Periodic Reporting for period 4 - GUTPEPTIDES (Novel therapeutic approaches to improve gastrointestinal wound healing)
Période du rapport: 2022-03-01 au 2023-08-31
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 (IBS) and inflammatory bowel diseases (IBD). A therapeutic strategy that controls inflammation and restores the barrier represents an innovative approach for the prevention and treatment of such diseases. This project focused on how gut peptides regulate epithelial protection and repair and explored novel therapeutic opportunities by targeting gut receptors that become accessible once the epithelium is compromised.
The project had three specific aims:
(I) Investigate the therapeutic potential of the oxytocin receptor during gastrointestinal inflammation
(II) Elucidate the mechanism of trefoil factor family induced gastrointestinal wound healing
(III) Discover and characterise novel ligands suitable for epithelial repair
We employed a multidisciplinary approach that included state-of-the-art peptide synthesis, scaffold grafting, pharmacology, gut stability and wound healing assays, and inflammatory mouse models. We developed probes to study the mechanisms of action at a molecular level and explored the biological diversity of venoms for novel therapeutic leads.
This project advanced our fundamental understanding of epithelial protection and repair and identified several new therapeutic leads and approaches for treating IBS, IBD and abdominal pain. Key outcomes included a new class of patented peptide therapeutics against chronic abdominal pain, the discovery that bacterial biofilms are highly prevalent in patients suffering from IBD and IBS, first chemical synthesis of all TFF peptides, and new mechanistic insights into their physiological role and therapeutic potential in the gut, and new gut-stable nature-derived ligands as promising starting points to tackle gut disorders.
The project had three specific aims:
(I) Investigate the therapeutic potential of the oxytocin receptor during gastrointestinal inflammation
(II) Elucidate the mechanism of trefoil factor family induced gastrointestinal wound healing
(III) Discover and characterise novel ligands suitable for epithelial repair
We employed a multidisciplinary approach that included state-of-the-art peptide synthesis, scaffold grafting, pharmacology, gut stability and wound healing assays, and inflammatory mouse models. We developed probes to study the mechanisms of action at a molecular level and explored the biological diversity of venoms for novel therapeutic leads.
This project advanced our fundamental understanding of epithelial protection and repair and identified several new therapeutic leads and approaches for treating IBS, IBD and abdominal pain. Key outcomes included a new class of patented peptide therapeutics against chronic abdominal pain, the discovery that bacterial biofilms are highly prevalent in patients suffering from IBD and IBS, first chemical synthesis of all TFF peptides, and new mechanistic insights into their physiological role and therapeutic potential in the gut, and new gut-stable nature-derived ligands as promising starting points to tackle gut disorders.
OBJECTIVE I:
We established gut stability assays and bioassays to test activity at OTR. Using cutting-edge medicinal chemistry, we produced leads equipotent to OT, yet with a gut stability half-life of >24 h compared to 8 min of OT. Our leads are potent analgesics when given orally in IBS mouse models with a gut specific mode of action. These first-in-class oral and non-opioid-based leads were patented and form the basis of discussions with biotech.
MAIN OUTCOMES:
- gut stability assays & bioassays
- gut-stable bioactive drug leads
- new knowledge about OTR in gut disorders
- high-impact publications and patent
- ERC PoC grant on advancing drug leads
OBJECTIVE II:
The trefoil factor family (TFF) are peptides (TFF1-3) expressed throughout the gut with roles in gut protection and repair. To investigate the molecular mechanisms, we first needed a reliable way of producing them. Using a combination of solid-phase peptide synthesis and native chemical ligation we, for the first time, synthesised TFF1, TFF3 and their corresponding homodimers. We demonstrated that they were folded correctly and that TFF1 interacts with mucins. We revealed that TFF1&3 get digested in the gut but yield a bioactive metabolite. We identified a receptor for TFF1 and TFF3. Production of TFF2 was more challenging, and we had to pursue recombinant expression, semi-synthetic methods, and total chemical synthesis. We have now also control over TFF2 production. Isotope labelling of TFF2 to determine its 3D structure, resulted in a new technology of producing isotope-labelled amino acids.
MAIN OUTCOMES:
- Reliable production of TFF1-3 and TFF1/3 homodimers
- Discovery of a gut-stable bioactive metabolite of TFF1/3
- Discovery of target receptor of TFF1/3
- New mechanistic insights
- Key reviews and high impact publications
- ERC PoC grant to develop isotope-labelled amino acid production technology
OBJECTIVE III:
Venoms are a rich source of bioactive peptides and a main source in this project to identify new ligands that could accelerate wound healing or be used as gut-stable scaffolds for drug development.
