RESUMO
INTRODUCTION: Despite considerable therapeutic advances in the last 20 years, metastatic cancers remain a major cause of death. We previously identified prominin-2 (PROM2) as a biomarker predictive of distant metastases and decreased survival, thus providing a promising bio-target. In this translational study, we set out to decipher the biological roles of PROM2 during the metastatic process and resistance to cell death, in particular for metastatic melanoma. METHODS AND RESULTS: Methods and results: We demonstrated that PROM2 overexpression was closely linked to an increased metastatic potential through the increase of epithelial-to-mesenchymal transition (EMT) marker expression and ferroptosis resistance. This was also found in renal cell carcinoma and triple negative breast cancer patient-derived xenograft models. Using an oligonucleotide anti-sense anti-PROM2, we efficaciously decreased PROM2 expression and prevented metastases in melanoma xenografts. We also demonstrated that PROM2 was implicated in an aggravation loop, contributing to increase the metastatic burden both in murine metastatic models and in patients with metastatic melanoma. The metastatic burden is closely linked to PROM2 expression through the expression of EMT markers and ferroptosis cell death resistance in a deterioration loop. CONCLUSION: Our results open the way for further studies using PROM2 as a bio-target in resort situations in human metastatic melanoma and also in other cancer types.
Assuntos
Ferroptose , Melanoma , Humanos , Animais , Camundongos , Ferroptose/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Glicoproteínas de MembranaRESUMO
The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells in vitro and in vivo to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.
Assuntos
COVID-19 , Vesículas Extracelulares , Humanos , Antivirais/farmacologia , Oligonucleotídeos , Pandemias , SARS-CoV-2 , EritrócitosRESUMO
INTRODUCTION: Acute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%-65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs). METHODS: In this study, we harness red blood cell-derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33-binding monoclonal antibody onto the EV surface. RESULTS: The conjugation of RBCEVs with the CD33-binding antibody significantly increases the uptake of RBCEVs by CD33-positive AML cells, but not by CD33-negative cells. We also load CD33-targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3-ITD or miR-125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML. CONCLUSION: Targeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.
Assuntos
Vesículas Extracelulares , Leucemia Mieloide Aguda , MicroRNAs , Adulto , Anticorpos Monoclonais/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Oligonucleotídeos Antissenso/uso terapêutico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/uso terapêuticoRESUMO
Dysregulated T-cell activation is a hallmark of several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The lymphocyte cytosolic protein 2 (LCP2), also known as SLP-76, is essential for the development and activation of T cells. Despite the critical role of LCP2 in T-cell activation and the need for developing drugs that modify T-cell activation, no LCP2 inhibitors have been developed. This can be explained by the "undruggable" nature of LCP2, lacking a structure permissive to standard small molecule inhibitor modalities. Here, we explored an alternative drug modality, developing antisense oligonucleotides (ASOs) targeting LCP2 mRNAs, and evaluated its activity in modulating T-cell activation. We identified a set of 3' UTR targeting LCP2 ASOs, which knocked down LCP2 in a human T-cell line and primary human T cells and found that these suppressed T-cell receptor mediated activation. We also found that the ASOs suppressed FcεR1-mediated mast cell activation, in line with the role of LCP2 in mast cells. Taken together, our data provide examples of how immunomodulatory ASOs that interfere with undruggable targets can be developed and propose that such drug modalities can be used to treat autoimmune diseases.
Assuntos
Doenças Autoimunes , Oligonucleotídeos Antissenso , Linhagem Celular , Humanos , Ativação Linfocitária , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Linfócitos TRESUMO
A four-stranded scaffold of nucleic acids termed G-quadruplex (G4) has found growing applications in nano- and biotechnology. Propeller loops are a hallmark of the most stable intramolecular parallel-stranded G4s. To date, propeller loops have been observed to span only a maximum of three G-tetrad layers. Going beyond that would allow creation of more stable scaffolds useful for building robust nanodevices. Here we investigate the formation of propeller loops spanning more than three layers. We show that native nucleotide sequences are incompatible toward this goal, and we report on synthetic non-nucleotide linkers that form a propeller loop across four layers. With the established linkers, we constructed a four-layered intramolecular parallel-stranded G4, which exhibited ultrahigh thermal stability. Control on loop design would augment the toolbox toward engineering of G4-based nanoscaffolds for diverse applications.
Assuntos
Quadruplex G , DNA/genética , Nucleotídeos , Sequência de Bases , Conformação de Ácido NucleicoRESUMO
G-quadruplex (G4) binding proteins regulate important biological processes, but their interaction networks are poorly understood. We report the first use of G4 as a warhead of a proteolysis-targeting chimera (G4-PROTAC) for targeted degradation of a G4-binding protein (RHAU/DHX36). G4-PROTAC provides a new way to explore G4-protein networks and to develop potential therapeutics.
Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/química , Quadruplex G , Humanos , ProteóliseRESUMO
Joining peptides and oligonucleotides offers potential benefits, but current methods remain laborious. Here we present a novel approach towards enzymatic ligation of the two modalities through the development of tag phosphoramidites as adaptors that can be readily incorporated onto oligonucleotides. This simple and highly efficient approach paves the way towards streamlined development and production of peptide/protein-oligonucleotide conjugates.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Oligonucleotídeos/química , Peptídeos/química , Proteínas/química , Sequência de Aminoácidos , Sequência de Bases , Compostos Organofosforados/química , Técnicas de Síntese em Fase SólidaRESUMO
Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor tyrosine kinase, together with JAK1, JAK2, and JAK3. JAKs are important signaling mediators of many proinflammatory cytokines and represent compelling pharmacological targets for autoimmune and inflammatory diseases. Pan-acting small-molecule JAK inhibitors were approved for the treatment of rheumatoid arthritis and ulcerative colitis. However, their limited selectivity among JAK members have led to undesirable side effects, driving a search toward specific JAK inhibitors. Recently, TYK2 has emerged as a target of choice for the treatment of autoimmune diseases and severe COVID-19 with an optimum balance between efficacy and safety, based on observations from human genetics studies and clinical outcomes of several agents targeting cytokine pathways for which TYK2 plays an essential role. In this article, we address selective targeting of TYK2 from the genetic sequence space through development of antisense oligonucleotides (ASOs) against TYK2 mRNA. Potent ASO candidates were identified from the screening of over 200 ASOs using locked nucleic acid gapmer design. The lead ASOs exhibited potent and selective knockdown of TYK2 mRNA and protein across a panel of model human cell lines in a dose-dependent manner, showing no reduction in the mRNA and protein expression levels of other JAK paralogs. In agreement with the depletion of TYK2 proteins, several TYK2-mediated cytokine signaling pathways, including IFN-α and IL-12, were inhibited upon ASO treatment. Our results established the TYK2 ASOs as investigational tool compound and potential therapeutic agent for the treatment of autoimmune diseases and severe COVID-19.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Tratamento Farmacológico da COVID-19 , Inibidores de Janus Quinases/uso terapêutico , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , SARS-CoV-2/fisiologia , TYK2 Quinase/genética , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Interferon-alfa/metabolismo , Interleucina-12/metabolismo , Células Jurkat , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso/uso terapêutico , Transdução de SinaisRESUMO
COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin >> rutin, cyanidin-3-O-glucoside > epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.
RESUMO
The triple-negative breast cancer (TNBC), a subtype of breast cancer which lacks of targeted therapies, exhibits a poor prognosis. It was shown recently that the PIM1 oncogene is highly related to the proliferation of TNBC cells. A quadruplex-duplex hybrid (QDH) forming sequence was recently found to exist near the transcription start site of PIM1. This structure could be an attractive target for regulation of the PIM1 gene expression and thus the treatment of TNBC. Here, we present the solution structures of two QDHs that could coexist in the human PIM1 gene. Form 1 is a three-G-tetrad-layered (3+1) G-quadruplex containing a propeller loop, a lateral loop and a stem-loop made up of three Gâ¢C Watson-Crick base pairs. On the other hand, Form 2 is an anti-parallel G-quadruplex comprising two G-tetrads and a Gâ¢Câ¢Gâ¢C tetrad; the structure has three lateral loops with the middle stem-loop made up of two Watson-Crick Gâ¢C base pairs. These structures provide valuable information for the design of G-quadruplex-specific ligands for PIM1 transcription regulation.
Assuntos
DNA/química , Quadruplex G , Proteínas Proto-Oncogênicas c-pim-1/genética , Humanos , Sítio de Iniciação de Transcrição , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Beyond the consensus definition of G-quadruplex-forming motifs with tracts of continuous guanines, G-quadruplexes harboring bulges in the G-tetrad core are prevalent in the human genome. Here, we study the incorporation of a duplex hairpin within a bulge of a G-quadruplex. The NMR solution structure of a G-quadruplex containing a duplex bulge was resolved, revealing the structural details of the junction between the duplex bulge and the G-quadruplex. Unexpectedly, instead of an orthogonal connection the duplex stem was observed to stack below the G-quadruplex forming a unique quadruplex-duplex junction. Breaking up of the immediate base pair step at the junction, coupled with a narrowing of the duplex groove within the context of the bulge, led to a progressive transition between the quadruplex and duplex segments. This study revealed that a duplex bulge can be formed at various positions of a G-quadruplex scaffold. In contrast to a non-structured bulge, the stability of a G-quadruplex slightly increases with an increase in the duplex bulge size. A G-quadruplex structure containing a duplex bulge of up to 33 nt in size was shown to form, which was much larger than the previously reported 7-nt bulge. With G-quadruplexes containing duplex bulges representing new structural motifs with potential biological significance, our findings would broaden the definition of potential G-quadruplex-forming sequences.
