RESUMO
DICER1 syndrome is a cancer pre-disposition disorder caused by mutations that disrupt the function of DICER1 in miRNA processing. Studying the molecular, cellular and oncogenic effects of these mutations can reveal novel mechanisms that control cell homeostasis and tumor biology. Here, we conduct the first analysis of pathogenic DICER1 syndrome allele from the DICER1 3'UTR. We find that the DICER1 syndrome allele, rs1252940486, abolishes interaction with the PUMILIO RNA binding protein with the DICER1 3'UTR, resulting in the degradation of the DICER1 mRNA by AUF1. This single mutational event leads to diminished DICER1 mRNA and protein levels, and widespread reprogramming of miRNA networks. The in-depth characterization of the rs1252940486 DICER1 allele, reveals important post-transcriptional regulatory events that control DICER1 levels.
Assuntos
MicroRNAs , RNA Mensageiro , MicroRNAs/genéticaRESUMO
RNA nanotechnology is the bottom-up self-assembly of nanometer-scale architectures, resembling LEGOs, composed mainly of RNA. The ideal building material should be (1) versatile and controllable in shape and stoichiometry, (2) spontaneously self-assemble, and (3) thermodynamically, chemically, and enzymatically stable with a long shelf life. RNA building blocks exhibit each of the above. RNA is a polynucleic acid, making it a polymer, and its negative-charge prevents nonspecific binding to negatively charged cell membranes. The thermostability makes it suitable for logic gates, resistive memory, sensor set-ups, and NEM devices. RNA can be designed and manipulated with a level of simplicity of DNA while displaying versatile structure and enzyme activity of proteins. RNA can fold into single-stranded loops or bulges to serve as mounting dovetails for intermolecular or domain interactions without external linking dowels. RNA nanoparticles display rubber- and amoeba-like properties and are stretchable and shrinkable through multiple repeats, leading to enhanced tumor targeting and fast renal excretion to reduce toxicities. It was predicted in 2014 that RNA would be the third milestone in pharmaceutical drug development. The recent approval of several RNA drugs and COVID-19 mRNA vaccines by FDA suggests that this milestone is being realized. Here, we review the unique properties of RNA nanotechnology, summarize its recent advancements, describe its distinct attributes inside or outside the body and discuss potential applications in nanotechnology, medicine, and material science.
Assuntos
Nanomedicina/métodos , Neoplasias/tratamento farmacológico , Estabilidade de RNA , RNA/química , Animais , Humanos , Terapia de Alvo Molecular , TermodinâmicaRESUMO
GOLD SELEX, a novel SELEX approach has been developed that obviates the need for target immobilization for aptamer development. The approach purely relies on the affinity of the aptamers towards its target, to get detached from the gold nanoparticle (GNP) surface (weak attraction) after binding with its target. Thus, only the completely detached aptamers are selected for the next round of SELEX. This, in-process, also addresses the issue of residual binding and thus improves the sensitivity of the developed aptamers. As a proof of concept for establishing the utility of the approach for small molecules, we have developed aptamers against dichlorvos (DV), a pesticide in just 8 rounds. Using these aptamer candidates, we have developed an aptamer-NanoZyme (GNP having peroxidase mimic activity) based colorimetric assay. The developed aptamer displayed high affinity (Kd in sub micromolar range) and selectivity for DV. The developed assay could detect as low as 15 µM DV. The best-performing aptamer was also able to work in real samples like river water and commercial apple juice. The GOLD SELEX approach developed in this study, we believe, can act as a template for future SELEX strategy development and can replace the conventional SELEX strategy.
