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Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R., Competing Interests: PMS and AC are co-founders and shareholders of Septerna Inc. and DACRA Tx. DW is a shareholder in Septerna Inc. and co-founder and shareholder of DACRA Tx., (Copyright: © 2024 Harikumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Class B1 G protein-coupled receptors (GPCRs), collectively, respond to a diverse repertoire of extracellular polypeptide agonists and transmit the encoded messages to cytosolic partners. To fulfill these tasks, these highly mobile receptors must interconvert among conformational states in response to agonists. We recently showed that conformational mobility in polypeptide agonists themselves plays a role in activation of one class B1 GPCR, the receptor for glucagon-like peptide-1 (GLP-1). Exchange between helical and nonhelical conformations near the N-termini of agonists bound to the GLP-1R was revealed to be critical for receptor activation. Here, we ask whether agonist conformational mobility plays a role in the activation of a related receptor, the GLP-2R. Using variants of the hormone GLP-2 and the designed clinical agonist glepaglutide (GLE), we find that the GLP-2R is quite tolerant of variations in α-helical propensity near the agonist N-terminus, which contrasts with signaling at the GLP-1R. A fully α-helical conformation of the bound agonist may be sufficient for GLP-2R signal transduction. GLE is a GLP-2R/GLP-1R dual agonist, and the GLE system therefore enables direct comparison of the responses of these two GPCRs to a single set of agonist variants. This comparison supports the conclusion that the GLP-1R and GLP-2R differ in their response to variations in helical propensity near the agonist N-terminus. The data offer a basis for development of new hormone analogues with distinctive and potentially useful activity profiles; for example, one of the GLE analogues is a potent agonist of the GLP-2R but also a potent antagonist of the GLP-1R, a novel form of polypharmacology.

The parathyroid hormone (PTH) 1 receptor (PTH1R) is a G protein-coupled receptor (GPCR) that regulates skeletal development and calcium homeostasis. Here, we describe cryo-EM structures of the PTH1R in complex with fragments of the two hormones, PTH and PTH-related protein, the drug abaloparatide, as well as the engineered tool compounds, long-acting PTH (LA-PTH) and the truncated peptide, M-PTH(1-14). We found that the critical N terminus of each agonist engages the transmembrane bundle in a topologically similar fashion, reflecting similarities in measures of Gαs activation. The full-length peptides induce subtly different extracellular domain (ECD) orientations relative to the transmembrane domain. In the structure bound to M-PTH, the ECD is unresolved, demonstrating that the ECD is highly dynamic when unconstrained by a peptide. High resolutions enabled identification of water molecules near peptide and G protein binding sites. Our results illuminate the action of orthosteric agonists of the PTH1R., Competing Interests: Declaration of interests P.M.S. is a co-founder and shareholder of Septerna Inc. D.W. is a shareholder of Septerna Inc., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Class B1 GPCRs constitute a subfamily of 15 receptors that characteristically contain large extracellular domains (ECDs) and respond to long polypeptide hormones. Class B1 GPCRs are critical regulators of homeostasis, and, as such, many are important drug targets. While most transmembrane proteins, including GPCRs, are recalcitrant to crystallization, recent advances in cryo-electron microscopy (cryo-EM) have facilitated a rapid expansion of the structural understanding of membrane proteins. As a testament to this success, structures for all the class B1 receptors bound to G proteins have been determined by cryo-EM in the past 5 years. Further advances in cryo-EM have uncovered dynamics of these receptors, ligands, and signaling partners. Here, we examine the recent structural underpinnings of the class B1 GPCRs with an emphasis on structure-function relationships., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Polypeptides that activate the parathyroid hormone receptor-1 (PTHR1) are important in human physiology and medicine. Most previous studies of peptide binding to this receptor have involved the displacement of a radiolabeled ligand. We report a new assay format based on bioluminescence resonance energy transfer (BRET). Fusion of a NanoLuc luciferase (nLuc) unit to the N-terminus of the PTHR1 allows the direct detection of binding by an agonist peptide bearing a tetramethylrhodamine (TMR) unit. Affinity measurements from the BRET assay align well with results previously obtained via radioligand displacement. The BRET assay offers substantial operational benefits relative to affinity measurements involving radioactive compounds. The convenience of the new assay allowed us to explore several questions raised by earlier reports. For example, we show that although the first two residues of PTH(1-34) (the drug teriparatide) are critical for PTHR1 activation, these two residues contribute little or nothing to affinity. Comparisons among the well-studied agonists PTH(1-34), PTHrP(1-34), and "long-acting PTH" (LA-PTH) reveal that the high affinity of LA-PTH arises largely from a diminished rate constant for dissociation relative to the other two. A D-peptide recently reported to be comparable to PTH(1-34) as an agonist of the PTHR1 was found not to bind detectably to the receptor and to be a very weak agonist.

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and G s heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We describe the synthesis and characterization of a new class of oligomers built from a terphenyl-based amino acid. These oligomeric amides are of interest because the adoption of specific conformations could potentially be driven by the coordinated formation of inter-residue hydrogen bonds and aromatic interactions. Although high-resolution structural data have proven inaccessible, circular dichroism and nuclear magnetic resonance studies suggest that the new oligomers fold concomitantly with discrete self-association in chloroform.

