Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells.

Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells. The competitive binding model enabled the determination of common CA inhibitors' dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy.

Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.

The constitutive activity of melanocortin-4 receptors in cAMP pathway is allosterically modulated by zinc and copper ions.

Melanocortin-4 receptors (MC4 R) are unique among G-protein-coupled receptors (GPCRs) as they have endogenous ligands that can exhibit inverse agonistic properties in the case of elevated basal activity. It is known that the constitutive activity of GPCRs strongly affects the ligand-dependent physiological responses, but little is known about these regulatory mechanisms. Since several metal ions have been shown to be important modulators of the signal transduction of GPCRs, we hypothesized that metal ions regulate the basal activity of MC4 Rs. Implementation of a fluorescence anisotropy assay and novel redshifted fluorescent peptides enabled kinetic characterization of ligand binding to MC4 R expressed on budded baculoviruses. We show that Ca2+ is required for high-affinity ligand binding, but Zn2+ and Cu2+ in the presence of Ca2+ behave as negative allosteric modulators of ligand binding to MC4 R. FRET-based cAMP biosensor was used to measure the activation of MC4 R stably expressed in CHO-K1 cells. At low micromolar concentrations, Zn2+ caused MC4 R-dependent activation of the cAMP pathway, whereas Cu2+ reduced the activity of MC4 R even below the basal level. These findings indicate that at physiologically relevant concentrations can Zn2+ and Cu2+ function as MC4 R agonists or inverse agonists, respectively. This means that depending on the level of constitutive activity induced by Zn2+ ions, the pharmacological effect of orthosteric ligands of MC4 R can be switched from a partial to an inverse agonist. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Science badges can be found at https://cos.io/our-services/open-science-badges/.

Design and synthesis of a library of tertiary amides: evaluation as mimetics of the melanocortins' active core.

Two hundred and ten tertiary amides were prepared on solid phase. Diamines were coupled to activated carboxylated Wang polymer, and the polymeric substituted benzyloxycarbonyl protected diamines obtained were reacted with aldehydes or ketones in trimethyl orthoformate giving resin attached Schiff bases. Coupled resins were then reduced to secondary amines by sodium cyanoborohydride in 4% acetic acid/trimethyl orthoformate, followed by acylation with the carboxylic acid in the presence of PyBroP and diisopropylethylamine. Cleavage of tertiary amides from the resin was made by trifluoroacetic acid in the presence of scavengers (mainly 1,2-ethanedithiol). When indole derivatives were prepared, parallel alkylation with the linker fragment occurred, giving derivatives of 2-(4-hydroxybenzyl)-indole as side products. Solution synthesis or mixed liquid/solid phase preparation of title substances proved to be advantageous in cases when the above method did not give acceptable results. According to this approach an efficient formation of Schiff bases was achieved in the presence of TiCl(4). Substances were isolated by reversed phase chromatography; in some cases isomers were additionally separated by chiral chromatography on Chirobiotic T. When tested on human recombinant melanocortin receptors all the tertiary amides showed some binding affinities; for the highest affinity compounds the K(i)s reached 400 nM on MC(1), 2 microM on MC(3) and 1 microM on MC(4) and MC(5) receptors. cAMP assays of some of the title compounds showed that the tertiary amides are melanocortin receptor antagonists on the four MC receptor subtypes.

Proteochemometric modeling reveals the interaction site for Trp9 modified alpha-MSH peptides in melanocortin receptors.

The interactions of alpha-MSH peptides with melanocortin receptors (MCRs) were located by proteochemometric modeling. Nine alpha-MSH peptide analogues were constructed by exchanging the Trp9 residue in the alpha-MSH core with the natural or artificial amino acids Arg, Asp, Cys, Gly, Leu, Nal, d-Nal, Pro, or d-Trp. The nine peptides created, and alpha-MSH itself, were evaluated for their interactions with the 4 wild-type MC(1,3-5)Rs and 15 multichimeric MCRs, each of the latter being constructed from three sequence segments, each taken from a different wild-type MC(1,3-5)R. The segments of the chimeric MCRs were selected according to the principles of statistical molecular design and were arranged so as to divide the receptors into five parts. By this approach, a set of 19 maximally diverse MC receptor proteins was obtained for which the interaction activity with the 10 peptides were measured by radioligand binding thus creating data for 190 ligand-protein pairs, which were subsequently analyzed by use of proteochemometric modeling. In proteochemometrics, the structural or physicochemical properties of both interaction partners, which represent the complementarity of the interacting entities, are used to create multivariate mathematical descriptions. (Here, physicochemical property descriptors of the receptors' and peptides' amino acids were used). A valid, highly predictive (Q2 = 0.74) and easily interpretable model was then obtained. The model was further validated by its ability to correctly predicting the affinity of alpha-MSH for new point and cassette-mutated MC4/MC1Rs, and it was then used to identify the receptor residues that are important for affording the high affinity and selectivity of alpha-MSH for the MC1R. It was revealed that these residues are located in several quite distant parts of the receptors' transmembrane cavity and must therefore cause their influence at various stages of the dynamic ligand-binding process, such as by affecting the conformation of the ligand at the vicinity of the receptor and taking part in the path of the ligand's entry into its binding pocket. Our study can be used as a template how to create high resolution proteochemometric models when there are a limited number of natural proteins and ligands available.

Co-operative regulation of ligand binding to melanocortin receptor subtypes: evidence for interacting binding sites.

This study evaluates the binding the melanocyte stimulating hormone peptide analogue [125I]NDP-MSH to melanocortin receptors MC1, MC3, MC4 and MC5 in insect cell membranes produced by baculovirus expression systems. The presence of Ca2+ was found to be mandatory to achieve specific [125I]NDP-MSH binding to the melanocortin receptors. Although association kinetics of [125I]NDP-MSH followed the regularities of simple bimolecular reactions, the dissociation of [125I]NDP-MSH from the melanocortin receptors was heterogeneous. Eleven linear and cyclic MSH peptides studied displaced the [125I]NDP-MSH binding to the studied melanocortin receptors, with the shapes of their competition curves varying from biphasic or shallow to super-steep (Hill coefficients ranging from 0.4 to 1.5). Notably the same peptide often gave highly different patterns on different melanocortin receptor subtypes; e.g. the MC4 receptor selective antagonist HS131 gave a Hill coefficient of 1.5 on the MC1 receptor but 0.5-0.7 on the MC(3-5) receptors. Adding a mask of one of the peptides to block its high affinity binding did not prevent other competing peptides to yield biphasic competition curves. The data indicate that the binding of MSH peptides to melanocortin receptors are governed by a complex dynamic homotropic co-operative regulations.