ABSTRACT
Fibroblast growth factors (FGFs) are a family of proteins possessing paracrine, autocrine, or endocrine functions in a variety of biological processes, including embryonic development, angiogenesis, tissue homeostasis, wound repair, and cancer. Canonical FGFs bind and activate tyrosine kinase FGF receptors (FGFRs), triggering intracellular signaling cascades that mediate their biological activity. Experimental evidence indicates that FGFs play a complex role in the physiopathology of the prostate gland that ranges from essential functions during embryonic development to modulation of neoplastic transformation. The use of ligand- and receptor-deleted mouse models has highlighted the requirement for FGF signaling in the normal development of the prostate gland. In adult prostate, the maintenance of a functional FGF/FGFR signaling axis is critical for organ homeostasis and function, as its disruption leads to prostate hyperplasia and may contribute to cancer progression and metastatic dissemination. Dissection of the molecular landscape modulated by the FGF family will facilitate ongoing translational efforts directed toward prostate cancer therapy.
Subject(s)
Fibroblast Growth Factors/physiology , Prostate/physiology , Prostate/physiopathology , Prostatic Diseases/physiopathology , Prostatic Neoplasms/physiopathology , Receptors, Fibroblast Growth Factor/physiology , Animals , Humans , Intercellular Signaling Peptides and Proteins/physiology , Male , Prostate/growth & developmentABSTRACT
Fibroblast growth factors (FGFs) act as proangiogenic and mitogenic cytokines in several cancers, including multiple myeloma (MM). Indeed, corrupted FGF autocrine and paracrine secretion induces an aberrant activation of the FGF receptor (FGFR) signaling sustaining cancer cell spreading and resistance to pharmacological treatments. Thus, FGF traps may represent a promising anti-cancer strategy to hamper the ligand-dependent activation of the FGF/FGFR system. We previously identified NSC12 as the first orally available small molecule FGF trap able to inhibit the growth and progression of several FGF-dependent tumor models. NSC12 is a pregnenolone derivative carrying a 1,1-bis-trifluoromethyl-1,3-propanediol chain in position 17 of the steroid nucleus. Investigation of structure-activity relationships (SARs) provided more potent and specific NSC12 steroid derivatives and highlighted that the C17-side chain is pivotal for the FGF trap activity. Here, a scaffold hopping approach allowed to obtain two FGF trap compounds (22 and 57) devoid of the steroid nucleus and able to efficiently bind FGF2 and to inhibit FGFR activation in MM cells. Accordingly, these compounds exert a potent anti-tumor activity on MM cell lines both in vitro and in vivo and on MM patient-derived primary cells, strongly affecting the survival of both proteasome-inhibitor sensitive and resistant MM cells. These results propose a new therapeutic option for relapsed/refractory MM patients and set the bases for the development of novel FGF traps prone to chemical diversification to be used in the clinic for the treatment of those tumors in which the FGF/FGFR system plays a pivotal role, including MM.
Subject(s)
Antineoplastic Agents , Fibroblast Growth Factors , Multiple Myeloma , Receptors, Fibroblast Growth Factor , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Humans , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/chemistry , Cell Line, Tumor , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Receptors, Fibroblast Growth Factor/metabolism , Fibroblast Growth Factors/metabolism , Structure-Activity Relationship , Drug Discovery , Mice , Fibroblast Growth Factor 2/metabolismABSTRACT
Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.
Subject(s)
COVID-19/metabolism , Heparin/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Binding Sites , Heparin/chemistry , Heparin/pharmacology , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , COVID-19 Drug TreatmentABSTRACT
The characterization of modifications of microbial proteins is of primary importance to dissect pathogen lifecycle mechanisms and could be useful in identifying therapeutic targets. Attempts to solve this issue yielded only partial and non-exhaustive results. We developed a multidisciplinary approach by coupling in vitro infection assay, mass spectrometry (MS), protein 3D modelling, and surface plasma resonance (SPR). As a proof of concept, the effect of low UV-C (273 nm) irradiation on SARS-CoV-2 spike (S) protein was investigated. Following UV-C exposure, MS analysis identified, among other modifications, the disruption of a disulphide bond within the conserved S2 subunit of S protein. Computational analyses revealed that this bond breakage associates with an allosteric effect resulting in the generation of a closed conformation with a reduced ability to bind the ACE2 receptor. The UV-C-induced reduced affinity of S protein for ACE2 was further confirmed by SPR analyses and in vitro infection assays. This comprehensive approach pinpoints the S2 domain of S protein as a potential therapeutic target to prevent SARS-CoV-2 infection. Notably, this workflow could be used to screen a wide variety of microbial protein domains, resulting in a precise molecular fingerprint and providing new insights to adequately address future epidemics.
