Prediction of HIV-1 entry inhibitors neomycin-arginine conjugates interaction with the CD4-gp120 binding site by molecular modeling and multistep docking procedure

Developing of multi-target HIV-1 entry inhibitors represents an important avenue of drug therapy. Two such inhibitors are hexa-arginine-neomycin-conjugate (NeoR6) and nona-d-arginine-neomycin-conjugate (Neo-r9). Our findings that NeoR6-resistant mutations appear in the gp120 constant regions; and NeoR6 is not CCR5 antagonist, but inhibits CXCR4 and CCR5 HIV-1 using isolates, led us to suggest that NeoR6 may inhibit HIV-1 entry by interfering with the CD4-gp120 binding. To support this notion, we constructed a homology model of unliganded HIV-1(IIIB) gp120 and docked NeoR6 and Neo-r9 to it, using a multistep docking procedure: geometric-electrostatic docking by MolFit; flexible ligand docking by Autodock3 and final refinement of the obtained complexes by Discover3. Binding free energies were calculated by MM-PBSA methodology. The model predicts competitive inhibition of CD4-gp120 binding by NeoR6 and Neo-r9. We determined plausible binding sites between constructed CD4-bound gp120 trimer and homology modeled membranal CXCR4, and tested NeoR6 and Neo-r9 interfering with this interaction. These models support our notion that another mechanism of anti-HIV-1 activity of NeoR6 is inhibition of gp120-CXCR4 binding. These structural models and interaction of NeoR6 and Neo-r9 with gp120 and CXCR4 provide a powerful approach for structural based drug design for selective targeting of HIV-1 entry and/or for inhibition of other retroviruses with similar mechanism of entry.     

Bone age advancement in prepubertal children with obesity and premature adrenarche: possible potentiating factors

Obesity and premature adrenarche (PA) are both associated with bone age (BA) advancement of unclear etiology, which may lead to earlier puberty, suboptimal final height and obesity in adulthood. Our objective was to understand the hormonal and anthropometric characteristics of BA advancement in a spectrum of prepubertal children with and without obesity and PA. In this cross-sectional study of 66 prepubertal children (35 PA, 31 control, 5-9 years), BMI z-score, hormonal values and response to an oral glucose tolerance test were the main outcome measures. Subjects were divided into tertiles by BA divided by chronological age (BA/CA), an index of BA advancement. Subjects in the top tertile for BA/CA had the highest dehydroepiandrosterone sulfate (DHEAS), free testosterone (%), hemoglobin A(1C), BMI z-score, and weight (P < 0.05). BMI z-score (r = 0.47), weight (r = 0.40), free testosterone (%) (r = 0.34), and DHEAS (r = 0.30) correlated with BA/CA (P < 0.02). Regression analysis showed greater BA/CA in PA compared to controls after controlling for weight (0.21 ± 0.56, P < 0.004). An exploratory stepwise regression model showed that weight, estradiol, and DHEAS were the strongest predictors of BA/CA accounting for 24% of its variance. Obesity was highly associated with BA advancement in this study of prepubertal children. In addition, children with PA had greater BA/CA at any given weight when compared to controls. These findings suggest a possible hormonal factor, which potentiates the effect of obesity on BA advancement in children with obesity and/or PA.     

Bacterial RNase P RNA is a drug target for aminoglycoside-arginine conjugates

The ribonuclease P (RNase P) holoenzymes are RNPs composed of RNase P RNA (PRNA) and a variable number of P protein subunits. Primary differences in structure and function between bacterial and eukaryotic RNase P and its indispensability for cell viability make the bacterial enzyme an attractive drug target. On the basis of our previous studies, aminoglycoside-arginine conjugates (AACs) bind to HIV-1 TAR and Rev responsive element (RRE) RNAs significantly more efficiently than neomycin B. Their specific inhibition of bacterial rRNA as well as the findings that the hexa-arginine neomycin derivative (NeoR6) is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, led us to explore the structure-function relationships of AACs in comparison to a new set of aminoglycoside-polyarginine conjugates (APACs). We here present predicted binding modes of AACs and APACs to PRNA. We used a multistep docking approach comprising rigid docking full scans and final refinement of the obtained complexes. Our docking results suggest three possible mechanisms of RNase P inhibition by AACs and APACs: competition with the P protein and pre-tRNA on binding to P1-P4 multihelix junction and to J19/4 region (probably including displacement of Mg2+ ions from the P4 helix) of PRNA; competition with Mg2+ ions near the P15 loop; and competition with the P protein and/or pre-tRNA near the P15 helix and interfering with interactions between the P protein and pre-tRNA at this region. The APACs revealed about 10-fold lower intermolecular energy than AACs, indicating stronger interactions of APACs than AACs with PRNA.     

