Biology subjects

Salgado-Polo, F.

Publications and source records attributed to Salgado-Polo, F..

8 recordsLinked to original sources

bioRxiv2026

De novo macrocyclic peptide discovery produces potent class A GPCR ligands with diverse pharmacology

G protein-coupled receptors (GPCRs) represent one of the largest protein families of therapeutic importance, as over one third of all currently approved drugs target GPCRs. A growing proportion of GPCR drugs are peptides. However, class A GPCRs (over 80% of clinically studied targets) are underexploited as peptide targets, presumably since their only structured domain is the core, seven-helix, integral membrane fold presenting a limited solvent-exposed surface area. Screening libraries of de novo macrocyclic peptides have allowed for the targeting of otherwise intractable proteins, but their potential to generate selective ligands for class A GPCRs has not been demonstrated. Here, we used random nonstandard peptides integrated discovery (RaPID) to identify de novo macrocyclic peptide ligands targeting the chemokine receptor CXCR4. CXCR4 regulates cell migration, immune surveillance, and inflammation, making it an important therapeutic target for multiple diseases. We discovered novel, potent CXCR4-selective antagonists binding within the orthosteric pocket with nanomolar affinity. The most active peptides effectively inhibited receptor signaling, suppressed chemokine-driven cell migration, and blocked CXCR4-driven HIV entry. Our results show that even the structurally minimal class A GPCRs are viable targets for de novo cyclic peptide discovery, highlighting the potential for uncovering ligands towards other members of the GPCR superfamily.

pharmacology and toxicology↗

bioRxiv2026

Chemokine receptor activity is differentially regulated by membrane cholesterol

Cholesterol is a key membrane component that regulates G protein-coupled receptor (GPCR) function, yet its molecular mechanisms remain unclear. Here, we combine chemical extraction of membrane sterols with functional signaling assays and single-molecule fluorescence resonance energy transfer (smFRET) to define how cholesterol controls activation of chemokine receptors. Reduction of membrane cholesterol in mammalian cells selectively decreased constitutive and agonist-induced signaling across CXCR1, CXCR2, CXCR4, while it activated ACKR3, and did not affect CXCR3, revealing receptor-specific dependence on membrane sterols. Mechanistically, cholesterol regulation partly required the conserved class A GPCR residue Trp4.50 and shifted agonist-bound CXCR4 toward active conformational states, providing a molecular explanation for its functional effects. In contrast, replenishment with oxidized cholesterol species failed to restore receptor activity, distinguishing cholesterol from oxysterols as modulators of receptor activation. Our findings identify cholesterol as an allosteric regulator of chemokine receptors and suggest that oxysterols may reshape inflammatory signaling by selectively modulating GPCR activity.

biochemistry↗

bioRxiv2026

Basal Internalization and Subcellular Localization of XCR1

Chemokines orchestrate immune cell trafficking through receptor-mediated signaling and are implicated in inflammatory, autoimmune, and neuropathic disorders. The XCL1-XCR1 axis is of particular interest because XCR1 is selectively expressed on mature conventional type 1 dendritic cells (cDC1s), where it supports communication with activated CD8+ T cells and NK cells and promotes antigen cross-presentation. This selectivity has made XCR1 an attractive target for dendritic cell-based cancer vaccines, while emerging evidence also links XCL1-XCR1 signaling to neuroinflammation and pain. Despite its therapeutic potential, the mechanisms governing XCR1 activation and trafficking remain understudied. Here, we characterize XCR1 expression, membrane trafficking, and basal internalization to define mechanisms that may influence therapeutic targeting. We show that XCR1 undergoes constitutive internalization through a {beta}-arrestin-independent but adaptor protein 2 (AP2)-dependent pathway, distinguishing it from other chemokine receptors with constitutive endocytosis. Furthermore, we identify specific sequence motifs critical for its subcellular localization and intracellular trafficking. These findings provide new mechanistic insights into XCR1 regulation and may inform the development of targeted therapeutics and antigen-delivery strategies in cancer and inflammation.

