Multifaceted nucleic acid probing with a rationally upgraded molecular rotor.

Probing the sequence alterations, structures, interactions, and other important aspects of nucleic acids serves as the cornerstone of understanding nucleic acid-mediated biology and etiology, as well as the development of nucleic acid-based therapeutics. New strategies capable of accommodating these imperatives without necessitating specialized instrument or skills and potentially complementing existing methods are highly desired. Herein, we describe a rationally designed molecular rotor CCVJ-H ((9-(2-carboxy-2-cyanovinyl)julolidine-hydrazide)) and its superior performances compared to the universal base excision reporter probe CCVJ-1 in applications such as nucleic acid detection and DNA glycosylase assays. Furthermore, we showcase that the CCVJ-H probe accurately profiles the interactions between nucleic acids and small molecules, providing binding affinity and binding site information in a single reaction. We subsequently demonstrate the feasibility of applying the CCVJ-H system in high-throughput screening to identify nucleic acid-binding small molecules such as DNA CTG repeat expansion binders, potentially providing therapeutic interventions for myotonic dystrophy type 1. Finally, we profile the recognition difference between DNA/DNA and DNA/RNA against a library of small molecules, uncovering two drug-like molecules that preferentially bind DNA/RNA. We anticipate the versatile CCVJ-H probe will be a useful tool for both fundamental and translational nucleic acid research and application.

Identification of selective plant-derived natural carotenoid and flavonoids as the potential inhibitors of DHHC-mediated protein S-palmitoylation: an in silico study.

Protein S-palmitoylation mediated by DHHCs is recognized as a distinct and reversible form of lipid modification connected with several health perturbations, including neurodegenerative disorders, cancer, and autoimmune conditions. However, the pharmacological characteristics of current pan-DHHC inhibitors, particularly their toxicity and off-target effects, have hindered their in-depth cellular investigations. The therapeutic properties of the natural compounds, with minimal side effects, allowed us to evaluate them as DHHC-targeting inhibitors. Here, we performed an insilico screening of 115 phytochemicals to assess their interactions with the DHHC20 binding site. Among these compounds, lutein, 5-hydroxyflavone, and 6-hydroxyflavone exhibited higher binding energy (-9.2, -8.5, and -8.5 kcal/mol) in the DHHC20 groove compared to pan-DHHC inhibitor 2-BP (-7.0 kcal/mol). Furthermore, we conducted a 100 ns MD simulation to evaluate the stability of these complexes under physiological conditions. The MDsimulation results indicated that DHHC20 formed a more stable conformation with lutein compared to 5-hydroxyflavone and 6-hyroxyflavone via hydrophobic and H-bond interactions. Conclusively, these results could serve as a promising starting point for exploring the use of these natural molecules as DHHC20 inhibitors.Communicated by Ramaswamy H. Sarma.

Immune landscape and redox imbalance during neurological disorders in COVID-19.

The outbreak of Coronavirus Disease 2019 (COVID-19) has prompted the scientific community to explore potential treatments or vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes the illness. While SARS-CoV-2 is mostly considered a respiratory pathogen, several neurological complications have been reported, raising questions about how it may enter the Central Nervous System (CNS). Receptors such as ACE2, CD147, TMPRSS2, and NRP1 have been identified in brain cells and may be involved in facilitating SARS-CoV-2 entry into the CNS. Moreover, proteins like P2X7 and Panx-1 may contribute to the pathogenesis of COVID-19. Additionally, the role of the immune system in the gravity of COVID-19 has been investigated with respect to both innate and adaptive immune responses caused by SARS-CoV-2 infection, which can lead to a cytokine storm, tissue damage, and neurological manifestations. A redox imbalance has also been linked to the pathogenesis of COVID-19, potentially causing mitochondrial dysfunction, and generating proinflammatory cytokines. This review summarizes different mechanisms of reactive oxygen species and neuro-inflammation that may contribute to the development of severe COVID-19, and recent progress in the study of immunological events and redox imbalance in neurological complications of COVID-19, and the role of bioinformatics in the study of neurological implications of COVID-19.