Subject(s)
COVID-19 , Humans , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Protein BindingABSTRACT
With the aim of identifying novel antagonists selective for the EphA receptor family, a combined experimental and computational approach was taken to investigate the molecular basis of the recognition between a prototypical Eph-ephrin antagonist (UniPR1447) and two representative receptors of the EphA and EphB subfamilies, namely, EphA2 and EphB2 receptors. The conformational free-energy surface (FES) of the binding state of UniPR1447 within the ligand binding domain of EphA2 and EphB2, reconstructed from molecular dynamics (MD) simulations performed on the microsecond time scale, was exploited to drive the design and synthesis of a novel antagonist selective for EphA2 over the EphB2 receptor. The availability of compounds with this pharmacological profile will help discriminate the importance of these two receptors in the insurgence and progression of cancer.
Subject(s)
Receptor, EphA2 , Receptor, EphB2 , Humans , Ligands , Molecular Dynamics Simulation , Protein Binding , Receptor, EphA2/antagonists & inhibitors , Receptor, EphB2/antagonists & inhibitorsABSTRACT
Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Humans , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Drug Repositioning , Proteasome Endopeptidase Complex/metabolism , Benzodioxoles/pharmacology , Benzodioxoles/therapeutic use , Aminopyridines/pharmacology , Aminopyridines/therapeutic use , Niacinamide/therapeutic use , Ubiquitins/metabolism , MutationABSTRACT
HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This "two-way" activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.
Subject(s)
Endothelium/metabolism , Heparan Sulfate Proteoglycans/metabolism , Lymphocytes/metabolism , tat Gene Products, Human Immunodeficiency Virus/metabolism , Cell Adhesion , Cell Movement , Endothelium/chemistry , Heparan Sulfate Proteoglycans/chemistry , Humans , Lymphocytes/chemistry , Models, Molecular , Molecular Structure , Stereoisomerism , Tumor Cells, Cultured , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug discovery process targeted to CFTR.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Drug Discovery/methods , Biosensing Techniques , Computational Biology , Cystic Fibrosis/drug therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Models, Molecular , Protein ConformationABSTRACT
Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.
Subject(s)
Fibroblast Growth Factor 2/chemistry , Surface Plasmon Resonance , Thrombospondins/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Fibroblast Growth Factor 2/metabolism , Humans , Protein Binding , Protein Domains , Repetitive Sequences, Amino Acid , Thrombospondins/metabolismABSTRACT
Cystic fibrosis (CF) is mainly caused by the deletion of Phe 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. New drugs able to rescue ΔF508-CFTR trafficking are eagerly awaited. An integrated bioinformatics and surface plasmon resonance (SPR) approach was here applied to investigate the rescue mechanism(s) of a series of CFTR-ligands including VX809, VX770 and some aminoarylthiazole derivatives (AAT). Computational studies tentatively identified a large binding pocket in the ΔF508-CFTR nucleotide binding domain-1 (NBD1) and predicted all the tested compounds to bind to three sub-regions of this main pocket. Noticeably, the known CFTR chaperone keratin-8 (K8) seems to interact with some residues located in one of these sub-pockets, potentially interfering with the binding of some ligands. SPR results corroborated all these computational findings. Moreover, for all the considered ligands, a statistically significant correlation was determined between their binding capability to ΔF508-NBD1 measured by SPR and the pockets availability measured by computational studies. Taken together, these results demonstrate a strong agreement between the in silico prediction and the SPR-generated binding data, suggesting a path to speed up the identification of new drugs for the treatment of cystic fibrosis.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Thiazoles/chemistry , Binding Sites , Computational Biology , Cystic Fibrosis/drug therapy , Drug Evaluation, Preclinical , Humans , Molecular Dynamics Simulation , Protein Binding , Surface Plasmon ResonanceABSTRACT
Fibroblast growth factors (FGFs) are a family of pleiotropic factors produced by stromal and parenchymal tumor cells. Even though FGFs have been firstly characterized as angiogenic factors, they exert autocrine and paracrine functions not only on endothelial cells but also on tumor cells and other stromal components. Thus, the FGF/FGF receptor (FGFR) pathway may represent a key player in tumor growth by regulating the complex cross-talk between stromal and tumor compartments. The ligand dependent or independent activation of the FGF/FGFR system by gene upregulation, oncogenic mutation or amplification occurs in a variety of human tumors and is implicated in various key steps of tumor growth and progression. In addition, FGF/FGFR activation has been described as a mechanism of tumor escape in response to antiangiogenic/anti-VEGF therapies. Experimental and clinical evidences provide a compelling biologic rationale for the development of anti-FGF/FGFR targeting agents in cancer therapy. However, the development of drugs specifically targeting the FGF/FGFR pathway proved to be difficult, also due to the high redundancy and pleiotropic effects of FGF and FGFR family members. On the other hand, the possibility to develop "two-compartment" targeting agents endowed with both antiangiogenic and antitumor activities remains promising. Here we will review the preclinical and clinical approaches and potential therapeutics currently available to block the FGF/FGFR system in human cancer.
