The recent emergence of the pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent for the coronavirus disease 2019 (COVID-19), is causing a global pandemic that poses enormous challenges to global public health and economies. SARS-CoV-2 host cell entry is mediated by the interaction of the viral transmembrane spike glycoprotein (S-protein) with the angiotensin-converting enzyme 2 gene (ACE2), an essential counter-regulatory carboxypeptidase of the renin-angiotensin hormone system that is a critical regulator of blood volume, systemic vascular resistance, and thus cardiovascular homeostasis. Accordingly, this work reports an atomistic-based, reliable in silico structural and energetic framework of the interactions between the receptor-binding domain of the SARS-CoV-2 S-protein and its host cellular receptor ACE2 that provides qualitative and quantitative insights into the main molecular determinants in virus/receptor recognition. In particular, residues D38, K31, E37, K353, and Y41 on ACE2 and Q498, T500, and R403 on the SARS-CoV-2 S-protein receptor-binding domain are determined as true hot spots, contributing to shaping and determining the stability of the relevant protein–protein interface. Overall, these results could be used to estimate the binding affinity of the viral protein to different allelic variants of ACE2 receptors discovered in COVID-19 patients and for the effective structure-based design and development of neutralizing antibodies, vaccines, and protein/protein inhibitors against this terrible new coronavirus.

Small interfering RNA (siRNA) is emerging as a novel therapeutic for treating various diseases, provided a safe and efficient delivery is available. In particular, specific delivery to target cells is critical for achieving high therapeutic efficacy while reducing toxicity. Amphiphilic dendrimers are emerging as novel promising carriers for siRNA delivery by virtue of the combined multivalent cooperativity of dendrimers with the self-assembling property of lipid vectors. Here, we report a ballistic approach for targeted siRNA delivery to cancer cells using an amphiphilic dendrimer equipped with a dual targeting peptide bearing an RGDK warhead. According to the molecular design, the amphiphilic dendrimer was expected to deliver siRNA effectively, while the aim of the targeting peptide was to home in on tumors via interaction of its warhead with integrin and the neuropilin-1 receptor on cancer cells. Coating the positively charged siRNA/dendrimer delivery complex with the negatively charged segment of the targeting peptide via electrostatic interactions led to small and stable nanoparticles which were able to protect siRNA from degradation while maintaining the accessibility of RGDK for targeting cancer cells and preserving the ability of the siRNA to escape from endosomes. The targeted system had enhanced siRNA delivery, stronger gene silencing, and more potent anticancer activity compared to nontargeted or covalent dendrimer-based systems. In addition, neither acute toxicity nor induced inflammation was observed. Consequently, this delivery system constitutes a promising nonviral vector for targeted delivery and can be further developed to provide RNAi-based personalized medicine against cancer. Our study also gives new perspectives on the use of nanotechnology based on self-assembling dendrimers in various biomedical applications.

Self-assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self-assembly remains challenging. In this work, through a combination of experimental and computational approaches, we report on the self-assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high-generation dendrimers. We demonstrate that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self-assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA (siRNA) and achieve effective gene silencing both in cells - including the highly refractory human hematopoietic CD34+ stem cells - and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self-assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self-assembling nanosystems for complex and functional applications.

Basal cell carcinomas (BCCs) and a subset of medulloblastomas are characterized by loss-of-function mutations in the tumor suppressor gene, PTCH1. PTCH1 normally functions by repressing the activity of the Smoothened (SMO) receptor. Inactivating PTCH1 mutations result in constitutive Hedgehog pathway activity through uncontrolled SMO signaling. Targeting this pathway with vismodegib, a novel SMO inhibitor, results in impressive tumor regression in patients harboring genetic defects in this pathway. However, a secondary mutation in SMO has been reported in medulloblastoma patients following relapse on vismodegib to date. This mutation preserves pathway activity, but appears to confer resistance by interfering with drug binding. Here we report for the first time on the molecular mechanisms of resistance to vismodegib in two BCC cases. The first case, showing progression after 2 months of continuous vismodegib (primary resistance), exhibited the new SMO G497W mutation. The second case, showing a complete clinical response after 5 months of treatment and a subsequent progression after 11 months on vismodegib (secondary resistance), exhibited a PTCH1 nonsense mutation in both the pre- and the post-treatment specimens, and the SMO D473Y mutation in the post-treatment specimens only. In silico analysis demonstrated that SMO(G497W) undergoes a conformational rearrangement resulting in a partial obstruction of the protein drug entry site, whereas the SMO D473Y mutation induces a direct effect on the binding site geometry leading to a total disruption of a stabilizing hydrogen bond network. Thus, the G497W and D473Y SMO mutations may represent two different mechanisms leading to primary and secondary resistance to vismodegib, respectively.

