RESUMO
Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with ß-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding ß-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the ß-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to ß-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides.
RESUMO
In this study, carbazole (MC) and dibenzofuran (MD) derivatives were synthesized to examine their effect on the biomolecular recognition of G-quadruplex (G4) targets. Biophysical studies revealed that MC-4, a carbazole derivative, exhibits a specific affinity and effectively stabilizes the c-KIT 1 G4. Molecular modeling suggests a stable interaction of MC-4 with the terminal G-tetrad of c-KIT 1 G4. Biological studies demonstrate that MC-4 efficiently enters cells, reduces c-KIT gene expression, and induces cell cycle arrest, DNA damage, and apoptosis in cancer cells. These findings demonstrate MC-4 as a selective c-KIT G4 ligand with therapeutic potential, providing insight into the structural basis of its anticancer mechanisms.
RESUMO
We designed a platform for monitoring the degradation of exogenous proteins in live cells. We engineered a semi-synthetic platform, which consists of Enhanced Green Fluorescent Protein tagged with SpyCatcher to enable its conjugation to a SpyTag peptide bearing a Von Hippel-Lindau E3 ligand, which was delivered to live cells to promote its degradation. This platform lays the ground for studying the degradation of endogenous proteins equipped with SpyTag and for tracking the degradation of post-translationally modified proteins in live cells.
Assuntos
Proteólise , Peptídeos , Processamento de Proteína Pós-TraducionalRESUMO
Nucleic acids play crucial roles in transferring cellular information and gene regulations. DNA and RNA molecules have been associated with multiple human diseases and thus offer opportunities for exploring small molecule-based therapeutics. However, developing target-selective molecules possessing well-defined biological activity, has always been challenging. In the current scenario, where the world is continuously experiencing outbreaks of new infectious diseases, it is always important to expand the scope of chemical toolsets to override conventional drug discovery strategies for developing therapeutically relevant drug candidates. The template-directed synthetic approach has emerged as a promising tool for rapid drug discovery. It allows a biological target to template the selection or synthesis of its ligands from a pool of reactive fragments. There are two main template-directed synthetic strategies: thermodynamically controlled dynamic combinatorial chemistry (DCC) and kinetically controlled target-guided in situ click chemistry. Though discovered only two decades ago, these techniques have proven their usefulness for nucleic acid targets, as exemplified by the increasing number of applications with therapeutically important DNA and RNA targets. However, nucleic acid templated synthetic techniques are relatively unexplored in drug discovery compared to protein targets. In this review article, we have presented a detailed discussion of all the reported nucleic acid templated synthetic studies to portray the great potential of this strategy for efficient hit discovery and lead optimisation. This article would assist in expanding the scope and utility of this strategy through a summary of the advancements and emerging applications. Additionally, a brief overview of the catalytic potential of nucleic acids in asymmetric synthesis has been provided to give a valuable vision of the use of nucleic acids to induce enantioselectivity in chiral drug-like candidates.
Assuntos
Ácidos Nucleicos , Humanos , Química Click , RNA , Estereoisomerismo , DNA/química , Técnicas de Química Combinatória/métodosRESUMO
In fundamental research and drug discovery, there is still a need for effective and straightforward chemical approaches for generating cyclic peptides. The divergent synthesis of cyclic peptides remains a challenge, in particular when cyclization is carried out in the presence of unprotected side chains and a nonpeptidic component within the cycle is needed. Herein, we describe a novel and efficient strategy based on Au(I)-mediated cyclization of unprotected peptides through rapid (30-60 min) amine addition on a propargyl group to generate an imine linkage. Mechanistic insights reveal that the reaction proceeds via regioselective Markovnikov's addition of the amine on the Au(I)-activated propargyl. This strategy was successfully applied to prepare efficiently (56-94%) over 35 diverse cyclic peptides having different sequences and lengths. We have also achieved stereoselective reduction of cyclic imines employing chiral ligands. The practicality of our method was extended for the synthesis of cyclic peptides that bind Lys48-linked di-ubiquitin chains with high affinity, leading to apoptosis of cancer cells.
