Binaphthyl-anchored antibacterial tripeptide derivatives with hydrophobic C-terminal amino acid variations.

The facile synthesis of seven new dicationic tripeptide benzyl ester derivatives, with hydrophobic group variations in the C-terminal amino acid component, is described. Moderate to good activity was seen against Gram-positive bacteria in vitro. One cyclohexyl-substituted compound 2c was tested more widely and showed good potency (MIC values ranging from 2-4 µg/mL) against antibiotic-resistant strains of Staphylococcus aureus and Enterococci (VRE, VSE), and against Staphylococcus epidermidis.

Crystal structures of novel allosteric peptide inhibitors of HIV integrase identify new interactions at the LEDGF binding site.

An optimised method of solution cyclisation gave us access to a series of peptides including SLKIDNLD (2). We investigated the crystallographic complexes of the HIV integrase (HIV-IN) catalytic core domain with 13 of the peptides and identified multiple interactions at the binding site, including hydrogen bonds with residues Thr125 and Gln95, that have not previously been described as being accessible within the binding site. We show that the peptides inhibit the interaction of lens epithelium-derived growth factor (LEDGF) with HIV-IN in a proximity AlphaScreen assay and in an assay for the LEDGF enhancement of HIV-IN strand transfer. The interactions identified represent a potential framework for the development of new HIV-IN inhibitors.

Structural basis for a new mechanism of inhibition of HIV-1 integrase identified by fragment screening and structure-based design.

BACKGROUND: HIV-1 integrase is a clinically validated therapeutic target for the treatment of HIV-1 infection, with one approved therapeutic currently on the market. This enzyme represents an attractive target for the development of new inhibitors to HIV-1 that are effective against the current resistance mutations. METHODS: A fragment-based screening method employing surface plasmon resonance and NMR was initially used to detect interactions between integrase and fragments. The binding sites of the fragments were elucidated by crystallography and the structural information used to design and synthesize improved ligands. RESULTS: The location of binding of fragments to the catalytic core of integrase was found to be in a previously undescribed binding site, adjacent to the mobile loop. Enzyme assays confirmed that formation of enzyme-fragment complexes inhibits the catalytic activity of integrase and the structural data was utilized to further develop these fragments into more potent novel enzyme inhibitors. CONCLUSIONS: We have defined a new site in integrase as a valid region for the structure-based design of allosteric integrase inhibitors. Using a structure-based design process we have improved the activity of the initial fragments 45-fold.

Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site.

HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

Modification of glycopeptidolipids by an O-methyltransferase of Mycobacterium smegmatis.

Glycopeptidolipids (GPLs) are a major component of the outer layers of the cell walls of several non-tuberculous mycobacteria. The Mycobacterium smegmatis GPLs consist of a diglycosylated lipopeptide core which is variably modified by acetylation and methylation. Analysis of a region of the M. smegmatis chromosome, upstream of the peptide synthetase gene, mps, revealed a GPL biosynthetic locus containing genes potentially involved in glycosylation, methylation, acetylation and transport of GPLs. Methyltransferases are required to modify rhamnose and the fatty acid of GPLs. Of the four methyltransferases encoded within the locus, one methyltransferase, Mtf2, was unlike sugar methyltransferases from other species. An mtf2 mutant was created and was shown to be unable to methylate the GPL fatty acids. Direct evidence is presented that Mtf2 is a methyltransferase that modifies the GPL fatty acid.

Mannose metabolism is required for mycobacterial growth.

Mycobacteria are the causative agents of tuberculosis and several other significant diseases in humans. All species of mycobacteria synthesize abundant cell-wall mannolipids (phosphatidylinositol mannosides, lipoarabinomannan), a cytoplasmic methylmannose polysaccharide and O-mannosylated glycoproteins. To investigate whether these molecules are essential for mycobacterial growth, we have generated a Mycobacterium smegmatis mannose auxotroph by targeted deletion of the gene encoding phosphomannose isomerase (PMI). The PMI deletion mutant displayed a mild hyperseptation phenotype, but grew normally in media containing an exogenous source of mannose. When this mutant was suspended in media without mannose, ongoing synthesis of both the mannolipids and methylmannose polysaccharides was halted and the hyperseptation phenotype became more pronounced. These changes preceded a dramatic loss of viability after 10 h in mannose-free media. Mannose starvation did not lead to detectable changes in cell-wall ultrastructure or permeability to hydrophobic drugs, or to changes in the rate of biosynthesis of other plasma-membrane or wall-associated phospholipids. These results show that mannose metabolism is required for growth of M. smegmatis and that one or more mannose-containing molecules may play a role in regulating septation and cell division in these bacteria.

Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium.

Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis.