Alvocidib is a cyclin dependent kinase inhibitor under clinical development for the treatment

by Selleckchem | Jan 17 2013

Lipopolysaccharide is the primary constituent of the outer leaflet of the outer membrane of Gram-negative bacteria . In addition to being the major surface molecule in Gram-negative bacteria, Alvocidib is also considered a major pathogenic factor. Lipid A, also referred to as endotoxin, is the hydrophobic membrane anchor of LPS and is known to be a potent inducer of the host innate immune system . Structurally, lipid A is characterized as a phosphoglycolipid defined by a conserved diglucosamine disaccharide with structural variations occurring by fatty acid position and identity, phosphorylation, and additional monosaccharide modification. Alteration of lipid A structure greatly affects the bacterium's virulence and can occur via a variety of environmental stimuli including divalent ion concentration, temperature, and other growth conditions . The phosphorylation pattern of lipid A has been shown to be important for its biological activity. For example, removal of a phosphate group has been shown to substantially reduce lipid A toxicity and interleukin-1 induction capacity . By contrast, masking of lipid A phosphate groups has been shown to affect bacterial resistance to host Anastrozole cationic antimicrobial peptides .
The biochemical effects of phosphate groups in lipid A have been attributed to their negative charge that affects recognition by the Toll-like receptor 4 and further LPS-induced signaling in the host immune response to bacterial infection . Furthermore, monosaccharide modification to lipid A is thought to occur via an ester linkage with the phosphate substituents. The biosynthesis of axitinib lipid A, as characterized in Escherichia coli, involves LPS intermediates that have a 1-position pyrophosphate and a 4_-position monophosphate . An inner membrane enzyme has been recently identified that transfers a phosphate group to the lipid A moiety to form the 1-position pyrophosphate structure . However, lipid A is commonly described as Bosutinib bisphosphorylated with monophosphate attachment at the 1- and 4_-positions of the glucosamine dimer backbone or monophosphorylated with phosphate attachment at either the 1- or 4_-position. We intend to demonstrate that several Gram-negative bacteria produce diphosphorylated lipid A that retains the pyrophosphate substituent. This finding is important for further studies of biochemical modifications of LPS that involve yet unknown mechanisms .
Our particular emphasis is centered on Yersinia pestis , the causative agent of the plague, which is a highly invasive and often lethal Gram-negative pathogen that is transmitted to the mammalian host by either fleabite or inhalation of an infectious droplet . Because of its potential use as a bioterrorism agent, Yp has been classified by the Center for Disease Control and Prevention as a Class A select agent. Transmission from the flea vector to the mammalian host induces temperature-dependent structural modification of Yp lipid A . At the mammalian host temperature of 37C, the primary structure of lipid A consists of a linked diglucosamine disaccharide with two phosphate moieties at the 1 and 4 positions, amide-linked 3-hydroxymyristic acids at the 2- and 2_-positions, and ester-linked 3-hydroxymyristic acids at the 3- and 3_-positions . However, at a temperature typical of the flea vector , the primary lipid A structure is hexa-acylated. The additional fatty acids, palmitoleic acid and lauric acid , are thought to be ester-linked via the 3-hydroxy position of the 3 and 3_ fatty acids, respectively, to form acyloxyacyl groups . Furthermore, Yp lipid A from both Apoptosis temperature variants has been reported to contain modifications of aminoarabinose and phosphoethanolamine . Despite extensive research, ambiguity remains to date as to the exact location of the acyloxyacyl fatty acids, phosphate moieties, and other modifications. Diphosphorylated lipid A is generally presumed to be phosphorylated at the 1- and 4_-positions. Our initial analysis of Yp lipid A grown at 37C and 21C suggested that several diphosphorylated lipid A structures from these two growth conditions were pyro- phosphorylated rather than bisphosphorylated.