Assuntos
Ouro , Iminas , Aminas , Ciclização , Peptídeos/química , Peptídeos Cíclicos/químicaRESUMO
Polymorphic G-quadruplex (G4) secondary DNA structures have received increasing attention in medicinal chemistry owing to their key involvement in the regulation of the maintenance of genomic stability, telomere length homeostasis and transcription of important proto-oncogenes. Different classes of G4 ligands have been developed for the potential treatment of several human diseases. Among them, the carbazole scaffold with appropriate side chain appendages has attracted much interest for designing G4 ligands. Because of its large and rigid π-conjugation system and ease of functionalization at three different positions, a variety of carbazole derivatives have been synthesized from various natural or synthetic sources for potential applications in G4-based therapeutics and biosensors. Herein, we provide an updated close-up of the literatures on carbazole-based G4 ligands with particular focus given on their detailed binding insights studied by NMR spectroscopy. The structure-activity relationships and the opportunities and challenges of their potential applications as biosensors and therapeutics are also discussed. This review will provide an overall picture of carbazole ligands with remarkable G4 topological preference, fluorescence properties and significant bioactivity; portraying carbazole as a very promising scaffold for assembling G4 ligands with a range of novel functional applications.
Assuntos
Quadruplex G , Carbazóis , DNA , Humanos , Ligantes , Relação Estrutura-AtividadeRESUMO
The assembly of guanosine and boronic acids produces anionic hydrogels (G-B hydrogels) that mimic the topology of the DNA G-quadruplex. We herein demonstrate an unconventional approach of using the G-B hydrogel as a supramolecular template that assembles the irreversible formation of DNA G-quadruplex-selective 1,4-triazole ligands from a pool of alkyne-azide building blocks. These generated ligands could also stabilize and strengthen the gel assembly.
Assuntos
Ácidos Borônicos/química , DNA/química , Alcinos/química , Azidas/síntese química , Azidas/química , Catálise , Química Click , Quadruplex G , Ligantes , Estrutura Molecular , Triazóis/síntese química , Triazóis/químicaRESUMO
Herein, we present a gold nanoparticle (GNP)-based DNA nanosensor to detect the formation of an i-motif from the random coil structure by small molecules at physiological pH. The nanosensor shows a distance dependent fluorescence turn-off response in the presence of a ligand, indicating conformational changes from the C-rich single stranded DNA into an i-motif.
Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Ligantes , Nanopartículas Metálicas/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Benzotiazóis/química , DNA/química , DNA/efeitos dos fármacos , Doxorrubicina/química , Corantes Fluorescentes/química , Ouro/química , Ácidos Nucleicos Imobilizados/química , Espectrometria de FluorescênciaRESUMO
Non-canonical DNA i-motifs and G-quadruplexes are postulated as genetic switches for the transcriptional regulation of proto-oncogenes. However, in comparison to G-quadruplexes, the therapeutic potential of i-motifs is less explored. The development of i-motif selective ligands by conventional approaches is challenging due to the structural complexity of i-motifs. The target guided synthetic (TGS) approach involving in situ cycloaddition could provide specific ligands for these dynamic DNA structures. Herein, we have used i-motif forming C-rich DNA and their complementary G-quadruplex forming DNA sequences of c-MYC and BCL2 promoter regions as well as a control self-complementary duplex DNA sequence as the templates to generate selective ligands from a pool of reactive azide-alkyne building blocks. In our approach, thiolated DNA targets are immobilized on the surface of gold-coated iron nanoparticles to enable efficient isolation of the newly generated ligands from the solution mixture by simple magnetic decantation. The combinatorial in situ cycloaddition generated cell-membrane permeable triazole leads for respective DNA targets (c-MYC and BCL2 i-motifs and G-quadruplexes) that selectively promote their formation. In vitro cellular studies reveal that the c-MYC i-motif and G-quadruplex leads downregulate c-MYC gene expression whereas the BCL2 i-motif lead upregulates and the BCL2 G-quadruplex lead represses BCL2 gene expression. The TGS strategy using i-motif DNA nanotemplates represents a promising platform for the direct in situ formation of i-motif specific ligands for therapeutic intervention.
