Oseltamivir (Tamiflu) and zanamivir (Relenza), two extensively used clinically effective anti-influenza drugs, are viral sialidase (also known as neuraminidase) inhibitors that prevent the release of progeny virions and thereby limit the spread of infection. Recently mortalities and neuropsychiatric events have been reported with the use of oseltamivir, especially in pediatric cases in Japan, suggesting that these drugs might also inhibit endogenous enzymes involved in sialic acid metabolism, including sialidase, sialyltransferase, and CMPsynthase, in addition to their inhibitory effects on the viral sialidase. The possible inhibition could account for some of the rare side effects of oseltamivir. However, there has been little direct evidence in regard to the sensitivities of animal sialidases to these drugs. Here, we examined whether these inhibitors might indeed affect the activities of human sialidases, which differ in primary structures and enzyme properties but possess tertiary structures similar to those of the viral enzymes. Using recombinant enzymes corresponding to the four human sialidases identified so far, we found that oseltamivir carboxylate scarcely affected the activities of any of the sialidases, even at 1 mM, while zanamivir significantly inhibited the human sialidases NEU3 and NEU2 in the micromolar range (K i , 3.7 ؎ 0.48 and 12.9 ؎ 0.07 M, respectively), providing a contrast to the low nanomolar concentrations at which these drugs block the activity of the viral sialidases.

Based on the human cDNA sequence predicted to represent the NEU4 sialidase gene in public databases, a cDNA covering the entire coding sequence was isolated from human brain and expressed in mammalian cells. The cDNA encodes two isoforms: one possessing an N-terminal 12-amino-acid sequence that is predicted to be a mitochondrial targeting sequence, and the other lacking these amino acids. Expression of the isoforms is tissue specific, as assessed by reverse transcription-PCR. Brain, muscle and kidney contained both isoforms; liver showed the highest expression, and the short form was predominant in this organ. In transiently transfected COS-1 cells, enzyme activity was markedly increased with gangliosides as well as with glycoproteins and oligosaccharides as substrates compared with the control levels. This differs from findings with other human sialidases. Although the isoforms were not distinguishable with regard to substrate specificity, they exhibited differential subcellular localizations. Immunofluorescence microscopy and biochemical fractionation demonstrated that an exogenously expressed haemagglutinin-tagged long form of NEU4 was concentrated in mitochondria in several human culture cell types, whereas the short form was present in intracellular membranes, indicating that the sequence comprising the N-terminal 12 amino acid residues acts as a targeting signal for mitochondria. Co-localization of the long form to mitochondria was further supported by efficient targeting of the N-terminal region fused to enhanced green fluorescent protein, and by the targeting failure of a mutant with an amino acid substitution in this region. NEU4 is possibly involved in regulation of apoptosis by modulation of ganglioside G(D3), which accumulates in mitochondria during apoptosis and is the best substrate for the sialidase.

GEX1A is a microbial product with antitumor activity. HeLa cells cultured with GEX1A accumulated p27(Kip) and its C-terminally truncated form p27*. GEX1A inhibited the pre-mRNA splicing of p27, producing p27* from the unspliced mRNA containing the first intron. p27* lacked the site required for E3 ligase-mediated proteolysis of p27, leading to its accumulation in GEX1A-treated cells. The accumulated p27* was able to bind to and inhibit the cyclin E-Cdk2 complex that causes E3 ligase-mediated degradation of p27, which probably triggers the accumulation of p27. By using a series of photoaffinity-labeling derivatives of GEX1A, we found that GEX1A targeted SAP155 protein, a subunit of SF3b responsible for pre-mRNA splicing. The linker length between the GEX1A pharmacophore and the photoreactive group was critical for detection of the GEX1A-binding protein. GEX1A serves as a novel splicing inhibitor that specifically impairs the SF3b function by binding to SAP155.

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.