A single-amino acid substitution in West Nile virus 2K peptide between NS4A and NS4B confers resistance to lycorine, a flavivirus inhibitor

AbstractLycorine potently inhibits flaviviruses in cell culture. At 1.2-μM concentration, lycorine reduced viral titers of West Nile (WNV), dengue, and yellow fever viruses by 102- to 104-fold. However, the compound did not inhibit an alphavirus (Western equine encephalitis virus) or a rhabdovirus (vesicular stomatitis virus), indicating a selective antiviral spectrum. The compound exerts its antiviral activity mainly through suppression of viral RNA replication. A Val→Met substitution at the 9th amino acid position of the viral 2K peptide (spanning the endoplasmic reticulum membrane between NS4A and NS4B proteins) confers WNV resistance to lycorine, through enhancement of viral RNA replication. Initial chemistry synthesis demonstrated that modifications of the two hydroxyl groups of lycorine can increase the compound's potency, while reducing its cytotoxicity. Taken together, the results have established lycorine as a flavivirus inhibitor for antiviral development. The lycorine-resistance results demonstrate a direct role of the 2K peptide in flavivirus RNA synthesis

PROBING BINDING REQUIREMENTS OF NAD KINASE WITH MODIFIED NAD ANALOGUES

NAD kinase (NADK) catalyzes a magnesium-dependent phosphorylation of the 2′-hydroxyl group of the adenosine ribose moiety of nicotinamide adenine dinucleotide (NAD) using ATP or inorganic polyphosphates as phosphoryl donors to give NADP. There are two classes of the enzyme, one is specific for NAD+ and the other also phosphorylates NADH. With the recent discovery of NADK from Mycobacterium tuberculosis and with the emergence of multidrug resistant and extensively drug resistant tuberculosis (TB) worldwide, NADK has became deeply important as attractive target for antibiotic discovery. Recently Boshoff et al. (1) reported that inhibition of enzymes involved in the early stages of NAD biosynthesis for the treatment of TB is often reversed by the unwanted involvement of alternative rescue pathways, whereas the enzymes comprising the last steps, such as NAD synthetase (NADS) and NADK, should be preferred. Furthermore, NADK cannot be complemented by rescue pathways, and being the only enzyme which supplies vital NADP in prokaryotic or eukaryotic cells, could be an even better target than NADS. As a proof of concept, following our recent discovery of NAD analogues, such as DTA 1 and 8-BrDTA 2 (2), that afforded potent but not selective inhibitors, we decided to design a new series of NAD analogues where the nicotinamide riboside moiety has been replaced by 2-thiopyridyl, benzyl and meta-nitro benzyl groups (compounds 3-5), maintaining the short di-sulfur linker instead of the pyrophosphate linkage of NAD. The synthesis and the biological evaluation against human and bacterial NADKs of these heterodisulfides will be presented in detail

Synthesis and Enzymatic Evaluation of NAD Mimics as Nicotinamide Adenine Dinucleotide Kinase (NADK) Inhibitors

NAD kinase (NADK) is a key enzyme that regulates supply of NADP in the cell. At this time no other pathway of NADP biosynthesis has been found in prokaryotic or eukaryotic cells. Human NAD kinase catalyzes a magnesium- dependent phosphorylation of the 2’-hydroxyl group of the adenosine ribose moiety of NAD using ATP as phosphoryl donor to give NADP. Bacterial enzymes can use inorganic polyphosphates as phosphoryl donors in addition to ATP. Significant differences between the human and the mycobacterium enzyme were found that might allow for construction of inhibitors with selectivity against these proteins. Therefore, M. tubercolosis NADK has became an appealing new target for the development of potential drugs against multi-drug resistant (MDR) and extensively drug resistant (XDR) tubercolosis (TB). Recently, we have reported that dinucleoside disulfide NAD mimics, such as diadenosine disulfide (DTA), were found to be moderate inhibitors of M. tubercolosis and human NADKs. A restriction of the conformation of adenine moiety to syn by substitution with a bulky bromine atom at the C8 of one or two adenine rings of DTA, furnished the most potent inhibitors of both human and mycobacterium NADK reported so far. On the contrary, fixing the sugar conformation in the “North” or “South” conformation by introduction of a methyl group at the 2’- or 3’-position of the ribose ring was detrimental for NADK inhibitory activity [1]. To further investigate the structural features of the ribose moiety, herein we report the synthesis and the NADK inhibitory activity of 2’-deoxy-, 3’-deoxy-, and 2’,3’-dideoxy-DTA. The results of this study will be presented. [1] R. Petrelli, Y.Y. Sham, L. Chen, K. Felczak, E. Bennett, D. Wilson, C. Aldrich, J.S. Yu, L. Cappellacci, P. Franchetti, M. Grifantini, F. Mazzola, M. Di Stefano, G. Magni, and K. Pankiewicz, Bioorg. Med. Chem., 17, 2009, 5656