OC 41.1 - Structural Identification of the Factor XII - uPAR Binding Interface For Selective Drug Development.

Monday, June 26, 2023

14:45 – 15:00 ET

Room: 516

Session Available As: Live-Stream and On-Demand

Presenting Author(s)

Dillon J. Bohinc (he/him/his)

Researcher
Department of Medicine, Hematology and Oncology Division, CWRU School of Medicine, Cleveland, OH, USA.
Cleveland, Ohio, United States

Co-Author(s)

KB

Kara L. Bane

Research Associate
Department of Medicine, Hematology and Oncology Division, CWRU School of Medicine, Cleveland, OH, USA.
Cleveland, Ohio, United States
Md

Maria de la Fuente

Research Associate
Department of Pharmacology, CWRU School of Medicine, Cleveland, OH, USA.
Cleveland, Ohio, United States
SR

Shruti Raghunathan

Researcher
Department of Biomedical Engineering, Case Western Reserve University
Cleveland, Ohio, United States
MN

Marvin T. Nieman

Associate Professor
Case Western Reserve University
Cleveland, Ohio, United States
Steven de Maat, PhD

Assistant Professor
Department of CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Utrecht, Utrecht, Netherlands
Helen Philippou, PhD

Professor
University of Leeds.
Leeds, England, United Kingdom
Jonas Emsley, Phd

Professor
Nottingham Biodiscovery Institute, School of Pharmacy
Nottingham, England, United Kingdom
Coen Maas, PhD

Associate Professor
CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
Utrecht, Utrecht, Netherlands
TR

Thomas Renné

Professor
Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Hamburg, Hamburg, Germany
Anirban Sen Gupta, PhD (he/him/his)

Professor of Biomedical Engineering
Case Western Reserve University
Cleveland, Ohio, United States
Evi X. Stavrou, MD

Associate Professor
Department of Medicine, Hematology and Oncology Division, CWRU School of Medicine, Cleveland, OH, USA.
Cleveland, Ohio, United States

Background: We previously identified the FXII-uPAR signaling axis in neutrophils and found that it upregulates neutrophil functions. In sterile inflammation, the axis can be targeted to improve wound healing and cancer progression. Current strategies primarily focus on inhibition of FXII protease functions. Similarly, uPAR inhibition with antibodies can impair access of other ligands and adversely impact tissue proteolysis and fibrinolysis. To circumvent these issues, we propose to selectively abrogate the FXII-uPAR interaction. However, the precise binding sites between FXII and uPAR are not known.

Aims: To elucidate the binding interface between FXII and uPAR and create therapeutics that abrogate FXII-uPAR mediated proinflammatory effects.

Methods: We used hydroxyl radical footprinting (HRF), site-directed mutagenesis, and neutrophil functional assays to uncover the binding sites between FXII and uPAR and developed novel inhibitory peptides that selectively abrogate FXII-uPAR complex formation.

Results: The schema we pursued in solving the FXII-uPAR interface is detailed in Figure 1A. FXII-uPAR HRF revealed three distinct regions on FXII that contained highly protected amino acid residues (Figure 1B). These residues were replaced with alanine through site-directed mutagenesis and FXII variants were tested for their ability to bind uPAR using microscale thermophoresis (Figure 1C). These studies provided the details for the development of FXII-derived peptide antagonists that interfered with FXII-mediated phosphorylation of pAkt2S474 (Figure 2A-B), ROS generation and NET formation in healthy human neutrophils, alone or in combination (Combo; Figure 2C-E). In a murine model of type I diabetes, combination of FXII-uPAR inhibitory peptides, delivered within neutrophil-targeted nanoparticles, led to increased rate of wound closure (Figure 2F-G) and significantly reduced the expression of proinflammatory mediators, determined by RNAseq.

Conclusion(s): Here we uncover the FXII-uPAR binding interface and present the design and testing of small molecule inhibitors that prevent FXII and uPAR effects in models of inflammation ex vivo and in vivo.

Learning Objectives:

Upon completion, the participants will be able to gain insight on novel zymogen Factor XII functions in chronic sterile inflammation.
Upon completion, the participants will be able to appreciate domain interactions within the heavy chain of Factor XII.
Upon completion, the participants will be able to learn about strategies to target specific Factor XII conformations and their therapeutic applicability.