Periodic Reporting for period 3 - GUTPEPTIDES (Novel therapeutic approaches to improve gastrointestinal wound healing)
Okres sprawozdawczy: 2020-09-01 do 2022-02-28
The gastrointestinal epithelium is a major physical barrier that protects us from diverse and potentially immunogenic or toxic content. A compromised epithelium results in increased permeability to such content, thus leading to inflammation, immune response, pain, and diseases, such as irritable bowel syndrome and inflammatory bowel disease. A therapeutic strategy that controls inflammation and restores the barrier represents an innovative approach for the prevention and treatment of such diseases. This proposal focuses on how gut peptides regulate epithelial protection and repair, and explores novel therapeutic opportunities by targeting gut receptors that become accessible once the epithelium is compromised.
We tackle the overall aim of improving gastrointestinal wound healing via three complementary objectives:
(I) to investigate the therapeutic potential of the oxytocin receptor during gastrointestinal inflammation,
(II) to elucidate the mechanism of trefoil factor peptide-induced gastrointestinal wound healing, and
(III) to discover and characterise novel ligands suitable for epithelial repair.
To achieve these objectives, we employ a multidisciplinary approach that includes state-of-the-art peptide synthesis, scaffold grafting, pharmacology, gut stability and wound healing assays, and inflammatory mouse models. We will develop probes to study the mechanisms of action at a molecular level that is not possible with current tools, and explore the biological diversity of venoms for novel therapeutic leads.
This project will significantly advance our understanding of epithelial protection and repair and might identify novel drug targets or therapeutic approaches that treat the source of the problems rather than the symptoms. This project has the potential to change the way we think about treating gut disorders and how to develop peptide therapeutics, and it will pave the way towards the intriguing and longer-term goal of modulating the central nervous system via the gut-brain axis.
We tackle the overall aim of improving gastrointestinal wound healing via three complementary objectives:
(I) to investigate the therapeutic potential of the oxytocin receptor during gastrointestinal inflammation,
(II) to elucidate the mechanism of trefoil factor peptide-induced gastrointestinal wound healing, and
(III) to discover and characterise novel ligands suitable for epithelial repair.
To achieve these objectives, we employ a multidisciplinary approach that includes state-of-the-art peptide synthesis, scaffold grafting, pharmacology, gut stability and wound healing assays, and inflammatory mouse models. We will develop probes to study the mechanisms of action at a molecular level that is not possible with current tools, and explore the biological diversity of venoms for novel therapeutic leads.
This project will significantly advance our understanding of epithelial protection and repair and might identify novel drug targets or therapeutic approaches that treat the source of the problems rather than the symptoms. This project has the potential to change the way we think about treating gut disorders and how to develop peptide therapeutics, and it will pave the way towards the intriguing and longer-term goal of modulating the central nervous system via the gut-brain axis.
OBJECTIVE I: INVESTIGATE THE THERAPEUTIC POTENTIAL OF THE OXYTOCIN RECEPTOR (OTR) DURING GASTROINTESTINAL INFLAMMATION
To investigate the therapeutic potential of OTR during gastrointestinal inflammation we first need ligands that are stable to gastrointestinal digestion while at the same time able to active the OTR in the same fashion as OTR’s endogenous ligand oxytocin (OT). This is not an easy task since the endogenous peptide OT is degraded in the gut within minutes and we need to have a half-life of multiple hours for relevant in vivo studies.
We thus successfully established gastrointestinal stability assays that mimic the environment of the stomach and the intestine. We also set up the pharmacological assays to test the ligands’ bioactivity at the OTR, including stable cell lines not just for OTR, but also for the closely-related vasopressin (VP) receptors (V1aR, V1bR, V2R). We then designed and synthesised a medium-sized library of ligands using three different grafting strategies with the aim to develop gut-stable OTR-active ligands and characterised them regarding gut stability and OTR bioactivity.
We demonstrated that ligand scaffold grafting strategies that provide high stability in human serum are not stable enough for the metabolically harsher gastrointestinal environment. We also demonstrated that the stability of these grafts is highly sequence dependent and that the steric constrictions imposed on OT grafted into these scaffolds leads to substantial drops in bioactivity at the OTR. We thus moved away from scaffold grafting and switched to OT-centred Medicinal Chemistry approaches. This allowed us to identify the metabolic cleavages sites of OT and to develop the first set of ligands that are active at the OTR and have improved gut stability properties. Our current lead ligand has 3 hours gut stability compared to 8 minutes of OT. A second generation of analogues has been designed and is currently being synthesised.
