Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus.

Triclosan, a widely used antibacterial agent, possesses potent activity against Staphylococcus aureus. This study reports on an investigation of the antibacterial target of triclosan in this pathogen. A strain of S. aureus overexpressing the enoyl-[acyl-carrier-protein] reductase (FabI), demonstrated by Western immunoblotting, gave rise to an increase in the MIC of triclosan, while susceptibilities to a range of unrelated antibacterials were unaffected. There are approximately 12 000 molecules of FabI per cell in mid-log phase growth. This number increased by approximately three- to four-fold in the S. aureus FabI overexpressor. Triclosan selectively inhibited the incorporation of [(14)C]acetate into TCA-precipitable product, an indicator of fatty acid biosynthesis. Furthermore, it inhibited de novo fatty acid biosynthesis in this organism. In vitro, triclosan inhibited recombinant, purified S. aureus FabI with an IC(50) of approximately 1 microM. The combination of these biochemical and genetic data provide further evidence that the mode of action of triclosan in S. aureus is via inhibition of FabI.

Identification, substrate specificity, and inhibition of the Streptococcus pneumoniae beta-ketoacyl-acyl carrier protein synthase III (FabH).

In the bacterial type II fatty acid synthase system, beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) catalyzes the condensation of acetyl-CoA with malonyl-ACP. We have identified, expressed, and characterized the Streptococcus pneumoniae homologue of Escherichia coli FabH. S. pneumoniae FabH is approximately 41, 39, and 38% identical in amino acid sequence to Bacillus subtilis, E. coli, and Hemophilus influenzae FabH, respectively. The His-Asn-Cys catalytic triad present in other FabH molecules is conserved in S. pneumoniae FabH. The apparent K(m) values for acetyl-CoA and malonyl-ACP were determined to be 40.3 and 18.6 microm, respectively. Purified S. pneumoniae FabH preferentially utilized straight short-chain CoA primers. Similar to E. coli FabH, S. pneumoniae FabH was weakly inhibited by thiolactomycin. In contrast, inhibition of S. pneumoniae FabH by the newly developed compound SB418011 was very potent, with an IC(50) value of 0.016 microm. SB418011 also inhibited E. coli and H. influenzae FabH with IC(50) values of 1.2 and 0.59 microm, respectively. The availability of purified and characterized S. pneumoniae FabH will greatly aid in structural studies of this class of essential bacterial enzymes and facilitate the identification of small molecule inhibitors of type II fatty acid synthase with the potential to be novel and potent antibacterial agents active against pathogenic bacteria.

Expression, purification, and crystallization of the Escherichia coli selenomethionyl beta-ketoacyl-acyl carrier protein synthase III.

Bacterial beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called FabH) catalyzes the condensation and transacylation of acetyl-CoA with malonyl-ACP. In order to understand the mode of enzyme/substrate interaction and design small molecule inhibitors, we have expressed, purified, and crystallized a selenomethionyl-derivative of E. coli KAS III. Several lines of evidence confirmed that purified selenomethionyl KAS III was homogenous, stably folded, and enzymatically active. Dynamic light scattering, size exclusion chromatography, and mass spectrometry results indicated that selenomethionyl KAS III is a noncovalent homodimer. Diffraction quality crystals of selenomethionyl KAS III/acetyl-CoA complex, which grew overnight to a size of 0.2 mm(3), belonged to the tetragonal space group P4(1)2(1)2.

Cyclic GMP potentiation by WIN 58237, a novel cyclic nucleotide phosphodiesterase inhibitor.

