Natural lead structures targeting influenza

The respiratory influenza viruses A and B remain a significant global health challenge by causing annual seasonal flu, occasional epidemics and pandemics. These threats in combination with the propensity of influenza viruses to develop resistance to commonly used drugs require the continued development of new antiviral agents. This project proposal aims at the identification of novel anti-influenza agents from natural sources targeting two influenza virus proteins, namely the neuraminidase (NA) and the nucleocapsid protein (NP). These two targets are highly conserved and cover various stages of viral life cycle. Novel agents against NA and NP shall help to overcome resistance and to develop multidrug strategies. Inhibition of the influenza virus NA impedes the release of newly formed virions from infected cells and causes viral aggregation, thus halting the spread of infection. The high amount of data and information of this established drug target and its ligands will be used as starting point to apply structure-based computational methods to cope with the flexibility of the NA binding site on the one hand, and to scrutinize its ligand-target interactions for the discovery of ligands that overcome the influence of mutational changes conferring drug resistance. In contrast to NA, the influenza NP only recently emerged as druggable target for potential small molecule therapies. Ligands of this abundantly expressed protein are found to impede influenza A virus replication by triggering the aggregation of NP and inhibition of its nuclear accumulation. Based on previously published compounds interacting on the NP, a ligand-based approach will be the rationale for the target-oriented identification of putative NP-ligands from the rich pool of secondary metabolites. The selection of natural starting materials for phytochemical investigations will depend on (i) virtually predicted hits form ligand- and structure- based approaches in cooperation with Ao.Univ. Prof. Dr. Klaus R. Liedl (Depart. of Theoretical Chemistry, University of Innsbruck), (ii) previously determined anti-influenza or anti-viral activities of herbal remedies or constituents thereof (with unknown mechanism of action) and (iii) empirical knowledge of antiviral herbal remedies form traditional medicine. Based on these selection criteria, in depth analytical and phytochemical studies will be performed with the ~10 most promising natural starting materials for each target, primarily from plant origin. Fractions enriched with virtually predicted hits and finally isolated and identified compounds will be provided for assaying on the respective anti-influenza targets and determination of their antiviral efficacy in cooperation with PD Dr. Michaela Schmidtke (Institute of Virology and Antiviral Therapy, University of Jena). Applying this strategy, we will (i) explain (at least partly) the anti-influenza molecular mechanism of traditionally approved herbal remedies, (ii) identify naturally derived drug leads for novel NA-inhibitors with lower risk to develop drug resistance, and (iii) enrich the pool of small molecule ligands for the innovative anti-influenza target NP in an effort to discover agents for a rapidly evolving pathogen.

Influenza A and B viruses remain a significant global health threat by causing annual seasonal flu, occasional epidemics and pandemics. Treatment options are limited to ion channel blockers (M2 inhibitors) and inhibitors of the key surface enzyme neuraminidase (NA). M2 inhibitors are highly prone to viral resistance and neuraminidase inhibitors (NAIs) are limited to oseltamivir and zanamivir being the only two representatives approved in most countries. Moreover, severe complications of influenza infections are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recent discoveries have shown that dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. In this project, based on the knowledge of antiviral herbal remedies from traditional medicine, starting materials from plants and fungi were selected for the production of 162 extracts. Antiviral testing of these extracts (in cooperation with PD Dr. Michaela Schmidtke (Jena University Hospital) revealed 20%, 11%, and 14% actives against influenza virus A/Hong Kong/68 (HK/68), and two related viruses involved in acute respiratory infections, i.e. rhinovirus 2 (RV-A2) and coxsackie virus B3 (CV-B3), respectively. Data from the phenotypic antiviral screening in combination with information from virtually predicted hits (in cooperation with Univ. Prof. Dr. Klaus R. Liedl, University of Innsbruck and Prof. Dr. Johannes Kirchmair, University of Hamburg) guided the analytical and phytochemical investigations for the identification of novel antiviral lead structures from nature. From the 10 most promising natural starting materials 73 pure compounds were isolated, identified and tested for their antiviral activity. For 38 bioactive isolates further assays on the target level were performed to identify their molecular mechanisms. Main findings of this project are the identification of (i) resistance-breaking anti-influenza lead structures, (ii) unique dual inhibitors of both viral and bacterial NAs, as well as the development of (iii) an assay protocol for the straightforward identification of anti-influenza molecular mechanisms, and (iv) a standard procedure for ruling out false positives in the screening of extracts and pure compounds.