In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using highaffinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.S ignal transducer and activator of transcription 3 (STAT3) is a key element in multiple signaling pathways and is aberrantly activated in many human cancers (1, 2). STAT3 promotes cell proliferation, survival, angiogenesis, and immune-evasion (1-3). Phosphorylation at Tyr705 (pTyr705), catalyzed by Janus kinases (JAK) and other tyrosine kinases, induces STAT3 dimerization through the interaction of the SH2 domain (SH2D), nuclear accumulation, and target gene transcription (1, 3, 4). Emerging evidence indicates that STAT3 also localizes to mitochondria and controls mitochondrial functions (2, 5-7). Mitochondrial localized STAT3 (mSTAT3) is critical for survival of RAStransformed mouse embryo fibroblasts (MEF) under glucosestarvation, reflecting a specific dependency of cancer cells on mitochondria in certain conditions (6). Interestingly, mSTAT3 is prevalently phosphorylated at Ser727 (pSer727), which enhances its mitochondrial functions (5, 6). Furthermore, constitutive pSer727 is found in many human cancers and is apparently sufficient to drive tumorigenesis in various model systems (8-10).STAT3 is an attractive cancer therapeutic target because of its central role in multiple oncogenic processes and great effort has been devoted in recent years to discover STAT3 inhibitors (STAT3i) (11,12). To date, small-molecule STAT3i have shown relevant activity in preclinical models and few of them are currently investigated in clinical trials (11,(13)(14)(15)(16)(17). However, an important gap persists in our knowledge of the biological mechanisms of antitumor activity, the critical cellular processes affected, and the factors determining sensitivity of cancer cells to STAT3i, hindering further clinical development of these highly promising anticancer drugs. Indeed, great atten...

This study presents for the first time the 3D model of the σ 1 receptor protein as obtained from homology modeling techniques, shows the applicability of this structure to docking-based virtual screening, defines a computational strategy to optimize the results based on a combination of 3D pharmacophore-based docking and MM/PBSA free energy of binding scoring, and provides evidence that these in silico models and recipes are powerful tools on which virtual screening of new σ 1 ligands can be based. In particular, the validation of the applicability of docking-based virtual screening to homology models is of utmost importance, since no crystal structure is available to date for the σ 1 receptor, and this missing information still constitutes a major hurdle for a rational ligand design for this important protein target.

Successful achievement of RNA interference in therapeutic applications requires safe and efficient vectors for siRNA delivery. In the present study, we demonstrate that a triethanolamine (TEA)-core PAMAM dendrimer of generation 5 (G(5)) is able to deliver sticky siRNAs bearing complementary A(n)/T(n) 3'-overhangs effectively to a prostate cancer model in vitro and in vivo and produce potent gene silencing of the heat shock protein 27, leading to a notable anticancer effect. The complementary A(n)/T(n) (n = 5 or 7) overhangs characteristic of these sticky siRNA molecules help the siRNA molecules self-assemble into "gene-like" longer double-stranded RNAs thus endowing a low generation dendrimer such as G(5) with greater delivery capacity. In addition, the A(n)/T(n) (n = 5 or 7) overhangs act as protruding molecular arms that allow the siRNA molecule to enwrap the dendrimer and promote a better interaction and stronger binding, ultimately contributing toward the improved delivery activity of G(5). Consequently, the low generation dendrimer G(5) in combination with sticky siRNA therapeutics may constitute a promising gene silencing-based approach for combating castration-resistant prostate tumors or other cancers and diseases, for which no effective treatment currently exists.

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