The objectives of this study were to determine the potency and selectivity of the structurally novel cyclic nucleotide phosphodiesterase (PDE) inhibitor, WIN 58237 (1-cyclopentyl-3-methyl-6-(4- pyridyl)pyrazolo[3,4-d]pyrimidin-4-(5H)-one), and to determine if this compound possesses cyclic GMP (cGMP) PDE inhibitory activity in vitro and in vivo. WIN 58237 is a competitive inhibitor of cGMP PDE V from canine aorta, with a Ki value of 170 nM. It is a relatively less potent inhibitor of calmodulin-sensitive PDE I and cGMP-inhibitable cyclic AMP PDE III; but does inhibit cyclic AMP PDE IV with an IC50 value of approximately 300 nM. In vitro, WIN 58237 is a functional cGMP PDE inhibitor at submicromolar concentrations as evident by potentiation of both sodium nitroprusside- and atrial natriuretic factor-mediated vasorelaxation of contracted, endothelial-denuded rat aortic rings. Moreover, WIN 58237 possesses vasorelaxant activity in the presence of an intact endothelium or nitric oxide. Similar results are evident in vivo, as WIN 58237 (0.3-3.0 mg/kg i.v.) decreases mean arterial pressure in conscious spontaneously hypertensive rats with an associated increase in vascular (aortic) cGMP content in vivo. Both the decrease in mean arterial blood pressure and increase in aortic cGMP content are attenuated by the nitric oxide synthase inhibitor, N omega-nitro-l-arginine. However, WIN 58237 may possess an additional depressor mechanism of action. WIN 58237 restores vasorelaxation responsiveness to nitroglycerin in vitro and in vivo in models of vascular tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of iodixanol and iopamidol on hemodynamic and cardiac electrophysiologic parameters in vitro and in vivo.

Iodixanol is a new, nonionic, dimeric contrast medium which, in concentrations appropriate for radiographic use, is hypotonic with respect to plasma. The purpose of these in vivo and in vitro studies was to compare the effects of iopamidol, iodixanol formulated to isotonicity with sodium salts (sodium formulation), and iodixanol formulated to isotonicity with sodium, calcium, and magnesium salts (cationic formulation) on hemodynamic and electrophysiologic parameters. In vitro, the spontaneous rate of contraction by guinea pig right atrial and force development by right ventricular papillary muscles were evaluated in the presence of 1% to 100% (v/v) of the three contrast media. Iopamidol significantly (P less than .05) decreased the rate of atrial contraction to a greater extent than either formulation of iodixanol. Iopamidol decreased papillary muscle force development more than the sodium formulation of iodixanol (P less than .05). The cationic formulation of iodixanol had little effect (less than 30% change) on papillary muscle force development at concentrations up to 100%. The contrast media were also injected into the left coronary arteries of open-chest, anesthetized dogs at 0.8 mL/second for 5 to 30 seconds. All contrast media increased (P less than .05) systolic blood pressure (SBP), mean arterial pressure (MAP), and peak left ventricular pressure (LVP). Iopamidol increased LVP and LV end diastolic pressure to a greater extent (P less than .05) than the cationic formulation of iodixanol. We conclude that iopamidol affected cardiovascular parameters more than iodixanol.

Inhibition of low Km cGMP phosphodiesterases and Ca+(+)-regulated protein kinases and relationship to vasorelaxation by cicletanine.

In the present studies we sought to determine if cicletanine, which is an antihypertensive agent of unknown mechanism, could alter cGMP metabolism via inhibition of cGMP phosphodiesterases (PDE) in vascular smooth muscle. Cicletanine was determined to be a mixed (competitive, noncompetitive) inhibitor of both calmodulin-regulated and cGMP-specific PDEs from monkey aortic smooth muscle with Ki values of 450 to 700 microM. Cicletanine also potentiated vasorelaxation by the guanylate cyclase activators sodium nitroprusside and atrial natriuretic peptide in isolated rat aortas. Potentiation was not dependent upon the contractile agonists nor was it indomethacin-sensitive. Neither potentiation nor inhibition of cGMP PDEs was stereoselective. Methylene blue attenuated a component of cicletanine-induced vasorelaxation, but did not completely obviate relaxation. Both cicletanine and the cGMP-PDE inhibitor zaprinast potentiated sodium nitroprusside-mediated cGMP formation and relaxation, although the increase in cGMP content was markedly greater with zaprinast compared to cicletanine. In further studies, cicletanine did not potentiate cGMP activation of cGMP-dependent protein kinase, but did inhibit calmodulin-activated myosin light chain kinase and protein kinase C at relatively high concentrations (approximately 1 mM). In summary, these data demonstrate that cicletanine inhibits vascular cGMP PDEs, potentiates vasorelaxation, and to a limited extent, cGMP formation by guanylate cyclase activators in vascular smooth muscle. However, these relationships for cicletanine are dissimilar from the reference cGMP PDE inhibitor, zaprinast. Thus, other mechanisms may also contribute to the vasorelaxant action of cicletanine.