RESUMO
In order to adapt in host tissues, microbial pathogens regulate their gene expression through a variety of transcription factors. Here, we have functionally characterized Rv0792c, a HutC homolog from Mycobacterium tuberculosis. In comparison to the parental strain, a strain of M. tuberculosis with a Rv0792c mutant was compromised for survival upon exposure to oxidative stress and infection in guinea pigs. RNA sequencing analysis revealed that Rv0792c regulates the expression of genes involved in stress adaptation and virulence of M. tuberculosis. Solution small-angle X-ray scattering (SAXS) data-steered model building confirmed that the C-terminal region plays a pivotal role in dimer formation. Systematic evolution of ligands by exponential enrichment (SELEX) resulted in the identification of single-strand DNA (ssDNA) aptamers that can be used as a tool to identify small-molecule inhibitors targeting Rv0792c. Using SELEX and SAXS data-based modeling, we identified residues essential for Rv0792c's aptamer binding activity. In this study, we also identified I-OMe-Tyrphostin as an inhibitor of Rv0792c's aptamer and DNA binding activity. The identified small molecule reduced the growth of intracellular M. tuberculosis in macrophages. The present study thus provides a detailed shape-function characterization of a HutC family of transcription factor from M. tuberculosis. IMPORTANCE Prokaryotes encode a large number of GntR family transcription factors that are involved in various fundamental biological processes, including stress adaptation and pathogenesis. Here, we investigated the structural and functional role of Rv0792c, a HutC homolog from M. tuberculosis. We demonstrated that Rv0792c is essential for M. tuberculosis to adapt to oxidative stress and establish disease in guinea pigs. Using a systematic evolution of ligands by exponential enrichment (SELEX) approach, we identified ssDNA aptamers from a random ssDNA library that bound to Rv0792c protein. These aptamers were thoroughly characterized using biochemical and biophysical assays. Using SAXS, we determined the structural model of Rv0792c in both the presence and absence of the aptamers. Further, using a combination of SELEX and SAXS methodologies, we identified I-OMe-Tyrphostin as a potential inhibitor of Rv0792c. Here we provide a detailed functional characterization of a transcription factor belonging to the HutC family from M. tuberculosis.
Assuntos
Aptâmeros de Nucleotídeos , Mycobacterium tuberculosis , Tuberculose , Animais , Cobaias , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Tirfostinas , Espalhamento a Baixo Ângulo , Aptâmeros de Nucleotídeos/química , Difração de Raios X , Fatores de Transcrição/metabolismo , DNA/metabolismoRESUMO
Expansion of CGG trinucleotide repeats in 5' untranslated region of the FMR1 gene is the causative mutation of neurological diseases such as fragile X syndrome (FXS), fragile X-associated tremor/ataxia syndrome (FXTAS), and ovarian disorder such as fragile X-associated primary ovarian insufficiency (FXPOI). CGG repeats containing FMR1 transcripts form the toxic ribonuclear aggregates, abrupt pre-mRNA splicing, and cause repeat-associated non-AUG translation, leading to the disease symptoms. Here, we utilized a small molecule library of ~ 250,000 members obtained from the National Cancer Institute (NCI) and implemented a shape-based screening approach to identify the candidate small molecules that mitigate toxic CGG RNA-mediated pathogenesis. The compounds obtained from screening were further assessed for their affinity and selectivity towards toxic CGG repeat RNA by employing fluorescence-binding experiment and isothermal calorimetry titration assay. Three candidate molecules B1, B4, and B11 showed high affinity and selectivity for expanded CGG repeats RNA. Further, NMR spectroscopy, gel mobility shift assay, CD spectroscopy, UV-thermal denaturation assay, and molecular docking affirmed their high affinity and selectivity for toxic CGG RNAs. Next, these lead compounds selectively improved the pre-mRNA alternative splicing defects with no perturbation in global splicing efficacy and simultaneously reduced the FMR1polyG protein aggregate formation without affecting the downstream expression of the gene. Taken together these findings, we addressed compound B1, B4, and B11 as potential lead molecules for developing promising therapeutics against FXTAS. Herein, this study, we have utilized shape similarity approach to screen the NCI library and found out the potential candidate which improves the pre-mRNA splicing defects and reduces FMR1polyG aggregations.
Assuntos
Síndrome do Cromossomo X Frágil , Ataxia/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Simulação de Acoplamento Molecular , Agregados Proteicos , Tremor/metabolismo , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Gallbladder cancer (GBC) is an aggressive and difficult to treat biliary tract carcinoma with a poor survival rate. The aim of this study was to design a peptide-based multi-epitope vaccine construct against GBC using immunoinformatics approaches. Three proteins implicated in the progression of GBC were selected for B and T cell epitope prediction and the designing of the potential vaccine construct. Seven CTL, four HTL and six Bcell epitopes along with a suitable adjuvant were selected and connected using linkers for designing the vaccine construct. The secondary and tertiary models of the designed vaccine were generated and satisfactorily validated. A Ramachandran plot of the final 3D model showed more than 90% of the residues in allowed regions and only 0.4% in disallowed regions. The binding affinity of a vaccine construct with TLR 2, 3 and 4 receptors was assessed through molecular docking and simulation. The average numbers of hydrogen bonds for vaccine-TLR 2, 3 and 4 complexes in the simulation were 15.36, 16.45, and 11.98, respectively, and remained consistent over a 100 ns simulation period, which is critical for their function. The results of this study provide a strong basis for further evaluation through in vitro/in vivo experimental validation of the safety and efficacy of the designed vaccine construct.
