Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity.

Novel muraminomicin derivatives with antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) were synthesized by esterification of the hydroxy group on the diazepanone ring of muraminomicin Z1. Compound 1b (DS14450354) possessed a diheptoxybenzyl-ß-Alanyl-ß-Alanyl group and exhibited minimum inhibitory concentrations (MICs) against MRSA comparable to those against methicillin-susceptible S. aureus (MSSA). The MICs that inhibited 50 and 90% of the strains were 1 and 2 µg/mL, respectively. Compound 1a (DS60182922) possessed an aminoethylbenzoyldodecylglycyl moiety and showed bactericidal activity against MSSA Smith. The bactericidal activity of 1a against MRSA 10925 was comparatively lower, whilst 1b exhibited dose-dependent bactericidal activity against MRSA 10925. The mutation frequency of 1b was lower than that of 1a. An amino acid substitution (F226I) was observed in MraY mutants isolated from culture plates containing 1a or 1b. Subcutaneous 1a and 1b administration showed good therapeutic efficacy in murine systemic infection models with MSSA Smith and MRSA 10925, comparable to that of vancomycin, suggesting that the novel muraminomicin derivatives may be effective therapeutic agents against MRSA that warrant further investigation. A scheme for the formulation of the key ester intermediate, requiring no HPLC preparation, was also established.

Muraminomicins, new lipo-nucleoside antibiotics from Streptosporangium sp. SANK 60501-structure elucidations of muraminomicins and supply of the core component for derivatization.

We screened for bacterial phospho-N-acetylmuramyl-pentapeptide-translocase (MraY: EC 2.7.8.13) inhibitors with the aim of discovering novel antibiotics and observed inhibitory activity in the culture broth of an actinomycete, SANK 60501. The active compounds, muraminomicins A, B, C, D, E1, E2, F, G, H, and I exhibited strong inhibitory activity against MraY with IC50 values of 0.0105, 0.0068, 0.0104, 0.0099, 0.0115, 0.0109, 0.0089, 0.0134, 0.0186, and 0.0094 µg ml-1, respectively. Although muraminomicin F exhibited favorable antibacterial activity against drug-resistant Gram-positive bacteria, this activity was reduced with the addition of serum. To efficiently supply the core component for chemical modification studies, production was carried out in a controlled trial by adding myristic acid to the medium, and a purification method suitable for large-scale production was successfully developed.

High and continuous exposure of laninamivir, an anti-influenza drug, may work suppressively to generate low-susceptibility mutants in animals.

Laninamivir octanoate (Inavir(®); Daiichi Sankyo, Tokyo, Japan) is an anti-influenza drug that provides complete treatment by a single inhalation. It works as a long-acting neuraminidase (NA) inhibitor by means of high and continuous exposure of laninamivir, its active metabolite, in the lungs of mice after intranasal administration. Even after 6 days after intranasal administration of 236 µg/kg laninamivir octanoate, the concentration of laninamivir in the lungs was maintained more than 2-3 orders higher than 50% inhibitory concentrations of laninamivir to N1 NAs, about 2 orders higher than N2 NA of seasonal influenza A viruses, and more than about 50 times higher than influenza B virus NA. From A/H1N1 influenza virus-infected and laninamivir octanoate-treated mice, no low-susceptibility mutants to laninamivir were obtained. In contrast, four different mutants to oseltamivir were obtained from mice administered oseltamivir phosphate, which required repeated administration for treatment under the experimental condition, showing similar virus load reduction between both compounds. This finding suggested the unique characteristics of laninamivir octanoate in mice may work suppressively to generate low-susceptibility mutants.

In vivo pharmacodynamic activity of tomopenem (formerly CS-023) against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in a murine thigh infection model.

Tomopenem (formerly CS-023) is a novel carbapenem with broad-spectrum activities against diverse hospital pathogens, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). We examined the in vivo pharmacodynamic characteristics of tomopenem against P. aeruginosa and MRSA by using a neutropenic murine thigh infection model with P. aeruginosa 12467 (MIC, 1 µg/ml) and MRSA 12372 (MIC, 2 µg/ml). The mice had 10(6) to 10(7) CFU/thigh of each strain 2 h after inoculation and were treated for 24 h with a fractionated administration of tomopenem given at intervals of 3, 6, 12, and 24 h. The serum protein binding of tomopenem was 17.4%. The efficacy of tomopenem in both infection models was enhanced by frequent dosing, which indicates that the efficacy is driven by the time above MIC (T(MIC)). In a sigmoid model, the cumulative percentages of the 24-h period that the concentrations of free, unbound fractions of the drug exceeded the MIC under steady-state pharmacokinetic conditions (f%T(MIC)s) were best correlated with efficacy when R(2) was 0.79 and 0.86 against P. aeruginosa and MRSA, respectively. Other pharmacokinetic and pharmacodynamic (PK-PD) indexes for the free, unbound fractions, the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC) and the maximum concentration of the drug in serum divided by the MIC (C(max)/MIC), showed poor correlation with efficacy when R(2) was ≤0.42. The f%T(MIC) values required for a static effect, 1-log kill, and 2-log kill against P. aeruginosa were 29, 39, and 51, respectively, which were similar to those for meropenem, for which the values were 24, 33, and 45, respectively. Against MRSA, the values for tomopenem were 27, 35, and 47. In conclusion, the pharmacodynamic characteristics of tomopenem were similar to those of meropenem against P. aeruginosa, and there was no difference between the target values for P. aeruginosa and MRSA required for efficacy in this study.