Subject(s)
Fibroblast Growth Factors/antagonists & inhibitors , Neoplasms/drug therapy , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Animals , Endothelial Cells/metabolism , Fibroblast Growth Factors/metabolism , Humans , Neoplasms/metabolism , Receptors, Fibroblast Growth Factor/metabolismABSTRACT
Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, low-cost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.
Subject(s)
Antigens/isolation & purification , Biomarkers/metabolism , Chemistry Techniques, Analytical/methods , Plastics/chemistry , Water/chemistry , Antibodies/metabolism , Antigens/metabolism , HIV Infections/diagnosis , Hepatitis B/diagnosis , Humans , Immunoassay , Light , Optical Phenomena , Protein Array AnalysisABSTRACT
Human immunodeficiency virus p17 matrix protein is released by infected cells and may accumulate within lymphoid tissues where it may deregulate the biological activities of different cell populations by binding to CXCR1 and CXCR2 cellular receptors. S75X, a natural p17 variant, was recently shown to enhance the malignant properties of lymphoma cells. We investigated a reference p17 protein and the S75X variant for their ability to bind to Epstein-Barr virus (EBV)-infected primary and fully transformed B-lymphocytes and trigger downstream effects of potential pathogenic relevance. We demonstrate that EBV infection of primary B-lymphocytes or the ectopic expression of the latent membrane protein-1 viral oncoprotein in EBV-negative B-cells up-regulates CXCR2, but not CXCR1. Multispectral imaging flow cytometry showed that EBV-infected primary B-cells more efficiently bind and internalize p17 proteins as compared with activated B-lymphocytes. The S75X variant bound more efficiently to EBV-infected primary and fully transformed B-lymphocytes compared with reference p17, because of a higher affinity to CXCR2, and enhanced the proliferation of these cells, an effect associated with cyclin D2 and D3 up-regulation and increased interleukin-6 production. Notably, the S75X variant markedly up-regulated latent membrane protein-1 expression at both mRNA and protein levels and enhanced the activation of Akt, ERK1/2 and STAT3 signaling, thereby contributing to EBV(+) B-cell growth promotion. These results indicate that EBV infection sensitizes B-lymphocytes to CXCR2-mediated effects of p17 proteins and provide evidence supporting a possible contribution of natural p17 variants to EBV-driven lymphomagenesis in the human immunodeficiency virus setting.
Subject(s)
B-Lymphocytes/metabolism , Epstein-Barr Virus Infections/genetics , Herpesvirus 4, Human/genetics , Oncogene Proteins/genetics , Up-Regulation/genetics , Viral Matrix Proteins/genetics , Cell Line , Cyclin D2/genetics , Cyclin D3/genetics , HIV Infections/genetics , HIV Infections/virology , Humans , Interleukin-6/genetics , Lymphocyte Activation/genetics , RNA, Messenger/genetics , Receptors, Interleukin-8B/genetics , Signal Transduction/genetics , Transcriptional Activation/genetics , Viral Proteins/geneticsABSTRACT
The agmatine-containing poly(amidoamine) polymer AGMA1 was recently shown to inhibit the infectivity of several viruses, including human papillomavirus 16 (HPV-16), that exploit cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The aim of this work was to assess the antiviral activity of AGMA1 and its spectrum of activity against a panel of low-risk and high-risk HPVs and to elucidate its mechanism of action. AGMA1 was found to be a potent inhibitor of mucosal HPV types (i.e., types 16, 31, 45, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 0.34 µg/ml and 0.73 µg/ml, and no evidence of cytotoxicity was observed. AGMA1 interacted with immobilized heparin and with cellular heparan sulfates, exerting its antiviral action by preventing virus attachment to the cell surface. The findings from this study indicate that AGMA1 is a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.