An immunoinformatics analysis: design of a multi-epitope vaccine against Cryptosporidium hominis by employing heat shock protein triggers the innate and adaptive immune responses.

Cryptosporidium hominis, an anthropologically transferred species in the Cryptosporidium genus, represents many clinical studies in several countries. Its growth in the recent decade is primarily owing to epidemiologic studies. This parasite has complicated life cycles that require differentiation through a variety of phases of development and passage across two or more hosts throughout their lifetimes. As they move from host to host and environment to environment, pathogenic organisms are continually exposed to unexpected changes in the circumstances under which they develop. Heat shock proteins (HSPs) are targets of the host immune response; they are involved in the progression of diseases and play a significant part in this process. It has been discovered that the immunodominant immunogenic antigens in parasite infections HSPs. In this study, we have generated a multi-epitope vaccine against Cryptosporidium hominis (C. hominis) by using heat shock proteins. The epitopes that were selected had a substantial binding affinity for the B- and T-cell reference set of alleles, a high antigenicity score, a nature that was not allergic, a high solubility, non-toxicity and good binders. The epitopes were incorporated into a chimeric vaccine by using appropriate linkers. In order to increase the immunogenicity of the connected epitopes and effectively activate both innate and adaptive immunity, an adjuvant was attached to the epitopes. We have also analyzed the physiochemical characteristics of the vaccine which were satisfactory and then lead to the development of a 3D model. In addition, the binding confirmation of the vaccine to the TLR-4 innate immune receptor was also determined using molecular docking and molecular dynamics (MD) simulation. The results of this simulation show that the vaccine has a strong binding affinity for TLR4, which indicates that the vaccine is highly effective. In general, the vaccine that has been described here has a good potential for inducing protective and targeted immunogenicity, however, this hypothesis is contingent upon more experimental testing.Communicated by Ramaswamy H. Sarma.

G-quadruplex-mediated specific recognition, stabilization and transcriptional repression of bcl-2 by small molecule.

The presence of the G-quadruplex (G4) structure in the promoter region of the human bcl-2 oncogenes makes it a promising target for developing anti-cancer therapeutics. Bcl-2 inhibits apoptosis, and its frequent overexpression in cancer cells contributes to tumor initiation, progression, and resistance to therapy. Small molecules that can specifically bind to bcl-2 G4 with high affinity and selectivity are remaining elusive. Here, we report that small molecule 1,3-bis-) furane-2yl-methylidene-amino) guanidine (BiGh) binds to bcl-2 G4 DNA structure with very high affinity and selectivity over other genomic G4 DNA structures and duplex DNA. BiGh stabilizes folded parallel conformation of bcl-2 G4 via non-covalent and electrostatic interactions and increases the thermal stabilization up to 15 °C. The ligand significantly suppresses the bcl-2 transcription in HeLa cells by a G4-dependent mechanism and induces cell cycle arrest which promotes apoptosis. The in silico ADME profiling confirms the potential 'drug-likeness' of BiGh. Our results showed that BiGh stabilizes the bcl-2 G-quadruplex motif, downregulates the bcl-2 gene transcription as well as translation process in cervical cancer cells, and exhibits potential anti-cancer activity. This work provides a potential platform for the development of lead compound(s) as G4 stabilizers with drug-like properties of BiGh for cancer therapeutics.

Discovery of a potent Guanidine derivative that selectively binds and stabilizes the human BCL-2 G-quadruplex DNA and downregulates the transcription.

