ABSTRACT
A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Indoleacetic Acids/metabolism , Indoleacetic Acids/pharmacology , Protein Prenylation/drug effects , ras Proteins/metabolism , Alkyl and Aryl Transferases/metabolism , Carrier Proteins/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , HSP40 Heat-Shock Proteins , Heat-Shock Proteins/metabolism , Humans , Indoleacetic Acids/chemical synthesis , Protein Prenylation/physiology , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-microm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-microm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1).
ABSTRACT
The Outer Membrane Protein Complex (OMPC) of the bacterium Neisseria meningitidis group B has been used successfully as a protein carrier in a Haemophilus influenza type b (Hib) polysaccharide conjugate vaccine and a Streptococcus pneumoniae (Pn) polysaccharide conjugate vaccine to elicit antipolysaccharide immune responses in young infants. The OMPC carrier is derived by detergent extraction of whole cells and, thus, the consistent generation of suitable biomass is central to an effective production process. Therefore, we have developed a large-scale, high-cell density (5 g/L dry cell weight) fermentation process for the cultivation of N. meningitidis B11. Since current requirements for the production of human biologics mandate strict control of all aspects of the manufacturing process, several key features of the process, including a chemically defined medium and a rational event-based harvest criterion, support current good manufacturing practice (cGMP) and increased productivity.
