Author: Oliveira, Gabriel S. de ; Huang, Fanglu ; Sun, Yuhui ; Leadlay, Peter F. ; dos S. Bury, Priscila ; Araújo, Natália C. de ; Leeper, Finian J. ; Zhou, Jiahai ; Li, Yuan ; Dias, Marcio V. B.

Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6'. GenB2 is the enzyme responsible for this epimerization and is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster. Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogues. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization but with a mechanism distinct from that of fold III PLP-dependent epimerases using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme, confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.

01 Dec 2015·Antiviral Chemistry and Chemotherapy

Isolation and characterization of hepatitis C virus resistant to a novel phenanthridinone derivative

Article

Author: Baba, Masanori ; Toyama, Masaaki ; Ito, Wataru ; Wakita, Takaji ; Watashi, Koichi ; Ikeda, Masanori ; Hashimoto, Yuichi ; Okamoto, Mika

Background The novel phenanthridinone derivative HA-719 has recently been identified as a highly potent and selective inhibitor of hepatitis C virus replication. To elucidate its mechanism of inhibition, we have isolated and analyzed a clone of hepatitis C virus replicon cells resistant to HA-719. Methods To isolate HA-719-resistant replicon cells, Huh-7 cells containing subgenomic hepatitis C virus replicons (genotype 1b) with a luciferase reporter (LucNeo#2) were cultured in the presence of G418 and escalating concentrations of HA-719. After several passages, total RNA was extracted from the growing cells, and Huh-7 cells were transfected with the extracted RNA. Limiting dilution of the transfected cells was performed to obtain an HA-719-resistant clone. Results The 50% effective concentration (EC50) of HA-719 for hepatitis C virus replication was 0.058 ± 0.012 µM in LucNeo#2 cells. The replicon cells capable of growing in the presence of G418 and 3 µM HA-719 were obtained after 18 passages (72 days). The HA-719-resistant clone LucNeo719R showed 98.3-fold resistant to the compound (EC50 = 5.66 ± 0.92 µM), but the clone had no cross-resistance to telaprevir (NS3 inhibitor), daclatasvir (NS5A inhibitor), and VX-222 (NS5B inhibitor). The sequence analysis for the wild-type and LucNeo719R identified 3, 2 and 7 mutations in NS3/4 A, NS4B, and NS5A, respectively, but no mutations in NS5B. Conclusion None of the amino acid mutations in the resistant clone corresponds to those reported to confer drug-resistance to current anti-hepatitis C virus agents, suggesting that the target of HA-719 for hepatitis C virus inhibition differs from those of the existing agents.

04 Jun 2009·International Journal of Molecular MedicineQ3 · MEDICINE

Conditional expression of type 2 angiotensin II receptor in rat vascular smooth muscle cells reveals the interplay of the angiotensin system in matrix metalloproteinase 2 expression and vascular remodeling

Q3 · MEDICINE

Article

Author: Liu, Jianping ; He, Yong ; Srivenugopal, Kalkunte S. ; Wang, Haidong ; Miao, Li ; Jing, Tao ; He, Guoxiang

Angiotensin II is well implicated in neointimal proliferation and the resulting restenosis, however, the mechanisms involved remain unclear. The type 2 angiotensin II (AT2) receptor, largely unexpressed in the adult vasculature, however, appears at significant levels after vascular injury. To investigate the specific contribution of AT2 receptor and the interplay of the angiotensin system to neointima, we engineered rat vascular smooth muscle cells (VSMCs) to express the AT2 receptor in a tetracycline-regulated system. Several VSMC clones resistant to both hygromycin and G418 were selected, many of which showed high, but regulatable levels of AT2R expression within 48 h of doxycycline (Dox) exposure. In untransfected VSMCs and stable transfectants with no AT2R induction, Ang II significantly increased the expression of matrix metalloproteinase 2 (MMP-2), which is linked to neointimal growth. However, induction of AT2R by Dox addition markedly decreased MMP-2 levels (P<0.01) and this downregulation was further promoted by CV-11974, a specific antagonist of AT1 receptor. In contrast, the PD123319 compound, which selectively curtails the AT2 receptor, reversed the inhibition caused by CV-11974. We conclude that Ang II enhances the MMP-2 expression via AT1R, and that enforces AT2R inhibited the same. These data confirm that AT2R functions to downregulate the effects elicited by Ang II + AT1R signaling and point to the role of MMP and extracellular matrix in vascular injury. The findings provide fresh experimental approaches to prevent or control restenosis through transduction of VSMCs expressing optimal levels of AT2R.

100 Deals associated with G-418

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Indication	Highest Phase	Country/Location	Organization	Date
Bacterial Infections	Preclinical	US	Schering Corp.	01 Aug 1974

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