Exploring the impact of trifluoromethyl (-CF3) functional group on the anti-cancer activity of isoxazole-based molecules: design, synthesis, biological evaluation and molecular docking analysis.

Herein we report the design and synthesis of a series of fully-substituted 4-(trifluoromethyl)isoxazoles and evaluation of their anti-cancer activities against MCF-7, 4T1 and PC-3 cell lines as a proof of concept study. 4-(Trifluoromethyl)isoxazole is a synthetically challenging class of molecules and very few synthetic methods have been developed so far and all of them suffered from several serious limitations. Recently we developed a novel, metal-free, and general synthetic strategy to access synthetically challenging 4-(trifluoromethyl)isoxazoles starting from readily available chalcones using cheap CF3SO2Na as the source of the -CF3 group and multitasking t BuONO as an oxidant as well as the source of N and O and thus we have overcome the limitations of the previous methods. Based on the structure of an isoxazole-based anti-cancer agent, 3-(3,4-dimethoxyphenyl)-5-(thiophen-2-yl)isoxazole 14, we designed a set of 4-(trifluoromethyl)isoxazoles for synthesis and further anti-cancer evaluation. Among various molecules, 3-(3,4-dimethoxyphenyl)-5-(thiophen-2-yl)-4-(trifluoromethyl)isoxazole 2g (IC50 = 2.63 µM) and 3-(thiophen-2-yl)-5-(4-(thiophen-2-yl)-1H-pyrrol-3-yl)-4-(trifluoromethyl)isoxazole 5 (IC50 = 3.09 µM) exhibited the best anti-cancer activity against the human breast cancer cell-lines (MCF-7), 2g being the lead molecule among all. Interestingly, 2g is found to be almost 8 times more active compared to its non-trifluoromethylated analogue, i.e., 3-(3,4-dimethoxyphenyl)-5-(thiophen-2-yl)isoxazole 14 (IC50 = 19.72 µM) which revealed the importance of a 'CF3' moiety in enhancing the anti-cancer activity of 14. Further studies such as apoptosis induction, cell cycle analysis, and nuclear staining revealed an apoptotic cell death mechanism. The in silico molecular docking, induced fit analysis, and ADME studies further supported the effect of a -CF3 moiety on the enhancement of anti-cancer activity of isoxazole-based anti-cancer molecules. Further exploration of the biodistribution and therapeutic efficacy of lead 2gin vivo holds significant promise, positioning it as a potential candidate for anticancer therapy.

Unleashing the power of porphyrin photosensitizers: Illuminating breakthroughs in photodynamic therapy.

This comprehensive review provides the current trends and recent developments of porphyrin-based photosensitizers. We discuss their evolution from first-generation to third-generation compounds, including cutting-edge nanoparticle-integrated derivatives, and explores their pivotal role in advancing photodynamic therapy (PDT) for enhanced cancer treatment. Integrating porphyrins with nanoparticles represents a promising avenue, offering improved selectivity, reduced toxicity, and heightened biocompatibility. By elucidating recent breakthroughs, innovative methodologies, and emerging applications, this review provides a panoramic snapshot of the dynamic field, addressing challenges and charting prospects. With a focus on harnessing reactive oxygen species (ROS) through light activation, PDT serves as a minimally invasive therapeutic approach. This article offers a valuable resource for researchers, clinicians, and PDT enthusiasts, highlighting the potential of porphyrin photosensitizers to improve the future of cancer therapy.

Synthesis of (4-Trifluoromethyl)isoxazoles through a Tandem Trifluoromethyloximation/Cyclization/Elimination Reaction of α,ß-Unsaturated Carbonyls.