Assuntos
DNA/ultraestrutura , Quadruplex G , Guanina/química , Conformação de Ácido Nucleico , Pareamento de Bases , Sequência de Bases , Dicroísmo Circular , DNA/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos MolecularesRESUMO
The folding kinetics is an important parameter affecting the relevance of DNA and RNA G-quadruplex (G4) structures in biological processes. Previous studies of the G4 folding kinetics mainly depended on fast measurement techniques. In previously available examples of G4s with loops up to three residues, the folding kinetics spanning several orders of magnitude has been reported, ranging from milliseconds to over 100 s. It is difficult to systematically and fundamentally understand the effect of multiple parameters, especially the loop properties, on the G4 folding kinetics, as the G4 fold is often altered when the sequence is varied. In this study, judicious choices of multiple parameters allowed us to bring various systems into the measurable window of a simple UV absorption technique. Using a well-controlled parallel-stranded G4 fold, we were able to systematically investigate the effect of seven different parameters of the environment and loop properties (temperature, K+ concentration, ionic strength, co-solute, loop length, loop sequence, and loop structure) on the G4 folding kinetics. We found that structured loops can drive up the G4 folding: for a long loop, the fast folding of a stem loop can guide the G4 folding and accelerate its folding kinetics by several orders of magnitude compared to an unstructured loop counterpart.
Assuntos
Quadruplex G , DNA , Cinética , RNARESUMO
Two separate structural elements of a G-quadruplex (G4), a vacant site and a flanking single-strand, provide an opportunity for specific targeting of a particular G4 structure via dual recognition. Here, we show that a short peptide nucleic acid (PNA) can specifically recognize and bind to a G4 at sub-micromolar affinity based on both G-tetrad vacant site filling and complementary duplex formation. This sequence-guided guanine-anchoring strategy can be further developed for specific targeting of G4 structures using short DNA, LNA and PNA strands.
Assuntos
DNA/química , Quadruplex G , Guanina/química , Nucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Ribose/análogos & derivados , Ribose/químicaRESUMO
G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure and the RHAU helicase has been identified to have high specificity for recognition of parallel-stranded G4s. We have designed and synthesized two stapled peptide analogues of the G4-specfic motif of RHAU, which preserve the G4 binding ability. Characterization of these peptides identified the stapled variants to exhibit higher helical formation propensity in aqueous buffer in comparison to the native RHAU sequence. Moreover, the stapled peptides exhibit superior enzymatic stability towards α-chymotrypsin. Our stapled RHAU peptides can serve as a new tool for targeting G4 nucleic acid structures.
Assuntos
RNA Helicases DEAD-box/química , Quadruplex G , Peptídeos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , RNA Helicases DEAD-box/síntese química , RNA Helicases DEAD-box/metabolismo , Humanos , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-HéliceRESUMO
G-quadruplexes (G4) are non-canonical nucleic acid structures with important implications in biology. Based on an α-helical fragment of the RHAU helicase that displays high specificity for parallel-stranded G-quadrplexes, herein we demonstrate its head-to-tail cyclization by a high-efficiency ligase. The cyclic peptide exhibits superior stability and binding affinity to a G-quadruplex, and can serve as an excellent investigational tool for chemical biology applications.
Assuntos
RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Quadruplex G , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Células A549 , Ciclização , RNA Helicases DEAD-box/química , DNA/genética , Humanos , Oldenlandia/enzimologia , Fragmentos de Peptídeos/química , Peptídeo Sintases/química , Peptídeos Cíclicos/síntese química , Ligação Proteica , Estabilidade ProteicaRESUMO
Small-molecule ligands targeting nucleic acids have been explored as potential therapeutic agents. Duplex groove-binding ligands have been shown to recognize DNA in a sequence-specific manner. On the other hand, quadruplex-binding ligands exhibit high selectivity between quadruplex and duplex, but show limited discrimination between different quadruplex structures. Here we propose a dual-specific approach through the simultaneous application of duplex- and quadruplex-binders. We demonstrated that a quadruplex-specific ligand and a duplex-specific ligand can simultaneously interact at two separate binding sites of a quadruplex-duplex hybrid harbouring both quadruplex and duplex structural elements. Such a dual-specific targeting strategy would combine the sequence specificity of duplex-binders and the strong binding affinity of quadruplex-binders, potentially allowing the specific targeting of unique quadruplex structures. Future research can be directed towards the development of conjugated compounds targeting specific genomic quadruplex-duplex sites, for which the linker would be highly context-dependent in terms of length and flexibility, as well as the attachment points onto both ligands.
