Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.     

National Use of Safety-Net Clinics for Primary Care among Adults with Non-Medicaid Insurance in the United States

Objective

To describe the prevalence, characteristics, and predictors of safety-net use for primary care among non-Medicaid insured adults (i.e., those with private insurance or Medicare).

Methods

Cross-sectional analysis using the 2006–2010 National Ambulatory Medical Care Surveys, annual probability samples of outpatient visits in the U.S. We estimated national prevalence of safety-net visits using weighted percentages to account for the complex survey design. We conducted bivariate and multivariate logistic regression analyses to examine characteristics associated with safety-net clinic use.

Results

More than one-third (35.0%) of all primary care safety-net clinic visits were among adults with non-Medicaid primary insurance, representing 6,642,000 annual visits nationally. The strongest predictors of safety-net use among non-Medicaid insured adults were: being from a high-poverty neighborhood (AOR 9.53, 95% CI 4.65–19.53), being dually eligible for Medicare and Medicaid (AOR 2.13, 95% CI 1.38–3.30), and being black (AOR 1.97, 95% CI 1.06–3.66) or Hispanic (AOR 2.28, 95% CI 1.32–3.93). Compared to non-safety-net users, non-Medicaid insured adults who used safety-net clinics had a higher prevalence of diabetes (23.5% vs. 15.0%, p<0.001), hypertension (49.4% vs. 36.0%, p<0.001), multimorbidity (≥2 chronic conditions; 53.5% vs. 40.9%, p<0.001) and polypharmacy (≥4 medications; 48.8% vs. 34.0%, p<0.001). Nearly one-third (28.9%) of Medicare beneficiaries in the safety-net were dual eligibles, compared to only 6.8% of Medicare beneficiaries in non-safety-net clinics (p<0.001).

Conclusions

Safety net clinics are important primary care delivery sites for non-Medicaid insured minority and low-income populations with a high burden of chronic illness. The critical role of safety-net clinics in care delivery is likely to persist despite expanded insurance coverage under the Affordable Care Act.     

Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT

The cooperation of Bacillus subtilis strain DKT and Comamonas testosteroni KT5 was investigated for biofilm development and toluenes and chlorobenzenes degradation. Bacillus subtilis strain DKT and C. testosteroni KT5 were co-cultured in liquid media with toluenes and chlorobenzenes to determine the degradation of these substrates and formation of dual-species biofilm used for the degradation process. Bacillus subtilis strain DKT utilized benzene, mono- and dichlorinated benzenes as carbon and energy sources. The catabolism of chlorobenzenes was via hydroxylation, in which chlorine atoms were replaced by hydroxyl groups to form catechol, followed by ring fission via the ortho-cleavage pathway. The investigation of the dual-species biofilm composed of B. subtilis DKT and C. testosteroni KT5 (a toluene and chlorotoluene-degrading isolate with low biofilm formation) showed that B. subtilis DKT synergistically promoted C. testosteroni KT5 to develop biofilm. The bacterial growth in dual-species biofilm overcame the inhibitory effects caused by monochlorobenzene and 2-chlorotoluene. Moreover, the dual-species biofilm showed effective degradability toward the mixture of these substrates. This study provides knowledge about the commensal relationships in a dual-culture biofilm for designing multispecies biofilms applied for the biodegradation of toxic organic substrates that cannot be metabolized by single-organism biofilms.

     

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.     

Ionizing radiation‐induced long‐term expression of senescence markers in mice is independent of p53 and immune status

Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR‐induced senescence markers could persist long‐term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16^INK4a expression, two hallmarks of cellular senescence and aging. BrdU‐labeling experiments revealed that IR‐induced damaged cells are preferentially eliminated, at least partially, in a tissue‐dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild‐type and Rag2^?/? γC^?/? mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long‐term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.     

Indel-II region deletion sizes in the white spot syndrome virus genome correlate with shrimp disease outbreaks in southern Vietnam

Sequence comparisons of the genomes of white spot syndrome virus (WSSV) strains have identified regions containing variable-length insertions/deletions (i.e. indels). Indel-I and Indel-II, positioned between open reading frames (ORFs) 14/15 and 23/24, respectively, are the largest and the most variable. Here we examined the nature of these 2 indel regions in 313 WSSV-infected Penaeus monodon shrimp collected between 2006 and 2009 from 76 aquaculture ponds in the Mekong Delta region of Vietnam. In the Indel-I region, 2 WSSV genotypes with deletions of either 5950 or 6031 bp in length compared with that of a reference strain from Thailand (WSSV-TH-96-II) were detected. In the Indel-II region, 4 WSSV genotypes with deletions of 8539, 10970, 11049 or 11866 bp in length compared with that of a reference strain from Taiwan (WSSV-TW) were detected, and the 8539 and 10970 bp genotypes predominated. Indel-II variants with longer deletions were found to correlate statistically with WSSV-diseased shrimp originating from more intensive farming systems. Like Indel-I lengths, Indel-II lengths also varied based on the Mekong Delta province from which farmed shrimp were collected.     

Avian influenza a virus in wild birds in highly urbanized areas

Avian influenza virus (AIV) surveillance studies in wild birds are usually conducted in rural areas and nature reserves. Less is known of avian influenza virus prevalence in wild birds located in densely populated urban areas, while these birds are more likely to be in close contact with humans. Influenza virus prevalence was investigated in 6059 wild birds sampled in cities in the Netherlands between 2006 and 2009, and compared with parallel AIV surveillance data from low urbanized areas in the Netherlands. Viral prevalence varied with the level of urbanization, with highest prevalence in low urbanized areas. Within cities virus was detected in 0.5% of birds, while seroprevalence exceeded 50%. Ring recoveries of urban wild birds sampled for virus detection demonstrated that most birds were sighted within the same city, while few were sighted in other cities or migrated up to 2659 km away from the sample location in the Netherlands. Here we show that urban birds were infected with AIVs and that urban birds were not separated completely from populations of long-distance migrants. The latter suggests that wild birds in cities may play a role in the introduction of AIVs into cities. Thus, urban bird populations should not be excluded as a human-animal interface for influenza viruses.     

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

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