RESUMO
The biomedical application of discrete supramolecular metal-based structures, specifically self-assembled metallacages, is still an emergent field of study. Capitalizing on the knowledge gained in recent years on the development of 3-dimensional (3D) metallacages as novel drug delivery systems and theranostic agents, we explore here the possibility to target [Pd2L4]4+ cages (L = 3,5-bis(3-ethynylpyridine)phenyl ligand) to the brain. In detail, a new water-soluble homoleptic cage (CPepH3) tethered to a blood brain barrier (BBB)-translocating peptide was synthesized by a combination of solid-phase peptide synthesis (SPPS) and self-assembly procedures. The cage translocation efficacy was assessed by inductively coupled mass spectrometry (ICP-MS) in a BBB cellular model in vitro. Biodistribution studies of the radiolabeled cage [[99mTcO4]- â CPepH3] in the CD1 mice model demonstrate its brain penetration properties in vivo. Further DFT studies were conducted to model the structure of the [[99mTcO4]- â cage] complex. Moreover, the encapsulation capabilities and stability of the cage were investigated using the [ReO4]- anion, the "cold" analogue of [99mTcO4]-, by 1H NMR spectroscopy. Overall, our study constitutes another proof-of-concept of the unique potential of supramolecular coordination complexes for modifying the physiochemical and biodistribution properties of diagnostic species.
Assuntos
Barreira Hematoencefálica , Paládio/química , Animais , Teoria da Densidade Funcional , Sistemas de Liberação de Medicamentos/métodos , Técnicas In Vitro , Ligantes , Espectrometria de Massas/métodos , Camundongos , Espectroscopia de Prótons por Ressonância Magnética/métodos , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared "classical" fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH2) and the novel peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the "classical" method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the "classical" SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a "working day" (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3.
Assuntos
Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Humanos , Peptídeos/química , Receptor Ativador de Fator Nuclear kappa-B/química , Receptores de Fatores de Crescimento de Fibroblastos/química , Sonicação/métodos , Fator 6 Associado a Receptor de TNF/químicaRESUMO
Blood-brain barrier (BBB) peptide-shuttles (BBBpS) are able to translocate the BBB and reach the brain. Despite the importance of brain targeting in pharmacology, BBBpS are poorly characterized. Currently, their development relies on the empiric assumption that cell-penetrating peptides (CPPs), with proven ability to traverse lipid membranes, will likewise behave as a BBBpS. The relationship between CPPs/BBBpS remains elusive and, to the best of our knowledge, has not hitherto been subject to thorough experimental scrutiny. In this work, we have identified/quantified the main physicochemical properties of BBBpS and then searched for CPPs with these properties, hence potential BBBpS. The specific features found for BBBpS are: (i) small size, (ii) none or few aromatic residues, (iii) hydrophobic, and (iv) slight cationic nature. Then, we selected the 10 scoring best in an ordinary least squares analysis, and tested them in vitro and in vivo. Overall, we identified the molecular determinants for brain targeting by peptides, devised a methodology that can be used to assist in the design of peptides with potential brain penetration from amino acid residue sequences, and found four new BBBpS within the CPP library.
Assuntos
Barreira Hematoencefálica , Encéfalo , Peptídeos Penetradores de Células , Barreira Hematoencefálica/metabolismo , Peptídeos Penetradores de Células/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Sistemas de Liberação de Medicamentos/métodosRESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of commonly targeted receptors. Unspecific chemotherapy is currently the main therapeutic option, with poor results. Another major challenge is the frequent appearance of brain metastasis (BM) associated with a significant decrease in patient overall survival. The treatment of BM is even more challenging due to the presence of the blood-brain barrier (BBB). Here, we present a dual-acting peptide (PepH3-vCPP2319) designed to tackle TNBC/BM, in which a TNBC-specific anticancer peptide (ACP) motif (vCPP2319) is joined to a BBB peptide shuttle (BBBpS) motif (PepH3). PepH3-vCPP2319 demonstrated selectivity and efficiency in eliminating TNBC both in monolayers (IC50≈5.0⯵M) and in spheroids (IC50≈25.0⯵M), with no stringent toxicity toward noncancerous cell lines and red blood cells (RBCs). PepH3-vCPP2319 was also able to cross the BBB in vitro and penetrate the brain in vivo, and was stable in serum with a half-life above 120â¯min. Tumor cell-peptide interaction is fast, with quick peptide internalization via clathrin-mediated endocytosis without membrane disruption. Upon internalization, the peptide is detected in the nucleus and the cytoplasm, indicating a multi-targeted mechanism of action that ultimately induces irreversible cell damage and apoptosis. In conclusion, we have designed a dual-acting peptide capable of brain penetration and TNBC cell elimination, thus expanding the drug arsenal to fight this BC subtype and its BM.