RESUMO
Herein, we develop a competitive screening method in which G-quadruplex DNA linked magnetic nanoparticles pull down selective ligands for a particular quadruplex topology from a series of small molecules. The screening strategy is first optimized with known G-quadruplex ligands and then used with a new series of G-quadruplex interactive bis-triazolyl ligands that are synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition. The assay enables the identification of c-MYC and BCL2 G-quadruplex selective bis-triazole ligands that specifically target promoter G-quadruplexes in cancer cells.
Assuntos
Quadruplex G , Ligantes , Nanopartículas de Magnetita/química , Alcinos/química , Azidas/química , Catálise , Linhagem Celular Tumoral , Cobre/química , Reação de Cicloadição , Óxido Ferroso-Férrico/química , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Triazóis/químicaRESUMO
The Cu(i)-catalyzed azide and alkyne 1,3-dipolar cycloaddition (CuAAC), commonly known as the "click reaction", has emerged as a powerful and versatile synthetic tool that finds a broad spectrum of applications in chemistry, biology and materials science. The efficiency, selectivity and versatility of the CuAAC reactions have enabled the preparation of vast arrays of triazole compounds with biological and pharmaceutical applications. In this feature article, we outline the applications and future prospects of click chemistry in the synthesis and development of small molecules that target G-quadruplex nucleic acids and show promising biological activities. Furthermore, this article highlights the template-assisted in situ click chemistry for developing G-quadruplex specific ligands and the use of click chemistry for enhancing drug specificity as well as designing imaging and sensor systems to elucidate the biological functions of G-quadruplex nucleic acids in live cells.
Assuntos
Química Click , Quadruplex G , Alcinos/química , Azidas/química , Catálise , Cobre/química , Reação de Cicloadição , DNA/síntese química , DNA/química , Imidas/química , Triazóis/químicaRESUMO
Dynamic combinatorial chemistry (DCC) has emerged as a promising strategy for template-driven selection of high-affinity ligands for biological targets from equilibrating combinatorial libraries. However, only a few examples using disulfide-exchange-based DCC are reported for nucleic acid targets. Herein, we have demonstrated that gold-coated magnetic nanoparticle-conjugated DNA targets can be used as templates for dynamic selection of ligands from an imine-based combinatorial library. The implementation of DCC using DNA nanotemplates enables efficient identification of the lead compounds, from the dynamic combinatorial library via magnetic decantation. It further allows quick separation of DNA nanotemplates for reuse in DCC reactions. The identified lead compound exhibits significant quadruplex versus duplex DNA selectivity and suppresses promoter activity of c-MYC gene that contains G-quadruplex DNA forming sequence in the upstream promoter region. Further cellular experiments indicated that the lead compound is able to permeate into cell nuclei and trigger a DNA damage response in cancer cells.
Assuntos
Técnicas de Química Combinatória/métodos , DNA/química , Quadruplex G , Ligantes , Nanopartículas Metálicas/química , Aldeídos/química , Aminas/química , Aminas/metabolismo , Aminas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Ouro/química , Humanos , Microscopia Confocal , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
G-quadruplexes have been considered attractive therapeutic targets for the development of anticancer agents. We herein report synthesis of a series of carbazole derivatives by employing modular one-pot Cu(I) catalyzed cycloaddition. These carbazole derivatives are easily synthesizable, soluble in aqueous media, and able to strongly interact with quadruplexes. FRET based melting assay and fluorescence titration experiments suggest that a carbazole derivative, Cz-1, preferentially binds c-MYC quadruplex DNA over other investigated quadruplex and duplex DNAs. The biological studies revealed that Cz-1 inhibits cancer cell proliferation by inducing apoptosis. Moreover, Cz-1 inhibits the expression of c-MYC at transcriptional as well as translational levels. Exon-specific-assay confirms that the downregulation of MYC expression is mainly driven by the binding of Cz-1 with the promoter G-quadruplex structures. Immunocytochemistry, using quadruplex binding antibody BG4, further suggests that Cz-1 induces and stabilizes G-quadruplexes in a cellular system.