RESUMO
Metastasis is one of the leading causes of mortality in cancer patients. As the firstly identified metastasis suppressor, NM23-H1 has been endowed with expectation as a potent target in metastatic cancer therapy during the past decades. However, many challenges impede its clinical use. Accumulating evidence shows that NM23-H1 has a dichotomous role in tumor metastasis as a suppressor and promoter. It has potentially attributed to its versatile biochemical characteristics such as nucleoside diphosphate kinase (NDPK) activity, histidine kinase activity (HPK), exonuclease activity, and protein scaffold, which further augment the complexity and uncertainty of its physiological function. Simultaneously, tumor cells have evolved multiple ways to regulate the expression and function of NM23-H1 during tumorigenesis and metastasis. This review summarized and discussed the regulatory mechanisms of NM23-H1 in cancer including transcriptional activation, subcellular location, enzymatic activity, and protein degradation, which significantly modulate its anti-metastatic function.
Assuntos
Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Metástase Neoplásica/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Curcumin exhibits a broad spectrum of beneficial health properties that include anti-tumor and anti-cancer activities. The down-regulation of c-myc transcription via stabilizing the G-quadruplex structure formed at the promoter region of the human c-myc gene allows the repression in cancer growth. Small molecules can bind and stabilize this structure to provide an exciting and promising strategy for anti-cancer therapeutics. Herein, we investigated the interaction of Curcumin and its synthetic analogs with G-quadruplex DNA formed at the c-myc promoter by using various biophysical and biochemical assays. Further, its cytotoxic effect and mechanistic insights were explored in various cancer cell lines as well as in multicellular tumor spheroid (MCTS) model. The MCTS possesses almost similar microenvironment as avascular tumors, and micro-metastases can be used as a suitable model for the small molecule-based therapeutics development. Our study provides an expanded overview of the anti-cancer effect of a new Curcumin analog via targeting G-quadruplex structures formed at the promoter region of the human c-myc gene.
Assuntos
Antineoplásicos/farmacologia , Curcumina/análogos & derivados , Curcumina/farmacologia , Quadruplex G/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Curcumina/química , Regulação para Baixo/efeitos dos fármacos , Humanos , Modelos Moleculares , Proteínas Proto-Oncogênicas c-myc/metabolismo , Esferoides Celulares/efeitos dos fármacosRESUMO
Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Animais , Bungarotoxinas , Bungarus , Venenos Elapídicos/toxicidade , HumanosRESUMO
Inactivation of RB is one of the hallmarks of cancer, however gaps remain in our understanding of how RB-loss changes human cells. Here we show that pRB-depletion results in cellular reprogramming, we quantitatively measured how RB-depletion altered the transcriptional, proteomic and metabolic output of non-tumorigenic RPE1 human cells. These profiles identified widespread changes in metabolic and cell stress response factors previously linked to E2F function. In addition, we find a number of additional pathways that are sensitive to RB-depletion that are not E2F-regulated that may represent compensatory mechanisms to support the growth of RB-depleted cells. To determine whether these molecular changes are also present in RB1-/- tumors, we compared these results to Retinoblastoma and Small Cell Lung Cancer data, and identified widespread conservation of alterations found in RPE1 cells. To define which of these changes contribute to the growth of cells with de-regulated E2F activity, we assayed how inhibiting or depleting these proteins affected the growth of RB1-/- cells and of Drosophila E2f1-RNAi models in vivo. From this analysis, we identify key metabolic pathways that are essential for the growth of pRB-deleted human cells.
