Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state

Disordered Structural Ensembles of Vasopressin and Oxytocin and Their Mutants

Vasopressin and oxytocin are intrinsically disordered cyclic nonapeptides belonging to a family of neurohypophysial hormones. Although unique in their functions, these peptides differ only by two residues and both feature a tocin ring formed by the disulfide bridge between first and sixth cysteine residues. This sequence and structural similarity are experimentally linked to oxytocin agonism at vasopressin receptors and vasopressin antagonism at oxytocin receptors. Yet single- or double-residue mutations in both peptides have been shown to have drastic impacts on their activities at either receptor, and possibly the ability to bind to their neurophysin carrier protein. In this study we perform molecular dynamics simulations of the unbound native and mutant sequences of the oxytocin and vasopressin hormones to characterize their structural ensembles. We classify the subpopulations of these structural ensembles on the basis of the distributions of radius of gyration and secondary structure and hydrogen-bonding features of the canonical tocin ring and disordered tail region. We then relate the structural changes observed in the unbound form of the different hormone sequences to experimental information about peptide receptor binding, and more indirectly, carrier protein binding affinity, receptor activity, and protease degradation. This study supports the hypothesis that the structural characteristics of the unbound form of an IDP can be used to predict structural or functional preferences of its functional bound form

Chemical proteomics approaches for identifying the cellular targets of natural products.

Covering: 2010 up to 2016. Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed

ROLE OF THE GUANIDINE GROUP IN POSITION 11 OF PTH(1-11) ANALOGUES

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recent studies have demonstrated that helicity-enhancing substitutions yielded potent analogues of PTH(1-11) and PTH(1-14). The role of alpha-helicity on biological potency is well known. In the context of searching the pharmacological properties of PTH(1-11) analogues, we studied the role of a positive charge at the C-terminal position, which was identified to play an essential role in bioactivity and binding. A series of modified peptide analogues of PTH(1-11) was synthesized and characterized. As a reference, we synthesized an analogue containing Lys in position 11. This analogue exhibited an activity similar to that of [Ala1,3, Gln10, Har11]PTH(1-11)NH2, reference peptide. The peptide series was synthesised by SPPS employing Fmoc protected amino acids and a small library of Fmoc-protected glutamic acid amides of variable chain length previously prepared in solution. A circular dichroism study of the synthesized peptides was performed to compare their conformation with biological activity. We found a reduced activity for modified analogues which could be justified by the role of the C-terminal amide group. New syntheses of analogues containing mimetics are underway to establish the correlation between the guanidine function and bio-activity of PTH(1-11) analogues

Design, conformational studies and analysis of structure\u2013function relationships of PTH (1\u201311) analogues: the essential role of Val in position 2

The N-terminal 1\u201334 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it elicits all the biological responses characteristic of the native intact PTH. Recent studies reported potent helical analogues of the PTH (1\u201311) with helicity-enhancing substitutions. This work describes the synthesis, biological activity, and conformational studies of analogues obtained from the most active non-natural PTH (1\u201311) peptide H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2; specifically, the replacement of Val in position 2 with d-Val, l-(\u3b1Me)-Val and N-isopropyl-Gly was studied. The synthesized analogues were characterized functionally by in-cell assays and their structures were determined by CD and NMR spectroscopy. To clarify the relationship between the structure and activity, the structural data were used to generate a pharmacophoric model, obtained overlapping all the analogues. This model underlines the fundamental functional role of the side chain of Val2 and, at the same time, reveals that the introduction of conformationally constrained C\u3b1-tetrasubstituted \u3b1-amino acids in the peptides increases their helical content, but does not necessarily ensure significant biological activity

Structure-function relationship studies of PTH(1-11) analogues containing D-amino acids

Parathyroid hormone (PTH) is an 84-amino acid peptide hormone. Produced in the parathyroid glands, it acts primarily on bone and kidney to maintain extracellular calcium levels within normal limits. It has been shown that the 1\u201334 amino acid fragment of PTH is sufficient to bind and activate the PTH type-I receptor. Recent investigations focusing on the interaction of N-terminal fragments of PTH with PTH type-I receptor showed that certain modifications can increase signalling potency in peptides as short as 11 amino acids. To understand the role of the side chains of all the amino acid residues in PTH(1\u201311), we synthesized all-D PTH, three retro-inverso analogues of the most active modified PTH(1\u201311), H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2, and we substituted every L-AA of the latter with the corresponding d-AA, obtaining a library of PTH(1\u201311) analogues that were tested as agonists. The library was synthesized by SPPS, employing the Fmoc protocol. The biological tests showed that the activity of the D-Har11 analogue is of the same order of magnitude of that of the most active modified PTH(1\u201311). This behaviour is paralleled by an increase of the helical content on going from the D-Val2 to the D-Har11 analogue. This is in agreement with previous work where a correlation between activity and helical content has been demonstrated. The importance of a positively charged group in the C-terminal position is shown to be independent of the configuration of the C\u3b1-carbon