Targeted Disruption of E6/p53 Binding Exerts Broad Activity and Synergism with Paclitaxel and Topotecan against HPV-Transformed Cancer Cells

High-risk human papillomaviruses (HR-HPV) are the etiological agents of almost all cervical cancer cases and a high percentage of head-and-neck malignancies. Although HPV vaccination can reduce cancer incidence, its coverage significantly differs among countries, and, therefore, in the next decades HPV-related tumors will not likely be eradicated worldwide. Thus, the need of specific treatments persists, since no anti-HPV drug is yet available. We recently discovered a small molecule (Cpd12) able to inhibit the E6-mediated degradation of p53 through the disruption of E6/p53 binding in HPV16- and HPV18-positive cervical cancer cells. By employing several biochemical and cellular assays, here we show that Cpd12 is also active against cervical cancer cells transformed by other HR-HPV strains, such as HPV68 and HPV45, and against a HPV16-transformed head-and-neck cancer cell line, suggesting the possibility to employ Cpd12 as a targeted drug against a broad range of HPV-induced cancers. In these cancer cell lines, the antitumoral mechanism of action of Cpd12 involves p53-dependent cell cycle arrest, a senescent response, and inhibition of cancer cell migration. Finally, we show that Cpd12 can strongly synergize with taxanes and topoisomerase inhibitors, encouraging the evaluation of Cpd12 in preclinical studies for the targeted treatment of HPV-related carcinomas

Potent and broad-spectrum cycloheptathiophene-3-carboxamide compounds that target the PA-PB1 interaction of influenza virus RNA polymerase and possess a high barrier to drug resistance

Influenza viruses are major respiratory pathogens responsible for both seasonal epidemics and occasional pandemics worldwide. The current available treatment options have limited efficacy and thus the development of new antivirals is highly needed. We previously reported the identification of a series of cycloheptathiophene-3-carboxamide compounds as influenza A virus inhibitors that act by targeting the protein-protein interactions between the PA-PB1 subunits of the viral polymerase. In this study, we characterized the antiviral properties of the most promising compounds as well as investigated their propensity to induce drug resistance. Our results show that some of the selected compounds possess potent, broad-spectrum anti-influenza activity as they efficiently inhibited the replication of several strains of influenza A and B viruses, including an oseltamivir-resistant clinical isolate, with nanomolar or low-micromolar potency. The most promising compounds specifically inhibited the PA−PB1 binding in vitro and interfered with the influenza A virus polymerase activity in a cellular context, without showing cytotoxicity. The most active PA-PB1 inhibitors showed to possess a drug resistance barrier higher than that of oseltamivir. Indeed, no viral variants with reduced susceptibility to the selected compounds emerged after serial passages of influenza A virus under drug selective pressure. Overall, our studies identified potent PA-PB1 inhibitors as promising candidates for the development of new anti-influenza drugs

The Dimeric Form of HPV16 E6 Is Crucial to Drive YAP/TAZ Upregulation through the Targeting of hScrib

Human papillomavirus is the most common viral infectious agent responsible for cancer development in humans. High-risk strains are known to induce cancer through the expression of the viral oncogenes E6 and E7, yet we have only a partial understanding of the precise mechanisms of action of these viral proteins. Here we investigated the molecular mechanism through which the oncoprotein E6 alters the Hippo-YAP/TAZ pathway to trigger YAP/TAZ induction in cancer cells. By employing E6 overexpression systems combined with protein–protein interaction studies and loss-of-function approaches, we discovered that the E6-mediated targeting of hScrib, which supports YAP/TAZ upregulation, intimately requires E6 homodimerization. We show that the self-association of E6, previously reported only in vitro, takes place in the cytoplasm and, as a dimer, E6 targets the fraction of hScrib at the cell cortex for proteasomal degradation. Thus, E6 homodimerization emerges as an important event in the mechanism of E6-mediated hScrib targeting to sustain downstream YAP/TAZ upregulation, unraveling for the first time the key role of E6 homodimerization in the context of its transforming functions and thus paving the way for the possible development of E6 dimerization inhibitors

A novel small-molecule inhibitor of the human papillomavirus E6-p53 interaction that reactivates p53 function and blocks cancer cells growth

