Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro.

Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with zinc demonstrate better clinical outcomes. The molecular targets and mechanistic details of the anti-coronaviral activity of zinc remain obscure. We show that zinc not only inhibits the SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of the Mpro-Zn2+ complex at 1.9 Å and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, the natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.

Extracellular vesicle-dependent effect of RNA-binding protein IGF2BP1 on melanoma metastasis.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is a multifunctional RNA-binding protein with an oncofetal pattern of expression shown to be implicated in the development of a variety of malignancies. In this study, we explored the role and mechanisms of IGF2BP1 in melanoma development and progression. In two different in vivo models, we showed that although genetic deletion or shRNA-mediated suppression of IGF2BP1 did not affect primary tumor formation, it drastically suppressed lung metastasis. Here we demonstrated that extracellular vesicles (EVs) secreted by melanoma cells mediate the effects of IGF2BP1 on metastasis: EVs from the IGF2BP1 knockdown melanoma cells failed to promote metastasis, whereas EVs isolated from IGF2BP1-overexpressed melanoma cells further accelerated EV-induced metastasis. Moreover, the EVs from IGF2BP1 knockdown melanoma cells inhibited fibronectin deposition and accumulation of CD45+ cells in the lungs compared with control EVs, thus blocking the pre-metastatic niche formation potential of EVs. IGF2BP1 knockdown did not affect size, number, or protein/RNA concentration of secreted EVs or their uptake by recipient cells in vitro or in vivo. However, RNA-sequencing and proteomics analysis of the EVs revealed differential expression in a number of mRNA, proteins, and miRNAs. This suggested that IGF2BP1 is intimately involved in the regulation of the cargo of EVs, thereby affecting the pro-metastatic function of melanoma-derived EVs. To the best of our knowledge, this is the first study that demonstrates the role of RNA-binding protein IGF2BP1 in EV-mediated promotion of melanoma metastasis and may provide novel avenues for the development of metastatic inhibitors.

dGTP-Templated Luminescent Gold Nanocluster-Based Composite Nanoparticles for Cancer Theranostics.

dGTP-templated facile synthesis of luminescent gold nanoclusters (Au NCs) in the presence of cisplatin following PEG coating developed spherical composite NPs. The composite NPs delivered cisplatin efficiently into HeLa cells to induce apoptosis-mediated cell death, and simultaneously bioimaged the cellular uptake.

Connexin-43 enhances tumor suppressing activity of artesunate via gap junction-dependent as well as independent pathways in human breast cancer cells.

The gap junction (GJ) protein connexin-43 (Cx43) is considered as a tumour suppressor protein for its role in reversing the phenotype of the cancer cells. In this study, we exploited the antitumor property of Cx43 in conjunction with the artesunate (ART), a plant-based active anti-malarial compound. The reactive oxygen species (ROS) generated by ART resulted in DNA damage, which in turn led to DNA damage response by activation of DNA damage repair proteins. GJ deficient MCF-7 cells transfected with Cx43 gene showed an increased sensitivity towards dose-dependent ART treatment and required a significantly lower dose of ART to attain its IC50, as compared to parental cells. This would ultimately result in reduced dose-dependent side effects of ART. The Co-culture experiments involving GJ intercellular communication (GJIC) deficient and GJIC enabled cells, established the transfer of ROS to the neighbouring cancer cells not exposed to ART. The ROS accumulated in the ART-treated cells induced the oxidative damage in neighbouring cells, leading to bystander cell death and inhibition of bystander cell proliferation. Thus, our study revealed that expression of Cx43 helped in reducing the dose-dependent cytotoxicity of ART as well as enhanced the bystander apoptosis of the neighbouring cells.

Antagonizing canonical Wnt signaling pathway by recombinant human sFRP4 purified from E. coli and its implications in cancer therapy.

The Wnt signaling pathway plays a predominant role in aberrant proliferation in myriad of cancers. In non-cancerous cells, Wnts are blocked by the secreted frizzled-related proteins (sFRPs) that are generally downregulated in cancer cells. We have purified and characterized bacterially expressed glutathione S-transferase-tagged SFRP4 from a novel clone generated from human cell origin. Cervical cancer (HeLa) and lung cancer (A549) cells, in which Wnt and associated genes were found to be expressed, were treated with the purified recombinant sFRP4, which revealed a significant dose-dependent cell growth inhibition up to 40 %. The current investigation on functionality of this bacterially produced recombinant sFRP4 in arresting cancer cell proliferation is the first of its kind, where G2/M phase arrest and early apoptosis were evident. Increase in phosphorylated ß-catenin in sFRP4 treatment indicated inhibition of Wnt pathway, which was further confirmed by downregulation of pro-proliferative genes, namely cyclin D1, c-myc, and survivin. Functional activity of recombinant sFRP4 was further exploited in co-therapy module with chemotherapeutic drugs to decipher molecular events. Collectively, our study on purified recombinant sFRP4 from bacterial host holds great promise in targeting Wnt signaling for exploring new strategies to combat cancer.

Targeting Wnt Canonical Signaling by Recombinant sFRP1 Bound Luminescent Au-Nanocluster Embedded Nanoparticles in Cancer Theranostics.

Secreted frizzled-related protein 1 (SFRP1) is a natural blocker of the Wnt signaling pathway in normal adult cells but is epigenetically silenced in cancer cells leading to aberrant proliferation. In this study, we have reported novel composite nanoparticles fabricated with gold nanocluster embedded chitosan and alginate, bound to bacterially expressed human recombinant sFRP1. The Wnt pathway, which is upregulated in cancer, has been specifically targeted with the nanoparticles to achieve an antiproliferative effect on cancer cells, as evident from reduced levels of downstream molecules, namely, ß-catenin, cyclin D1, and survivin. The nanoparticles enabled sustained release of sFRP1 outside the cells, where it is functional. Moreover, remarkable luminescence properties of gold nanoclusters were exploited for binding, imaging, and tracking studies. Co-therapy of sFRP1-loaded nanoparticles with the drug cisplatin targeted two independent pathways to induce apoptosis, as documented by flow cytometry based assays. Overall, this nanosystem is promising for tracking, imaging, and targeting cancer signaling with therapeutic protein.

Expression, purification, and therapeutic implications of recombinant sFRP1.

Secreted frizzled-related proteins (sFRPs) constitute a family of proteins, which impede the Wnt signaling pathway. Upregulation of the Wnt cascade is one of the multiple facets of carcinogenesis. Herein, we report the expression, solubilization, purification, characterization, and anti-cell proliferative activity of a novel recombinant GST-tagged sFRP1 of human origin. sFRP1 was cloned into pGEX-4T2 bacterial expression vector, and the recombinant protein was overexpressed in Escherichia coli BL21 (DE3). It was solubilized from inclusion bodies with N-lauroylsarcosine and Triton X-100, before being purified to homogeneity using glutathione agarose affinity chromatography column. The purified protein was characterized using Western blotting, MALDI TOF-TOF, and circular dichroism spectroscopy analysis. Homology modeling and docking studies revealed that tagging GST with sFRP1 does not change the binding conformation of the cysteine-rich domain and hence, possibly does not alter its function. The novel anti-proliferative activity of GST-sFRP1 was demonstrated in a dose-dependent manner on two cancer cell lines, viz., HeLa (cervical cancer) and MCF-7 (breast cancer). Also, combination therapy of the protein with chemotherapeutic drugs resulted in enhanced anti-cancer activity. This opens up a new avenue in the application of recombinant sFRP1 for cancer therapeutics.