We disclose a metal-free, cascade regio- and stereoselective trifluormethyloximation, cyclization, and elimination strategy with readily available α,ß-unsaturated carbonyl compounds to access a wide variety of pharmaceutically potential heteroaromatics, i.e., 4-(trifluoromethyl)isoxazoles including a trifluoromethyl analogue of an anticancer agent. The transformation requires only a couple of commercially available and cheap reagents i.e., CF3SO2Na as the trifluoromethyl source, and tBuONO as an oxidant as well as a source of N and O. Notably, 5-alkenyl-4-(trifluoromethyl)isoxazoles were further synthetically diversified to a new class of biheteroaryls, i.e., 5-(3-pyrrolyl)-4-(trifluoromethyl)isoxazoles. Mechanistic studies revealed a radical pathway for the reaction.

Highly sensitive protein detection by aptamer-based single-molecule kinetic fingerprinting.

Sensitive assays of protein biomarkers play critical roles in clinical diagnostics and biomedical research. Such assays typically employ immunoreagents such as monoclonal antibodies that suffer from several drawbacks, including relatively tedious production, significant batch-to-batch variability, and challenges in site-specific, stoichiometric modification with fluorophores or other labels. One proposed alternative to such immunoreagents, nucleic acid aptamers generated by systematic evolution of ligand by exponential enrichment (SELEX), can be chemically synthesized with much greater ease, precision, and reproducibility than antibodies. However, most aptamers exhibit relatively poor affinity, yielding low sensitivity in the assays employing them. Recently, single molecule recognition through equilibrium Poisson sampling (SiMREPS) has emerged as a platform for detecting proteins and other biomarkers with high sensitivity without requiring high-affinity detection probes. In this manuscript, we demonstrate the applicability and advantages of aptamers as detection probes in SiMREPS as applied to two clinically relevant biomarkers, VEGF165 and IL-8, using a wash-free protocol with limits of detection in the low femtomolar range (3-9 fM). We show that the kinetics of existing RNA aptamers can be rationally optimized for use as SiMREPS detection probes by mutating a single nucleotide in the conserved binding region or by shortening the aptamer sequence. Finally, we demonstrate the detection of endogenous IL-8 from human serum at a concentration below the detection limit of commercial ELISAs.

Predominance of genomically defined A lineage of HPV16 over D lineage in Indian patients from eastern India with squamous cell carcinoma of the cervix in association with distinct oncogenic phenotypes.

Human papillomavirus type-16 (HPV16) is classified into lineages, A, B, C and D and 10 sub-lineages portraying variable infectivity, persistence, and cytological outcomes, however, with geographical variations. Our objective was to delineate the distinctive features of lineages among cervical squamous cell carcinoma (SCC) in the eastern region of India. A total of 145 SCC cases and 24 non-malignant specimens, harboring episomal HPV16, were included. The presence of higher proportion of lineage A over D was observed among SCC cases (86.89% A1, 8.97% D1 and 4.14% D2), while only A1 sub-lineage viruses were found among control specimens. Among the A1 viruses, an association of variants in the E5 (Y44L, I65V), E6 (L83V) genes and LCR: C7577T with SCC, with combined Odd's ratio (95% CI) of 20.5(4.61-91.25) was observed. Network analyses revealed the presence of 10 clades of lineage A viruses comprising of 64 HPV16 genomes harboring the risk alleles. High episomal HPV16 DNA copy numbers (adjusted p-value= 0.0271) and E7 mRNA expression (p-value=0.000017) predominated in SCC with lineage A, over D. Our study highlights the distinctive modalities of oncogenicity among different HPV16 lineages.

Cervical cancer subtypes harbouring integrated and/or episomal HPV16 portray distinct molecular phenotypes based on transcriptome profiling of mRNAs and miRNAs.