MAIN OUTCOMES OF OBJECTIVE I:
- gut stability assays and OTR bioassays established
- library of OTR ligands designed and synthesised, leading to first set of gut-stable and bioactive lead ligands
- new collaborations established in the OT field
- Science Signaling publication on OT peptidomimetics
- Scientific Reports publication on OT-like ligands as pharmacological tools
- Biomedicines publication on testing and improving stability of bioactive peptides
- FASEB Journal publication on OT/OTR signalling system
- Book chapter on peptide synthesis
- Book chapter on OT synthesis
- Invited talks to national and international conferences
- Multiple press releases covering OT/OTR research
OBJECTIVE II: ELUCIDATE THE MECHANISM OF TREFOIL FACTOR-INDUCED GASTROINTESTINAL WOUND HEALING
The trefoil factor family (TFF) are large peptides that are expressed throughout the gastrointestinal tract and play an important role in epithelial protection and repair. Their mechanism of action remain however unknown and no TFF receptors or interaction proteins have been reliably identified and validated. The TFF comprises three members, TFF1, TFF2 and TFF3. They are large and complex peptides ranging from 59 to 106 amino acids in length with 3–7 disulfide bonds and rigid secondary structure. TFF1 and TFF3 can further from homo- and heteromers, while TFF2 is already made of two trefoil domains.
To investigate the mechanisms of actions of the TFF, we first needed to establish a reliable way of producing them. Using a combination of solid phase peptide synthesis and native chemical ligation we were able , for the first time, to chemically synthesise, fold TFF1, TFF3 and their corresponding homodimers, and characterise them structurally. We demonstrated that they were folded correctly and that TFF1 can interact with mucins. TFF1 and TFF3 were however inactive in our gastrointestinal wound healing assays, raising the question in how they mediate epithelial repair.
MAIN OUTCOMES OF OBJECTIVE II:
- Successfully achieved the total chemical synthesis and correct folding of TFF1 and TFF1 homodimer
- Successfully achieved the total chemical synthesis and correct folding of TFF3 and TFF3 homodimer
- Established gut stability and wound healing assays
- Trends in Biomedical Sciences review on TFF
- Cell Chemical Biology publication on chemical protein synthesis
- Two manuscripts on chemical synthesis and mechanistic studies of TFF1
- Established NMR assignments to characterise TFF1 and TFF3 structurally
- Established new TFF collaborations with Imperial College London and University of Magdeburg
- Invited talks to national and international conferences
- Multiple press releases regarding TFF
OBJECTIVE III: DISCOVER AND CHARACTERISE NOVEL LIGANDS SUITABLE FOR EPITHELIAL REPAIR
Venoms are a rich source of bioactive peptides and are the main source that we study in this project to identify new ligands that could accelerate wound healing or be used as gut-stable scaffold for ligand grafting experiments.
We purchased and set up a state-of-the-art instrument (Incucyte Zoom) to carry out the epithelial repair assays and established the assay protocols and analysis. We obtained venoms from various venomous animals, fractionated it, and characterised the venom fractions by HPLC and mass spectrometry. These fractions are currently being tested to identify novel ligands suitable for epithelial repair.
MAIN OUTCOMES OF OBJECTIVE III:
- Established epithelial repair assays and set up cell lab and Incucyte Zoom
- Fractionated and characterised various venoms using HPLC and mass spectrometry
- Chemical Review publication on conotoxins
- Australian Journal of Chemistry publication on fluorescently labelling of venom peptides
- Invited talks to national and international conferences
- Multiple press releases regarding venom peptide drug discovery
To investigate the therapeutic potential of OTR during gastrointestinal inflammation we first need ligands that are stable to gastrointestinal digestion while at the same time able to active the OTR in the same fashion as OTR’s endogenous ligand oxytocin (OT). This is not an easy task since the endogenous peptide OT is degraded in the gut within minutes and we need to have a half-life of multiple hours for relevant in vivo studies.
We thus successfully established gastrointestinal stability assays that mimic the environment of the stomach and the intestine. We also set up the pharmacological assays to test the ligands’ bioactivity at the OTR, including stable cell lines not just for OTR, but also for the closely-related vasopressin (VP) receptors (V1aR, V1bR, V2R). We then designed and synthesised a medium-sized library of ligands using three different grafting strategies with the aim to develop gut-stable OTR-active ligands and characterised them regarding gut stability and OTR bioactivity.
We demonstrated that ligand scaffold grafting strategies that provide high stability in human serum are not stable enough for the metabolically harsher gastrointestinal environment. We also demonstrated that the stability of these grafts is highly sequence dependent and that the steric constrictions imposed on OT grafted into these scaffolds leads to substantial drops in bioactivity at the OTR. We thus moved away from scaffold grafting and switched to OT-centred Medicinal Chemistry approaches. This allowed us to identify the metabolic cleavages sites of OT and to develop the first set of ligands that are active at the OTR and have improved gut stability properties. Our current lead ligand has 3 hours gut stability compared to 8 minutes of OT. A second generation of analogues has been designed and is currently being synthesised.
MAIN OUTCOMES OF OBJECTIVE I:
- gut stability assays and OTR bioassays established
- library of OTR ligands designed and synthesised, leading to first set of gut-stable and bioactive lead ligands
- new collaborations established in the OT field
- Science Signaling publication on OT peptidomimetics
- Scientific Reports publication on OT-like ligands as pharmacological tools
- Biomedicines publication on testing and improving stability of bioactive peptides
- FASEB Journal publication on OT/OTR signalling system
- Book chapter on peptide synthesis
- Book chapter on OT synthesis
- Invited talks to national and international conferences
- Multiple press releases covering OT/OTR research
OBJECTIVE II: ELUCIDATE THE MECHANISM OF TREFOIL FACTOR-INDUCED GASTROINTESTINAL WOUND HEALING
The trefoil factor family (TFF) are large peptides that are expressed throughout the gastrointestinal tract and play an important role in epithelial protection and repair. Their mechanism of action remain however unknown and no TFF receptors or interaction proteins have been reliably identified and validated. The TFF comprises three members, TFF1, TFF2 and TFF3. They are large and complex peptides ranging from 59 to 106 amino acids in length with 3–7 disulfide bonds and rigid secondary structure. TFF1 and TFF3 can further from homo- and heteromers, while TFF2 is already made of two trefoil domains.
To investigate the mechanisms of actions of the TFF, we first needed to establish a reliable way of producing them. Using a combination of solid phase peptide synthesis and native chemical ligation we were able , for the first time, to chemically synthesise, fold TFF1, TFF3 and their corresponding homodimers, and characterise them structurally. We demonstrated that they were folded correctly and that TFF1 can interact with mucins. TFF1 and TFF3 were however inactive in our gastrointestinal wound healing assays, raising the question in how they mediate epithelial repair.
MAIN OUTCOMES OF OBJECTIVE II:
- Successfully achieved the total chemical synthesis and correct folding of TFF1 and TFF1 homodimer
- Successfully achieved the total chemical synthesis and correct folding of TFF3 and TFF3 homodimer
- Established gut stability and wound healing assays
- Trends in Biomedical Sciences review on TFF
- Cell Chemical Biology publication on chemical protein synthesis
- Two manuscripts on chemical synthesis and mechanistic studies of TFF1
- Established NMR assignments to characterise TFF1 and TFF3 structurally
- Established new TFF collaborations with Imperial College London and University of Magdeburg
- Invited talks to national and international conferences
- Multiple press releases regarding TFF
OBJECTIVE III: DISCOVER AND CHARACTERISE NOVEL LIGANDS SUITABLE FOR EPITHELIAL REPAIR
Venoms are a rich source of bioactive peptides and are the main source that we study in this project to identify new ligands that could accelerate wound healing or be used as gut-stable scaffold for ligand grafting experiments.
We purchased and set up a state-of-the-art instrument (Incucyte Zoom) to carry out the epithelial repair assays and established the assay protocols and analysis. We obtained venoms from various venomous animals, fractionated it, and characterised the venom fractions by HPLC and mass spectrometry. These fractions are currently being tested to identify novel ligands suitable for epithelial repair.
MAIN OUTCOMES OF OBJECTIVE III:
- Established epithelial repair assays and set up cell lab and Incucyte Zoom
- Fractionated and characterised various venoms using HPLC and mass spectrometry
- Chemical Review publication on conotoxins
- Australian Journal of Chemistry publication on fluorescently labelling of venom peptides
- Invited talks to national and international conferences
- Multiple press releases regarding venom peptide drug discovery
The ERC Starting Grant and my establishment at the University of Vienna generated many new research opportunities. It helped to set up a range of novel collaborations that will be beneficial for the overall project progress and will provide new and innovative angles to our research. For example, we are working now together with gastroenterologist and microbiologist on novel treatment strategies for gastrointestinal disorders, as well as with a nanomaterial specialist to develop a nanoparticle delivery systems to get OT (or therapeutic peptides in general) to the colon, the desired site of action.
We expect to test our lead compounds of objectives I-III in different animal models of gastrointestinal disorders to determine if oral administration is possible and if our leads can elicit therapeutic responses. This has the potential to identify new therapeutic leads and drug targets, and validate new treatment strategies for gastrointestinal disorders.
We expect to test our lead compounds of objectives I-III in different animal models of gastrointestinal disorders to determine if oral administration is possible and if our leads can elicit therapeutic responses. This has the potential to identify new therapeutic leads and drug targets, and validate new treatment strategies for gastrointestinal disorders.