RESUMO
Duplex stem-loops and four-stranded G-quadruplexes have been implicated in (patho)biological processes. Overlap of stem-loop- and quadruplex-forming sequences could give rise to quadruplex-duplex hybrids (QDH), which combine features of both structural forms and could exhibit unique properties. Here, we present a combined genomic and structural study of stem-loop-containing quadruplex sequences (SLQS) in the human genome. Based on a maximum loop length of 20 nt, our survey identified 80 307 SLQS, embedded within 60 172 unique clusters. Our analysis suggested that these should cover close to half of total SLQS in the entire genome. Among these, 48 508 SLQS were strand-specifically located in genic/promoter regions, with the majority of genes displaying a low number of SLQS. Notably, genes containing abundant SLQS clusters were strongly associated with brain tissues. Enrichment analysis of SLQS-positive genes and mapping of SLQS onto transcriptional/mutagenesis hotspots and cancer-associated genes, provided a statistical framework supporting the biological involvements of SLQS. In vitro formation of diverse QDH by selective SLQS hits were successfully verified by nuclear magnetic resonance spectroscopy. Folding topologies of two SLQS were elucidated in detail. We also demonstrated that sequence changes at mutation/single-nucleotide polymorphism loci could affect the structural conformations adopted by SLQS. Thus, our predicted SLQS offer novel insights into the potential involvement of QDH in diverse (patho)biological processes and could represent novel regulatory signals.
Assuntos
Quadruplex G , Genoma Humano , Mapeamento Cromossômico , Ontologia Genética , Genômica , Humanos , Modelos Estatísticos , Mutação , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismoRESUMO
Aside from the well-known double helix, DNA can also adopt an alternative four-stranded structure known as G-quadruplex. Implications of such a structure in cellular processes, as well as its therapeutic and diagnostic applications, have been reported. The G-quadruplex structure is highly polymorphic, but so far, only right-handed helical forms have been observed. Here we present the NMR solution and X-ray crystal structures of a left-handed DNA G-quadruplex. The structure displays unprecedented features that can be exploited as unique recognition elements.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Cristalografia por Raios X , Espectroscopia de Ressonância MagnéticaRESUMO
We report here the formation of stable DNA quadruplex-duplex hybrid complexes harboring multiple duplex stems within the same loop of a quadruplex structure. The folding topology of a two-stem quadruplex-duplex hybrid construct was validated using nuclear magnetic resonance (NMR) spectroscopy. This multi-stem incorporation principle is applicable at different loop positions of the same quadruplex construct and could be extended to three or more duplex stems, giving rise to a diverse range of possible structures. These multi-stem complexes offer new design principles for the assembly of DNA architectures. The potential existence of such complex motifs in genomic sequences could have biological implications and would represent novel targets for drug development.
Assuntos
DNA/química , Quadruplex G , Sequência de Bases , DNA/genética , Modelos Moleculares , TemperaturaRESUMO
DNA has the capacity to adopt several distinct structural forms, such as duplex and quadruplex helices, which have been implicated in cellular processes and shown to exhibit important functional properties. Quadruplex-duplex hybrids, generated from the juxtaposition of these two structural elements, could find applications in therapeutics and nanotechnology. Here we used NMR and CD spectroscopy to investigate the thermal stability of two classes of quadruplex-duplex hybrids comprising fundamentally distinct modes of duplex and quadruplex connectivity: Construct I involves the coaxial orientation of the duplex and quadruplex helices with continual base stacking across the two components; Construct II involves the orthogonal orientation of the duplex and quadruplex helices with no base stacking between the two components. We have found that for both constructs, the stability of the quadruplex generally increases with the length of the stem-loop incorporated, with respect to quadruplexes comprising nonstructured loops of the same length, which showed a continuous drop in stability with increasing loop length. The stability of these complexes, particularly Construct I, can be substantially influenced by the base-pair steps proximal to the quadruplex-duplex junction. Bulges at the junction are largely detrimental to the adoption of the desired G-quadruplex topology for Construct I but not for Construct II. These findings should facilitate future design and prediction of quadruplex-duplex hybrids.