We obtained venoms from venomous animals, fractionated them, and characterised the fractions by HPLC and mass spectrometry. We developed a medium-throughput pipeline to discover novel peptide ligands that are stable to gastric and intestinal digestion. Identified leads were synthesised and tested for gut wound healing and mode-of-action. While no direct wound healing activity was discovered, some of these ligands might serve as gut-stable scaffolds for drug development.
MAIN OUTCOMES:
- Establishment of cell lab and epithelial repair assays
- Identified gut-stable ligands using our new discovery platform
- PeptideMiner, a new tool for in-silico screening of peptide homologues
- Reviews and publications in venom peptide drug discovery
Our research outcomes resulted in invitations at international conferences, new collaborations, prizes and awards, and several articles covering our research.
We established gut stability assays and bioassays to test activity at OTR. Using cutting-edge medicinal chemistry, we produced leads equipotent to OT, yet with a gut stability half-life of >24 h compared to 8 min of OT. Our leads are potent analgesics when given orally in IBS mouse models with a gut specific mode of action. These first-in-class oral and non-opioid-based leads were patented and form the basis of discussions with biotech.
MAIN OUTCOMES:
- gut stability assays & bioassays
- gut-stable bioactive drug leads
- new knowledge about OTR in gut disorders
- high-impact publications and patent
- ERC PoC grant on advancing drug leads
OBJECTIVE II:
The trefoil factor family (TFF) are peptides (TFF1-3) expressed throughout the gut with roles in gut protection and repair. To investigate the molecular mechanisms, we first needed a reliable way of producing them. Using a combination of solid-phase peptide synthesis and native chemical ligation we, for the first time, synthesised TFF1, TFF3 and their corresponding homodimers. We demonstrated that they were folded correctly and that TFF1 interacts with mucins. We revealed that TFF1&3 get digested in the gut but yield a bioactive metabolite. We identified a receptor for TFF1 and TFF3. Production of TFF2 was more challenging, and we had to pursue recombinant expression, semi-synthetic methods, and total chemical synthesis. We have now also control over TFF2 production. Isotope labelling of TFF2 to determine its 3D structure, resulted in a new technology of producing isotope-labelled amino acids.
MAIN OUTCOMES:
- Reliable production of TFF1-3 and TFF1/3 homodimers
- Discovery of a gut-stable bioactive metabolite of TFF1/3
- Discovery of target receptor of TFF1/3
- New mechanistic insights
- Key reviews and high impact publications
- ERC PoC grant to develop isotope-labelled amino acid production technology
OBJECTIVE III:
Venoms are a rich source of bioactive peptides and a main source in this project to identify new ligands that could accelerate wound healing or be used as gut-stable scaffolds for drug development.
We obtained venoms from venomous animals, fractionated them, and characterised the fractions by HPLC and mass spectrometry. We developed a medium-throughput pipeline to discover novel peptide ligands that are stable to gastric and intestinal digestion. Identified leads were synthesised and tested for gut wound healing and mode-of-action. While no direct wound healing activity was discovered, some of these ligands might serve as gut-stable scaffolds for drug development.
MAIN OUTCOMES:
- Establishment of cell lab and epithelial repair assays
- Identified gut-stable ligands using our new discovery platform
- PeptideMiner, a new tool for in-silico screening of peptide homologues
- Reviews and publications in venom peptide drug discovery
Our research outcomes resulted in invitations at international conferences, new collaborations, prizes and awards, and several articles covering our research.
The ERC Starting Grant and my establishment at the University of Vienna generated many new research opportunities and avenues. It resulted in several novel collaborations that will be beneficial for the overall project progress and will provide complementary and innovative angles to our research. For example, we are working now together with gastroenterologist and microbiologist on novel treatment strategies for gastrointestinal disorders; we revealed that gastrointestinal biofilms play a significant role in irritable bowel syndrome and inflammatory bowel diseases and our ERC program and established knowledge on developing gut-stable peptide ligands for oral delivery to target such biofilms is critical and unique in developing new drug leads. Another example is our latest collaboration with nanomaterial specialist to develop a nanoparticle delivery system to deliver therapeutic peptides into the colon, without having to modify the peptide. This could have a substantial impact on peptide drug delivery and boosting drug development and delivery for treating gastrointestinal disorders and metabolic diseases such as diabetes and obesity. In addition, this program along with our results has opened up new avenues of targeting the gut-brain axis via activating receptors within the gut or enteric nervous system. This is a very intriguing new direction that my lab wants to expand on.