Family B G protein-coupled receptors play important physiological roles and possess large extracellular domains (ECDs) that aid in binding the long polypeptide hormones that are their natural agonists. We have previously shown that agonist analogues in which subsets of native α-amino acid residues are replaced with β-amino acid residues can retain activity while avoiding proteolytic degradation. This study focuses on eight new α/β analogues of glucagon-like peptide 1 (GLP-1) that each contain five α-to-β replacements in the C-terminal half of the peptide. This portion of GLP-1 is known to adopt an α-helical conformation and contact the ECD. All four registries of the αααβ backbone pattern were evaluated; previous work has shown that the αααβ pattern supports adoption of an α-helix-like conformation. Two α-to-β replacement formats were employed, one involving β 3 homologues of the native residues replaced and the other involving a cyclic β residue. GLP-1R response was characterized in terms of stimulation of cAMP production and β-arrestin recruitment. Some of the backbone-modified GLP-1 analogues display biased agonism of the GLP-1R. This study helps to establish the scope of the α→β backbone modification strategy., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Synthesis of the C-C bonds of ketones relies upon one high-availability reagent (carboxylic acids) and one low-availability reagent (organometallic reagents or alkyl iodides). We demonstrate here a ketone synthesis that couples two different carboxylic acid esters, N-hydroxyphthalimide esters and S-2-pyridyl thioesters, to form aryl alkyl and dialkyl ketones in high yields. The keys to this approach are the use of a nickel catalyst with an electron-poor bipyridine or terpyridine ligand, a THF/DMA mixed solvent system, and ZnCl 2 to enhance the reactivity of the NHP ester. The resulting reaction can be used to form ketones that have previously been difficult to access, such as hindered tertiary/tertiary ketones with strained rings and ketones with α-heteroatoms. The conditions can be employed in the coupling of complex fragments, including a 20-mer peptide fragment analog of Exendin(9-39) on solid support., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phenol-soluble modulin α3 (PSMα3) is a cytotoxic peptide secreted by virulent strains of Staphylococcus aureus. We used a stereochemical strategy to examine the mechanism of PSMα3-mediated toxicity. One hypothesis is that PSMα3 toxicity requires fibril formation; an alternative is that toxicity is caused by soluble forms of PSMα3, possibly oligomeric. We find that the unnatural enantiomer (D residues) displays cytotoxicity comparable to that of L-PSMα3. Racemic PSMα3 is similarly toxic to enantiopure PSMα3 (L or D) under some conditions, but the toxicity is lost under conditions that cause racemic PSMα3 to aggregate. A crystal structure of racemic PSMα3-NH 2 displays an α-helical secondary structure and a packing pattern that is reminiscent of the cross-α arrangement recently discovered in crystals of L-PSMα3. Our data suggest that the cytotoxicity of PSMα3 does not depend on stereospecific engagement of a target protein or other chiral macromolecule, an observation that supports a mechanism based on membrane disruption. In addition, our data support the hypothesis that toxicity is exerted by a soluble form rather than an insoluble fibrillar form.

The receptor for advanced glycation endproducts (RAGE) is a 35 kDa transmembrane receptor that belongs to the immunoglobulin superfamily of cell surface molecules. Its role in Alzheimer's disease (AD) is complex, but it is thought to mediate influx of circulating amyloid-β into the brain as well as amplify Aβ-induced pathogenic responses. RAGE is therefore of considerable interest as both a diagnostic and a therapeutic target in AD. Herein we report the synthesis and preliminary preclinical evaluation of [(18)F]RAGER, the first small molecule PET radiotracer for RAGE (Kd = 15 nM). Docking studies proposed a likely binding interaction between RAGE and RAGER, [(18)F]RAGER autoradiography showed colocalization with RAGE identified by immunohistochemistry in AD brain samples, and [(18)F]RAGER microPET confirmed CNS penetration and increased uptake in areas of the brain known to express RAGE. This first generation radiotracer represents initial proof-of-concept and a promising first step toward quantifying CNS RAGE activity using PET. However, there were high levels of nonspecific [(18)F]RAGER binding in vitro, likely due to its high log P (experimental log P = 3.5), and rapid metabolism of [(18)F]RAGER in rat liver microsome studies. Therefore, development of second generation ligands with improved imaging properties would be advantageous prior to anticipated translation into clinical PET imaging studies.

The most commonly utilized PET radionuclide is fluorine-18 ((18)F) because of its convenient half-life and excellent imaging properties. In this review, we present the first analysis of patents issued for radiotracers labeled with fluorine-18 (between 2009 and 2015), and provide perspective on current trends and future directions in PET radiotracer development.

Interest in quantifying metal-Aβ species in vivo led to the synthesis and evaluation of [ 11 C]L2-b and [ 18 F]FL2-b as radiopharmaceuticals for studying the metallobiology of Alzheimer's disease (AD) using positron emission tomography (PET) imaging. [ 11 C]L2-b was synthesized in 3.6% radiochemical yield (nondecay corrected, n = 3), >95% radiochemical purity, from the corresponding desmethyl precursor. [ 18 F]FL2-b was synthesized in 1.0% radiochemical yield (nondecay corrected, n = 3), >99% radiochemical purity, from a 6-chloro pyridine precursor. Autoradiography experiments with AD positive and healthy control brain samples were used to determine the specificity of binding for the radioligands compared to [ 11 C]PiB, a known imaging agent for β-amyloid (Aβ) aggregates. The K d for [ 11 C]L2-b and [ 18 F]FL2-b were found to be 3.5 and 9.4 nM, respectively, from those tissue studies. Displacement studies of [ 11 C]L2-b and [ 18 F]FL2-b with PiB and AV-45 determined that L2-b binds to Aβ aggregates differently from known radiopharmaceuticals. Finally, brain uptake of [ 11 C]L2-b was examined through microPET imaging in healthy rhesus macaque, which revealed a maximum uptake at 2.5 min (peak SUV = 2.0) followed by rapid egress ( n = 2).