Unlike many interacting proteins NRG was the only

Unlike many interacting proteins, NRG1 was the only one observed to undergo cleavage by NS3/4A. NRG1 is one of four proteins in the neuregulin family that act on the ErbB member of the EGFR (EGF receptor) family. NRG1 has several isoforms produced by alternative splicing of pro-NRG1, which allows it to perform a wide variety of functions. Pro-NRG1 is cleaved by matrix metalloproteinases (MMPs) generating the mature NRG1 [56] with an activated EGF-like domain, which in turn increases the phosphorylation of ErbB receptor tyrosine kinase family. NRG1 is known as a central protein in neural proliferation and more recent studies have shown its involvement in HCV infection and HCC [53]. In addition, previous studies suggested that the NRG1 gene serves both as an oncogene as well as a tumor suppressor gene [57]. It was also proposed to play a role in epithelial cancer. Although NRG1 encoded proteins are usually considered as mitogens, they can also serve as powerful pro-apoptotic agents [58]. A recent study shows that HCV infection triggers down-regulation of ErbB3 expression. This affects downstream signaling of members of the ErbB receptor family, such as Akt, Ras and MKNK1 which play roles in the viral life naloxone hydrochloride mg [59]. They also show that HCV infection upregulates the mRNA levels of the ErbB3 ligands NRG1 and Epiregulin but not of NRG2. Our results show that it is the NS3/4A viral protein that is responsible for the 5-fold increase in NRG1 mRNA levels. Furthermore, this increase is independent of the NS3/4A protease activity, as it was not modulated by a NS3/4A protease inhibitor.
Author contributions
Acknowledgments The authors would like to thank Dr. Mushit Lindzen and Prof. Yossi Yarden for their valuable advice, and for providing the NRG1 antibody. The authors thank Dr. Shirit Einav for her expert advice on HCV. The authors also thank the Israel Science Foundation (ISF grant no. 715/11) and the European Research Council (ERC grant no. 3309600) for their support.
Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease. It is estimated that approximately four million people are infected with HCV in the United States while 177 million people are infected worldwide. HCV is an insidious disease as early symptoms are somewhat benign and disease progression is relatively slow. However, over time, infected individuals progress through various stages of cirrhosis which can lead to hepatocellular carcinoma., There is a significant mortality rate associated with HCV, as this disease is responsible for approximately 399,000 deaths per year., The genetic diversity of HCV is broad, consisting of seven genotypes (GTs) with GTs 1–4 contributing to the largest percentage of infections worldwide. The HCV genome comprises of a single strand of RNA that encodes a polyprotein that is ∼3000 amino acids in length. The polyprotein is divided into structural and non-structural (NS) proteins. The non-structural proteins NS3/4A, NS4B, NS5A, and NS5B are involved in the virus replication process. A complex of the NS3 protease and co-factor NS4A is responsible for cleaving the polyprotein at four sites to release functional non-structural proteins that are essential for replication of the HCV genome. Due to its critical role in virus replication, the NS3 protease has been an attractive target for drug discovery and to date efforts targeting this protein have led to six approved NS3 protease inhibitors that are used in combination therapies to treat HCV infection., We have previously reported on the discovery of BMS-605339 (, ) which demonstrated a robust antiviral effect following a single oral dose to HCV GT 1-infected patients. However, clinical studies with were terminated due to the observation of cardiac effects which were unanticipated based on pre-clinical toxicology studies., These drug-related effects included mild bradycardia, PR interval prolongation, and junctional escape rhythms. This cardiovascular observation in humans prompted the employment of the isolated heart model (Langendorff model) as a pre-clinical tool to more effectively assess potential cardiovascular (CV) liabilities. As previously described, perfusing isolated hearts with , at concentrations similar to those which precipitated CV events in humans, triggered a similar response in this model. The isolated heart model was integral to the program screening tier and enabled the discovery of asunaprevir () which demonstrates low nM antiviral activity against replicons representing HCV GT 1a (H77) and 1b (Con1) (). In clinical studies with , a robust antiviral response was observed in HCV GT 1-infected patients after a single dose. However, the pharmacokinetic-pharmacodynamic relationship observed in humans was not supportive of once-daily (QD) dosing but was instead suggestive of a twice-daily (BID) dosing profile. Follow-up efforts targeted the identification of a back-up compound with an antiviral profile similar to , but with the potential for QD dosing in humans. While this approach provided as a back-up molecule, a parallel workstream pursued a structurally distinct chemical series based on a P2-P4 macrocycle chemotype., This manuscript describes the results of that study and the properties of these P2-P4 macrocycles with respect to antiviral activity, pharmacokinetic properties and cardiac effects.