G-quadruplex also known as G4 (GQ) structures, are a non-canonical kind of DNA or RNA secondary structure that may develop inside guanine-rich nucleic acid sequences. They may be found in a variety of locations in the human genome, such as gene promoters, 5' untranslated region, and telomeres, among others. Because of their significance in biology, G4 structures are recognized as promising pharmacological targets, particularly for therapeutics against cancer. This has led to the discovery of small molecules that can stabilize G4 structures. Small molecules that interact with quadruplexes offer a wide range of potential applications, including not just as medications but also as sensors for quadruplexes structures. The BCL-2 is a proto-oncogene that often gets mutated in lethal cancer and could be an interesting target for developing an anti-cancer drug. In the present study, we have employed various biophysical techniques such as fluorescence, CD, Isothermal calorimetry, gel retardation, and PCR stop assay, indicating that Guanidine derivatives GD-1 and GD-2 selectively interact with high affinity with BCL-2 GQ over other G-quadruplex DNA and duplex DNA. The most promising small molecule GD-1 increases the thermostability of the BCL-2 GQ structure by 12°C. Our biological experiments such as ROS generation, qRT-PCR, western blot, TFP based reporter assay, show that the GD-1 ligand causes a synthetic lethal interaction by suppressing the expression of BCL-2 genes via interaction and stabilization of its promoter GQ strucure in HeLa cells and act as a potential anti-cancer agent.

Immunoinformatics analysis for design of multi-epitope subunit vaccine by using heat shock proteins against Schistosoma mansoni.

The development of T cell and B cell that able provide long-term immune response against the schistosomiasisis to the people belongs to the epidemic area. Heat Shock Proteins (HSPs) are up-regulated in schistosomes as their environment changes owing to the developmental cycle, assisting the parasite in living with the adverse circumstances related with its life cycle. Schistosomiasis is still a severe health problem in the people of many countries in worldwide. In this work, to develop a chimeric antigen, we used an advanced and powerful immunoinformatics technique that targeted Schistosoma mansoni (S. mansoni) Heat shock protein (HSPs). Antigenicity, immunogenicity, allergenicity, and physicochemical characteristics were all assessed in silico for the developed subunit vaccine. The 3D structure of the vaccine was constructed and the stability of the vaccine construct was increased by using disulphide engineering. The protein-protein docking and simulation were performed between the vaccine construct and Toll-like receptor-4. The antigenicity probability value obtained for the vaccine construct was 0.93, which indicates that vaccine is non-allergenic and safe for human consumption. Communicated by Ramaswamy H. Sarma.

A multi-epitope vaccine candidate developed from unique immunogenic epitopes against Cryptosporidium hominis by utilizing an immunoinformatics-driven approach.

An immunoinformatics-based strategy is being investigated to identify prospective multi-subunit vaccine candidates against Cryptosporidium hominis (C. hominis). We used a systematic technique based on protein structure to create a competent multi-subunit vaccine candidate against C. hominis, with the likelihood of antigenicity, allergenicity, and transmembrane helices as the screening criteria. Using the suitable linkers, the best-screened epitopes such as B-cell epitopes (BCL), Helper T-lymphocytes (HTL), and cytotoxic T-lymphocytes (CTL) were linked together to intensify and develop the presentation and processing of the antigenic molecules. The greatest 3 D model of the component protein was created with the help of modeling software called Raptorax. The validation of the modeled protein was accomplished via the use of PROCHECK. Furthermore, using the ClusPro web server, the projected modeled structure was docked with known receptor TLR-4 to determine their interactions. A molecular dynamics (MD) simulation was used to investigate the stability of the multi-subunit vaccine bound with TLR-4 based on the docking score. Aside from that, the codon optimization and in silico expression demonstrate the possibility of high expression and simple purification of the vaccine product resulting from codon optimization. The overall findings indicated that the multi-subunit vaccine might be a viable vaccination candidate against C. hominis.Communicated by Ramaswamy H. Sarma.

Identification of substrates of MBL Associated Serine Protease-1 (MASP-1) from human plasma using N-terminomics strategy.

