Thammaiah Viswanatha

N-Arylsulfonyl Hydrazones as Inhibitors of IMP-1 Metallo-β-Lactamase

ABSTRACT Members of a family of N-arylsulfonyl hydrazones have been identified as novel inhibitors of IMP-1, a metallo-β-lactamase of increasing prevalence. Structure-activity relationship studies have indicated a requirement for bulky aromatic substituents on each side of the sulfonyl hydrazone backbone for these compounds to serve as efficient inhibitors of IMP-1. Molecular modeling has provided insight into the structural basis for the anti-metallo-β-lactamase activity exhibited by this class of compounds.

A novel role for calcite in calcium homeostasis

Calcium carbonate (CaCO3) minerals are known to be deposited in a wide array of different organisms, ranging from microbes to vertebrates [(1989) On Biomineralization, Oxford University Press, New York]. Calcite, aragonite and vaterite are the major crystalline structural polymorphs of CaCO3 associated with living systems, and participate in a variety of biological functions [(1989) Biomineralization: Chemical and Biochemical Perspectives. VCH Publishers, Weinham, Germany; (1991) Advances in Inorganic Chemistry 36, 137–200]. Here we report on the ability of a soil bacterium to synthesize calcite in a calcium‐stressed environment. The elaboration of this exocellular crystalline residue enables the organism to regulate its calcium content. The attainment of calcium homeostasis via the exocellular deposition of bacterial calcite with unique crystal habits is a novel biological phenomenon.

IMP-1 metallo-β-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry

Metallo-beta-lactamases have attracted considerable attention due to their role in microbial resistance to beta-lactam antibiotics. IMP-1, the binuclear Zn-dependent beta-lactamase produced by Pseudomonas aeruginosa and other microorganisms, is of particular interest in view of its increasing prevalence. An examination of the susceptibility of IMP-1 to inactivation by six different divalent metal ion chelators has revealed that all except Zincon cause inhibition by forming a complex with the holoenzyme. Exposure of the enzyme to dipicolinic acid (DPA), the most potent inhibitor, results in the production of the mononuclear Zn form of the protein as determined by electrospray ionization mass spectrometry (ESI-MS) under nondenaturing conditions. This mononuclear Zn species was found to be catalytically competent. Studies with the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) show that the two zinc centers in IMP-1 differ in their accessibility, a feature that could be overcome in the presence of guanidine hydrochloride (GdnHCl, 1.5 M).

Calcium involvement in mediating the action of octopamine and hypertrehalosemic peptides on insect haemocytes

Octopamine, the hypertrehalosemic peptides HT‐I and HT‐II and the calcium ionophore A23187 elevate intracellular calcium levels in cultured haemocytes of Malacosoma disstria (Lepidoptera: Lasiocampidae). The octopamine‐mediated response is dose‐dependent and the magnitude of the response is influenced by the concentration of calcium in the incubation medium. Mianserin, an inhibitor of octopamine‐mediated elevation of cyclic AMP production, blocks the octopamine‐mediated increase in intracellular calcium but has no effect on the HT‐I‐ and HT‐II‐mediated responses. The results indicate that some effects of octopamine are mediated through agonist‐dependent calcium gating whereas others are expressed through receptors coupled to adenylate cyclase. The study also confirms previous suggestions that HT‐I and HT‐II elevate intracellular calcium concentrations.

Influence of octopamine on trehalase activity in muscle and hemolymph of the American cockroach, Periplaneta americana L.

Injection of adult male cockroaches (Periplaneta americana) with 10 microliter 1 microM octopamine causes elevated activity of trehalase (alpha, alpha-trehalose glucohydrolase; EC 3.2.1.28) in hemolymph and muscle but not in gut. Tyramine, dopamine and glutamate, at the same concentration, failed to elicit any effect on trehalase activity. Determination of some kinetic parameters for muscle and hemolymph trehalases reveal that octopamine causes an increase in Vmax without any significant alteration in the Km of the enzyme for trehalose. The results are discussed in terms of the physiological significance of octopamine-mediated activation of tissue trehalases.

Studies on the formation of N6-hydroxylysine in cell-free extracts of Aerobacter aerogenes 62-1.

We have investigated conditions optimal for the conversion of L-lysine to its N6-hydroxy derivative by partially purified cell-free extracts of Aerobacter aerogenes 62-1. The enzyme system was highly specific to L-lysine: the D-isomer and, the N2- or N6-derivatives of lysine, and alpha-amino acids were not hydroxylated. Most of the latter compounds had little effect onthe hydroxylation of L-lysine. However, -l-glutamic acid and L-glutamine enhanced the hydroxylation, with half-maximal activation achieved at 100 micrometers concentration of the effector. The Km values for pyruvate and L-(+)-lactate (compounds known to stimulate N-hydroxylysine formation) were found to be approx. 100 micrometers. The data show that N-hydroxylation of the amino acid precedes acylation in the biosynthesis of hydroxamic acid in A. aerogenes 62-1.

Synthesis of 4-carboxy-2-thiabicyclo [3.2.0] Heptan-6-ones via 3-carboxy-2,3-dihydrothiophenes: potential β-lactamase inhibitors

3-Carboalkoxy-2,3-dihydrothiophenes, available by Birch reduction of thiophene-3-carboxylic acid or more efficiently by deconjugation of 2,5-dihydrothiophene-3-carboxylic acid by reaction with ethyl chloroformate and triethylamine, undergo cycloaddition reactions with dichloroketene leading to 4-carboxy-7,7-dichloro-2-thiabicyclo[3.2.0]heptan-6-ones which are of interest as potential β-lactamase inhibitors.

The use of bis(mercaptoacetato-S,O)hydroxoiron(III) complex for the determination of hydroxamates

A rapid, indirect, spectrophotometric procedure for the determination of hydroxamates, based on the competition for ferric ions of the bis(mercaptoacetato-S,O)hydroxoiron(III) complex, has been developed. The assay is remarkably free of interferences by common ions, thus rendering it useful in the quantitative determination of hydroxamates in culture fluids and crude preparations.

Physico-chemical characterization of a recombinant cytoplasmic form of lysine: N6-hydroxylase

A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity. IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH. The enzyme is stringently specific with L-lysine and (S)-2-aminoethyl-L-cysteine serving as substrates. Protonophores, FCCP and CCCP, as well as cinnamylidene, have been found to serve as potent inhibitors of lysine: N6-hydroxylation by virtue of their ability to interfere in the reduction of the flavin cofactor.

6-β-(Trifluoromethane sulfonyl)-amido-penicillanic acid sulfone: A potent inhibitor for β-lactamases

P-Lactamases have attracted considerable attention in recent years in view of their ability to confer upon microorganisms resistance to penicillins [ 11. It is not surprising to find the emphasis being placed on the development of potent inhibitors of these enzymes. The finding that clavulanic acid, a naturally occurring p-lactam, could inactivate /3-lactamase [2-41 provided an impetus for the development of penicillin analogs capable of serving as suicide substrates. Such compounds include: 6+bromopenicillanic acid [5,6], penicillanic acid sulfone [7-91, 6-a-chloropenicillanic acid sulfone [lo] and quinacillin sulfone [ 111. The presence of a sufficiently acidic hydrogen at C6 and/or a good leaving substituent at C5 (features favoring facile elimination across the C6-C5 bond of the molecule) render penams as potent suicide substrates for /3-lactamases. Here, the synthesis of 6-/I-(trifluoromethane sulfonyl)-amido-penicillanic acid sulfone, IV, tias accomplished so as to incorporate both of the above mentioned features in the penicillin molecule. Compound IV proved to be a more potent inhibitor of /