Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones.

Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide(-/-) mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE's physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.

The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αß T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 α1 and α2 helices, similar to classical αß TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the α-chain, focused on the MR1 α2 helix. TCR ß-chain contacts are mostly through the variable CDR3ß loop that is positioned proximal to the CDR3α loop directly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of αß T-cell recognition of MHC and MHC-like ligands.

MAIT recognition of a stimulatory bacterial antigen bound to MR1.

MR1-restricted mucosal-associated invariant T (MAIT) cells represent a subpopulation of αß T cells with innate-like properties and limited TCR diversity. MAIT cells are of interest because of their reactivity against bacterial and yeast species, suggesting that they play a role in defense against pathogenic microbes. Despite the advances in understanding MAIT cell biology, the molecular and structural basis behind their ability to detect MR1-Ag complexes is unclear. In this study, we present our structural and biochemical characterization of MAIT TCR engagement of MR1 presenting an Escherichia coli-derived stimulatory ligand, rRL-6-CH2OH, previously found in Salmonella typhimurium. We show a clear enhancement of MAIT TCR binding to MR1 due to the presentation of this ligand. Our structure of a MAIT TCR/MR1/rRL-6-CH2OH complex shows an evolutionarily conserved binding orientation, with a clear role for both the CDR3α and CDR3ß loops in recognizing the rRL-6-CH2OH stimulatory ligand. We also present two additional xenoreactive MAIT TCR/MR1 complexes that recapitulate the docking orientation documented previously, despite having variation in the CDR2ß and CDR3ß loop sequences. Our data support a model by which MAIT TCRs engage MR1 in a conserved fashion, with their binding affinities modulated by the nature of the MR1-presented Ag or diversity introduced by alternate Vß usage or CDR3ß sequences.

A potent α/ß-peptide analogue of GLP-1 with prolonged action in vivo.

Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic ß cells. GLP-1R agoinsts are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. We describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained ß-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogues of other peptide hormones.

Deletion of PREPl causes growth impairment and hypotonia in mice.

Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL(-/-)) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL(-/-) mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL(+/+)) counterparts. A righting assay revealed that PREPL(-/-) pups took significantly longer than PREPL(+/+) pups to right themselves when placed on their backs. This deficit indicates that PREPL(-/-) mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease.

Methods for the elucidation of protein-small molecule interactions.

Understanding the interactions between small molecules and proteins can be approached from different perspectives and is important for the advancement of basic science and drug development. Chemists often use bioactive small molecules, such as natural products or synthetic compounds, as probes to identify therapeutically relevant protein targets. Biochemists and biologists often begin with a specific protein and seek to identify the endogenous metabolites that bind to it. These interests have led to the development of methodology that relies heavily on synthetic and analytical chemistry to identify protein-small molecule and protein-metabolite interactions. Here, we survey these strategies, highlighting key findings, to demonstrate the value of these approaches in answering important chemical and biological questions.