Assuntos
Neoplasias da Retina/fisiopatologia , Proteínas de Ligação a Retinoblastoma/genética , Retinoblastoma/fisiopatologia , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In recent years, neurological disorders have globally become a leading cause of disability and death. Neurological disorders are very common in both high- and low-income countries, and the number of patients is predicted to increase in the coming decades. Disorders caused by the expanded trinucleotide repeats (CAG, CGG, CCG, CTG, CUG, GAA, and GCN) in the genome, also described as trinucleotide repeat expansion disorders (TREDs), comprise of the major class of neurological diseases. Various TREDs have different modes of pathogenesis, but the severity and time of onset of disease depends on the trinucleotide repeat numbers. Numerous therapeutic strategies, including symptomatic treatment, blockage of mutant protein synthesis, targeting the toxic protein aggregates and degradation of RNA transcripts have been developed for the treatment of these diseases. However, various limitations to these therapeutic strategies have been reported, and therefore, researchers are exploring different avenues of therapeutics development. One of the recent developments include targeting the expanded repeats with small molecules. Small molecule binds with the secondary/tertiary structure of RNA (like bulges, loops, and hairpins) irrespective of its sequences. Altogether, small molecule-based therapeutics may have the advantage over others to be able to overcome the hurdles of the blood-brain barrier, poor absorption, and allergic reactions. In this review, we have summarized various TREDs and envisage the potential of small molecule-based therapeutics for targeting these hitherto incurable neurological disorders.
Assuntos
Doenças do Sistema Nervoso/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Expansão das Repetições de Trinucleotídeos/genética , Animais , Humanos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Fragile X-associated tremor ataxia syndrome is an untreatable neurological and neuromuscular disorder caused by unstable expansion of 55-200 CGG nucleotide repeats in 5' UTR of Fragile X intellectual disability 1 (FMR1) gene. The expansion of CGG repeats in the FMR1 mRNA elicits neuronal cell toxicity through two main pathogenic mechanisms. First, mRNA with CGG expanded repeats sequester specific RNA regulatory proteins resulting in splicing alterations and formation of ribonuclear inclusions. Second, repeat-associated non-canonical translation (RANT) of the CGG expansion produces a toxic homopolymeric protein, FMRpolyG. Very few small molecules are known to modulate these pathogenic events, limiting the therapeutic possibilities for FXTAS. Here, we found that a naturally available biologically active small molecule, Curcumin, selectively binds to CGG RNA repeats. Interestingly, Curcumin improves FXTAS associated alternative splicing defects and decreases the production and accumulation of FMRpolyG protein inclusion. Furthermore, Curcumin decreases cell cytotoxicity promptly by expression of CGG RNA in FXTAS cell models. In conclusion, our data suggest that small molecules like Curcumin and its derivatives may be explored as a potential therapeutic strategy against the debilitating repeats associated neurodegenerative disorders.
RESUMO
An expansion of CGG tandem repeats in the 5' untranslated region (5'-UTR) of fragile X mental retardation 1 (FMR1) gene causes fragile X-associated tremor/ataxia syndrome (FXTAS). The transcripts of these expanded repeats r(CGG)exp either form RNA foci or undergo the repeat-associated non-ATG (RAN) translation that produces toxic homopolymeric proteins in neuronal cells. The discovery of small molecule modulators that possess a strong binding affinity and high selectivity to these toxic expanded repeats RNA could be a promising therapeutic approach to cure the expanded repeat-associated neurological diseases. Therefore, here we sought to test the therapeutic potential of a natural alkaloid, piperine, by assessing its ability to bind and neutralize the toxicity of r(CGG)exp RNA motif. To accomplish this first, we have determined the affinity of piperine to r(CGG)exp RNA using fluorescence-based binding assay and isothermal titration calorimetry assay. These assays showed that piperine forms a thermodynamically favorable interaction with r(CGG)exp RNA with high selectivity to the G-rich RNA motif. Interaction of piperine with r(CGG)exp motif was further validated using several biophysical techniques such as CD, CD melting, NMR spectroscopy, and gel retardation assay. Moreover, piperine was also found to be effective for improving the r(CGG)exp associated splicing defects and RAN translation in a FXTAS cell model system. Our results effectively provided the evidence that piperine strongly interacts with r(CGG)exp RNA and could be used as a suitable candidate for therapeutic development against FXTAS.