Heterogeneity in cervical cancers (CaCx) in terms of HPV16 physical status prompted us to investigate the mRNA and miRNA signatures among the different categories of CaCx samples. We performed microarray-based mRNA expression profiling and quantitative real-time PCR-based expression analysis of some prioritised miRNAs implicated in cancer-related pathways among various categories of cervical samples. Such samples included HPV16-positive CaCx cases that harboured either purely integrated HPV16 genomes (integrated) and those that harboured episomal viral genomes, either pure or concomitant with integrated viral genomes (episomal), which were compared with normal cervical samples that were either HPV negative or positive for HPV16. The mRNA expression profile differed characteristically between integrated and episomal CaCx cases for enriched biological pathways. miRNA expression profiles also differed among CaCx cases compared with controls (upregulation-miR-21, miR-16, miR-205, miR-323; downregulation-miR-143, miR-196b, miR-203, miR-34a; progressive upregulation-miR-21 and progressive downregulation-miR-143, miR-34a, miR-196b and miR-203) in the order of HPV-negative controls, HPV16-positive non-malignant samples and HPV16-positive CaCx cases. miR-200a was upregulated in HPV16-positive cervical tissues irrespective of histopathological status. Expression of majority of the predicted target genes was negatively correlated with their corresponding miRNAs, irrespective of the CaCx subtypes. E7 mRNA expression correlated positively with miR-323 expression among episomal cases and miR-203, among integrated cases. miR-181c expression was downregulated only among the episomal CaCx cases and negatively correlated with protein coding transcript of the proliferative target gene, CKS1B of the significantly enriched "G2/M DNA Damage Checkpoint Regulation" pathway among CaCx cases. Thus, the two CaCx subtypes are distinct entities at the molecular level, which could be differentially targeted for therapy. In fact, availability of a small molecule inhibitor of CKS1B, suggests that drugging CKS1B could be a potential avenue of treating the large majority of CaCx cases harbouring episomal HPV16.

Impact of viral and host DNA methylations on HPV16-related cervical cancer pathogenesis.

Epigenetic alterations within human papillomavirus (HPV) and host cellular genomes are known to occur during cervical carcinogenesis. Our objective was to analyse the influence of (1) methylation within two immunostimulatory CpG motifs within HPV16 E6 and E7 genes around the viral late promoter and their correlation, if any, with expression deregulation of host receptor (TLR9) and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and (2) global DNA methylation levels within CpGs of the repetitive Alu sequences, on cervical cancer (CaCx) pathogenesis. Significantly higher proportions of CaCx samples portrayed methylation in immunostimulatory CpG motifs, compared to HPV16-positive non-malignant samples, with cases harbouring episomal HPV16 showing decreased methylation compared to those with viral integration. A significant linear trend of TLR9 upregulation was recorded in the order of HPV-negative controls < HPV16-positive non-malignant samples < HPV16-positive CaCx cases. TLR9 upregulation in cases with episomal HPV16 was again higher among those with non-methylated immunostimulatory CpG motifs. Comparison of cases with HPV-negative controls revealed that DNMT3A was significantly downregulated only among integrated cases, DNMT3B was significantly overexpressed among both categories of cases, although at variable levels, while DNMT1 failed to show any deregulated expression among the cases. Global host DNA hypomethylation, also showed a significant linear increasing trend through the progressive CaCx development stages mentioned above and was most prominently higher among cases with episomal HPV16 as opposed to viral integration. Thus, HPV16 and host methylations appear to influence CaCx pathogenesis, with differential molecular signatures among CaCx cases with episomal and integrated HPV16.

Identification of genetic variation in the lncRNA HOTAIR associated with HPV16-related cervical cancer pathogenesis.

