Smitha Kota

Notch1 Is Required for Kras-Induced Lung Adenocarcinoma and Controls Tumor Cell Survival via p53

The Notch pathway has been implicated in a number of malignancies with different roles that are cell- and tissue-type dependent. Notch1 is a putative oncogene in non-small cell lung cancer (NSCLC) and activation of the pathway represents a negative prognostic factor. To establish the role of Notch1 in lung adenocarcinoma, we directly assessed its requirement in Kras-induced tumorigenesis in vivo using an autochthonous model of lung adenocarcinoma with concomitant expression of oncogenic Kras and deletion of Notch1. We found that Notch1 function is required for tumor initiation via suppression of p53-mediated apoptosis through the regulation of p53 stability. These findings implicate Notch1 as a critical effector in Kras-driven lung adenocarcinoma and as a regulator of p53 at a posttranslational level. Moreover, our study provides new insights to explain, at a molecular level, the correlation between Notch1 activity and poor prognosis in patients with NSCLC carrying wild-type p53. This information is critical for design and implementation of new therapeutic strategies in this cohort of patients representing 50% of NSCLC cases.

Dimerization-driven interaction of hepatitis C virus core protein with NS3 helicase

Hepatitis C virus (HCV) infects over 130 million people causing a worldwide epidemic of liver cirrhosis and hepatocellular-carcinoma. Because current HCV treatments are only partially effective, molecular mechanisms involved in HCV propagation are actively being pursued as possible drug targets. Here, we report on a new macromolecular interaction between the HCV capsid core protein and the helicase portion of HCV non-structural protein 3 (NS3h), confirmed by four different biochemical methods. The protease portion of NS3 is not required. Interaction between the two proteins could be disrupted by two types of specific inhibitors of core dimerization, the small molecule SL201 and core106, a C-terminally truncated core protein. Cross-linking experiments suggest that the physical interaction with NS3h is probably driven by core oligomerization. Moreover, SL201 blocks the production of infectious virus, but not the production of a subgenomic HCV replicon by hepatoma cells. Time-of-addition experiments confirm that SL201 has no effect on entry of the virus. These data underline the essential role of core as a key organizer of HCV particle assembly, confirm the importance of oligomerization, reveal the interaction with viral helicase and support a new molecular understanding of the formation of the viral particle at the level of the lipid droplets, before its migration to the site of release and budding.

Small molecule inhibitors of hepatitis c virus

New small molecule inhibitors of HCV were discovered by screening a small library of indoline alkaloid-type compounds. An automated assay format was employed which allowed identification of dimerization inhibitors of core, the capsid protein of the virus. These compounds were subsequently shown to block production of infectious virus in hepatoma cells.

A Time-Resolved Fluorescence–Resonance Energy Transfer Assay for Identifying Inhibitors of Hepatitis C Virus Core Dimerization

Binding of hepatitis C virus (HCV) RNA to core, the capsid protein, results in the formation of the nucleocapsid, the first step in the assembly of the viral particle. A novel assay was developed to discover small molecule inhibitors of core dimerization. This assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) between anti-tag antibodies labeled with either europium cryptate (Eu) or allophycocyanin (XL-665). The N-terminal 106-residue portion of core protein (core106) was tagged with either glutathione-S-transferase (GST) or a Flag peptide. Tag-free core106 was selected as the reference inhibitor. The assay was used to screen the library of pharmacologically active compounds (LOPAC) consisting of 1,280 compounds and a 2,240-compound library from the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU). Ten of the 28 hits from the primary TR-FRET run were confirmed in a secondary amplified luminescent proximity homogeneous assay (ALPHA screen). One hit was further characterized by dose-response analysis yielding an IC(50) of 9.3 microM. This 513 Da compound was shown to inhibit HCV production in cultured hepatoma cells.

Peptide inhibitors of hepatitis C virus core oligomerization and virus production.

Hepatitis C virus (HCV) nucleocapsid assembly requires dimerization of the core protein, an essential step in the formation of the virus particle. We developed a novel quantitative assay for monitoring this protein-protein interaction, with the goal of identifying inhibitors of core dimerization that might block HCV production in infected Huh-7.5 hepatoma cells. Two core-derived, 18-residue peptides were found that inhibited the dimerization of a fragment of core comprising residues 1-106 (core106) by 68 and 63%, respectively. A third, related 15-residue peptide displayed 50% inhibition, with an IC50 of 21.9 microM. This peptide was shown, by fluorescence polarization, to bind directly to core106 with a Kd of 1.9 microM and was displaced by the unlabelled peptide with an IC50 of 18.7 microM. When measured by surface plasmon resonance, the same peptide bound core169 with a Kd of 7.2 microM. When added to HCV-infected cells, each of the three peptides blocked release, but not replication, of infectious virus. When measured by real-time RT-PCR, the RNA levels were reduced by 7-fold. The 15-residue peptide had no effect on HIV propagation. Such inhibitors may constitute useful tools to investigate the role of core dimerization in the virus cycle.

Core as a Novel Viral Target for Hepatitis C Drugs

Hepatitis C virus (HCV) infects over 130 million people worldwide and is a major cause of liver disease. No vaccine is available. Novel specific drugs for HCV are urgently required, since the standard-of-care treatment of pegylated interferon combined with ribavirin is poorly tolerated and cures less than half of the treated patients. Promising, effective direct-acting drugs currently in the clinic have been described for three of the ten potential HCV target proteins: NS3/NS4A protease, NS5B polymerase and NS5A, a regulatory phosphoprotein. We here present core, the viral capsid protein, as another attractive, non-enzymatic target, against which a new class of anti-HCV drugs can be raised. Core plays a major role in the virion’s formation, and interacts with several cellular proteins, some of which are involved in host defense mechanisms against the virus. This most conserved of all HCV proteins requires oligomerization to function as the organizer of viral particle assembly. Using core dimerization as the basis of transfer-of-energy screening assays, peptides and small molecules were identified which not only inhibit core-core interaction, but also block viral production in cell culture. Initial chemical optimization resulted in compounds active in single digit micromolar concentrations. Core inhibitors could be used in combination with other HCV drugs in order to provide novel treatments of Hepatitis C.

Direct binding of a hepatitis C virus inhibitor to the viral capsid protein.

Over 130 million people are infected chronically with hepatitis C virus (HCV), which, together with HBV, is the leading cause of liver disease. Novel small molecule inhibitors of Hepatitis C virus (HCV) are needed to complement or replace current treatments based on pegylated interferon and ribavirin, which are only partially successful and plagued with side-effects. Assembly of the virion is initiated by the oligomerization of core, the capsid protein, followed by the interaction with NS5A and other HCV proteins. By screening for inhibitors of core dimerization, we previously discovered peptides and drug-like compounds that disrupt interactions between core and other HCV proteins, NS3 and NS5A, and block HCV production. Here we report that a biotinylated derivative of SL209, a prototype small molecule inhibitor of core dimerization (IC50 of 2.80 µM) that inhibits HCV production with an EC50 of 3.20 µM, is capable of penetrating HCV-infected cells and tracking with core. Interaction between the inhibitors, core and other viral proteins was demonstrated by SL209–mediated affinity-isolation of HCV proteins from lysates of infected cells, or of the corresponding recombinant HCV proteins. SL209-like inhibitors of HCV core may form the basis of novel treatments of Hepatitis C in combination with other target-specific HCV drugs such as inhibitors of the NS3 protease, the NS5B polymerase, or the NS5A regulatory protein. More generally, our work supports the hypothesis that inhibitors of viral capsid formation might constitute a new class of potent antiviral agents, as was recently also shown for HIV capsid inhibitors.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280 in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.

Potent inhibitors of hepatitis C core dimerization as new leads for anti-hepatitis C agents

New indoline alkaloid-type compounds which inhibit HCV production by infected hepatoma cells have been identified. These compounds, dimeric-type compounds of previously known inhibitors, display double digit nanomolar IC(50) and EC(50) values, with cytotoxicity CC(50) indexes higher than 36 μM, thus providing ample therapeutic windows for further development of HCV drugs.

YAP Mediates Tumorigenesis in Neurofibromatosis Type 2 by Promoting Cell Survival and Proliferation through a COX-2–EGFR Signaling Axis

The Hippo-YAP pathway has emerged as a major driver of tumorigenesis in many human cancers. YAP is a transcriptional coactivator and while details of YAP regulation are quickly emerging, it remains unknown what downstream targets are critical for the oncogenic functions of YAP. To determine the mechanisms involved and to identify disease-relevant targets, we examined the role of YAP in neurofibromatosis type 2 (NF2) using cell and animal models. We found that YAP function is required for NF2-null Schwann cell survival, proliferation, and tumor growth in vivo Moreover, YAP promotes transcription of several targets including PTGS2, which codes for COX-2, a key enzyme in prostaglandin biosynthesis, and AREG, which codes for the EGFR ligand, amphiregulin. Both AREG and prostaglandin E2 converge to activate signaling through EGFR. Importantly, treatment with the COX-2 inhibitor celecoxib significantly inhibited the growth of NF2-null Schwann cells and tumor growth in a mouse model of NF2. Cancer Res; 76(12); 3507-19. ©2016 AACR.