Assuntos
Alcaloides/farmacologia , Ataxia/metabolismo , Benzodioxóis/farmacologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Tremor/metabolismo , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacos , HumanosRESUMO
CAG repeats RNA causes various fatal neurodegenerative diseases exemplified by Huntington's disease (HD) and several spinocerebellar ataxias (SCAs). Although there are differences in the pathogenic mechanisms, these diseases share the common cause, i.e., expansion of CAG repeats. The shared cause of these diseases raises the possibility for the exploiting the common target as a potential therapeutic approach. Oligonucleotide-based therapeutics are designed earlier with the help of the base pairing rule but are not very promiscuous, considering the nonspecific stimulation of the immune system and the poor cellular delivery. Therefore, small molecules-based therapeutics are preferred for targeting the repeats expansion disorders. Here, we have used the chemical similarity search approach to discern the small molecules that selectively target toxic CAG RNA. The lead compounds showed the specificity towards AA mismatch in biophysical studies including CD, ITC, and NMR spectroscopy and thus aided to forestall the polyQ mediated pathogenicity. Furthermore, the lead compounds also explicitly alleviate the polyQ mediated toxicity in HD cell models and patient-derived cells. These findings suggest that the lead compound could act as a chemical probe for AA mismatch containing RNA as well as plays a neuroprotective role in fatal neurodegenerative diseases like HD and SCAs.
Assuntos
Fibroblastos/efeitos dos fármacos , Flavonoides/farmacologia , Fármacos Neuroprotetores/farmacologia , Peptídeos/antagonistas & inibidores , RNA/química , Bibliotecas de Moléculas Pequenas/farmacologia , Benzotiazóis/química , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Fibroblastos/metabolismo , Fibroblastos/patologia , Flavonoides/química , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/química , Conformação de Ácido Nucleico , Peptídeos/química , Peptídeos/metabolismo , Cultura Primária de Células , Agregados Proteicos/efeitos dos fármacos , RNA/genética , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/química , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacosRESUMO
Huntington's diseases (HD) is a very devastating disease caused by r(CAG) expansion in HTT gene, encoding the huntingtin protein. r(CAG) expansion causes disease via multiple pathways including, 1) loss of normal protein function like sequestration of RNA binding protein such as Muscleblind-like (MBNL) and nucleolin, 2) Gain of function for mutant proteins and 3) repeat-associated non-ATG (RAN) translation; in which expanded r(CAG) translates into toxic poly glu, poly ser, or poly ala without the use of any canonical start codon. Herein, we have rationally designed and synthesized a unique class of pyridocoumarin derivatives that target the r(CAG)exp involved in HD and spinocerebellar ataxia (SCA) pathogenesis. Notably, compounds 3 and 15 showed higher affinity (nanomolar Kd) and selectivity for diseased r(CAG)exp RNA compared to regular duplex AU-paired RNA. Interestingly, both scaffolds are cell permeable, exhibit low toxicity to healthy fibroblast cells and are also capable of reducing the level of poly Q aggregation in cellular models. Indeed, our current study offers promising facet for selectively targeting repeats containing RNAs that cause severe diseases like HD and SCAs.
Assuntos
Cumarínicos/química , Doença de Huntington/tratamento farmacológico , Proteínas Mutantes/genética , RNA Mensageiro/química , Ataxias Espinocerebelares/tratamento farmacológico , Células Cultivadas , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Desenho de Fármacos , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Cinética , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/efeitos dos fármacos , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
In majority of snakebite cases, the snake responsible for the bite remains unidentified. The traditional snakebite diagnostics method relies upon clinical symptoms and blood coagulation assays that do not provide accurate diagnosis which is important for epidemiological as well as diagnostics point of view. On the other hand, high batch-to-batch variations in antibody performance limit its application for diagnostic assays. In recent years, nucleic acid aptamers have emerged as a strong chemical rival of antibodies due to several obvious advantages, including but not limited to in vitro generation, synthetic nature, ease of functionalization, high stability and adaptability to various diagnostic formats. In the current study, we have rationally truncated an aptamer developed for α-Toxin of Bungarus multicinctus and demonstrated its utility for the detection of venom of Bungarus caeruleus. The truncated aptamer α-Tox-T2 (26mer) is found to have greater affinity than its 40-mer parent counterpart α-Tox-FL. The truncated aptamers are characterized and compared with parent aptamer for their binding, selectivity, affinity, alteration in secondary structure and limit of detection. Altogether, our findings establish the cross-species application of a DNA aptamer generated for α-Toxin of Bungarus multicinctus (a snake found in Taiwan and China) for the reliable detection of venom of Bungarus caeruleus (a snake found in the Indian subcontinent).
