Analogs of iso-azepinomycin as potential transition-state analog inhibitors of guanase: Synthesis,biochemical screening,and structure–activity correlations of various selectively substituted imidazo[4,5-e][1,4]diazepines

Guanase is an important enzyme of the purine salvage pathway of nucleic acid metabolism and its inhibition has beneficial implications in viral, bacterial, and cancer therapy. The work described herein is based on a hypothesis that azepinomycin, a heterocyclic natural product and a purported transition state analog inhibitor of guanase, does not represent the true transition state of the enzyme-catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogs of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogs were designed to explore the hydrophobic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O⁵ oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogs were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O⁵ results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N3 or N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at either N3 or N4 with a simultaneous increase in hydrophobicity at O⁵ considerably diminishes any gain in inhibition made by solely enhancing hydrophobicity at N3 or N4, and (5) finally, increasing the hydrophilic character near N3 has also a deleterious effect on inhibition. The most potent compound in the series has a K_i value of 8.0 ± 1.5 μM against rabbit liver guanase.     

Incidence and Prevalence of Tuberculosis among Household Contacts of Pulmonary Tuberculosis Patients in a Peri-Urban Population of South Delhi,India

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of mortality and morbidity across all age groups throughout the world, especially in developing countries.

Methodology/Principal Findings

In this study, we have included 432 open index cases with their 1608 household contacts in a prospective cohort study conducted from May 2007 to March 2009. The follow-up period was 2 years. All Index cases were diagnosed on the basis of suggestive signs and symptoms and sputum being AFB positive. Among the 432 index patients, 250 (57.9%) were males and 182 (42.1%) females; with mean age of 34±14.4 yr and 26±11.1 yr, respectively. Out of 1608 household contacts, 866 (53.9%) were males and 742 (46.1%) females; with mean age of 26.5±15.8 and 26.5±16.0 yr, respectively. Of the total 432 households, 304 (70.4%) had ≤4 members and 128 (29.6%) had ≥5 members. The median size of the family was four. Of the 1608 contacts, 1206 were able to provide sputum samples, of whom 83 (6.9%) were found MTB culture positive. Household contacts belonging to adult age group were predominantly (74, 89.2%) infected as compared to the children (9, 10.8%). On screening the contact relationship status with index patients, 52 (62.7%) were first-degree relatives, 18 (34.6%) second-degree relatives and 12 (14.5%) spouses who got infected from their respective index patients. Co-prevalent and incident tuberculosis was found in 52 (4.3%) and 31 (2.6%) contacts, respectively. In incident cases, the diagnosis could be made between 4 to 24 months of follow-up, after their baseline evaluation.

Conclusion

Active household contact investigation is a powerful tool to detect and treat tuberculosis at early stages and the only method to control TB in high-TB-burden countries.     

National mass drug administration costs for lymphatic filariasis elimination

Background

Because lymphatic filariasis (LF) elimination efforts are hampered by a dearth of economic information about the cost of mass drug administration (MDA) programs (using either albendazole with diethylcarbamazine [DEC] or albendazole with ivermectin), a multicenter study was undertaken to determine the costs of MDA programs to interrupt transmission of infection with LF. Such results are particularly important because LF programs have the necessary diagnostic and treatment tools to eliminate the disease as a public health problem globally, and already by 2006, the Global Programme to Eliminate LF had initiated treatment programs covering over 400 million of the 1.3 billion people at risk.

Methodology/Principal Findings

To obtain annual costs to carry out the MDA strategy, researchers from seven countries developed and followed a common cost analysis protocol designed to estimate 1) the total annual cost of the LF program, 2) the average cost per person treated, and 3) the relative contributions of the endemic countries and the external partners. Costs per person treated ranged from $0.06 to $2.23. Principal reasons for the variation were 1) the age (newness) of the MDA program, 2) the use of volunteers, and 3) the size of the population treated. Substantial contributions by governments were documented – generally 60%–90% of program operation costs, excluding costs of donated medications.

Conclusions/Significance

MDA for LF elimination is comparatively inexpensive in relation to most other public health programs. Governments and communities make the predominant financial contributions to actual MDA implementation, not counting the cost of the drugs themselves. The results highlight the impact of the use of volunteers on program costs and provide specific cost data for 7 different countries that can be used as a basis both for modifying current programs and for developing new ones.     