MBL Associated Serine Protease-1 (MASP-1) is an abundant enzyme of the lectin complement pathway. MASP-1 cleaves numerous substrates like MASP-2, MASP-3, C2, C3i, fibrinogen, FXIII and prothrombin. It has thrombin-like specificity and can cleave thrombin substrates. Owing to its high concentration and relaxed substrate specificity, MASP-1 has substrates outside the complement system and can influence other proteolytic cascades and physiological processes. The unidentified substrates may assist us to ascertain the role(s) of MASP-1. In this study, we used a high-throughput N-terminomics method to identify substrates of MASP-1 from human plasma. We have identified 35 putative substrates of MASP-1. Among the identified proteins, alpha 2-antiplasmin, alpha-1-acid glycoprotein, antithrombin III, and siglec-6 were demonstrated to be cleaved by MASP-1. We have discussed the physiological relevance of cleavage of these substrates by MASP-1. The expression of Siglec-6 and MASP-1 has been reported in the B cells. Alpha-1-acid glycoprotein cleavage by MASP-1 may occur in the acute phase as it is known to be an inhibitor of platelet aggregation, whereas MASP-1 triggers platelet aggregation. The cleavage alpha2 antiplasmin by MASP-1 implies that MASP-1 may be promoting plasmin-mediated fibrinolysis. Our study supports that MASP-1 may be implicated in thrombosis as well as thrombolysis.

Piperine analogs arrest c-myc gene leading to downregulation of transcription for targeting cancer.

G-quadruplex (G4) structures are considered a promising therapeutic target in cancer. Since Ayurveda, Piperine has been known for its medicinal properties. Piperine shows anticancer properties by stabilizing the G4 motif present upstream of the c-myc gene. This gene belongs to a group of proto-oncogenes, and its aberrant transcription drives tumorigenesis. The transcriptional regulation of the c-myc gene is an interesting approach for anticancer drug design. The present study employed a chemical similarity approach to identify Piperine similar compounds and analyzed their interaction with cancer-associated G-quadruplex motifs. Among all Piperine analogs, PIP-2 exhibited strong selectivity, specificity, and affinity towards c-myc G4 DNA as elaborated through biophysical studies such as fluorescence emission, isothermal calorimetry, and circular dichroism. Moreover, our biophysical observations are supported by molecular dynamics analysis and cellular-based studies. Our study showed that PIP-2 showed higher toxicity against the A549 lung cancer cell line but lower toxicity towards normal HEK 293 cells, indicating increased efficacy of the drug at the cellular level. Biological evaluation assays such as TFP reporter assay, quantitative real-time PCR (qRT- PCR), and western blotting suggest that the Piperine analog-2 (PIP-2) stabilizes the G-quadruplex motif located at the promoter site of c-myc oncogene and downregulates its expression. In conclusion, Piperine analog PIP-2 may be used as anticancer therapeutics as it affects the c-myc oncogene expression via G-quadruplex mediated mechanism.

Regulatory role of Non-canonical DNA Polymorphisms in human genome and their relevance in Cancer.

DNA has the ability to form polymorphic structures like canonical duplex DNA and non-canonical triplex DNA, Cruciform, Z-DNA, G-quadruplex (G4), i-motifs, and hairpin structures. The alteration in the form of DNA polymorphism in the response to environmental changes influences the gene expression. Non-canonical structures are engaged in various biological functions, including chromatin epigenetic and gene expression regulation via transcription and translation, as well as DNA repair and recombination. The presence of non-canonical structures in the regulatory region of the gene alters the gene expression and affects the cellular machinery. Formation of non-canonical structure in the regulatory site of cancer-related genes either inhibits or dysregulate the gene function and promote tumour formation. In the current article, we review the influence of non-canonical structure on the regulatory mechanisms in human genome. Moreover, we have also discussed the relevance of non-canonical structures in cancer and provided information on the drugs used for their treatment by targeting these structures.

Curcumin analogs exhibit anti-cancer activity by selectively targeting G-quadruplex forming c-myc promoter sequence.

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.

Identification and characterization of two conserved G-quadruplex forming motifs in the Nipah virus genome and their interaction with G-quadruplex specific ligands.

