Design, synthesis, and evaluation of novel small molecule inhibitors of the influenza virus protein NS1

doi:10.1016/j.bmc.2011.10.026

Bioorganic & Medicinal Chemistry

Volume 20, Issue 1, 1 January 2012, Pages 487-497

https://doi.org/10.1016/j.bmc.2011.10.026 Get rights and content

Abstract

Influenza is a continuing world-wide public health problem that causes significant morbidity and mortality during seasonal epidemics and sporadic pandemics. The existing vaccination program is variably effective from year to year, and drug resistance to available antivirals is a growing problem, making the development of additional antivirals an important challenge. Influenza virus non-structural protein 1 (NS1) is the centerpiece of the viral response to the host interferon (IFN) system. NS1 was demonstrated previously to be a potential therapeutic target for antiviral therapy by the identification of specific small-molecule inhibitors. One inhibitory compound, NSC125044, was subjected to chemical evaluation. Initial synthetic work comprised simplifying the core structure by removing unwanted functionality and determination of key features important for activity. Several subclasses of molecules were designed and synthesized to further probe activity and develop the basis for a structure–activity relationship. Apparent potency, as judged by activity in virus replication assays, increased dramatically for some analogs, without cytotoxicity. Results suggest that the target binding site tolerates hydrophobic bulk as well as having a preference for weakly basic substituents.

Graphical abstract

Introduction

Influenza is a continuing worldwide public health problem. The World Health Organization estimate of annual mortality due to seasonal influenza is 250,000–500,000, and there are approximately 30,000 influenza associated deaths and 200,000 hospitalizations in the United States per year.¹ Severe infections are most commonly seen in the elderly, the very young, and the chronically ill.2, 3, 4

In addition to seasonal epidemics, there have been four documented influenza pandemics during this and the previous century, the most serious occurring in 1918. During an eight month period the 1918 Spanish influenza claimed 20–40 million lives.⁵ Two subsequent pandemics in 1957 and 1968 were less severe but shared the characteristic of having avian origin.⁶ There is concern that another devastating pandemic is inevitable, perpetuated by the spread of the avian H5N1 virus through multiple bird populations in Asia, Africa and Europe, which periodically causes human infections.7, 8, 9, 10 Although avian H5N1 has not acquired the ability to transmit from person to person, those who are infected through direct contact with birds are at a very high risk for mortality, approximately 60%.11, 12 The H1N1 swine influenza pandemic of 2009 has added weight to concerns regarding the pandemic threat.13, 14, 15

The most common method for combating influenza virus is vaccination. However, viral antigenic drift dictates that a new vaccine be developed each year, and the effectiveness of the vaccine is variable.¹⁶ In addition there are obstacles to worldwide vaccine distribution, and reluctance on the part of some to vaccine administration.¹⁷ Two classes of drugs have been used to treat influenza infections, including one that targets the viral M2 ion channel and one that inhibits the viral neuraminidase protein. Due to the emergence of viral strains that are resistant to M2 and neuraminidase inhibitors, the variable effectiveness of seasonal vaccines, and the high probability of future pandemics, it is important to identify additional viable viral targets to treat the influenza infections.¹⁸

Previously we identified non-structural protein 1 (NS1) of influenza virus as a novel drugable antiviral target.1, 19 NS1 is encoded by all strains of influenza A and is highly conserved.20, 21, 22 It is a multifunctional protein with essential roles in viral replication and evasion of the cellular innate immune response. The central role of NS1 in virus propagation and spread make it an attractive drug target.20, 21 Numerous studies have shown that NS1 participates in a wide range of functions. NS1 binds to double stranded RNA in a non-sequence specific manner.23, 24 In doing so NS1 is able to shield dsRNA from detection by the 2′–5′ oligo(A) synthetase (OAS)/RNase L pathway, which is responsible for degradation of viral RNA.²⁵ NS1 also binds to the 30 kDa subunit of cleavage and polyadenylation specificity factor (CPSF30), and poly (A)-binding protein II.26, 27 In doing this the 3′-end processing of cellular mRNAs is inhibited, impeding the metabolism of essential RNAs. Among these are mRNAs for interferons (IFNs), which are produced in response to viral infection.26, 27, 28, 29 Additionally NS1 associates with cellular protein kinase R (PKR), thereby preventing it from phosphorylating translation elongation factor eIF-2α and allowing viral protein synthesis to continue.30, 31 Recently, it has been demonstrated that NS1 interacts with TRIM25. This interaction prevents the activation of retinoic acid-inducible gene I (RIG-I), thereby suppressing the production of cellular IFN.32, 33, 34

In a recent study we identified several compounds that specifically inhibit NS1 function in cells.¹ Importantly, four of the compounds with anti-NS1 activity also demonstrated significant antiviral activity in cell culture assays and reversed NS1-dependent blockade of the cellular interferon synthesis pathway.¹ In this report we describe the design, synthesis and biological testing of a series of compounds based on one of the compounds reported previously, NSC125044. These studies have resulted in several highly potent antiviral compounds.

Section snippets

Method validation

Efficacy of an antiviral compound in cell culture is a complex function of its activity, permeability, and other factors. In addition, virus replication is a multi-dimensional non-linear process, so the overall effect of chemical inhibition of a viral target may be complex as well. In the case of inhibition of NS1 function there is the added complexity of the interplay between NS1 and the host cell innate immune response, which involves modulation of cellular interferon production and the

Discussion

Starting from hit NSC125044 (compound A1), obtained from a screen as reported previously,¹ a systematic analoging approach has yielded a simple molecule that inhibits virus replication in cells by greater than three orders of magnitude (Figure 5, Figure 10 and Supplementary Fig. S5). This is virtually the same magnitude of effect on replication observed with a virus carrying a deletion of the NS1 gene (‘delNS1’) in the A/PR/8/34 background, the same used in this study.³⁵ This strongly suggests

Mammalian cells and viruses

MDCK cells were maintained in Iscove medium supplemented with 10% fetal bovine serum and 2 mM l-glutamine. Medium and serum were from Invitrogen. For infections, viral stocks were diluted in growth medium supplemented with 0.3% bovine serum albumin, 0.22% sodium bicarbonate, and 0.25 U of TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-trypsin (Invitrogen)/mL. Influenza virus A/PR/8/34 was propagated in 10-day-old embryonated chicken eggs at 37 °C. Titers of influenza virus were determined

Acknowledgement

This study was supported by Public Health Service grants R01AI071341 to D.A.E. and 5R43AI084244 to D.B.

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Design, synthesis, and evaluation of novel small molecule inhibitors of the influenza virus protein NS1

Abstract

Graphical abstract

Introduction

Section snippets

Method validation

Discussion

Mammalian cells and viruses

Acknowledgement

Microb. Infect.

Vaccine

Trends Pharmacol. Sci.

Virology

Virology

J. Biol. Chem.

Mol. Cell

Virology

Pharmacol. Ther.

Virology

Cell Host Microbe

Cell Host Microbe

Virology

J. Virol.

Arch. Intern. Med.

JAMA-J. Am. Med. Assoc.

JAMA-J. Am. Med. Assoc.

Angew. Chem., Int. Ed.