Despite prophylactic vaccination campaigns, human papillomavirus (HPV)-induced cancers still represent a major medical issue for global population, thus specific anti-HPV drugs are needed. Since the ability of HPV E6 oncoprotein to promote p53 degradation is linked to tumor progression, E6 has been proposed as an ideal target for cancer treatment. Using the crystal structure of the E6/E6AP/p53 complex, we performed an in silico screening of small-molecule libraries against a highly conserved alpha-helix in the N-terminal domain of E6 involved in the E6-p53 interaction. We discovered a compound able to inhibit the E6-mediated degradation of p53 through disruption of E6-p53 binding both in vitro and in cells. This compound could restore p53 intracellular levels and transcriptional activity, reduce the viability and proliferation of HPV-positive cancer cells, and block 3D cervospheres formation. Mechanistic studies revealed that the compound anti-tumor activity mainly relies on induction of cell cycle arrest and senescence. Our data demonstrate that the disruption of the direct E6-p53 interaction can be obtained with a small-molecule compound leading to specific antitumoral activity in HPV-positive cancer cells and thus represents a new approach for anti-HPV drug development.Fil: Celegato, Marta. Università di Padova; ItaliaFil: Messa, Lorenzo. Università di Padova; ItaliaFil: Goracci, Laura. Università di Perugia; ItaliaFil: Mercorelli, Beatrice. Università di Padova; ItaliaFil: Bertagnin, Chiara. Università di Padova; ItaliaFil: Spyrakis, Francesca. Università di Torino; ItaliaFil: Suarez, Irina Paula. Centre National de la Recherche Scientifique. Igbmc; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cousido Siah, Alexandra. Centre National de la Recherche Scientifique. Igbmc; FranciaFil: Travé, Gilles. Centre National de la Recherche Scientifique. Igbmc; FranciaFil: Banks, Lawrence. International Centre for Genetic Engineering and Biotechnology; ItaliaFil: Cruciani, Gabriele. Università di Perugia; ItaliaFil: Palù, Giorgio. Università di Padova; ItaliaFil: Loregian, Arianna. Università di Padova; Itali

Synthesis and characterization of 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide-based compounds targeting the PA-PB1 interface of influenza A virus polymerase

Influenza viruses (Flu) are responsible for seasonal epidemics causing high rates of morbidity, which can dramatically increase during severe pandemic outbreaks. Antiviral drugs are an indispensable weapon to treat infected people and reduce the impact on human health, nevertheless anti-Flu armamentarium still remains inadequate. In search for new anti-Flu drugs, our group has focused on viral RNA-dependent RNA polymerase (RdRP) developing disruptors of PA-PB1 subunits interface with the best compounds characterized by cycloheptathiophene-3-carboxamide and 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide scaffolds. By merging these moieties, two very interesting hybrid compounds were recently identified, starting from which, in this paper, a series of analogues were designed and synthesized. In particular, a thorough exploration of the cycloheptathiophene-3-carboxamide moiety led to acquire important SAR insight and identify new active compounds showing both the ability to inhibit PA-PB1 interaction and viral replication in the micromolar range and at non-toxic concentrations. For few compounds, the ability to efficiently inhibit PA-PB1 subunits interaction did not translate into anti-Flu activity. Chemical/physical properties were investigated for a couple of compounds suggesting that the low solubility of compound 14, due to a strong crystal lattice, may have impaired its antiviral activity. Finally, computational studies performed on compound 23, in which the phenyl ring suitably replaced the cycloheptathiophene, suggested that, in addition to hydrophobic interactions, H-bonds enhanced its binding within the PAC cavity

A novel N-heterocycles substituted oseltamivir derivatives as potent inhibitors of influenza virus neuraminidase: discovery, synthesis and biological evaluation

AbstractOur previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop

Discovery of Novel Boron-Containing N-Substituted Oseltamivir Derivatives as Anti-Influenza A Virus Agents for Overcoming N1-H274Y Oseltamivir-Resistant