Subject(s)
Agmatine/analogs & derivatives , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Heparitin Sulfate/metabolism , Papillomaviridae/drug effects , Polyamines/metabolism , Polyamines/pharmacology , Agmatine/metabolism , Agmatine/pharmacology , Cell Line , Humans , Virus Attachment/drug effectsABSTRACT
UNLABELLED: Exogenous HIV-1 matrix protein p17 (p17) deregulates the function of different cells after its N-terminal loop (AT20) binding to the chemokine receptors CXCR1 and CXCR2. One site within AT20 has been recently found to be the major determinant of viral fitness following transmission of simian immunodeficiency virus (SIV) to the human host. Therefore, we sought to determine whether SIV matrix protein (MA) was already capable of interacting with CXCR1 and CXCR2 and mimic p17 biological activities rather than this being a newly acquired function during host adaptation. We show here that SIV MA binds with the same affinity of p17 to CXCR1 and CXCR2 and displays both p17 proangiogenic on human primary endothelial cells and chemotactic activity on human primary monocytes and B cells. However, SIV MA exhibited a higher degree of plasticity than p17 in the C terminus, a region known to play a role in modulating B cell growth. Indeed, in contrast to p17, SIV MA was found to activate the phosphatidylinositol 3-kinase/Akt signaling pathway and strongly promote B cell proliferation and clonogenic activity. Interestingly, we have recently highlighted the existence of a Ugandan HIV-1 strain-derived p17 variant (S75X) with the same B cell growth-promoting activity of SIV MA. Computational modeling allowed us to hypothesize an altered C terminus/core region interaction behind SIV MA and S75X activity. Our findings suggest the appearance of a structural constraint in the p17 C terminus that controls B cell growth, which may help to elucidate the evolutionary trajectory of HIV-1. IMPORTANCE: The HIV-1 matrix protein p17 (p17) deregulates the biological activities of different cells after binding to the chemokine receptors CXCR1 and CXCR2. The p17 functional domain responsible for receptors interaction includes an amino acid which is considered the major determinant of SIV replication in humans. Therefore, we sought to determine whether SIV matrix protein (SIV MA) already had the ability to bind to both chemokine receptors rather than being a function newly acquired during host adaptation. We show here that SIV MA binds to CXCR1 and CXCR2 and fully mimics the p17 proangiogenic and chemokine activity. However, it differs from p17 in its ability to signal into B cells and promote B cell growth and clonogenicity. Computational analysis suggests that the accumulation of mutations in the C-terminal region may have led to a further SIV MA adaptation to the human host. This finding in turn sheds light on the evolutionary trajectory of HIV-1.
Subject(s)
B-Lymphocytes/virology , Cell Proliferation , Gene Products, gag/metabolism , HIV Antigens/metabolism , HIV-1/physiology , Host-Pathogen Interactions , Simian Immunodeficiency Virus/physiology , Viral Matrix Proteins/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , B-Lymphocytes/physiology , Humans , Models, Molecular , Protein Conformation , Signal TransductionABSTRACT
Despite decades of antiviral drug research and development, viruses still remain a top global healthcare problem. Compared to eukaryotic cells, viruses are composed by a limited numbers of proteins that, nevertheless, set up multiple interactions with cellular components, allowing the virus to take control of the infected cell. Each virus/host interaction can be considered as a therapeutical target for new antiviral drugs but, unfortunately, the systematic study of a so huge number of interactions is time-consuming and expensive, calling for models overcoming these drawbacks. Surface plasmon resonance (SPR) is a label-free optical technique to study biomolecular interactions in real time by detecting reflected light from a prism-gold film interface. Launched 20 years ago, SPR has become a nearly irreplaceable technology for the study of biomolecular interactions. Accordingly, SPR is increasingly used in the field of virology, spanning from the study of biological interactions to the identification of putative antiviral drugs. From the literature available, SPR emerges as an ideal link between conventional biological experimentation and system biology studies functional to the identification of highly connected viral or host proteins that act as nodal points in virus life cycle and thus considerable as therapeutical targets for the development of innovative antiviral strategies.
