RESUMO
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.
Assuntos
Antineoplásicos , Fatores de Crescimento de Fibroblastos , Mieloma Múltiplo , Receptores de Fatores de Crescimento de Fibroblastos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Humanos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Linhagem Celular Tumoral , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Descoberta de Drogas , Camundongos , Fator 2 de Crescimento de Fibroblastos/metabolismoRESUMO
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.
Assuntos
Receptor EphA2 , Receptor EphB2 , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Receptor EphA2/antagonistas & inibidores , Receptor EphB2/antagonistas & inibidoresRESUMO
The Bcl-2-associated athanogene 3 (BAG3) protein plays multiple roles in controlling cellular homeostasis, and it has been reported to be deregulated in many cancers, leading tumor cell apoptosis escape. BAG3 protein is then an emerging target for its oncogenic activities in both leukemia and solid cancers, such as medulloblastoma. In this work a series of forty-four compounds were designed and successfully synthesized by the modification and optimization of a previously reported 2,4-thiazolidinedione derivative 28. Using an efficient cloning and transfection in human embryonic kidney HEK-293T cells, BAG3 was collected and purified by chromatographic techniques such as IMAC and SEC, respectively. Subsequently, through Surface Plasmon Resonance (SPR) all the compounds were evaluated for their binding ability to BAG3, highlighting the compound FB49 as the one having the greatest affinity for the protein (Kd = 45 ± 6 µM) also against the reference compound 28. Further analysis carried out by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) spectroscopy further confirmed the highest affinity of FB49 for the protein. In vitro biological investigation showed that compound FB49 is endowed with an antiproliferative activity in the micromolar range in three human tumoral cell lines and more importantly is devoid of toxicity in human peripheral mononuclear cell deriving from healthy donors. Moreover, FB49 was able to block cell cycle in G1 phase and to induce apoptosis as well as autophagy in medulloblastoma HD-MB03 treated cells. In addition, FB49 demonstrated a synergistic effect when combined with a chemotherapy cocktail of Vincristine, Etoposide, Cisplatin, Cyclophosphamide (VECC). In conclusion we have demonstrated that FB49 is a new derivative able to bind human BAG3 with high affinity and could be used as BAG3 modulator in cancers correlated with overexpression of this protein.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Tiazolidinedionas , Humanos , Meduloblastoma/tratamento farmacológico , Apoptose , Tiazolidinedionas/farmacologia , Linhagem Celular Tumoral , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de ApoptoseRESUMO
To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as extracellular vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, to date, studies dealing with the functionalization of the EV surface with proteins have never considered the protein corona "variable", namely the fact that extrinsic proteins may spontaneously adsorb on the EV surface, contributing to determine the surface, and in turn the biological identity of the EV. In this work, we explore and compare the two edge cases of EVs modified with the antibody Cetuximab (CTX) by chemisorption of CTX (through covalent binding via biorthogonal click-chemistry) and by formation of a physisorbed CTX corona. The results indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significant differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells, very likely due to the higher robustness of the CTX anchorage.
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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.
Assuntos
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Ligação ProteicaRESUMO
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.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Reposicionamento de Medicamentos , Complexo de Endopeptidases do Proteassoma/metabolismo , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Niacinamida/uso terapêutico , Ubiquitinas/metabolismo , MutaçãoRESUMO
Infections caused by viruses from the Herpesviridae family produce some of the most prevalent transmitted diseases in the world, constituting a serious global public health issue. Some of the virus properties such as latency and the appearance of resistance to antiviral treatments complicate the development of effective therapies capable of facing the infection. In this context, dendrimers present themselves as promising alternatives to current treatments. In this study, we propose the use of PEGylated cationic carbosilane dendrimers as inhibitors of herpes simplex virus 2 (HSV-2) and human cytomegalovirus (HCMV)infections. Studies of mitochondrial toxicity, membrane integrity, internalization and viral infection inhibition indicated that G2-SN15-PEG, G3-SN31-PEG, G2-SN15-PEG fluorescein isothiocyanate (FITC) labeled and G3-SN31-PEG-FITC dendrimers are valid candidates to target HSV-2 and HCMV infections since they are biocompatible, can be effectively internalized and are able to significantly inhibit both infections. Later studies (including viral inactivation, binding inhibition, heparan sulphate proteoglycans (HSPG)binding and surface plasmon resonance assays) confirmed that inhibition takes place at first infection stages. More precisely, these studies established that their attachment to cell membrane heparan sulphate proteoglycans impede the interaction between viral glycoproteins and these cell receptors, thus preventing infection. Altogether, our research confirmed the high capacity of these PEGylated carbosilane dendrimers to prevent HSV-2 and HCMV infections, making them valid candidates as antiviral agents against Herpesviridae infections.
