Anti-influenza activity of phenethylphenylphthalimide analogs derived from thalidomide

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Abstract

Swine-origin influenza A virus has caused pandemics throughout the world and influenza A is regarded as a serious global health issue. Hence, novel drugs that will target these viruses are very desirable. Influenza A expresses an RNA polymerase essential for its transcription and replication which comprises PA, PB1, and PB2 subunits. We identified potential novel anti-influenza agents from a screen of 34 synthesized phenethylphenylphthalimide analogs derived from thalidomide (PPT analogs). For this screen we used a PA endonuclease inhibition assay, a PB2 pathogenicity-determinant domain-binding assay, and an anti-influenza A virus assay. Three PPT analogs, PPT-65, PPT-66, and PPT-67, were found to both inhibit PA endonuclease activity and retard the growth of influenza A, suggesting a correlation between their activities. PPT-28 was also found to inhibit the growth of influenza A. These four analogs have a 3,4-dihydroxyphenethyl group in common. We also discuss the possibility that 3,4-dihydroxyphenethyl group flexibility may play an important functional role in PA endonuclease inhibition. Another analog harboring a dimethoxyphenethyl group, PPT-62, showed PB2 pathogenicity-determinant domain-binding activity, but did not inhibit the growth of the virus. Our present results indicate the utility of the PA endonuclease assay in the screening of anti-influenza drugs and are therefore useful for future strategies to develop novel anti-influenza A drugs and for mapping the function of the influenza A RNA polymerase subunits.

Introduction

Thalidomide, a hypnotic/sedative drug, was originally launched in the 1950s but was subsequently withdrawn from the market in the 1960s because of its teratogenic properties.1, 2 Thalidomide has been subsequently shown however to be useful in the treatment of Hansen’s disease, multiple myeloma, cancer, rheumatoid arthritis, graft-versus-host diseases, and acquired immunodeficiency syndrome.1, 2, 3, 4 Pharmacologically, thalidomide has anti-cachexia, anti-inflammatory, anti-tumor-promoting, anti-angiogenic, tumor cell invasion-inhibiting, anti-viral, and hypoglycemic activities. Hence, thalidomide is a multi-target drug and is thought to be useful as a template in the development of other biologically active compounds. Indeed, Hashimoto and colleagues have previously developed various thalidomide analogs2, 3, 5, 6, 7 and based on the target molecules have created tumor necrosis factor-production-regulating agents, cyclooxygenase inhibitors, nitric oxide synthase inhibitors, histone deacetylase inhibitors, anti-angiogenic agents, and tubulin polymerization inhibitors.2, 3, 5, 6, 7 These researchers have also developed the structure of thalidomide based on its pharmacological effects and thereby produced androgen antagonists, progesterone antagonists, cell differentiation inducers, aminopeptidase inhibitors, thymidine phosphorylase inhibitors, μ-calpain inhibitors, α-glucosidase inhibitors, and nuclear liver X receptor antagonists.2, 3, 5, 6, 7

In 1918, an influenza A pandemic caused ten million deaths worldwide8 and strategies to prevent any future expansions of this virus are therefore an important endeavor.9, 10 The avian H5N1 influenza A virus is highly pathogenic to humans11 and the emergence of a new strain of this virus in 2009, the swine-origin A/H1N1 pdm influenza virus (SOIV), emphasizes this issue further as it has become a serious global health issue.12 Although inhibitors of influenza A such as the neuraminidase-like compound oseltamivir are widely used as anti-viral drugs,13, 14 some adverse effects of these agents and also the emergence of viral strains that are resistant to these drugs have now been reported.15, 16, 17

For the prevention and control of influenza outbreaks, the development of novel anti-viral drugs that are not based on neuraminidase inhibition is now regarded as critical.12 The influenza A genome consists of a segmented single stranded RNA (−) and its transcription and replication require the activity of a highly conserved RNA-dependent RNA polymerase.18, 19 This polymerase is essential for the influenza A virus to propagate and thus represents a very promising target for anti-viral drug development. The influenza A virus RNA-dependent RNA polymerase is composed of three subunits, PA, PB1, and PB2, and synthesizes viral mRNAs using short capped primers derived from host cellular pre-mRNAs cleaved by the viral endonuclease.18, 19 Yuan et al. and Dias et al. have shown that the N-terminal domain of the PA subunit contains the endonuclease active site and that this domain also harbors RNA/DNA endonuclease activity.20, 21, 22 The PB2 subunit includes K627, which plays a role in the high pathogenicity and host range restriction of the virus.23, 24, 25, 26 We and others have elucidated the tertiary structure of the C-terminal domain (627 domain) of PB2 by X-ray crystallography and identified a unique loop and basic groove proximal to the K627 residue.27, 28 D701 N has also been shown to be associated with the viral pathogenicity levels and is also contained in this domain. Hence, we speculated that the PA endonuclease and PB2 627 domains would be very effective targets in the development of novel anti-influenza A drugs.

Our preliminary results in this regard suggested that phenethylphenylphthalimide (PPT) analogs derived from thalidomide are possible lead compounds (Supplementary Fig. 1) and we thus screened a further cohort of PPT analogs using a PA endonuclease inhibition assay, PB2 627 domain-binding assay and anti-influenza A virus assay. The results of these assays and also of our analysis of the structure–activity relationships of PPT analogs are described.

Section snippets

Synthesis of PPT analogs

PPT derivatives were synthesized as previously described29 in which diphenylethene derivatives were prepared as E/Z mixtures via a Wittig reaction of nitrobenzaldehyde with appropriately substituted benzyl ylide (Fig. 1A). After reduction of the nitro group and/or olefin moiety of the adducts, PPT derivatives were obtained by condensation with phthalic anhydride (Fig. 1A). We denoted our PPT analog series as PPT-n (n = sequential numbering from 1; Fig. 1B). We obtained 37 PPT analogs, three of

Discussion

We demonstrate that three PPT analogs, PPT-65, PPT-66, and PPT-67, inhibit PA endonuclease and the viral growth. Significantly, all of these analogs contain a 3,4-dihydroxyphenethyl group at the ortho or meta position of the N-phenyl moiety (Fig. 1B). The PPT analogs possessing methoxyphenethyl group(s) (PPT-27, -32, -59, -60, -62, -63, -80, -91, -95, -121, -122, -123, and -124), a monohydroxyphenethyl group (PPT-85, -94, -125, -136, -137, and -138) or an unsubstituted phenethyl group (PPT-21,

Synthesis of PPT analogs

Synthesis of the PPT analogs is described in the text and depicted also in Figure 1A. Their chemical structures are depicted in Figure 1B.

Acknowledgments

The influenza virus (A/PR/8/34) RNA polymerase PA and PB2 plasmids, pBMSA-PA and pBMSA-PB2, were provided by the DNA Bank, RIKEN BioResource Center (Tsukuba, Japan; originally deposited by Dr. Susumu Nakada) with the support of National Bio-Resources Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). This work was supported in part by a Grant-in-Aid for Young Scientists (Start-up) 20870037 (D.H.) from the Japan Society for the Promotion of Science

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