ABSTRACT
Intestinal organoids have widespread research and biomedical applications, such as disease modeling, drug testing and regenerative medicine. However, the transition towards clinical use has in part been hampered by the dependency on animal tumor-derived basement membrane extracts (BMEs), which are poorly defined and ill-suited for regulatory approval due to their origin and batch-to-batch variability. In order to overcome these limitations, and to enable clinical translation, we tested the use of a fully defined hydrogel matrix, QGel CN99, to establish and expand intestinal organoids directly from human colonic biopsies. We achieved efficient de novo establishment, expansion and organoid maintenance, while also demonstrating sustained genetic stability. Additionally, we were able to preserve stemness and differentiation capacity, with transcriptomic profiles resembling normal colonic epithelium. All data proved comparable to organoids cultured in the BME-benchmark Matrigel. The application of a fully defined hydrogel, completely bypassing the use of BMEs, will drastically improve the reproducibility and scalability of organoid studies, but also advance translational applications in personalized medicine and stem cell-based regenerative therapies.
Subject(s)
Organoids , Stem Cells , Animals , Biopsy , Humans , Intestines , Reproducibility of ResultsABSTRACT
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallow substrate binding sites, selective, small-molecule inhibitors of MMPs have remained elusive. In a tour-de-force peptide engineering approach combining phage-display selections, rational design of enhanced zinc chelation, and d-amino acid screening, we succeeded in developing a first synthetic MMP-2 inhibitor that combines high potency (Ki =1.9±0.5â nm), high target selectivity, and proteolytic stability, and thus fulfills all the required qualities for in cell culture and inâ vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics.
Subject(s)
Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Engineering , Amino Acid Sequence , Binding Sites , Catalytic Domain , Humans , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase Inhibitors/chemical synthesis , Peptide Library , Proteolysis , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Chemical modification of phage libraries has allowed the in vitro evolution of ligands having properties not provided by natural polypeptides. The development of novel and more diverse chemical reactions on phage was hampered by the lack of analytical methods to efficiently monitor the reaction products on the more than 10 000 kDa large filamentous phage particles. Herein, we present a strategy to detect chemically modified peptides on phage based on enzymatic release of peptide from phage and mass spectrometry analysis.
Subject(s)
Bacteriophages/metabolism , Capsid Proteins/metabolism , Peptide Library , Peptides/chemistry , Peptides/metabolism , Directed Molecular Evolution , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Bicyclic peptide ligands were found to have good binding affinity and target specificity. However, the method applied to generate bicyclic ligands based on phage-peptide alkylation is technically complex and limits its application to specialized laboratories. Herein, we report a method that involves a simpler and more robust procedure that additionally allows screening of structurally more diverse bicyclic peptide libraries. In brief, phage-encoded combinatorial peptide libraries of the format X(m)CX(n)CX(o)CX(p) are oxidized to connect two pairs of cysteines (C). This allows the generation of 3 × (m + n + o + p) different peptide topologies because the fourth cysteine can appear in any of the (m + n + o + p) randomized amino acid positions (X). Panning of such libraries enriched strongly peptides with four cysteines and yielded tight binders to protein targets. X-ray structure analysis revealed an important structural role of the disulfide bridges. In summary, the presented approach offers facile access to bicyclic peptide ligands with good binding affinities.
Subject(s)
Bridged Bicyclo Compounds/chemistry , Cysteine/chemistry , Peptides/chemistry , Alkylation , Amino Acid Sequence , Amino Acids/chemistry , Combinatorial Chemistry Techniques , Crystallization , Disulfides , Electrophoresis, Polyacrylamide Gel , Ligands , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Peptide Library , X-Ray DiffractionABSTRACT
From a large combinatorial library of chemically constrained bicyclic peptides we isolated a selective and potent (K(i) = 53 nM) inhibitor of human urokinase-type plasminogen activator (uPA) and crystallized the complex. This revealed an extended structure of the peptide with both peptide loops engaging the target to form a large interaction surface of 701 Å(2) with multiple hydrogen bonds and complementary charge interactions, explaining the high affinity and specificity of the inhibitor. The interface resembles that between two proteins and suggests that these constrained peptides have the potential to act as small protein mimics.