Scaling Up Family Planning to Reduce Maternal and Child Mortality: The Potential Costs and Benefits of Modern Contraceptive Use in South Africa

Introduction

Family planning contributes significantly to the prevention of maternal and child mortality. However, many women still do not use modern contraception and the numbers of unintended pregnancies, abortions and subsequent deaths are high. In this paper, we estimate the service delivery costs of scaling up modern contraception, and the potential impact on maternal, newborn and child survival in South Africa.

Methods

The Family Planning model in Spectrum was used to project the impact of modern contraception on pregnancies, abortions and births in South Africa (2015-2030). The contraceptive prevalence rate (CPR) was increased annually by 0.68 percentage points. The Lives Saved Tool was used to estimate maternal and child deaths, with coverage of essential maternal and child health interventions increasing by 5% annually. A scenario analysis was done to test impacts when: the change in CPR was 0.1% annually; and intervention coverage increased linearly to 99% in 2030.

Results

If CPR increased by 0.68% annually, the number of pregnancies would reduce from 1.3 million in 2014 to one million in 2030. Unintended pregnancies, abortions and births decrease by approximately 20%. Family planning can avert approximately 7,000 newborn and child and 600 maternal deaths. The total annual costs of providing modern contraception in 2030 are estimated to be US$33 million and the cost per user of modern contraception is US$7 per year. The incremental cost per life year gained is US$40 for children and US$1,000 for mothers.

Conclusion

Maternal and child mortality remain high in South Africa, and scaling up family planning together with optimal maternal, newborn and child care is crucial. A huge impact can be made on maternal and child mortality, with a minimal investment per user of modern contraception.     

Insight into the mechanisms of aminoglycoside derivatives interaction with HIV-1 entry steps and viral gene transcription

In recent years, based on peptide models of HIV-1 RNA binding, NMR structures of Tat-responsive element-ligand complexes and aminoglycoside-RNA interactions, and HIV-1 Tat structure, we have designed and synthesized aminoglycoside-arginine conjugates (AACs) and aminoglycoside poly-arginine conjugates (APACs), to serve as Tat mimetics. These novel molecules inhibit HIV-1 infectivity with 50% effective concentration values in the low micromolar range, the most potent compounds being the hexa-arginine-neomycin B and nona-D-arginine-neomycin conjugates. Importantly, these compounds, in addition to acting as Tat antagonists, inhibit HIV-1 infectivity by blocking several steps in HIV-1 cell entry. The AACs and APACs inhibit HIV-1 cell entry by interacting with gp120 at the CD4-binding site, by interacting with CXCR4 at the binding site of the CXCR4 mAb 12G5, and apparently by interacting with transient structures of the ectodomain of gp41. In the current review, we discuss the mechanisms of anti-HIV-1 activities of these AACs, APACs and other aminoglycoside derivatives in detail. Targeting several key processes in the viral life cycle by the same compound not only may increase its antiviral efficacy, but more importantly, may reduce the capacity of the virus to develop resistance to the compound. AACs and APACs may thus serve as leading compounds for the development of multitargeting novel HIV-1 inhibitors.     

CDD: a curated Entrez database of conserved domain alignments

The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE(R). This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein-protein queries submitted to BLAST(R) at http://www.ncbi.nlm.nih.gov/BLAST. CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate 'columns' corresponding to functional sites conserved among family members.     

Val-BoroPro Accelerates T Cell Priming via Modulation of Dendritic Cell Trafficking Resulting in Complete Regression of Established Murine Tumors

Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE) modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine.     

Localized egg-cell expression of effector proteins for targeted modification of the Arabidopsis genome

Targeted modification of the genome is an important genetic tool, which can be achieved via homologous, non-homologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.