Assuntos
Carbazóis/síntese química , Carbazóis/farmacologia , Quadruplex G , Regulação da Expressão Gênica/efeitos dos fármacos , Genes myc , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Carbazóis/metabolismo , Proliferação de Células/efeitos dos fármacos , Cobre/química , Reação de Cicloadição , DNA/química , Regulação para Baixo , Transferência Ressonante de Energia de Fluorescência , Células HCT116 , Células HeLa , Humanos , Relação Estrutura-AtividadeRESUMO
Herein, we report a carbazole (Cz) ligand that displays distinct turn-on fluorescence signals upon interaction with human telomeric G-quadruplex ( h-TELO) and nuclease enzymes. Interestingly, Cz selectively binds and stabilizes the mixed hybrid topology of h-TELO G-quadruplex that withstands digestion by exonucleases and nuclease S1. The distinct fluorescence signatures of Cz-stabilized h-TELO with nucleases are used to design conceptually novel DNA devices for selectively detecting the enzymatic activity of DNase I as well as performing logic operations. An INHIBIT logic gate is constructed using h-TELO and DNase I as the inputs while the inputs of h-TELO and nuclease S1 form a YES logic gate. Furthermore, a two-input two-output reusable logic device with "multireset" function is developed by using h-TELO and DNase I as inputs. On the basis of this platform, combinatorial logic systems (INHIBIT-INHIBIT and NOR-OR) have been successfully installed using different combinations of nucleases as inputs. Moreover, this new strategy of using a synthetic dual emissive probe and enzyme/DNA inputs for constructing reusable logic device may find important applications in biological computing and information processing.
Assuntos
Carbazóis/química , Computadores Moleculares , Desoxirribonuclease I/metabolismo , Quadruplex G , Carbazóis/metabolismo , Desoxirribonuclease I/análise , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Proteínas Fúngicas/metabolismo , Humanos , Lógica , Conformação de Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismoRESUMO
The development of small molecules is essential to modulate the cellular functions of biological targets in living system. Target Guided Synthesis (TGS) approaches have been used for the identification of potent small molecules for biological targets. We herein demonstrate an innovative example of TGS using DNA nano-templates that promote Huisgen cycloaddition from an array of azide and alkyne fragments. A G-quadruplex and a control duplex DNA nano-template have been prepared by assembling the DNA structures on gold-coated magnetic nanoparticles. The DNA nano-templates facilitate the regioselective formation of 1,4-substituted triazole products, which are easily isolated by magnetic decantation. The G-quadruplex nano-template can be easily recovered and reused for five reaction cycles. The major triazole product, generated by the G-quadruplex inhibits c-MYC expression by directly targeting the c-MYC promoter G-quadruplex. This work highlights that the nano-TGS approach may serve as a valuable strategy to generate target-selective ligands for drug discovery.
Assuntos
Alcinos/química , Azidas/química , Reação de Cicloadição , Quadruplex G , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Triazóis/síntese química , DNA/química , Ouro , Nanopartículas de Magnetita , Triazóis/farmacologiaRESUMO
The changes in structure and dynamics of oncogenic (c-MYC) and telomeric (h-TELO) G-rich DNA sequences due to the binding of a novel carbazole derivative (BTC) are elucidated using single-molecule Förster resonance energy transfer (sm-FRET), fluorescence correlation spectroscopy (FCS) and NMR spectroscopy. In contrast to the previous reports on the binding of ligands to pre-folded G-quadruplexes, this work illustrates how ligand binding changes the conformational equilibria of both unstructured G-rich DNA sequences and K+-folded G-quadruplexes. The results demonstrate that K+ free c-MYC and h-TELO exist as unfolded and partially folded conformations. The binding of BTC shifts the equilibria of both investigated DNA sequences towards the folded G-quadruplex structure, increases the diffusion coefficients and induces faster end-to-end contact formation. BTC recognizes a minor conformation of the c-MYC quadruplex and the two-tetrad basket conformations of the h-TELO quadruplex.