The G-quadruplex (GQ) motifs are considered as potential drug-target sites for several human pathogenic viruses such as Zika, Hepatitis, Ebola, and Human Herpesviruses. The recent outbreaks of Nipah virus (NiV) in India, the highly fatal emerging zoonotic virus is a potential threat to global health security as no anti-viral drug or vaccine in currently available. Therefore, here in the present study, we sought to assess the ability of the putative G-quadruplex forming sequences in the NiV genome to form G-quadruplex structures and act as targets for anti-viral compounds. Bioinformatics analysis underpinned by various biophysical and biochemical techniques (such as NMR, CD, EMSA, DMS footprinting assay) confirmed the presence of two highly conserved G-quadruplex forming sequences (HGQs) in the G and L genes of NiV. These genes encode the cell attachment glycoprotein and RNA-dependent RNA polymerase, respectively and are essential for the virus entry and replication within the host cell. It remains possible that stabilization of these HGQs by the known G-quadruplex binding ligands like TMPyP4 and Braco-19 represents a promising strategy to inhibit the expression of the HGQ harboring genes and thereby stop the viral entry and replication inside the host cell. Accordingly, we report for the first time, that HGQs in Nipah virus genome are targets for G-quadruplex specific ligands; therefore, could serve as potential targets for anti-viral therapy.

DNA binding, antioxidant activity, and DNA damage protection of chiral macrocyclic Mn(III) salen complexes.

We are reporting the synthesis, characterization, and calf thymus DNA binding studies of novel chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2. These chiral complexes showed ability to bind with DNA, where complex S-1 exhibits the highest DNA binding constant 1.20 × 10(6) M(-1). All the compounds were screened for superoxide and hydroxyl radical scavenging activities; among them, complex S-1 exhibited significant activity with IC(50) 1.36 and 2.37 µM, respectively. Further, comet assay was used to evaluate the DNA damage protection in white blood cells against the reactive oxygen species wherein complex S-1 was found effective in protecting the hydroxyl radicals mediated plasmid and white blood cells DNA damage.

Influence of chirality of V(V) Schiff base complexes on DNA, BSA binding and cleavage activity.

New chiral V(V) Schiff base complexes (S)-[VO(OMe)L] and (R)-[VO(OMe)L] were synthesized and characterized by microanalysis, infrared (IR), UV-Visible, Circular dichroism (CD) spectroscopy and single crystal X-ray studies. The interaction of these complexes with calf thymus (CT) DNA and bovine serum albumin (BSA) protein showed chiral expression DNA/protein binding strength. The influence of chirality was also observed in cytotoxicity assay of Hep 2 cells. (R)-[VO(OMe)L] enantiomer exhibited higher binding constant (5 ± 1 × 10(5) M(-1)) as compared to (S)-[VO(OMe)L] (8 ± 1 × 10(4) M(-1)). The fluorescence quenching, thermal melting and viscosity data suggest DNA surface and/or groove binding nature of the complexes and electrophoresis studies also showed greater activity for (R)-[VO(OMe)L] in cleaving DNA and protein as against (S)-[VO(OMe)L].

Chiral discrimination asserted by enantiomers of Ni (II), Cu (II) and Zn (II) Schiff base complexes in DNA binding, antioxidant and antibacterial activities.

Chiral Schiff base ligands (S)-H(2)L and (R)-H(2)L and their complexes (S-Ni-L, R-Ni-L, S-Cu-L, R-Cu-L, S-Zn-L and R-Zn-L) were synthesized, characterized and examined for their DNA binding, antioxidant and antibacterial activities. The complexes showed higher binding affinity to calf thymus DNA with binding constant ranging from 2.0×10(5) to 4.5×10(6) M(-1). All the complexes also exhibited remarkable superoxide (56-99%) and hydroxyl scavenging (45-89%) activities as well as antibacterial activities against gram (+) and gram (-) bacteria. However, none of the complexes showed antifungal activity. Conclusively, S enantiomers of the complexes were found to be relatively more efficient for DNA interaction, antioxidant and antibacterial activities than their R enantiomers. This study reveals the possible utilization of chiral Schiff base complexes for pharmaceutical applications.

