In vitro activity of LK-157, a novel tricyclic carbapenem as broad-spectrum {beta}-lactamase inhibitor.

LK-157 is a novel tricyclic carbapenem with potent activity against class A and class C beta-lactamases. When tested against the purified TEM-1 and SHV-1 enzymes, LK-157 exhibited 50% inhibitory concentrations (IC(50)s) in the ranges of the clavulanic acid and tazobactam IC(50)s (55 nM and 151 nM, respectively). Moreover, LK-157 significantly inhibited AmpC beta-lactamase (IC(50), 62 nM), as LK-157 was >2,000-fold more potent than clavulanic acid and approximately 28-fold more active than tazobactam. The in vitro activities of LK-157 in combination with amoxicillin, piperacillin, ceftazidime, cefotaxime, ceftriaxone, cefepime, cefpirome, and aztreonam against an array of Ambler class A (TEM-, SHV-, CTX-M-, KPC-, PER-, BRO-, and PC-type)- and class C-producing bacterial strains derived from clinical settings were evaluated in synergism experiments and compared with those of clavulanic acid, tazobactam, and sulbactam. In vitro MICs against ESBL-producing strains (except CTX-M-containing strains) were reduced 2- to >256-fold, and those against AmpC-producing strains were reduced even up to >32-fold. The lowest MICs (< or =0.025 to 1.6 microg/ml) were observed for the combination of cefepime and cefpirome with a constant LK-157 concentration of 4 microg/ml, thus raising an interest for further development. LK-157 proved to be a potent beta-lactamase inhibitor, combining activity against class A and class C beta-lactamases, which is an absolute necessity for use in the clinical setting due to the worldwide increasing prevalence of bacterial strains resistant to beta-lactam antibiotics.

4-Substituted trinems as broad spectrum beta-lactamase inhibitors: structure-based design, synthesis, and biological activity.

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.

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity.

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

Macrocyclic inhibitors of the bacterial cell wall biosynthesis enzyme MurD.

Computer-based molecular design has been used to produce a series of new macrocyclic systems targeted against the bacterial cell wall biosynthetic enzyme MurD. Following their preparation, which involved a novel metathesis-based cyclisation as the key step, these systems were found to show good inhibition when assayed against the MurD enzyme.

Human non-pregnancy ribonuclease with anti-Kaposi's sarcoma activity.

Our demonstration of a 19kDa anti-Kaposi's sarcoma (KS) ribonuclease (RNase) in urine from a non-pregnant female may provide at least part of the explanation for the low incidence of KS in human females. N-terminal sequence analysis and isoelectric focusing of the purified RNase, coupled with the very low levels of anti-KS activity noted for recombinant forms of human eosinophil derived neurotoxin and human eosinophil cationic protein, suggest that the 19kDa enzyme is an eosinophilic protein whose potent anti-KS activity is dependent upon post-translational modifications that occur only in human cells.