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Bungarotoxinas/metabolismo , Bungarus/metabolismo , Venenos Elapídicos/metabolismo , Mordeduras de Serpentes/metabolismo , Animais , Anticorpos/metabolismo , Antivenenos/metabolismo , China , Índia , TaiwanRESUMO
Huntington's disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of CAG repeats in the HTT gene. The transcribed mutant RNA contains expanded CAG repeats that translate into a mutant huntingtin protein. This expanded CAG repeat also causes mis-splicing of pre-mRNA due to sequestration of muscle blind like-1 splicing factor (MBNL1), and thus both of these elicit the pathogenesis of HD. Targeting the onset as well as progression of HD by small molecules could be a potent therapeutic approach. We have screened a set of small molecules to target this transcript and found Myricetin, a flavonoid, as a lead molecule that interacts with the CAG motif and thus prevents the translation of mutant huntingtin protein as well as sequestration of MBNL1. Here, we report the first solution structure of the complex formed between Myricetin and RNA containing the 5'CAG/3'GAC motif. Myricetin interacts with this RNA via base stacking at the AA mismatch. Moreover, Myricetin was also found reducing the proteo-toxicity generated due to the aggregation of polyglutamine, and further, its supplementation also improves neurobehavioral deficits in the HD mouse model. Our study provides the structural and mechanistic basis of Myricetin as an effective therapeutic candidate for HD and other polyQ related disorders.
Assuntos
Flavonoides/química , Flavonoides/farmacologia , Doença de Huntington/tratamento farmacológico , Expansão das Repetições de Trinucleotídeos , Animais , Células COS , Ataxia Cerebelar/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Espectroscopia de Ressonância Magnética , Mutação , Conformação de Ácido Nucleico , Estresse Oxidativo/efeitos dos fármacos , Peptídeos , Ratos Wistar , Bibliotecas de Moléculas PequenasRESUMO
Protein misfolding and aggregation is a key attribute of different neurodegenerative diseases. Misfolded and aggregated proteins are intrinsically disordered and rule out structure based drug design. The comprehensive characterization of misfolded proteins and associated aggregation pathway is prerequisite to develop therapeutics for neurodegenerative diseases caused due to the protein aggregation. Visible protein aggregates used to be the final stage during aggregation mechanism. The structural analysis of intermediate steps in such protein aggregates will help us to discern the conformational role and subsequently involved pathways. The structural analysis of protein aggregation using various biophysical methods may aid for improved therapeutics for protein misfolding and aggregation related neurodegenerative diseases. In this mini review, we have summarized different spectroscopic methods such as fluorescence spectroscopy, circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy for structural analysis of protein aggregation. We believe that the understanding of invisible intermediate of misfolded proteins and the key steps involved during protein aggregation mechanisms may advance the therapeutic approaches for targeting neurological diseases that are caused due to misfolded proteins.
Assuntos
Amiloide , Modelos Moleculares , Agregados Proteicos , Análise Espectral/métodos , Amiloide/análise , Amiloide/química , Amiloide/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Conformação ProteicaRESUMO
BACKGROUND: Small cell lung cancer (SCLC) frequently presents as metastatic disease. It would be useful to detect serum tumor biomarkers at an earlier stage in order to improve the overall survival. MATERIALS AND METHODS: Serum samples from SCLC patients (6 limited disease, 7 extensive disease, 4 relapsed disease, 4 no evidence of disease post-treatment) and 5 normal controls were used to identify tumor biomarkers utilizing proteomics. Serum hepatocyte growth factor was also studied using standard ELISA. RESULTS: Utilizing MALDI-TOF-Mass Spectrometry (MS) based protein identification techniques, a SCLC specific overexpressed protein was identified to be haptoglobin alpha-subunit, with its serum level correlating with the disease stage. The mean level of alpha-haptoglobin was increased in SCLC serum as compared to the normal controls. Serum HGF was also studied as potential tumor biomarker and was found to correlate with the disease status. Either serum alpha-haptoglobin relative level (above 1.9 U), or HGF level (above 500 pg/ml) was associated with a trend towards worse survival. CONCLUSION: Our current findings suggest that serum levels of alpha-haptoglobin and HGF may serve as useful serum tumor biomarkers in SCLC. It would now also be useful to determine if these serum biomarkers are altered in response to therapy for this disease.