PURPOSE: Previously, over-expression of the long noncoding RNA (lncRNA) HOTAIR has been found to be associated with the invasive and metastatic capacities of several epithelial cancers, including cervical cancer (CaCx). Here, we aimed at identifying functionally relevant genetic variants that may be employed to differentiate between clinically distinct CaCx subtypes, i.e., those exhibiting high HOTAIR levels and molecular signatures of metastasis and those lacking such signatures in the presence of low HOTAIR expression levels. METHODS: Genomic DNA isolated from various cervical tissue samples (characterized by histopathology and HPV status) was used for HOTAIR amplicon sequencing, followed by validation of the findings by Sanger sequencing. The impact of the genetic variants found on the secondary structure of HOTAIR and the concomitant alterations in miRNA binding sites were determined through in silico analysis, followed by miRNA expression analysis by quantitative real-time PCR and confirmation of miRNA binding using a luciferase reporter assay. RESULTS: We found that rs2366152C was over-represented [ORage-adjusted = 2.58 (1.23-5.57); p = 0.014] in low HOTAIR expressing HPV positive CaCx cases compared to HPV negative controls. This genetic variant showed the propensity of a secondary structure alteration and gain of a miR-22 binding site in HOTAIR, which was found to be concordant with miR-22 over-expression in low HOTAIR CaCx cases compared to controls. We found that miR-22 expression negatively correlated with HOTAIR and E7 expression in HPV16 positive cases and in an E7 transfected HPV negative CaCx-derived cell line (C33A), but was not altered in high HOTAIR cases compared to controls. Reduced luciferase activity of a HOTAIR rs2366152C expression plasmid in C33A cells through miR-22 co-transfection confirmed the ability of miR-22 to specifically target rs2366152C-harbouring HOTAIR lncRNA in CaCx cells, ultimately leading to its down-regulation. CONCLUSIONS: Our data indicate that rs2366152C not only has the potential to serve as a marker for singling out CaCx cases lacking metastatic molecular signatures, but also to explain the functional inactivation of HOTAIR in these cases, including the mechanism of its down-regulation.

Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis.

Human Papillomavirus (HPV) type 16 oncoprotein E7 plays a major role in cervical carcinogenesis by interacting with and functionally inactivating various host regulatory molecules. Long noncoding RNA (lncRNA) HOTAIR is one such regulator that recruits chromatin remodelling complex PRC2, creating gene silencing H3K27 me3 marks. Hence, we hypothesized that HOTAIR could be a potential target of E7, in HPV16 related cervical cancers (CaCx). We identified significant linear trend of progressive HOTAIR down-regulation through HPV negative controls, HPV16 positive non-malignants and CaCx samples. Majority of CaCx cases portrayed HOTAIR down-regulation in comparison to HPV negative controls, with corresponding up-regulation of HOTAIR target, HOXD10, and enrichment of cancer related pathways. However, a small subset had significantly higher HOTAIR expression, concomitant with high E7 expression and enrichment of metastatic pathways. Expression of HOTAIR and PRC2-complex members (EZH2 and SUZ12), showed significant positive correlation with E7 expression in CaCx cases and E7 transfected C33A cell line, suggestive of interplay between E7 and HOTAIR. Functional inactivation of HOTAIR by direct interaction with E7 could also be predicted by in silico analysis and confirmed by RNA-Immunoprecipitation. Our study depicts one of the causal mechanisms of cervical carcinogenesis by HPV16 E7, through modulation of HOTAIR expression and function.

Ligand-centered redox in nickel(II) complexes of 2-(arylazo)pyridine and isolation of 2-pyridyl-substituted triaryl hydrazines via catalytic N-arylation of azo-function.

A series of nickel complexes of 2-(arylazo)pyridine have been synthesized, and the precise structure and stoichiometry of the complexes are controlled by the use of different metal precursors. Molecular and electronic structures of the isolated complexes are scrutinized thoroughly by various spectroscopic techniques, single crystal X-ray crystallography, and density functional theory (DFT). Two different classes of Ni(II) complexes are identified where the ligands bind as neutral or anion radicals in the respective metal complexes. These are shown to be chemically interconvertible, and their characterization confirmed that the redox series is entirely ligand-centered without affecting the bivalent oxidation state of the metal ion. An efficient method of Ni(II) catalyzed N-arylation of 2-(arylazo)pyridine substrates has been elaborated. The chemical reactions have led to isolation of strongly fluorescent 2-pyridyl-substituted hydrazine derivatives, which have been characterized thoroughly. Three-dimensional X-ray structure of a hydrazine molecule, 2-(2-(naphthalen-1-yl)-2-phenylhydrazinyl)pyridine, is reported. Isolated hydrazines satisfy all the prerequisites of an ideal dye with moderate absorptive property, large Stokes shift, high quantum yields, and high photostability.