Biology subjects

Franco, R.

Publications and source records attributed to Franco, R..

6 recordsLinked to original sources

bioRxiv2024

Homodimerization of CB2 cannabinoid receptor triggered by a bivalent ligand enhances cellular signaling

G protein-coupled receptors (GPCRs) exist within a landscape of interconvertible conformational states and in dynamic equilibrium between monomers and higher-order oligomers, both influenced by ligand binding. Here, we have shown that a homobivalent ligand formed by equal chromenopyrazole moieties as pharmacophores, connected by 14 methylene units, can modulate the dynamics of the cannabinoid CB2 receptor (CB2R) homodimerization by simultaneously binding both protomers of the CB2R-CB2R homodimer. Computational and pharmacological experimentals showed that one of the ligand pharmacophores binds to the orthosteric site of one protomer, and the other pharmacophore to a membrane-oriented pocket between transmembranes 1 and 7 of the partner protomer. This provides unique pharmacological properties, such as increased potency in Gi binding and increased recruitment of {beta}-arrestin. Thus, by modulating dimerization dynamics, it may be possible to fine-tune CB2R activity with potentially improved therapeutic outcomes. HIGHLIGHTSO_LIA homobivalent ligand of CB2R (PM369) modulates the dynamics of receptor homodimerization C_LIO_LIPM369 binds to the orthosteric site of one protomer and to a complementary, membrane-facing, site of the other protomer C_LIO_LIPM369 triggers CB2R homodimerization via the TM 1/7 interface that provides unique pharmacological properties C_LIO_LIPM369 potentiates signaling, increased potency in Gi binding and increased recruitment of {beta}-arrestin C_LIO_LIThese results highlight new approaches to control GPCR signaling C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/593612v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1736aeborg.highwire.dtl.DTLVardef@707654org.highwire.dtl.DTLVardef@168925borg.highwire.dtl.DTLVardef@60bc11_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Shedding Light on the D1-Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors

Dopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi (D1R) = 8.34, pKi (D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.

molecular biology↗

bioRxiv2023

Fluorescent Tools for Imaging and Ligand Screening of Dopamine D2-Like Receptors

The family of dopamine D2-like receptors represent an interesting target for a variety of neurological diseases, e.g. Parkinsons disease (PD), addiction or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2-like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2-like receptors (pKi (D2longR) = 8.24, pKi (D3R) = 8.58, pKi (D4R) = 7.78) with decent selectivity towards D1-like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2longR, D3R and D4R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2longR in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligands binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2-like receptors in a broad variety of experimental setups.

molecular biology↗

bioRxiv2019

DIMERBOW: exploring possible GPCR dimer interfaces

G protein-coupled receptors (GPCRs) can form homo-, heterodimers and larger order oligomers that exert different functions than monomers. The pharmacological potential of such complexes is hampered by the limited information available on the type of complex formed and its quaternary structure. Several GPCR structures in the Protein Data Bank display crystallographic interfaces potentially compatible with physiological interactions. Here we present DIMERBOW, a database and web application aimed to visually browse the complete repertoire of potential GPCR dimers present in solved structures. The tool is suited to help finding the best possible structural template to model GPCR homomers. DIMERBOW is available at http://lmc.uab.es/dimerbow/.

bioinformatics↗

bioRxiv2019

Adrenergic-melatonin heteroreceptor complexes are key in controlling ion homeostasis and intraocular eye pressure and their disruption contributes to hypertensive glaucoma

Melatonin regulates intraocular pressure (IOP) whose increase leads to glaucoma and eye nerve degeneration. Aiming at elucidating the role of melatonin receptors in humour production and IOP maintenance, we here demonstrate that glaucoma correlates with disassembly of 1-adrenergic/melatonin receptor functional units in cells producing the aqueous humour. Remarkably, 1-adrenoceptor-containing complexes do not coupled to the cognate Gq protein and, hence, phenylephrine activation of these receptors does not lead to Ca2+ mobilization. Functional complexes are significantly decreased in models of glaucoma and, more importantly, in human samples of glaucoma patients (GP). In such glaucomatous conditions phenylephrine produces, via 1-adrenoceptor activation, an increase in cytoplasmic [Ca2+] that is detrimental in glaucoma. The results led to hypothesize that using melatonin, a hypotensive agent, plus blockade of 1-adrenergic receptors may normalize pressure in glaucoma. Remarkably, co-instillation of melatonin and prazosin, a 1-adrenergic receptor antagonist, results in long-term decreases in IOP in a well-established animal model of glaucoma. The findings are instrumental to understand the physiological function of melatonin in the eye and its potential to address eye pathologies by targeting melatonin receptors and their complexes.

physiology↗

bioRxiv2019

The Michaelis-Menten paradox: Km is not an equilibrium constant but a steady-state constant.

Often, in vitro or in vivo enzyme-mediated catalytic events occur far from equilibrium and, therefore, substrate affinity measured as the inverse of ES {rightleftarrows} E+S dissociation equilibrium constant (Kd) has a doubtful physiological meaning; in practice it is almost impossible to determine Kd (except using stopped-flow or other sophisticated methodologies). The Michaelis-Menten constant (Km), the concentration of substrate ([S]) providing half of enzyme maximal activity, is not the (Kd). In the simple E+S {rightleftarrows} ES [->] E+P or in more complex models describing S conversion into P, Km must be considered the constant defining the steady state at any substrate concentration. Enzyme kinetics is based on initial rate determination, i.e. in the linear part of the S to P conversion when the concentration of [ES] remains constant while steady state occurs. We also show that Systems Biology issues such as the time required to respond to a system perturbation, is more dependent on k1, the kinetic constant defining substrateenzyme association, than on Km. Whereas Km is instrumental for biochemical basic and applied approaches, in any physiological condition, an important parameter to be considered is the substrate association rate (k1).

biochemistry↗