organic chemistry
• language: English
• id: 9163494
• date added to LUP: 2024-06-18 11:26:40
• date last changed: 2024-06-18 11:26:40