Inhibition of MCF-7 breast cancer cell proliferation and in vivo steroid sulphatase activity by 2-methoxyoestradiol-bis-sulphamate

The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2) inhibits the growth of breast cancer cells and is also a potent anti-angiogenic agent. We have previously shown that the 3-sulphamoylated derivatives of 2-methoxyoestrogens are more potent than the non-sulphamoylated compounds. In this study, we have compared the abilities of 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-MeOE2 to inhibit the growth of MCF-7 breast cancer cells. Both compounds inhibited cell growth with the IC(50) for 2-MeOE2bisMATE (0.4 microM) being six-fold lower than that for 2-MeOE2 (2.5 microM). Oestrogen sulphamates are potent inhibitors of steroid sulphatase (STS) activity. 2-MeOE2bisMATE was found to retain its STS inhibitory activity and in a placental microsome assay system it was equipotent with oestrone-3-O-sulphamate (EMATE). An in vivo study was also carried out to compare the potency of 2-MeOE2bisMATE with that of EMATE and the non-steroidal STS inhibitor, 667 coumarin sulphamate (667 COUMATE). After a single oral dose (10mg/kg) some recovery of STS activity was detected by day 3 (10%) with activity partially restored (55%) by day 7 after administration of 667 COUMATE. For the other two steroidal compounds, STS activity remained almost completely inactivated for up to 5 days with complete restoration of activity occurring by day 15. The anti-proliferative and STS inhibitory properties of 2-MeOE2bisMATE suggest that it has considerable potential for development as a novel anti-cancer drug.     

Integrative approach to pain genetics identifies pain sensitivity loci across diseases

Identifying human genes relevant for the processing of pain requires difficult-to-conduct and expensive large-scale clinical trials. Here, we examine a novel integrative paradigm for data-driven discovery of pain gene candidates, taking advantage of the vast amount of existing disease-related clinical literature and gene expression microarray data stored in large international repositories. First, thousands of diseases were ranked according to a disease-specific pain index (DSPI), derived from Medical Subject Heading (MESH) annotations in MEDLINE. Second, gene expression profiles of 121 of these human diseases were obtained from public sources. Third, genes with expression variation significantly correlated with DSPI across diseases were selected as candidate pain genes. Finally, selected candidate pain genes were genotyped in an independent human cohort and prospectively evaluated for significant association between variants and measures of pain sensitivity. The strongest signal was with rs4512126 (5q32, ABLIM3, P = 1.3×10⁻¹⁰) for the sensitivity to cold pressor pain in males, but not in females. Significant associations were also observed with rs12548828, rs7826700 and rs1075791 on 8q22.2 within NCALD (P = 1.7×10⁻⁴, 1.8×10⁻⁴, and 2.2×10⁻⁴ respectively). Our results demonstrate the utility of a novel paradigm that integrates publicly available disease-specific gene expression data with clinical data curated from MEDLINE to facilitate the discovery of pain-relevant genes. This data-derived list of pain gene candidates enables additional focused and efficient biological studies validating additional candidates.     

Inhibition of 5′-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

Background

The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.

Methods and Results

We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.

Conclusions

Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.     

An Analysis of Regulatory T-Cell and Th-17 Cell Dynamics during Cytomegalovirus Replication in Solid Organ Transplant Recipients

Background

CMV-specific T-cells are crucial to control CMV-replication post-transplant. Regulatory T-cells (T-regs) are associated with a tolerant immune state and may contribute to CMV-replication. However, T-cell subsets such as T-regs and IL-17 producing T-cells (Th-17) are not well studied in this context. We explored T-regs and Th-17 frequencies during CMV-replication after transplantation.

Methods

We prospectively evaluated 30 transplant patients with CMV-viremia. We quantified CMV-specific CD4⁺ and CD8⁺ T-cells, T-regs (CD4⁺CD25⁺FoxP3⁺) and Th-17 frequencies using flow-cytometry and followed patients requiring anti-viral treatment. Two subsets were compared: anti-viral treatment requirement (n = 20) vs. spontaneous clearance of viremia (n = 10).