RESUMO
The c-MYC proto-oncogene is a regulator of fundamental cellular processes such as cell cycle progression and apoptosis. The development of novel c-MYC inhibitors that can act by targeting the c-MYC DNA G-quadruplex at the level of transcription would provide potential insight into structure-based design of small molecules and lead to a promising arena for cancer therapy. Herein we report our finding that two simple bis-triazolylcarbazole derivatives can inhibit c-MYC transcription, possibly by stabilizing the c-MYC G-quadruplex. These compounds are prepared using a facile and modular approach based on Cu(I) catalysed azide and alkyne cycloaddition. A carbazole ligand with carboxamide side chains is found to be microenvironment-sensitive and highly selective for "turn-on" detection of c-MYC quadruplex over duplex DNA. This fluorescent probe is applicable to visualize the cellular nucleus in living cells. Interestingly, the ligand binds to c-MYC in an asymmetric fashion and selects the minor-populated conformer via conformational selection.
Assuntos
Núcleo Celular/metabolismo , Regulação para Baixo , Sondas Moleculares/metabolismo , Neoplasias/genética , Transcrição Gênica , Apoptose/efeitos dos fármacos , Carbazóis/síntese química , Carbazóis/química , Carbazóis/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Quadruplex G , Humanos , Ligantes , Sondas Moleculares/química , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
An uncatalyzed aldol reaction of N-substituted thiazolidinediones with isatin derivatives has been developed "on water" to afford a new class of pharmacologically important thiazolidinedione-isatin conjugates in excellent yields and diastereoselectivities. The isatin-thiazolidine conjugate undergoes a catalyst-free stereoselective transfer aldol reaction on water. Single-crystal X-ray studies reveal that the aldol products can self-assemble to form supramolecular DNA "zipper" like structures through intermolecular hydrogen bonds and aromatic π-π interactions.
Assuntos
DNA/química , DNA/síntese química , Isatina/química , Tiazolidinedionas/química , Catálise , Cristalografia por Raios X , Ligação de Hidrogênio , Estrutura Molecular , Estereoisomerismo , ÁguaRESUMO
BACKGROUND: We analysed data on patients of Hodgkin and non-Hodgkin lymphoma treated with high dose chemotherapy followed by autologous stem cell transplantation to determine the toxicity, pattern of infections and long-term outcome. METHODS: There were 34 male and 10 female patients (median age 35 years, range 15-67 years). Before transplantation, 31 patients (70.5%) had chemosensitive disease and 13 (29.5%) had chemoresistant disease. Granulocyte-colony stimulating factor mobilized peripheral blood stem cells were used as the source of stem cells. The patients received high dose chemotherapy using CBV (cyclophosphamide, BCNU and VP16 [etoposide] n = 38), BEAM (BCNU, etoposide, cytosine arabinoside and melphalan, n = 3), cytosine arabinoside, etoposide and melphalan (n = 2) and melphalan alone (n = 1). Prophylaxis with antifungal drugs (fluconazole/itraconazole) and acyclovir was used. RESULTS: Following transplant, 32 patients (72.7%) responded; complete response was achieved in 25 patients (56.8%) and partial response in 7 (15.9%). The rate of complete response was higher for patients with pre-transplant chemosensitive disease (23/31 [74.2%] v. 2/13 [15.4%], p < 0.001). Gastrointestinal toxicity, and renal and liver dysfunctions were major non-haematological toxicities; 3 patients (7%) died of regimen-related toxicity. Infections (predominantly Gram-negative) accounted for 2 deaths (4.5%) seen before day 30. At a median follow up of 79 months (range 14-168 months), median overall and event-free survival were 78 months and 28 months, respectively. Estimated mean (SE) overall and event-free survival at 60 months were 54.34% (0.07) and 34.3% (9.88), respectively. CONCLUSION: Patients with pre-transplant chemosensitive disease and those who achieved complete response following transplant had a significantly better chance of survival.