Design and Characterization of Macrocyclic Peptide Inhibitors Targeting Protease Activity in Wound Healing

Introduction

Proteases play multiple roles in the wound healing process, as well as in processes crucial for bacterial growth, dissemination, and virulence. Their detection can be achieved using antibodies, which indicate their presence and expression levels, but do not provide information about their enzymatic activity. Importantly, it is the active forms of proteases that carry out effector functions. This means that a comprehensive understanding of the role of proteases requires assessing both their expression and their activity.

Protease activity is typically studied using peptide-based compounds that contain a sequence recognized by the target protease, as well as a detectable reporter group, such as a fluorescent moiety. In the search for structures that interact with proteases, both linear and cyclic compounds can be explored. It has been demonstrated that macrocyclic compounds are more stable and often more specific than their linear analogues, which is why they are the focus of our research.

The lab of P. Kasperkiewicz is interested in development of chemical tools for proteases study and their application in the biological systems, mainly for study of immune system.

Background

Our previous studies have demonstrated that the incorporation of non-natural amino acids facilitates the identification of more specific structures for probing proteases. Moreover, it has been shown that macrocyclic compounds are more stable than their linear analogues and enable the discovery of structures with reduced cross-reactivity.

The main objectives of the PhD project will be (i) to develop chemical tools for selected proteases detection and (ii) to validate utility of these molecules in biological samples in wound healing.

Methodology

To search for selective structures targeting the studied proteases, we will employ peptide libraries containing unnatural amino acids. For peptide synthesis in our studies, we will use the solid-phase peptide synthesis (SPPS) method with a Rink amide resin. Hydrolysis of peptides will be detected using the fluorophore ACC, a coumarin derivative. Compound purification and analysis will be performed by HPLC and LC-MS. Fluorescence measurements will be carried out with a plate-based spectrofluorimeter. Biological studies will include, among others, the use of confocal microscopy.

Expected Results

We will develop activity-based probes that selectively detect protease activity, rather than merely their expression levels. This distinction is crucial, as protease abundance can be misleading due to the presence of inactive forms, such as pro-enzymes or enzymes bound to endogenous inhibitors. The probes designed by our team will enable the detection of active neutrophil and bacterial serine proteases—key players in inflammation and infection—in wound samples.

In addition, we aim to target Staphylococcus aureus proteases using a new generation of macrocyclic peptide inhibitors. These inhibitors, built from unnatural amino acids, are expected to offer enhanced stability and specificity compared to their linear counterparts, and may effectively prevent bacterial dissemination within tissues.

The successful candidate will be responsible for conducting independent and collaborative research within the scope of the project, including the design, synthesis, and characterization of macrocyclic compounds. The role involves planning and performing experiments, analyzing and interpreting data, and maintaining accurate documentation of research activities.

The candidate will contribute to the development and optimization of experimental protocols, carry out biochemical and cell-based assays, and apply advanced analytical and imaging techniques. They will also be expected to critically review scientific literature, prepare reports and publications, and present research findings at international conferences.

In addition, the candidate will participate in research stays (secondments) at partner institutions, collaborate with interdisciplinary teams, and actively contribute to the overall progress and dissemination of the project.

https://remod-healing.univ-lyon1.fr/consortium-supervisors/

https://wch.pwr.edu.pl/

Eligibility for enrollment in the Doctoral School at Wrocław University of Science and Technology

Master’s degree in chemistry, biotechnology, or a related field

Basic knowledge of biochemistry

Strong motivation to pursue a scientific career

Interest in and attitude for experimental research

Ability to learn quickly and effectively assimilate new knowledge

Good teamwork and communication skills Proficiency in English (written and spoken)

Computer literacy, including MS Office and scientific data analysis software (e.g., GraphPad Prism)- Commitment to comply with the regulations of the Department of Biological Chemistry and Bioimaging, Faculty of Chemistry, WUST, regarding research and work organization, as well as the Doctoral Student Code of Ethics

Applicants should not be in possession of a doctoral degree at the date of the recruitment and need to comply with the mobility rule (no residence or main activity (work, studies, etc.) in the country of the recruiting beneficiary for more than 12 months in the 36 months before their recruitment date).