pharmacology and toxicology↗

bioRxiv2022

A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis in mice

An important but rather underexplored pathway implicated in liver disease is the lysophosphatidic acid (LPA) signaling axis. LPA acts through G-protein coupled receptors inducing downstream signaling pathways related to cell proliferation, differentiation, and migration, and is predominantly produced by the extracellular phosphodiesterase, Autotaxin (ATX). ATX has gained significant attention lately with an impressive number of ATX inhibitors (type I-IV) reported. Here, we aim to evaluate the therapeutic potential of a (yet unexplored) type IV ATX inhibitor, Cpd17, in liver injury. In this study, we first confirmed the involvement of the ATX/LPA signaling axis in human and murine diseased livers. Thereafter, we evaluated the effects of Cpd17, in comparison with the classic type I ATX inhibitor PF8380, in vitro. While both inhibitors attenuated induced cell injury phenotypes as assessed using various assays and specific readout parameters in hepatocytes, macrophages, and hepatic stellate cells (HSCs), Cpd17 appeared more effective. This prompted us to characterize the mechanism of action of both inhibitors in situ and in vitro in macrophages and HSCs, demonstrating that Cpd17 was more potent in inhibiting relevant signaling pathways, namely RhoA-mediated cytoskeletal remodeling, and phosphorylation of MAPK/ERK and AKT/PKB. Finally, we investigated the therapeutic potential of Cpd17 in two liver disease mouse models, CCl4-induced acute liver injury and diet-induced non-alcoholic steatohepatitis. We demonstrate that Cpd17 has an excellent potential for reducing liver injury in both disease models in vivo. We conclude that ATX inhibition, by type IV inhibitor in particular, has an excellent potential for clinical application in liver diseases.

molecular biology↗

bioRxiv2022

Autotaxin facilitates selective LPA receptor signaling

Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA1-3) and P2Y (LPA4-6) G protein-coupled receptors. ATX/LPA promote several (patho)physiological processes, including in pulmonary fibrosis, thus serving as attractive drug targets. However, it remains unclear if clinical outcome depends on how different ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that inhibitors binding to the ATX "tunnel" specifically abrogate key aspects of ATX/LPA signaling. We find that the tunnel is essential for signaling efficacy and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. These responses are abrogated by tunnel-binding inhibitors, such as ziritaxestat, but not by inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors. HighlightsATX is a dual-function protein acting as an LPA-producing enzyme and LPA chaperone. Structural integrity of the ATX tunnel is essential to mediate signaling functions. ATX-bound LPA signals preferentially via P2Y family LPA receptors. Occupancy of the ATX tunnel is crucial for ziritaxestat to exert inhibition in vivo.

cell biology↗

bioRxiv2020

Secreted autotaxin through LPA suppresses chemotaxis and tumor infiltration of CD8+ T Cells

Autotaxin (ATX) is secreted by diverse cell types to produce lysophosphatidic acid (LPA) that regulates multiple biological functions via G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis mainly via LPAR1; however, its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T-cell repulsion predominantly involves G12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T-cell responses but suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from patient samples are consistent with intra-tumor ATX acting as a T-cell repellent. These studies highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T-cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

cancer biology↗

bioRxiv2019

Sequence-dependent trafficking of GDE2, a GPI-specific phospholipase promoting neuronal differentiation

GDE2 is a six-transmembrane glycerophosphodiesterase with phospholipase D-like activity that cleaves select glycosylphosphatidylinositol (GPI)-anchored proteins and thereby influences biological signaling cascades. GDE2 promotes neuronal differentiation cell-autonomously through glypican cleavage and is a prognostic marker in neuroblastoma, while GDE2 deficiency causes progressive neurodegeneration in mice and developmental defects in zebrafish. However, the regulation of GDE2 remains unclear. Here we show that in undifferentiated neuronal cells, GDE2 undergoes constitutive internalization and traffics back along both fast and slow recycling routes, while a small percentage is sorted to late endosomes. GDE2 trafficking is dictated by distinctive C-terminal tail sequences that determine secretion, endocytosis and recycling preference, respectively, and thereby regulate GDE2 function both positively and negatively. Our study reveals the sequence determinants of GDE2 trafficking and surface localization, and provides insight into the control of GPI-anchored protein activities with potential implications for nervous system disorders associated with impaired trafficking and beyond.

neuroscience↗

bioRxiv2018

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Autotaxin is a secreted phosphodiesterase that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key cellular responses such as migration, proliferation and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, the \"pocket\" and the \"tunnel\". Here, we revisit the kinetics of the ATX catalytic cycle in light of allosteric regulation, dissecting the different steps and pathways that lead to LPC hydrolysis. Consolidating all experimental kinetics data to a comprehensive catalytic model supported by molecular modelling simulations, suggests a positive feedback mechanism, regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend current understanding of ATX hydrolysis in light of the allosteric regulation by produced LPA species, and have implications for the design and application of orthosteric and allosteric ATX inhibitors.

biochemistry↗