RESUMO
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.
Assuntos
COVID-19/metabolismo , Heparina/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , Heparina/química , Heparina/farmacologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Tratamento Farmacológico da COVID-19RESUMO
Angiogenesis, the formation of new blood vessels from preexisting ones, is crucial for tumor growth and metastatization, and is considered a promising therapeutic target. Unfortunately, drugs directed against a specific proangiogenic growth factor or receptor turned out to be of limited benefit for oncology patients, likely due to the high biochemical redundancy of the neovascularization process. In this scenario, multitarget compounds that are able to simultaneously tackle different proangiogenic pathways are eagerly awaited. UniPR1331 is a 3ß-hydroxy-Δ5-cholenic acid derivative, which is already known to inhibit Eph-ephrin interaction. Here, we employed an analysis pipeline consisting of molecular modeling and simulation, surface plasmon resonance spectrometry, biochemical assays, and endothelial cell models to demonstrate that UniPR1331 directly interacts with the vascular endothelial growth factor receptor 2 (VEGFR2) too. The binding of UniPR1331 to VEGFR2 prevents its interaction with the natural ligand vascular endothelial growth factor and subsequent autophosphorylation, signal transduction, and in vitro proangiogenic activation of endothelial cells. In vivo, UniPR1331 inhibits tumor cell-driven angiogenesis in zebrafish. Taken together, these data shed light on the pleiotropic pharmacological effect of UniPR1331, and point to Δ5-cholenic acid as a promising molecular scaffold for the development of multitarget antiangiogenic compounds.
Assuntos
Efrinas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Células Endoteliais/metabolismo , Efrinas/metabolismo , Efrinas/farmacologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Endotélio/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Linfócitos/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Adesão Celular , Movimento Celular , Endotélio/química , Proteoglicanas de Heparan Sulfato/química , Humanos , Linfócitos/química , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Células Tumorais Cultivadas , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached to a core protein. GAGs and PGs can be found as free molecules, associated with the extracellular matrix or expressed on the cell membrane. They play a role in the regulation of a wide array of physiological and pathological processes by binding to different proteins, thus modulating their structure and function, and their concentration and availability in the microenvironment. Unfortunately, the enormous structural diversity of GAGs/PGs has hampered the development of dedicated analytical technologies and experimental models. Similarly, computational approaches (in particular, molecular modeling, docking and dynamics simulations) have not been fully exploited in glycobiology, despite their potential to demystify the complexity of GAGs/PGs at a structural and functional level. Here, we review the state-of-the art of computational approaches to studying GAGs/PGs with the aim of pointing out the "bitter" and "sweet" aspects of this field of research. Furthermore, we attempt to bridge the gap between bioinformatics and glycobiology, which have so far been kept apart by conceptual and technical differences. For this purpose, we provide computational scientists and glycobiologists with the fundamentals of these two fields of research, with the aim of creating opportunities for their combined exploitation, and thereby contributing to a substantial improvement in scientific knowledge.
Assuntos
Membrana Celular/metabolismo , Biologia Computacional/métodos , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Animais , Glicosaminoglicanos/química , Humanos , Proteoglicanas/química , Relação Estrutura-AtividadeRESUMO
Computational drug repositioning is of growing interest to academia and industry, for its ability to rapidly screen a huge number of candidates in silico (exploiting comprehensive drug datasets) together with reduced development cost and time. The potential of drug repositioning has not been fully evaluated yet for cystic fibrosis (CF), a disease mainly caused by deletion of Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. F508del-CFTR is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. CF is still a fatal disease. Nowadays, it is treatable by some CFTR-rescuing drugs, but new-generation drugs with stronger therapeutic benefits and fewer side effects are still awaited. In this manuscript we report about the results of a pilot computational drug repositioning screening in search of F508del-CFTR-targeted drugs performed on AIFA library by means of a dedicated computational pipeline and surface plasmon resonance binding assay to experimentally validate the computational findings.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Fenilalanina/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Conjuntos de Dados como Assunto , Relação Dose-Resposta a Droga , Reposicionamento de Medicamentos , Humanos , Estrutura Molecular , Fenilalanina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
UniPR129, an L-ß-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph-ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2-M7, mono-hydroxylated metabolites, M8-M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph-ephrin antagonists.
RESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Próstata/fisiologia , Próstata/fisiopatologia , Doenças Prostáticas/fisiopatologia , Neoplasias da Próstata/fisiopatologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Masculino , Próstata/crescimento & desenvolvimentoRESUMO
Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. It is caused by different mutations in the cystic fibrosis transmembrane regulator protein (CFTR) gene, with F508del being the most common. During the last years, small-molecule therapy chosen to contrast CF relied on compounds that correct CFTR misfolding and ER retention (correctors such as VX-809), or defective channel gating (potentiators such as VX-770). Combination therapy with the two series of drugs has been applied, leading to the approval of several multi-drugs such as Orkambi. Despite this, this treatment proved to be only partially effective making the search for novel modulators an urgent need to contrast CF. Recently, we reported compound 2a as reference compound of a series of aminoarylthiazole-VX-809 hybrid derivatives exhibiting promising F508del-CFTR corrector ability. Herein, we report exploring the docking mode of the prototype VX-809 and of 2a in order to derive useful guidelines for the rational design of novel optimized analogues. To demonstrate experimentally their effective F508del-CFTR-binding and rescuing potential, the most promising derivatives had been synthesized and evaluated in biological assays including YFP functional assay on F508del-CFTR CFBE41o-cells, trans epithelial electrical resistance (TEER) and surface plasmon resonance (SPR). This multidisciplinary strategy led to the discovery of a second series of hybrids including 7j and 7m endowed with higher potency than the prototype.
Assuntos
Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Benzodioxóis/metabolismo , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Aminopiridinas/síntese química , Benzodioxóis/síntese química , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Domínios ProteicosRESUMO
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.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Descoberta de Drogas/métodos , Técnicas Biossensoriais , Biologia Computacional , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
The EphA2 receptor has been validated in animal models as new target for treating tumors depending on angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor, namely 5ß-cholan-24-oic acids and 5ß-cholan-24-oyl l-ß-homotryptophan conjugates, with the aim to develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a result of our exploration, we identified UniPR505, N-[3α-(Ethylcarbamoyl)oxy-5ß-cholan-24-oyl]-l-ß-homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay.
Assuntos
Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/normas , Ácido Litocólico/química , Neovascularização Fisiológica/efeitos dos fármacos , Compostos Policíclicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor EphA2/antagonistas & inibidores , Inibidores da Angiogênese/química , Animais , Proliferação de Células , Embrião de Galinha , Galinhas , Membrana Corioalantoide , Humanos , Masculino , Modelos Moleculares , Fosforilação , Compostos Policíclicos/química , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/normas , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
p17 matrix protein released by HIV+ cells interacts with leukocytes heparan sulfate proteoglycans (HSPGs), CXCR1 and CXCR2 exerting different cytokine-like activities that contribute to AIDS pathogenesis. Since the bioactive form of several cytokines is represented by dimers/oligomers and oligomerization is promoted by binding to heparin or HSPGs, here we evaluated if heparin/HSPGs also promote p17 oligomerization. Heparin favours p17 dimer, trimer and tetramer assembly, in a time- and biphasic dose-dependent way. Heparin-induced p17 oligomerization is of electrostatic nature, being it prevented by NaCl, by removing negative sulfated groups of heparin and by neutralizing positive lysine residues in the p17 N-terminus. A new computational protocol has been implemented to study heparin chains up to 24-mer accommodating a p17 dimer. Molecular dynamics show that, in the presence of heparin, two p17 molecules undergo conformational modifications creating a continuous "electropositive channel" in which heparin sulfated groups interact with p17 basic amino acids, promoting its dimerization. At the cell surface, HSPGs induce p17 oligomerization, as demonstrated by using B-lymphoblastoid Namalwa cells overexpressing the HSPG Syndecan-1. Also, HSPGs on the surface of BJAB and Raji human B-lymphoblastoid cells are required to p17 to induce ERK1/2 activation, suggesting that HS-induced oligomerization plays a role in p17-induced lymphoid dysregulation during AIDS.
Assuntos
Síndrome da Imunodeficiência Adquirida/metabolismo , Antígenos HIV , HIV-1 , Sistema de Sinalização das MAP Quinases , Multimerização Proteica , Sindecana-1 , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Linhagem Celular Tumoral , Antígenos HIV/química , Antígenos HIV/metabolismo , HIV-1/química , HIV-1/metabolismo , Heparina/química , Heparina/metabolismo , Humanos , Sindecana-1/química , Sindecana-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. METHODS: We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. RESULTS: By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. CONCLUSIONS: The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. GENERAL SIGNIFICANCE: Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.