Moxifloxacin Concentration and Proteomic Analysis of Aqueous Humor in Human Uveitis Associated with Oral Moxifloxacin Therapy

PURPOSE: The aim was to report the aqueous humor moxifloxacin concentration and proteome profile of an individual with bilateral uveitis-like syndrome with pigment dispersion. METHODS: Multiple reactions monitoring mass spectrometry quantified the aqueous concentration of moxifloxacin in the affected individual. Shotgun proteomic analysis performed via liquid chromatography tandem mass spectrometry (LC-MS/MS) defined the protein profile in the affected individual and unaffected control samples. RESULTS: Moxifloxacin was present at higher than expected levels in aqueous humor 18 days following oral administration. One-third of the proteins were identified by significantly lower spectral counts in the aqueous of the individual with moxifloxacin associated uveitis compared to the unaffected control. CONCLUSION: Moxifloxacin was detected in aqueous humor 18 days following the completion of oral administration. These results suggest that moxifloxacin toxicity may be responsible for the uveitis-like syndrome with pigment dispersion syndrome induced by moxifloxacin therapy

Crystal Structures of ABL-Related Gene (ABL2) in Complex with Imatinib, Tozasertib (VX-680), and a Type I Inhibitor of the Triazole Carbothioamide Class†

ABL2 (also known as ARG (ABL related gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in human neoplastic diseases and is deregulated in solid tumors. Oncogenic gene translocations occur in acute leukemia. So far no structural information for ABL2 has been reported. To elucidate structural determinants for inhibitor interaction, we determined the cocrystal structure of ABL2 with the oncology drug imatinib. Interestingly, imatinib not only interacted with the ATP binding site of the inactive kinase but was also bound to the regulatory myristate binding site. This structure may therefore serve as a tool for the development of allosteric ABL inhibitors. In addition, we determined the structures of ABL2 in complex with VX-680 and with an ATP-mimetic type I inhibitor, which revealed an interesting position of the DFG motif intermediate between active and inactive conformations, that may also serve as a template for future inhibitor design

Combined effects of GSTP1 and MRP1 in melanoma drug resistance

Glutathione-S-transferase Pi1 (GSTP1) and multidrug resistance protein 1 (MRP1) are overexpressed in melanoma, a skin cancer notoriously resistant to all current modalities of cancer therapy. To investigate the involvement of these detoxifying enzymes in the drug resistance of melanoma, an inducible (Tet-On™ system) antisense (AS) RNA strategy was used to specifically inhibit GSTP1 expression in A375 cells, a human melanoma cell line expressing high levels of GSTP1 and MRP1. Stable transfectant clones were established and analysed for GSTP1 inhibition by AS RNA. The clone A375-ASPi1, presenting a specific 40% inhibition of GSTP1 expression in the presence of doxycycline, was selected. Lowering the GSTP1 level significantly increased (about 3.3-fold) the sensitivity of A375-ASPi1 cells to etoposide. Inhibitors of glutathione synthesis (BSO), GSTs (curcumin, ethacrynic acid), and also of MRPs (MK571, sulphinpyrazone) improved the sensitising effect of GSTP1 AS RNA. All these inhibitors had stronger sensitising effects in control cells expressing high GSTP1 level (A375-ASPi1 cells in the absence of doxycycline). In conclusion, GSTP1 can act in a combined fashion with MRP1 to protect melanoma cells from toxic effects of etoposide

Farnesol-Induced Apoptosis in Candida albicans Is Mediated by Cdr1-p Extrusion and Depletion of Intracellular Glutathione

Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent

MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

<p>Abstract</p> <p>Background</p> <p>Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3).</p> <p>Methods</p> <p>We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS.</p> <p>Results</p> <p>Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed <it>MRP3 </it>mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined <it>MRP3</it>-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of <it>MRP3 </it>RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002).</p> <p>Conclusions</p> <p>Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.</p

Proteins with Complex Architecture as Potential Targets for Drug Design: A Case Study of Mycobacterium tuberculosis

Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel drug targets and drugs with new mechanisms of action. However, further insights are needed to establish automated protocols for target selection based on the available complete genome sequences. In the present study, we perform complete proteome level comparisons between M. tuberculosis, mycobacteria, other prokaryotes and available eukaryotes based on protein domains, local sequence similarities and protein disorder. We show that the enrichment of certain domains in the genome can indicate an important function specific to M. tuberculosis. We identified two families, termed pkn and PE/PPE that stand out in this respect. The common property of these two protein families is a complex domain organization that combines species-specific regions, commonly occurring domains and disordered segments. Besides highlighting promising novel drug target candidates in M. tuberculosis, the presented analysis can also be viewed as a general protocol to identify proteins involved in species-specific functions in a given organism. We conclude that target selection protocols should be extended to include proteins with complex domain architectures instead of focusing on sequentially unique and essential proteins only

BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure

The anti-proliferative and anti-angiogenic properties of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2), are enhanced in a series of sulphamoylated derivatives of 2-MeOE2. To investigate possible mechanisms of resistance to these compounds, a cell line, A2780.140, eightfold less sensitive to the 3,17-O,O-bis-sulphamoylated derivative, STX140, was derived from the A2780 ovarian cancer cell line by dose escalation. Other cell lines tested did not develop STX140 resistance. RT–PCR and immunoblot analysis demonstrated that breast cancer resistance protein (BCRP) expression is dramatically increased in A2780.140 cells. The cells are cross-resistant to the most structurally similar bis-sulphamates, and to BCRP substrates, mitoxantrone and doxorubicin; but they remain sensitive to taxol, an MDR1 substrate, and to all other sulphamates tested. Sensitivity can be restored using a BCRP inhibitor, and this pattern of resistance is also seen in a BCRP-expressing MCF-7-derived cell line, MCF-7.MR. In mice bearing wild-type (wt) and BCRP-expressing tumours on either flank, both STX140 and mitoxantrone inhibited the growth of the MCF-7wt xenografts, but only STX140 inhibited growth of the MCF-7.MR tumours. In conclusion, STX140, a promising orally bioavailable anti-cancer agent in pre-clinical development, is highly efficacious in BCRP-expressing xenografts. This is despite an increase in BCRP expression in A2780 cells in vitro after chronic dosing with STX140