Influence of chirality using Mn(III) salen complexes on DNA binding and antioxidant activity.

Chiral Mn(iii) salen complexes S-1, R-1, S-2, R-2, S-3 and R-3 derived from the respective chiral salen ligands, viz., (1S,2S)-N,N'-bis-[3-tert-butyl-5-chloromethyl-salicylidine]-1,2-cyclohexanediamine S-1'/(1R,2R)-N,N'-bis-[3-tert-butyl-5-chloromethyl-salicylidine]-1,2-cyclohexanediamine R-1'/(1S,2S)-N,N'-bis-[3-tert-butyl-5-N,N'N'triethylaminomethyl-salicylidine]-1,2-cyclohexanediamine dichloride S-2'/(1R,2R)-N,N'-bis-[3-tert-butyl-5-N,N'N'triethylaminomethyl-salicylidine]-1,2-cyclohexanediamine dichloride R-2'/(1S,2S)-N,N'-bis-[3,5-di-tert-butylsalicylidene]-1,2-cyclohexanediamine S-3' and (1R,2R)-N,N'-bis-[3,5-di-tert-butyl-salicylidene]-1,2-cyclohexanediamine R-3', were synthesized. Characterization of the complexes was done by microanalysis, IR, LC-MS, UV-vis. and circular dichroism (CD) spectroscopy. Binding of these complexes with calf thymus DNA (CT-DNA) was studied by absorption spectroscopy, competitive binding study, viscosity measurements, circular dichroism measurements, thermal denaturation study and observation of their different antioxidant activities. Among all the complexes used, the best result in terms of binding constant (intercalative) (130.4 x 10(4)) was achieved with the complex S-1 by spectroscopic titration. The complex S-1 showed strong antioxidant activity as well.

Synthesis, characterization, DNA binding and cleavage studies of chiral Ru(II) salen complexes.

Interaction of chiral Ru(II) salen complexes (S)-1 and (R)-1 with Calf Thymus DNA (CT-DNA) was studied by absorption spectroscopy, competitive binding study, viscosity measurements, CD measurements, thermal denaturation study and cleavage studies by agarose gel electrophoresis. The DNA binding affinity of (S)-1 (6.25 x 10(3)M(-1)) was found to be greater than (R)-1 (3.0 x 10(3)M(-1)). The antimicrobial studies of these complexes on five different gram (+)/(-) bacteria and three different fungal organisms showed selective inhibition of the growth of gram (+) bacteria and were not affective against gram (-) and fungal organisms. Further, the (S)-1 enantiomer inhibited the growth of organisms to a greater extent as compared to (R)-1 enantiomer.

Comparative study of interspecific genetic divergence and phylogenic analysis of genus Jatropha by RAPD and AFLP: genetic divergence and phylogenic analysis of genus Jatropha.

Genus Jatropha with 172 species having significant economic importance belongs to the family Euphorbiaceae. There are no reports on molecular characterization and phylogenetic relationship among the species of Jatropha. Hence, the present study was undertaken to assess the extent of genetic variability that exist and also to establish phylogenetic relationship among Jatropha curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica and J. tanjorensis using RAPD and AFLP. The percentage of loci that are polymorphic among the species studied was found to be 97.74% by RAPD and 97.25% by AFLP. The mean percentage of polymorphism (PP) was found to be 68.48 by RAPD and 71.33 by AFLP. The phylogram generated with RAPD and AFLP data showed maximum similarity. With the generated data maximum relatedness was found between J. curcas and J. integerrima this may be the reason for the success of inter hybrid crosses between these two species. Neither RAPD nor AFLP data generated in this study supports the view of J. tanjorensis, a natural interspecific hybrid between J. curcas and J. gossypifolia. The present study concludes that both RAPD and AFLP techniques are comparable in divergence studies of Jatropha species. The markers generated by RAPD and AFLP can be employed efficiently for interspecific hybrids identification, marker assisted selection and genetic resource management.