Laninamivir prodrug CS-8958, a long-acting neuraminidase inhibitor, shows superior anti-influenza virus activity after a single administration.

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for use for the treatment and prophylaxis of influenza. We have reported on laninamivir (code name, R-125489), a novel neuraminidase inhibitor, and have discovered that the laninamivir prodrug CS-8958 worked as a long-acting neuraminidase inhibitor in a mouse influenza virus infection model when it is intranasally administered. In this study, CS-8958 was administered just once 7 days before infection and showed significant efficacy in vivo. The efficacy of a single administration of CS-8958 after viral infection was then compared with that of repeated administrations of oseltamivir phosphate or zanamivir in mice and ferrets. CS-8958 showed efficacy superior or similar to the efficacies of the two licensed NA inhibitors. CS-8958 also significantly reduced the titers of an oseltamivir-resistant H1N1 virus with a neuraminidase H274Y substitution in a mouse infection model. These results suggest that since CS-8958 is characteristically long lasting in the lungs, it may be ideal for the prophylaxis and treatment of influenza.

CS-8958, a prodrug of the new neuraminidase inhibitor R-125489, shows long-acting anti-influenza virus activity.

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for the treatment of and prophylaxis against influenza. In this paper, the new potent NA inhibitor R-125489 is reported for the first time. R-125489 inhibited the NA activities of various type A and B influenza viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses. The survival effect of R-125489 was shown to be similar to that of zanamivir when administered intranasally in a mouse influenza virus A/Puerto Rico/8/34 infection model. Moreover, we found that the esterified form of R-125489 showed improved efficacy compared to R-125489 and zanamivir, depending on the acyl chain length, and that 3-(O)-octanoyl R-125489 (CS-8958) was the best compound in terms of its life-prolonging effect (P < 0.0001, compared to zanamivir) in the same infection model. A prolonged survival effect was observed after a single administration of CS-8958, even if it was given 7 days before infection. It is suggested that intranasally administered CS-8958 works as a long-acting NA inhibitor and shows in vivo efficacy as a result of a single intranasal administration.

The ClpXP ATP-dependent protease regulates flagellum synthesis in Salmonella enterica serovar typhimurium.

The ClpXP protease is a member of the ATP-dependent protease family and plays a dynamic role in the control of availability of regulatory proteins and the breakdown of abnormal and misfolded proteins. The proteolytic activity is rendered by the ClpP component, while the substrate specificity is determined by the ClpX component that has ATPase activity. We describe here a new role of the ClpXP protease in Salmonella enterica serovar Typhimurium in which ClpXP is involved in the regulation of flagellum synthesis. Cells deleted for ClpXP show "hyperflagellate phenotype," exhibit overproduction of the flagellar protein, and show a fourfold increase in the rate of transcription of the fliC encoding flagellar filament. The assay for promoter activity of the genes responsible for expression of the fliC showed that the depletion of ClpXP results in dramatic enhancement of the expression of the fliA encoding sigma factor final sigma(28), leaving the expression level of the flhD master operon lying at the top of the transcription hierarchy of flagellar regulon almost normal. These results suggest that the ClpXP may be responsible for repressing the expression of flagellar regulon through the control of the FlhD/FlhC master regulators at the posttranscriptional and/or posttranslational levels. Proteome analysis of proteins secreted from the mutant cells deficient for flhDC and clpXP genes demonstrated that the DeltaflhD mutation abolished the enhanced effect by DeltaclpXP mutation on the production of flagellar proteins, suggesting that the ClpXP possibly defines a regulatory pathway affecting the expression of flagellar regulon that is dependent on FlhD/FlhC master regulators.

The ATP-dependent lon protease of Salmonella enterica serovar Typhimurium regulates invasion and expression of genes carried on Salmonella pathogenicity island 1.

An early step in the pathogenesis of Salmonella enterica serovar Typhimurium infection is bacterial penetration of the intestinal epithelium. Penetration requires the expression of invasion genes found in Salmonella pathogenicity island 1 (SPI1). These genes are controlled in a complex manner by regulators in SPI1, including HilA and InvF, and those outside SPI1, such as two-component regulatory systems and small DNA-binding proteins. We report here that the expression of invasion genes and the invasive phenotype of S. enterica serovar Typhimurium are negatively regulated by the ATP-dependent Lon protease, which is known to be a major contributor to proteolysis in Escherichia coli. A disrupted mutant of lon was able to efficiently invade cultured epithelial cells and showed increased production and secretion of three identified SPI1 proteins, SipA, SipC, and SipD. The lon mutant also showed a dramatic enhancement in transcription of the SPI1 genes hilA, invF, sipA, and sipC. The increases ranged from 10-fold to almost 40-fold. It is well known that the expression of SPI1 genes is also regulated in response to several environmental conditions. We found that the disruption of lon does not abolish the repression of hilA and sipC expression by high-oxygen or low-osmolarity conditions, suggesting that Lon represses SPI1 gene expression by a regulatory pathway independent of these environmental signals. Since HilA is thought to function as a central regulator of SPI1 gene expression, it is speculated that Lon may regulate SPI1 gene expression by proteolysis of putative factors required for activation of hilA expression.