ABSTRACT
A wide variety of pathogens have acquired antimicrobial resistance as an inevitable evolutionary response to the extensive use of antibacterial agents. In particular, one of the most widely used antibiotic structural classes is the beta-lactams, in which the most common and the most efficient mechanism of bacterial resistance is the synthesis of beta-lactamases. Class C beta-lactamase enzymes are primarily cephalosporinases, mostly chromosomally encoded, and are inducible by exposure to some beta-lactam agents and resistant to inhibition by marketed beta-lactamase inhibitors. In an ongoing effort to alleviate this problem a series of novel 4-substituted trinems was designed and synthesized. Significant in vitro inhibitory activity was measured against the bacterial beta-lactamases of class C and additionally against class A. The lead compound LK-157 was shown to be a potent mechanism-based inactivator. Acylation of the active site Ser 64 of the class C enzyme beta-lactamase was observed in the solved crystal structures of two inhibitors complexes to AmpC enzyme from E. cloacae. Structure-activity relationships in the series reveal the importance of the trinem scaffold for inhibitory activity and the interesting potential of the series for further development.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Azetidines/chemical synthesis , Bacterial Proteins/antagonists & inhibitors , Drug Resistance, Bacterial , Heterocyclic Compounds, 3-Ring/chemical synthesis , beta-Lactamase Inhibitors , Acylation , Anti-Bacterial Agents/chemistry , Azetidines/chemistry , Bacterial Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Enterobacter cloacae/enzymology , Heterocyclic Compounds, 3-Ring/chemistry , Models, Molecular , Molecular Structure , Stereoisomerism , Structure-Activity Relationship , beta-Lactamases/chemistryABSTRACT
Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible.
Subject(s)
Bacteriophage lambda/genetics , Cloning, Molecular/methods , Escherichia coli/genetics , Escherichia coli/virology , Promoter Regions, Genetic/genetics , Escherichia coli/growth & development , Genes, Bacterial , Mutagenesis , Plasmids/genetics , Protein Biosynthesis , RNA, Messenger/genetics , Recombinant Proteins/biosynthesis , Repressor Proteins/metabolism , Restriction Mapping , Transcription, Genetic , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Known carbapenem compounds with inhibitory effect towards beta-lactamase enzymes are formed from bicyclical beta lactam structural scaffolds. On the basis of results from theoretical computational methods and molecular modelling we have designed and developed a synthetic route towards novel, biologically active tricyclic derivatives of carbapenems.
