Diphenyl Urea Derivatives as Inhibitors of Transketolase: A Structure-Based Virtual Screening

Transketolase is an enzyme involved in a critical step of the non-oxidative branch of the pentose phosphate pathway whose inhibition could lead to new anticancer drugs. Here, we report new human transketolase inhibitors, based on the phenyl urea scaffold, found by applying structure-based virtual screening. These inhibitors are designed to cover a hot spot in the dimerization interface of the homodimer of the enzyme, providing for the first time compounds with a suggested novel binding mode not based on mimicking the thiamine pyrophosphate cofactor

Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals

Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling

Enumerating Pathways of Proton Abstraction Based on a Spatial and Electrostatic Analysis of Residues in the Catalytic Site

The pathways of proton abstraction (PA), a key aspect of most catalytic reactions, is often controversial and highly debated. Ultrahigh-resolution diffraction studies, molecular dynamics, quantum mechanics and molecular mechanic simulations are often adopted to gain insights in the PA mechanisms in enzymes. These methods require expertise and effort to setup and can be computationally intensive. We present a push button methodology – Proton abstraction Simulation (PRISM) – to enumerate the possible pathways of PA in a protein with known 3D structure based on the spatial and electrostatic properties of residues in the proximity of a given nucleophilic residue. Proton movements are evaluated in the vicinity of this nucleophilic residue based on distances, potential differences, spatial channels and characteristics of the individual residues (polarity, acidic, basic, etc). Modulating these parameters eliminates their empirical nature and also might reveal pathways that originate from conformational changes. We have validated our method using serine proteases and concurred with the dichotomy in PA in Class A β-lactamases, both of which are hydrolases. The PA mechanism in a transferase has also been corroborated. The source code is made available at www.sanchak.com/prism

Population Response to Habitat Fragmentation in a Stream-Dwelling Brook Trout Population

Fragmentation can strongly influence population persistence and expression of life-history strategies in spatially-structured populations. In this study, we directly estimated size-specific dispersal, growth, and survival of stream-dwelling brook trout in a stream network with connected and naturally-isolated tributaries. We used multiple-generation, individual-based data to develop and parameterize a size-class and location-based population projection model, allowing us to test effects of fragmentation on population dynamics at local (i.e., subpopulation) and system-wide (i.e., metapopulation) scales, and to identify demographic rates which influence the persistence of isolated and fragmented populations. In the naturally-isolated tributary, persistence was associated with higher early juvenile survival (∼45% greater), shorter generation time (one-half) and strong selection against large body size compared to the open system, resulting in a stage-distribution skewed towards younger, smaller fish. Simulating barriers to upstream migration into two currently-connected tributary populations caused rapid (2–6 generations) local extinction. These local extinctions in turn increased the likelihood of system-wide extinction, as tributaries could no longer function as population sources. Extinction could be prevented in the open system if sufficient immigrants from downstream areas were available, but the influx of individuals necessary to counteract fragmentation effects was high (7–46% of the total population annually). In the absence of sufficient immigration, a demographic change (higher early survival characteristic of the isolated tributary) was also sufficient to rescue the population from fragmentation, suggesting that the observed differences in size distributions between the naturally-isolated and open system may reflect an evolutionary response to isolation. Combined with strong genetic divergence between the isolated tributary and open system, these results suggest that local adaptation can ‘rescue’ isolated populations, particularly in one-dimensional stream networks where both natural and anthropogenically-mediated isolation is common. However, whether rescue will occur before extinction depends critically on the race between adaptation and reduced survival in response to fragmentation

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3′-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

Abstract\ud \ud \ud \ud Background\ud \ud Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus.\ud \ud \ud \ud Methods\ud \ud One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay.\ud \ud \ud \ud Results\ud \ud We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls.\ud \ud \ud \ud Conclusion\ud \ud In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/000090; 00/030722; 01/142381; 02/113402; 03/099982; 04/116068; 04/157044 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/20078. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process.Work supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 01/00009-0; 00/03072-2; 01/14238-1; 02/11340-2; 03/09998-2; 04/11606-8; 04/15704-4 and Conselho Nacional de Desenvolvimento Científico e Tecnológico 481872/2007-8. We would like to thank the careful work and thoughtful suggestions of the two reviewers responsible for the reviewing editorial process

Inhibition of alpha-synuclein fibrillization by dopamine is mediated by interactions with five C-terminal residues and with E83 in the NAC region

The interplay between dopamine and alpha-synuclein (AS) plays a central role in Parkinson's disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the (125)YEMPS(129) region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the (125)YEMPS(129) region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization