G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

The effect of side chain variations on quinazoline-pyrimidine G-quadruplex DNA ligands.

G-quadruplex (G4) DNA structures are involved in central biological processes such as DNA replication and transcription. These DNA structures are enriched in promotor regions of oncogenes and are thus promising as novel gene silencing therapeutic targets that can be used to regulate expression of oncoproteins and in particular those that has proven hard to drug with conventional strategies. G4 DNA structures in general have a well-defined and hydrophobic binding area that also is very flat and featureless and there are ample examples of G4 ligands but their further progression towards drug development is limited. In this study, we use synthetic organic chemistry to equip a drug-like and low molecular weight central fragment with different side chains and evaluate how this affect the compound's selectivity and ability to bind and stabilize G4 DNA. Furthermore, we study the binding interactions of the compounds and connect the experimental observations with the compound's structural conformations and electrostatic potentials to understand the basis for the observed improvements. Finally, we evaluate the top candidates' ability to selectively reduce cancer cell growth in a 3D co-culture model of pancreatic cancer which show that this is a powerful approach to generate highly active and selective low molecular weight G4 ligands with a promising therapeutic window.

Bifunctional 3-Hydroxy-4-Pyridinones as Potential Selective Iron(III) Chelators: Solution Studies and Comparison with Other Metals of Biological and Environmental Relevance.

The binding ability of five bifunctional 3-hydroxy-4-pyridinones towards Cu2+ and Fe3+ was studied by means of potentiometric and UV-Vis spectrophotometric measurements carried out at I = 0.15 mol L-1 in NaCl(aq),T = 298.15 K and 310.15 K. The data treatments allowed us to determine speciation schemes featured by metal-ligand species with different stoichiometry and stability, owing to the various functional groups present in the 3-hydroxy-4-pyridinones structures, which could potentially participate in the metal complexation, and in the Cu2+ and Fe3+ behaviour in aqueous solution. Furthermore, the sequestering ability and metal chelating affinity of the ligands were investigated by the determination of pL0.5 and pM parameters at different pH conditions. Finally, a comparison between the Cu2+ and Fe3+/3-hydroxy-4-pyridinones data herein presented with those already reported in the literature on the interaction of Zn2+ and Al3+ with the same ligands showed that, from the thermodynamic point of view, the 3-hydroxy-4-pyridinones are particularly selective towards Fe3+ and could therefore be considered promising iron-chelating agents, also avoiding the possibility of competition, and eventually the depletion, of essential metal cations of biological and environmental relevance, such as Cu2+ and Zn2+.

Development of a rapid lateral flow immunochromatography assay for the detection of group-specific antibodies against VP7 protein of bluetongue virus in multiple species.

Bluetongue virus (BTV), the causative agent of bluetongue disease infects many domestic and wild ruminants. In the present study, colloidal gold nanoparticle-based lateral flow immunochromatography assay (LFIA) was developed to detect the group-specific antibodies to BTV in serum samples of sheep, goats, cattle, and camel. The recombinant VP7 protein of BTV conjugated to colloidal gold nanoparticles (GNPs) was used as a detector reagent. Recombinant streptococcal protein G and monoclonal antibody to BTV group-specific antigen were immobilized as the test and the control line, respectively on a nitrocellulose membrane. The protein G could capture the specific antibodies to BTV present in the serum of multiple ruminant species susceptible to BTV in a common test format and could eliminate the requirement of multiple anti-species antibodies. Upon addition of serum sample, GNP-rVP7 protein-serum complex migrated laterally onto the strip via capillary action and results were analyzed based on appearance of red colour band at test and control line. Serum samples (n = 481) of sheep, goats, cattle, and camel segregated as positive and negative by the commercial competitive-ELISA (c-ELISA) kit were tested in the fabricated LFIA strips to analyze the performance of the assay. In comparison with c-ELISA, the relative diagnostic sensitivity (DSn) of 95.2% with 91.6-97.6 (95%)) confidence interval and relative diagnostic specificity (DSp) of 99.6% 97.8-100.0 (95%) confidence interval were obtained for the optimized LFIA. The agreement between the LFIA and the c-ELISA was excellent as indicated by the kappa coefficient value of 0.949 (SE = 0.0142) with 0.9219 to 0.9779 (95%) confidence interval. The recombinant protein G based LFIA is a sensitive, specific, rapid, one-step test that can be used in the field or poorly equipped laboratories for serological diagnosis and serosurveillance of bluetongue in multiple susceptible species.

A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures.

G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small "druglike" optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure-activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

A site-specific self-assembled light-up rotor probe for selective recognition and stabilization of c-MYC G-quadruplex DNA.

Direct and unambiguous evidence of the formation of G-quadruplexes (G4s) in human cells have shown their implication in several key biological events and has emphasized their role as important targets for small-molecule cancer therapeutics. Here, we report on the first example of a self-assembled molecular-rotor G4-binder able to discriminate between an extensive panel of G4 and non-G4 structures and to selectively light-up (up to 64-fold), bind (nanomolar range), and stabilize the c-MYC promoter G4 DNA. In particular, association with the c-MYC G4 triggers the disassembly of its supramolecular state (disaggregation-induced emission, DIE) and induces geometrical restrictions (motion-induced change in emission, MICE) leading to a significant enhancement of its emission yield. Moreover, this optical reporter is able to selectively stabilize the c-MYC G4 and inhibit DNA synthesis. Finally, by using confocal laser-scanning microscopy (CLSM) we show the ability of this compound to localize primarily in the subnuclear G4-rich compartments of cancer cells. This work provides a benchmark for the future design and development of a new generation of smart sequence-selective supramolecular G4-binders that combine outstanding sensing and stability properties, to be utilized in anti-cancer therapy.

Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization.

The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes.

G-quadruplex (G4) DNA structures are linked to key biological processes and human diseases. Small molecules that target specific G4 DNA structures and signal their presence would therefore be of great value as chemical research tools with potential to further advance towards diagnostic and therapeutic developments. However, the development of these types of specific compounds remain as a great challenge. In here, we have developed a compound with ability to specifically signal a certain c-MYC G4 DNA structure through a fluorescence light-up mechanism. Despite the compound's two binding sites on the G4 DNA structure, only one of them result in the fluorescence light-up effect. This G-tetrad selectivity proved to originate from a difference in flexibility that affected the binding affinity and tilt the compound out of the planar conformation required for the fluorescence light-up mechanism. The intertwined relation between the presented factors is likely the reason for the lack of examples using rational design to develop compounds with turn-on emission that specifically target certain G4 DNA structures. However, this study shows that it is indeed possible to develop such compounds and present insights into the molecular details of specific G4 DNA recognition and signaling to advance future studies of G4 biology.

Novel tacrine-benzofuran hybrids as potential multi-target drug candidates for the treatment of Alzheimer's Disease.

Pursuing the widespread interest on multi-target drugs to combat Alzheimer´s disease (AD), a new series of hybrids was designed and developed based on the repositioning of the well-known acetylcholinesterase (AChE) inhibitor, tacrine (TAC), by its coupling to benzofuran (BF) derivatives. The BF framework aims to endow the conjugate molecules with ability for inhibition of AChE (bimodal way) and of amyloid-beta peptide aggregation, besides providing metal (Fe, Cu) chelating ability and concomitant extra anti-oxidant activity, for the hybrids with hydroxyl substitution. The new TAC-BF conjugates showed very good activity for AChE inhibition (sub-micromolar range) and good capacity for the inhibition of self- and Cu-mediated Aß aggregation, with dependence on the linker size and substituent groups of each main moiety. Neuroprotective effects were also found for the compounds through viability assays of neuroblastoma cells, after Aß1-42 induced toxicity. Structure-activity relationship analysis provides insights on the best structural parameters, to take in consideration for future studies in view of potential applications in AD therapy.

A Light-up Logic Platform for Selective Recognition of Parallel G-Quadruplex Structures via Disaggregation-Induced Emission.

The design of turn-on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G-quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition-driven disaggregation (on-signal) of an ultrabright coumarin-quinazoline conjugate. The synthesized probe selectively lights-up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label-free molecular logic system. Finally, our molecule preferentially stains the G4-rich nucleoli of cancer cells.

New Multitarget Hybrids Bearing Tacrine and Phenylbenzothiazole Motifs as Potential Drug Candidates for Alzheimer's Disease.

Research on neurodegenerative brain disorders, namely the age-dependent Alzheimer's disease (AD), has been intensified in the last decade due to the absence of a cure and the recognized increasing of life expectancy for populations. To address the multifactorial nature and complexity of AD, a multi-target-directed ligand approach was herein employed, by designing a set of six selected hybrids (14⁻19) that combine in the same entity two pharmacophores: tacrine (TAC) and 2-phenylbenzothiazole (PhBTA). The compounds contain a methoxy substituent at the PhBTA moiety and have a variable length linker between that and the TAC moiety. The docking studies showed that all the compounds assure a dual-binding mode of acetylcholinesterase (AChE) inhibition, establishing π-stacking and H-bond interactions with aminoacid residues at both active binding sites of the enzyme (CAS and PAS). The bioassays revealed that the designed compounds display excellent AChE inhibitory activity in the sub-micromolar range (0.06⁻0.27 µM) and moderate inhibition values for amyloid-ß (Aß) self-aggregation (27⁻44.6%), compounds 14 and 15 being the lead compounds. Regarding neuroprotective effects in neuroblastoma cells, compounds 15, 16 and 19 revealed the capacity to prevent Aß-induced toxicity, but compound 16 showed the highest neuroprotective effect. Overall these hybrid compounds, in particular 15 and 16, with promising multitarget anti-AD ability, encourage further pursuing studies on this type of TAC-PhBTA derivatives for potential AD therapy.

Tacrine-deferiprone hybrids as multi-target-directed metal chelators against Alzheimer's disease: a two-in-one drug.

Alzheimer's disease (AD) is a severe age-dependent neurodegenerative disorder affecting several million people worldwide. So far, there is no adequate medication to prevent or slow down the progression of the disease, only medication with palliative effects allowing temporary symptomatic reliefs. As part of our continuing efforts into the development of innovative drugs following a polypharmacological strategy, we decided to use a former anti-AD palliative drug (tacrine) and to reposition it by hybridization with a metal chelating drug (deferiprone, DFP). This combination endows the hybrids with good capacity to inhibit acetylcholinesterase (low micromolar range) and self-/Cu-induced Aß aggregation (up to ca. 90%) as well as a good radical scavenging ability (micromolar range) and metal (M) chelating capacity, with pM (pM = -log[M], CL/CM = 10, CM = 10-6 M at pH = 7.4, M = Fe, Cu, Zn) values close to those of DFP. The most promising compounds have 2-hydroxypropyl linkers, and a selection of compounds have demonstrated neuroprotective roles in neuroblastoma cells treated with Aß1-42 and ascorbate/iron stressors. Consequently, these hybrids can be considered as attractive multipotent therapeutic molecules that will eventually play key roles against AD progression, namely in the control of cholinergic dysfunction, amyloid peptide aggregation, oxidative stress, and metal modulation, besides presenting a good pharmacokinetic profile.

An overview of benzo[b]thiophene-based medicinal chemistry.

Among sulfur containing heterocycles, benzothiophene and its derivatives are at the focus as these candidates have structural similarities with active compounds to develop new potent lead molecules in drug design. Benzo[b]thiophene scaffold is one of the privileged structures in drug discovery as this core exhibits various biological activities allowing them to act as anti-microbial, anti-cancer, anti-inflammatory, anti-oxidant, anti-tubercular, anti-diabetic, anti-convulsant agents and many more. Further, numerous benzothiophene-based compounds as clinical drugs have been extensively used to treat various types of diseases with high therapeutic potency, which has led to their extensive developments. Due to the wide range of biological activities of benzothiophene, their structure activity relationships (SAR) have generated interest among medicinal chemists, and this has culminated in the discovery of several lead molecules against numerous diseases. The present review is endeavoring to highlight the progress in the various pharmacological activities of benzo[b]thiophene derivatives. It is hoped that this review will be helpful for new thoughts in the quest for rational designs of more active and less toxic benzothiophene-based medicinal drugs, as well as more effective diagnostic agents and pathologic probes. Also, SAR studies that highlight the chemical groups responsible for evoking the potential activities of benzothiophene derivatives are studied and compared.

Acetamide Derivatives of Chromen-2-ones as Potent Cholinesterase Inhibitors.

Alzheimer's disease (AD), a neurodegenerative disorder, is a serious medical issue worldwide with drastic social consequences. Inhibition of cholinesterase is one of the rational and effective approaches to retard the symptoms of AD and, hence, consistent efforts are being made to develop efficient anti-cholinesterase agents. In pursuit of this, a series of 19 acetamide derivatives of chromen-2-ones were synthesized and evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potential. All the synthesized compounds exhibited significant anti-AChE and anti-BChE activity, with IC50 values in the range of 0.24-10.19 µM and 0.64-30.08 µM, respectively, using donepezil hydrochloride as the standard. Out of 19 compounds screened, 3 compounds, viz. 22, 40, and 43, caused 50% inhibition of AChE at 0.24, 0.25, and 0.25 µM, respectively. A kinetic study revealed them to be mixed-type inhibitors, binding with both the CAS and PAS sites of AChE. The above-selected compounds were found to be effective inhibitors of AChE-induced and self-mediated Aß1-42 aggregation. ADMET predictions demonstrated that these compounds may possess suitable blood-brain barrier (BBB) permeability. Hemolytic assay results revealed that these compounds did not lyse human RBCs up to a thousand times of their IC50 value. MTT assays performed for the shortlisted compounds showed them to be negligibly toxic after 24 h of treatment with the SH-SY5Y neuroblastoma cells. These results provide insights for further optimization of the scaffolds for designing the next generation of compounds as lead cholinesterase inhibitors.

A review on antioxidant potential of bioactive heterocycle benzofuran: Natural and synthetic derivatives.

The majority of heterocycle compounds and typically common heterocycle fragments present in most pharmaceuticals currently marketed, alongside with their intrinsic versatility and unique physicochemical properties, have poised them as true cornerstones of medicinal chemistry. In this context, oxygen heterocycles exhibit diverse biological and pharmacological activities due in part to the similarities with many natural and synthetic molecules with known biological activity. Among oxygen containing heterocycles, benzofuran (synthetic and natural isolated) and its derivatives have attracted medicinal chemists and pharmacologists due to their pronounced biological activities and their potential applications as pharmacological agents such as antioxidant, antitumor, antiplatelet, antimalarial, antiinflammatory, antidepressant and anticonvulsant properties. There are also an amazing number of approved benzofuran-containing drugs in the market as well as compounds currently going through different clinical phases or registration statuses. Due to the wide range of biological activities of benzofurans, their structure activity relationships have generated interest among medicinal chemists, and this has culminated in the discovery of several lead molecules in numerous disease conditions. Recently, this scaffold has emerged as a pharmacophore of choice for designing antioxidant drug development as their derivatives have shown excellent results through different mechanism of action. This review focused on the recent development of benzofuran derivatives as antioxidant agents (including natural products) and their antioxidant activities; summarize the structure property, hoping to inspire new and even more creative approaches. Also, this study systematically provides a comprehensive report on current developments in benzofuran-based compounds as antioxidant agents and is also helpful for the researchers working on a substitution pattern around the nucleus, with an aim to help medicinal chemists to develop structure activity relationships (SAR) on these derivatives as antioxidant drugs.

A competitive ELISA for detection of group specific antibody to bluetongue virus using anti-core antibody.

Bluetongue virus (BTV) is transmitted by biting midges, which infects domestic and wild ruminants. In present study, a competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of serogroup-specific antibodies against VP7 protein of BTV has been developed. The assay measures the competition between a group specific antibody against core protein of BTV and a test serum to an optimized concentration of BTV recombinant-VP7 (r-VP7) antigen. Serum samples (n = 895) collected from small and large ruminants were used to optimize the C-ELISA. Percent inhibition (PI) values were used for estimation of the cut-off value for the C-ELISA. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >50% PI value was accepted as cut-off at which DSn and Dsp was achieved as 97.6% and 98.0% respectively, at >95% confidence interval. Results show the present C-ELISA assay described to be sensitive, specific and reliable and could be adopted for serological investigation of small and large ruminants.

Full genome sequencing of the bluetongue virus-1 isolate MKD20/08/Ind from goat in India

ABSTRACT This communication reports full genome sequencing of the bluetongue virus-1 (BTV-1) isolate MKD20/08/Ind from goat in northern India. The total BTV-1 genome size was found to be 19,190 bp. A comparison study between the Indian isolate and other global isolates revealed that it belongs to the 'Eastern' BTV topotype. The full genome sequence of BTV-1 will provide vital information on its geographical origin and it will also be proved useful for comparing the Indian isolate with global isolates from other host species.

Synthesis and evaluation of c-Src kinase inhibitory activity of pyridin-2(1H)-one derivatives.

Src kinase, a prototype member of the Src family of kinases (SFKs), is over-expressed in various human tumors, and has become a target for anticancer drug design. In this perspective, a series of eighteen 2-pyridone derivatives were synthesized and evaluated for their c-Src kinase inhibitory activity. Among them, eight compounds exhibited c-Src kinase inhibitory activity with IC50 value of less than 25µM. Compound 1-[2-(dimethylamino)ethyl]-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-one (36) exhibited the highest c-Src kinase inhibition with an IC50 value of 12.5µM. Furthermore, the kinase inhibitory activity of compound 36 was studied against EGFR, MAPK and PDK, however no significant activity was observed at the highest tested concentration (300µM). These results provide insights for further optimization of this scaffold for designing the next generation of 2-pyridone derivatives as candidate Src kinase inhibitors.

Substrate specificity of acetoxy derivatives of coumarins and quinolones towards Calreticulin mediated transacetylation: investigations on antiplatelet function.

Calreticulin transacetylase (CRTAase) is known to catalyze the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP), thus modulating their activity. Herein, we studied for the first time the substrate specificity of CRTAase towards N-acetylamino derivatives of coumarins and quinolones. This study is endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. Among all the N-acetylamino/acetoxy coumarins and quinolones screened, 7-N-acetylamino-4-methylcoumarin (7-AAMC, 17) was found to be the superior substrate to platelet CRTAase and emerged as the most promising antiplatelet agent both in vitro and in vivo. Further it caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2), modulation of tissue factor and the inhibition of platelet aggregation. It was also found effective in the inhibition of LPS induced pro-thrombotic conditions.