Results

Higher initial CMV-specific CD4⁺ T-cells and lower T-regs were observed in patients with spontaneous clearance (p = 0.043; p = 0.021 respectively). Using a ratio of CMV-specific CD4⁺ T-cells to T-regs allowed prediction of viral clearance with 80% sensitivity and 90% specificity (p = 0.001). One month after stop of treatment, the same correlation was observed in patients protected from CMV-relapse. The ratio of CMV-specific CD4⁺ T-cells to T-regs allowed prediction of relapse with 85% sensitivity and 86% specificity (p = 0.004). Th-17 responses were not correlated with virologic outcomes.

Conclusions

This study provides novel insights into T-regs and Th-17 subpopulations during CMV-replication after transplantation. These preliminary data suggest that measurement of CMV-specific CD4⁺ T-cells together with T-regs has value in predicting spontaneous clearance of viremia and relapse.     

Phasic respiratory modulation of pharyngeal collapsibility via neuromuscular mechanisms in rats

Obstructive sleep apnea patients experience recurrent upper airway (UA) collapse due to decreases in the UA dilator muscle activity during sleep. In contrast, activation of UA dilators reduces pharyngeal critical pressure (Pcrit, an index of pharyngeal collapsibility), suggesting an inverse relationship between pharyngeal collapsibility and dilator activity. Since most UA muscles display phasic respiratory activity, we hypothesized that pharyngeal collapsibility is modulated by respiratory drive via neuromuscular mechanisms. Adult male Sprague-Dawley rats were anesthetized, vagotomized, and ventilated (normocapnia). In one group, integrated genioglossal activity, Pcrit, and maximal airflow (V(max)) were measured at three expiration and five inspiration time points within the breathing cycle. Pcrit was closely and inversely related to phasic genioglossal activity, with the value measured at peak inspiration being the lowest. In other groups, the variables were measured during expiration and peak inspiration, before and after each of five manipulations. Pcrit was 26% more negative (-15.0 ± 1.0 cmH(2)O, -18.9 ± 1.2 cmH(2)O; n = 23), V(max) was 7% larger (31.0 ± 1.0 ml/s, 33.2 ± 1.1 ml/s), nasal resistance was 12% bigger [0.49 ± 0.05 cmH(2)O/(ml/s), 0.59 ± 0.05 cmH(2)O/(ml/s)], and latency to induced UA closure was 14% longer (55 ± 4 ms, 63 ± 5 ms) during peak inspiration vs. expiration (all P < 0.005). The expiration-inspiration difference in Pcrit was abolished with neuromuscular blockade, hypocapnic apnea, or death but was not reduced by the superior laryngeal nerve transection or altered by tracheal displacement. Collectively, these results suggest that pharyngeal collapsibility is moment-by-moment modulated by respiratory drive and this phasic modulation requires neuromuscular mechanisms, but not the UA negative pressure reflex or tracheal displacement by phasic lung inflation.     

Safety and pharmacodynamic effects of a pharmacological chaperone on α-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies

Background

Fabry disease (FD) is a genetic disorder resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A), which leads to globotriaosylceramide (GL-3) accumulation in multiple tissues. We report on the safety and pharmacodynamics of migalastat hydrochloride, an investigational pharmacological chaperone given orally at 150 mg every-other-day.

Methods

Two open-label uncontrolled phase 2 studies of 12 and 24 weeks (NCT00283959 and NCT00283933) in 9 males with FD were combined. At multiple time points, α-Gal A activity and GL-3 levels were quantified in blood cells, kidney and skin. GL-3 levels were also evaluated through skin and renal histology.

Results

Compared to baseline, increased α-Gal A activity of at least 50% was demonstrated in blood, skin and kidney in 6 of 9 patients. Patients’ increased α-Gal A activities paralleled the α-Gal A increases observed in vitro in HEK-293 cells transfected with the corresponding mutant form of the enzyme. The same 6 patients who demonstrated increases of α-Gal A activity also had GL-3 reduction in skin, urine and/or kidney, and had α-Gal A mutations that responded in transfected cells incubated with the drug. The 3 patients who did not show a consistent response in vivo had α-Gal A mutations that did not respond to migalastat HCl in transfected cells. Migalastat HCl was well tolerated.

Conclusions

Migalastat HCl is a candidate pharmacological chaperone that provides a novel genotype-specific treatment for FD. It enhanced α-Gal A activity and resulted in GL-3 substrate decrease in patients with responsive GLA mutations. Phase 3 studies are ongoing.

Trial registration

Clinicaltrial.gov: NCT00283959 and NCT00283933

     

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.