Bactericidal activity and mechanism of action of AZD5847, a novel oxazolidinone for treatment of tuberculosis.

Treatment of tuberculosis (TB) is impaired by the long duration and complexity of therapy and the rising incidence of drug resistance. There is an urgent need for new agents with improved efficacy, safety, and compatibility with combination chemotherapies. Oxazolidinones offer a potential new class of TB drugs, and linezolid-the only currently approved oxazolidinone-has proven highly effective against extensively drug-resistant (XDR) TB in experimental trials. However, widespread use of linezolid is prohibited by its significant toxicities. AZD5847, a novel oxazolidinone, demonstrates improved in vitro bactericidal activity against both extracellular and intracellular M. tuberculosis compared to that of linezolid. Killing kinetics in broth media and in macrophages indicate that the rate and extent of kill obtained with AZD5847 are superior to those obtained with linezolid. Moreover, the efficacy of AZD5847 was additive when tested along with a variety of conventional TB agents, indicating that AZD5847 may function well in combination therapies. AZD5847 appears to function similarly to linezolid through impairment of the mycobacterial 50S ribosomal subunit. Future studies should be undertaken to further characterize the pharmacodynamics and pharmacokinetics of AZD5847 in both in vitro and animal models as well is in human clinical trials.

Protein A-activated rat splenic lymphocyte proliferation involves tyrosine kinase-phospholipase C-protein kinase C pathway.

Protein A (PA) of Staphylococcus aureus was long been known for its affinity towards the Fc domain of immunoglobulin G. It is now well established that PA is a potent biological response modifier showing simultaneously antitumor, antitoxic, and anticarcinogenic properties. This bacterial protein was also observed to stimulate production of cytokines. But the molecular mechanism(s) of immunocyte activation by PA still remained essentially unknown. In this report, we demonstrate a hitherto undescribed role of PA as a signal inducer in rat splenic lymphocytes. Our studies describe that PA induces transition of G0/G1 to S and G2/M phases of cell cycle, thus ultimately stimulating splenic lymphocyte proliferation. It has also been revealed that PA binds to rat splenic lymphocytes in a dose dependent manner and stimulates proliferation via tyrosine kinase-phospholipase C (PLC)-Ca2+-dependent protein kinase C (PKC) pathway. These observations will be of valid help in correlating the immunostimulatory activities of PA with the molecular mechanism(s) of its action.

Protein kinase calpha regulates human monocyte O-2 production and low density lipoprotein lipid oxidation.

Our previous studies have shown that human native low density lipoprotein (LDL) can be oxidized by activated human monocytes. In this process, both activation of protein kinase C (PKC) and induction of superoxide anion (O-2) production are required. PKC is a family of isoenzymes, and the functional roles of individual PKC isoenzymes are believed to differ based on subcellular location and distinct responses to regulatory signals. We have shown that the PKC isoenzyme that is required for both monocyte O-2 production and oxidation of LDL is a member of the conventional PKC group of PKC isoenzymes (Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508-17515). The conventional PKC group includes PKCalpha, PKCbetaI, PKCbetaII, and PKCgamma. With the exception of PKCgamma, each of these isoenzymes was detected in human monocytes. In these studies, we investigated the requirement for select PKC isoenzymes in the process of monocyte-mediated LDL lipid oxidation. Our data indicate that PKC activity was rapidly induced upon monocyte activation with the majority of the activity residing in the membrane/particulate fraction. This enhanced PKC activity was sustained for up to 24 h after activation. PKCalpha, PKCbetaI, and PKCbetaII protein levels were induced upon monocyte activation, and PKCalpha and PKCbetaII substantially shifted their location from the cytosol to the particulate/membrane fraction. To distinguish between these isoenzymes for regulating monocyte O-2 production and LDL oxidation, PKCalpha or PKCbeta isoenzyme-specific antisense oligonucleotides were used to selectively suppress isoenzyme expression. We found that suppression of PKCalpha expression inhibited both monocyte-mediated O-2 production and LDL lipid oxidation by activated human monocytes. In contrast, inhibition of PKCbeta expression (including both PKCbetaI and PKCbetaII) did not affect O-2 production or LDL lipid oxidation. Further studies demonstrated that the respiratory burst oxidase responsible for O-2 production remained functionally intact in monocytes with depressed levels of PKCalpha because O-2 production could be restored by treating the monocytes with arachidonic acid. Taken together, our data reveal that PKCalpha, and not PKCbetaI or PKCbetaII, is the predominant isoenzyme required for O-2 production and maximal oxidation of LDL by activated human monocytes.

Mechanism of protein A-induced amelioration of toxicity of anti-AIDS drug, zidovudine.

Long-term treatment with 3-azido-3-deoxy thymidine (AZT) is often associated with myelosuppression. In AZT-treated Swiss mice, similar toxicological manifestations in terms of reduction of red blood and white blood cell counts and hemoglobin content had been observed as in AZT-treated AIDS patients. Pretreatment of animals with Protein A (PA) of Staphylococcus aureus Cowan I (1 microgram/ml), twice a week for two weeks, alleviated such hematopoietic toxicity due to AZT. AZT-induced reduction in colony-forming unit-erythroid (CFU-E) and colony-forming unit-granulocyte monocyte (CFU-GM) were also reversed by the combined treatment of AZT and PA. PA treatment showed an increased level of erythropoietin in the blood plasma, and cellularity of spleen, thymus, and bonemarrow was also increased in the group receiving combined treatment (PA+AZT), higher than that in the AZT group. AZT or its metabolites inhibited the activities of liver microsomal monooxygenases, which, however, could be regenerated in an accelerated manner by pretreatment of mice with PA. Moreover, the PA-treated group showed an accelerated clearance of AZT and/or its metabolites. These results suggest that such an immunopharmacologic approach might substantially reduce the toxic effects of drugs, such as AZT.