Identification of novel macrocyclic inhibitors of PSD-95 using tailored mRNA encoded libraries

Abstract

Macrocyclic peptides are promising therapeutic modalities due to their ability to target protein-protein interactions (PPIs) with high specificity and affinity. In contrast to their linear analogues, macrocycles are conformationally constrained and the lack of N- and C-terminal make them more resistant to metabolic degradation. Advances in de novo peptide drug discovery, such as the Random non-standard peptides integrated discovery (RaPID) system, provides a powerful mRNA display technology and facilitates generation of large (10^12) genetically diverse macrocyclic peptide libraries for development of therapeutic entities. The post-synaptic density protein 95 (PSD-95) is an abundant scaffolding protein present within the post synaptic density (PSD) of excitatory synapses. Inhibiting the neuronal nitric oxide synthase (nNOS)/PSD-95 interaction is a promising therapeutic strategy for preventing brain damage from acute ischemic stroke, a disease responsible as the second leading cause of death in the world. This thesis focused on the screening of a randomized cyclic peptide library, specifically comprising nine to thirteen amino acids (AAs), expressed using the RaPID platform. Employing next generation sequencing led to the discovery of a potential 17-residue macrocyclic peptide binder towards the PDZ2 domain of PSD-95 similar to previously discovered second-generation peptides. The 17 AAs containing peptide exhibited certain internal binding motifs previously described crucial for binding in the literature. Lastly, structure-activity relationships predicted with machine learning tools yielded a foundation for future optimization-efforts towards a lead-candidate.

Popular Abstract

Stroke is the second leading cause of death in the world. This health issue is caused by blockage of blood vessels in the brain, which causes parts of the brain to die, leaving the patient with brain-damage that can affect speech, the ability to walk and move, or in the worst-case scenario, death. There are only a few existing drugs today that treat brain-damage caused by stroke. However, they need to be given to the patient in a very narrow time-period from disease start to be effective.
The history of drug development towards all kinds of diseases have long been dominated by small molecules or larger drugs like antibodies (proteins that protect you when unwanted substances enter the body). Today’s drug development methods are exploring the functions of peptides. Peptides are short chains of amino acids linked by peptide bonds. The human body naturally contain peptides, where maybe the most commonly known peptide is insulin, that controls the bodies energy supply. Peptides advantage as drugs are their ability to bind to protein surfaces that small molecules are unable to, expanding the ways of how to treat diseases. Their disadvantage is that they are broken down fast by the body’s own enzymes. They also have difficulties entering cells and passing the barrier surrounding the brain. Cyclic peptides are peptide chains with a ring structure. Development of cyclic peptides is a possible solution for slower breakdown by the body’s enzyme. However, to develop successful cyclic peptides as drugs they need to have structural characteristics different from natural amino acids. There are different methods to do this, but one way is to reprogram the genetic code from which peptides are produced so that they contain non-natural amino acids. Creation of such peptides is done by using a cell-free translation machinery (the process in which peptides are produced using RNA molecules as templates) and man-made ribosome-related enzymes. Imagine being able to use this method to create a library containing more a trillion unique cyclic peptides with non-natural amino acids, and then displaying this library against a protein target with the aim to find a binder. This is a relatively new and very powerful method that expands the way for how to discover cyclic peptides as drugs.
In this thesis, performed at University of Copenhagen, this powerful method was used to create a library of a trillion cyclic peptides containing nine to thirteen amino acids. The library was displayed against a protein present in the brain and that is known to be related to the disease-mechanism of stroke. The aim of the study was to identify a cyclic peptide with structural characteristics so that it would not only bind to the stroke-related protein, but also be small enough to pass the barrier surrounding the brain compared with previously discovered peptides. The overall purpose of the study was to get further down the road towards developing a new, more effective drug for treatment of brain-damage caused by stroke.
Surprisingly, a longer peptide than in the initial library was discovered using the described method. However, this peptide was almost identical to previously identified peptides, using the same method. This points to the method being powerful for drug discovery. Key amino acid patterns of the peptide were identified as important for binding to the stroke-related protein. These key amino acids agreed with earlier studies. Lastly, this study provides an additional base for future attempts in finding a cyclic peptide drug-candidate against stroke, a devastating disease.

• author: Walse, Ellen
• supervisor: Johan Svensson Bonde; Kristian Strømgaard
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Acute ischemic stroke, macrocyclic peptides, mRNA display, applied biochemistry
• language: English
• id: 9163504
• date added to LUP: 2024-06-20 14:12:22
• date last changed: 2024-06-20 14:12:22