Subject(s)
Host-Pathogen Interactions , Surface Plasmon Resonance/methods , Virology/methods , Virus Attachment , Virus Physiological Phenomena , Viruses/drug effects , Protein BindingABSTRACT
Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65â Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/chemistry , Benzofurans/chemistry , Chaperonins/chemistry , HSP90 Heat-Shock Proteins/chemistry , Adenosine Triphosphatases/metabolism , Allosteric Site , Biochemical Phenomena , Cell Line, Tumor , HSP90 Heat-Shock Proteins/metabolism , Humans , Hydrolysis , Ligands , Protein Binding , Protein ConformationABSTRACT
Vascular diseases supported by aberrant angiogenesis have increased incidence in HIV-1-infected patients. Several data suggest that endothelium dysfunction relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. The HIV-1 matrix protein p17 is known to deregulate the biological activity of different immune cells. Recently, p17 was found to mimic IL-8 chemokine activity by binding to the IL-8 receptor CXCR1. Here we show that p17 binds with high affinity to CXCR2, a CXCR1-related receptor, and promotes the formation of capillary-like structures on human endothelial cells (ECs) by interacting with both CXCR1 and CXCR2 expressed on the EC surface. ERK signaling via Akt was defined as the pathway responsible for p17-induced tube formation. Ex vivo and in vivo experimental models confirmed the provasculogenic activity of p17, which was comparable to that induced by VEGF-A. The hypothesis of a major role for p17 in HIV-1-induced aberrant angiogenesis is enforced by the finding that p17 is detected, as a single protein, in blood vessels of HIV-1-patients and in particular in the nucleus of ECs. Localization of p17 in the nucleus of ECs was evidenced also in in vitro experiments, suggesting the internalization of exogenous p17 in ECs by mechanisms of receptor-mediated endocytosis. Recognizing p17 interaction with CXCR1 and CXCR2 as the key event in sustaining EC aberrant angiogenesis could help us to identify new treatment strategies in combating AIDS-related vascular diseases.
Subject(s)
Endothelium/blood supply , HIV Antigens/metabolism , HIV Infections/complications , Neovascularization, Pathologic/metabolism , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Vascular Diseases/etiology , gag Gene Products, Human Immunodeficiency Virus/metabolism , Analysis of Variance , Antibodies, Monoclonal/immunology , Blotting, Western , Cell Nucleus/virology , Endothelium/metabolism , HIV Infections/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Immunohistochemistry , Surface Plasmon Resonance , Vascular Diseases/metabolism , Vascular Diseases/virologyABSTRACT
Angiogenesis, the process of formation of new blood vessel from pre-existing ones, is involved in various intertwined pathological processes including virus infection, inflammation and oncogenesis, making it a promising target for the development of novel strategies for various interventions. To induce angiogenesis, angiogenic growth factors (AGFs) must interact with pro-angiogenic receptors to induce proliferation, protease production and migration of endothelial cells (ECs). The action of AGFs is counteracted by antiangiogenic modulators whose main mechanism of action is to bind (thus sequestering or masking) AGFs or their receptors. Many sugars, either free or associated to proteins, are involved in these interactions, thus exerting a tight regulation of the neovascularization process. Heparin and heparan sulfate proteoglycans undoubtedly play a pivotal role in this context since they bind to almost all the known AGFs, to several pro-angiogenic receptors and even to angiogenic inhibitors, originating an intricate network of interaction, the so called "angiogenesis glycomic interactome". The decoding of the angiogenesis glycomic interactome, achievable by a systematic study of the interactions occurring among angiogenic modulators and sugars, may help to design novel antiangiogenic therapies with implications in the cure of angiogenesis-dependent diseases.
Subject(s)
Glycomics , Heparan Sulfate Proteoglycans/metabolism , Heparin/metabolism , Neovascularization, Physiologic , Angiogenesis Modulating Agents/metabolism , Angiogenesis Modulating Agents/pharmacology , Angiogenesis Modulating Agents/therapeutic use , Animals , Heparan Sulfate Proteoglycans/therapeutic use , Heparin/therapeutic use , Humans , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Physiologic/drug effects , Protein BindingABSTRACT
Once released by HIV(+) cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (K(d) = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (ArgâAla substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.