Co‐evolutionary analysis implies auxiliary functions of HSP110 in Plasmodium falciparum

Plasmodium falciparum encounters frequent environmental challenges during its life cycle which makes productive protein folding immensely challenging for its metastable proteome. To identify the important components of protein folding machinery involved in maintaining P. falciparum proteome, we performed a proteome‐wide phylogenetic profiling across various species. We found that except HSP110, the parasite lost all other cytosolic nucleotide exchange factors essential for regulating HSP70 which is the centrum of the protein folding network. Evolutionary and structural analysis shows that besides its canonical interaction with HSP70, PfHSP110 has acquired sequence insertions for additional dynamic interactions. Molecular co‐evolution profile depicts that the co‐evolving proteins of PfHSP110 belong to distinct pathways like genetic variation, DNA repair, fatty acid biosynthesis, protein modification/trafficking, molecular motions, and apoptosis. These proteins exhibit unique physiochemical properties like large size, high iso‐electric point, low solubility, and antigenicity, hence require PfHSP110 chaperoning to attain functional state. Co‐evolving protein interaction network suggests that PfHSP110 serves as an important hub to coordinate protein quality control, survival, and immune evasion pathways in the parasite. Overall, our findings highlight potential accessory roles of PfHSP110 that may provide survival advantage to the parasite during its lifecycle and febrile conditions. The data also open avenues for experimental validation of auxiliary functions of PfHSP110 and their exploration for design of better antimalarial strategies. Proteins 2015; 83:1513–1525. © 2015 Wiley Periodicals, Inc.     

A novel transition state analog inhibitor of guanase based on azepinomycin ring structure: Synthesis and biochemical assessment of enzyme inhibition

Synthesis and biochemical inhibition studies of a novel transition state analog inhibitor of guanase bearing the ring structure of azepinomycin have been reported. The compound was synthesized in five-steps from a known compound and biochemically screened against the rabbit liver guanase. The compound exhibited competitive inhibition profile with a K_i of 16.7 ± 0.5 μM.     

Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K(+) and Na(+) ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15mer deoxynucleotide, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K(+) ions bind with higher affinity than the Na(+) ions. The enthalpy changes, DeltaH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.     

A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes

Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5–18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (ΔG₃₇) ranging from −1.7 to −15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex–duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.     

Effect of loop length variation on quadruplex-Watson Crick duplex competition

The effect of loop length on quadruplex stability has been studied when the G-rich strand is present along with its complementary C-rich strand, thereby resulting in competition between quadruplex and duplex structures. Using model sequences with loop lengths varying from T to T5, we carried out extensive FRET to discover the influence of loop length on the quadruplex-Watson Crick duplex competition. The binding data show an increase in the binding affinity of quadruplexes towards their complementary strands upon increasing the loop length. Our kinetic data reveal that unfolding of the quadruplex in presence of a complementary strand involves a contribution from a predominant slow and a small population of fast opening conformer. The contribution from the fast opening conformer increases upon increasing the loop length leading to faster duplex formation. FCS data show an increase in the interconversion between the quadruplex conformers in presence of the complementary strand, which shifts the equilibrium towards the fast opening conformer with an increase in loop length. The relative free-energy difference (Delta DeltaG(o)) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex.     

Certolizumab pegol

Certolizumab pegol (Cimzia^®) is currently the only PEGylated anti-TNFα biologic approved for the treatment of rheumatoid arthritis and Crohn disease. The product, developed by UCB, is a humanized antigen-binding fragment (Fab’) of a monoclonal antibody that has been conjugated to polyethylene glycol. Certolizumab pegol was approved as a treatment for rheumatoid arthritis in the EU, US and Canada in 2009, and as a treatment for Crohn disease in Switzerland in 2007 and the US in 2008. Certolizumab pegol is entering into an increasingly competitive marketplace, especially in rheumatoid arthritis, but clinical data demonstrate benefits across a range of clinical, radiographic and patient reported outcomes.Key words: certolizumab pegol, rheumatoid arthritis, Crohn disease, TNFα, PEGylated, methotrexate     

Long‐term digital mobile phone use and cognitive decline in the elderly

Research on long‐term exposure to electromagnetic fields on cognition is lacking. We investigated the associations between frequent digital mobile phone use and global and domain‐specific cognitive changes in older persons, a vulnerable group experiencing age‐associated cognitive decline. We assessed 871 non‐demented Chinese participants in the Singapore Longitudinal Ageing Studies cohort on the frequency of digital mobile phone use, neurocognitive performance and confounding variables at baseline, and neurocognitive performance at the 4‐year follow‐up. Findings showed that digital mobile phone users were typically self‐selected to possess characteristics favoring better cognitive functioning and concomitantly demonstrate better performance on cognitive tasks. There was evidently no significant deleterious effect of digital mobile phone use on cognitive functioning in older people. Findings suggest, however, that digital mobile phone use may have an independent facilitating effect on global and executive functioning. Bioelectromagnetics 33:176–185, 2012. © 2011 Wiley Periodicals, Inc.