To address drug resistance to influenza virus neuraminidase inhibitors (NAIs), a series of novel boron-containing N-substituted oseltamivir derivatives were designed and synthesized to target the 150-cavity of neuraminidase (NA). In NA inhibitory assays, it was found that most of the new compounds exhibited moderate inhibitory potency against the wild-type NAs. Among them, compound 2c bearing 4-(3-boronic acid benzyloxy)benzyl group displayed weaker or slightly improved activities against group-1 NAs (H1N1, H5N1, H5N8 and H5N1-H274Y) compared to that of oseltamivir carboxylate (OSC). Encouragingly, 2c showed 4.6 times greater activity than OSC toward H5N1-H274Y NA. Moreover, 2c exerted equivalent or more potent antiviral activities than OSC against H1N1, H5N1 and H5N8. Additionally, 2c demonstrated low cytotoxicity in vitro and no acute toxicity at the dose of 1000 mg/kg in mice. Molecular docking of 2c was employed to provide a possible explanation for the improved anti-H274Y NA activity, which may be due to the formation of key additional hydrogen bonds with surrounding amino acid residues, such as Arg152, Gln136 and Val149. Taken together, 2c appeared to be a promising lead compound for further optimization

Iterative Optimization and Structure-Activity Relationship Studies of Oseltamivir Amino Derivatives as Potent and Selective Neuraminidase Inhibitors via Targeting 150-Cavity

With our continuous endeavors in seeking neuraminidase (NA) inhibitors, we reported herein three series of novel oseltamivir amino derivatives with the goal of exploring the druggable chemical space inside the 150-cavity of influenza virus NAs. Among them, around half of the compounds in series C were demonstrated to be better inhibitors against both wild-type and oseltamivir-resistant group-1 NAs than oseltamivir carboxylate (OSC). Notably, compounds 12d, 12e, 15e, and 15i showed more potent or equipotent antiviral activity against H1N1, H5N1, and H5N8 viruses compared to OSC in cellular assays. Furthermore, compounds 12e and 15e exhibited high metabolic stability in human liver microsomes (HLMs) and low inhibitory effect on main cytochrome P450 (CYP) enzymes, as well as low acute/subacute toxicity and certain antiviral efficacy in vivo. Also, pharmacokinetic (PK) and molecular docking studies were performed. Overall, 12e and 15e possess great potential to serve as anti-influenza candidates and are worthy of further investigation

Structure-Based Optimization of N-Substituted Oseltamivir Derivatives as Potent Anti-Influenza A Virus Agents with Significantly Improved Potency against Oseltamivir-Resistant N1-H274Y Variant

Due to the emergence of highly pathogenic and oseltamivir-resistant influenza viruses, there is an urgent need to develop new anti-influenza agents. Herein, five subseries of oseltamivir derivatives were designed and synthesized to improve their activity toward drug-resistant viral strains by further exploiting the 150-cavity in the neuraminidases (NAs). The bioassay results showed that compound 21h exhibited antiviral activities similar to or better than those of oseltamivir carboxylate (OSC) against H5N1, H5N2, H5N6, and H5N8. Besides, 21h was 5- to 86-fold more potent than OSC toward N1, N8, and N1-H274Y mutant NAs in the inhibitory assays. Computational studies provided a plausible rationale for the high potency of 21h against group-1 and N1-H274Y NAs. In addition, 21h demonstrated acceptable oral bioavailability, low acute toxicity, potent antiviral activity in vivo, and high metabolic stability. Overall, the above excellent profiles make 21h a promising drug candidate for the treatment of influenza virus infection

Optimization of N-Substituted Oseltamivir Derivatives as Potent Inhibitors of Group-1 and -2 Influenza A Neuraminidases, Including a Drug-Resistant Variant

none25simixedZhang, Jian; Poongavanam, Vasanthanathan; Kang, Dongwei; Bertagnin, Chiara; Lu, Huamei; Kong, Xiujie; Ju, Han; Lu, Xueyi; Gao, Ping; Tian, Ye; Jia, Haiyong; Desta, Samuel; Ding, Xiao; Sun, Lin; Fang, Zengjun; Huang, Boshi*; Liang, Xuewu; Jia, Ruifang; Ma, Xiuli; Xu, Wenfang; Murugan, Natarajan Arul; Loregian, Arianna; Huang, Bing; Zhan, Peng; Liu, XinyongZhang, Jian; Poongavanam, Vasanthanathan; Kang, Dongwei; Bertagnin, Chiara; Lu, Huamei; Kong, Xiujie; Ju, Han; Lu, Xueyi; Gao, Ping; Tian, Ye; Jia, Haiyong; Desta, Samuel; Ding, Xiao; Sun, Lin; Fang, Zengjun; Huang, Boshi; Liang, Xuewu; Jia, Ruifang; Ma, Xiuli; Xu, Wenfang; Murugan, Natarajan Arul; Loregian, Arianna; Huang, Bing; Zhan, Peng; Liu, Xinyon