Assuntos
Barreira Hematoencefálica , Neoplasias Encefálicas , Peptídeos , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/patologia , Feminino , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Peptídeos/farmacologia , Antineoplásicos/farmacologia , Endocitose/efeitos dos fármacosRESUMO
New approaches aimed at identifying patient-specific drug targets and addressing unmet clinical needs in the framework of precision medicine are a strong motivation for researchers worldwide. As scientists learn more about proteins that drive known diseases, they are better able to design promising therapeutic approaches to target those proteins. The field of nanotechnology has been extensively explored in the past years, and nanoparticles (NPs) have emerged as promising systems for target-specific delivery of drugs. Virus-like particles (VLPs) arise as auspicious NPs due to their intrinsic properties. The lack of viral genetic material and the inability to replicate, together with tropism conservation and antigenicity characteristic of the native virus prompted extensive interest in their use as vaccines or as delivery systems for therapeutic and/or imaging agents. Owing to its simplicity and non-complex structure, one of the viruses currently under study for the construction of VLPs is the human immunodeficiency virus type 1 (HIV-1). Typically, HIV-1-based VLPs are used for antibody discovery, vaccines, diagnostic reagent development and protein-based assays. This review will be centered on the use of HIV-1-based VLPs and their potential biomedical applications.
Assuntos
HIV-1 , Nanopartículas , Humanos , HIV-1/genéticaRESUMO
There is an urgent need for the development of new anti-HIV drugs that can complement existing medicines to be used against resistant strains. Here, we report the anti-HIV-1 peptide pepRF1, a human serum-resistant peptide derived from the Dengue virus capsid protein. In vitro, pepRF1 shows a 50% inhibitory concentration of 1.5 nM with a potential therapeutic window higher than 53â¯000. This peptide is specific for CXCR4-tropic strains, preventing viral entry into target cells by binding to the viral coreceptor CXCR4, acting as an antagonist of this receptor. pepRF1 is more effective than T20, the only peptide-based HIV-1 entry inhibitor approved, and excels in inhibiting a HIV-1 strain resistant to T20. Potentially, pepRF1 can be used alone or in combination with other anti-HIV drugs. Furthermore, one can also envisage its use as a novel therapeutic strategy for other CXCR4-related diseases.
Assuntos
Vírus da Dengue , Infecções por HIV , HIV-1 , Proteínas do Capsídeo/genética , Humanos , Proteólise , Receptores CXCR4RESUMO
Paediatric Acquired ImmunoDeficiency Syndrome (AIDS) is a life-threatening and infectious disease in which the Human Immunodeficiency Virus (HIV) is mainly transmitted through Mother-To- Child Transmission (MTCT) during pregnancy, labour and delivery, or breastfeeding. This review provides an overview of the distinct therapeutic alternatives to abolish the systemic viral replication in paediatric HIV-1 infection. Numerous classes of antiretroviral agents have emerged as therapeutic tools for downregulation of different steps in the HIV replication process. These classes encompass Non- Nucleoside Analogue Reverse Transcriptase Inhibitors (NNRTIs), Nucleoside/Nucleotide Analogue Reverse Transcriptase Inhibitors (NRTIs/NtRTIs), INtegrase Inhibitors (INIs), Protease Inhibitors (PIs), and Entry Inhibitors (EIs). Co-administration of certain antiretroviral drugs with Pharmacokinetic Enhancers (PEs) may boost the effectiveness of the primary therapeutic agent. The combination of multiple antiretroviral drug regimens (Highly Active AntiRetroviral Therapy - HAART) is currently the standard therapeutic approach for HIV infection. So far, the use of HAART offers the best opportunity for prolonged and maximal viral suppression, and preservation of the immune system upon HIV infection. Still, the frequent administration of high doses of multiple drugs, their inefficient ability to reach the viral reservoirs in adequate doses, the development of drug resistance, and the lack of patient compliance compromise the complete HIV elimination. The development of nanotechnology-based drug delivery systems may enable targeted delivery of antiretroviral agents to inaccessible viral reservoir sites at therapeutic concentrations. In addition, the application of Computer-Aided Drug Design (CADD) approaches has provided valuable tools for the development of anti-HIV drug candidates with favourable pharmacodynamics and pharmacokinetic properties.
Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV , Criança , Infecções por HIV/tratamento farmacológico , Humanos , Inibidores da Transcriptase ReversaRESUMO
The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood-brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides.