BRCA1 regulation on ß-hCG: a mechanism for tumorigenicity in BRCA1 defective breast cancer.

Human chorionic gonadotropin ß (ß-hCG) has been implicated in breast tumorigenesis. However, the role of this hormone is highly controversial as certain studies suggest it has anti-tumor properties while others have found it to be pro-tumorigenic. To unveil the truth, we have analyzed the expression of ß-hCG in breast cancer. We identified for the first time that ß-hCG expression is linked to BRCA1 status and its overexpression is seen in BRCA1 mutated breast cancer cells, BRCA1 conditional knockout mouse breast cancer tissues and BRCA1 floxed basal cell carcinoma (BCC) tissues. An analysis of three large, transcriptomic data sets from TCGA (The Cancer Genome Atlas) expression profile confirmed the inverse correlation between BRCA1 and ß-hCG in human breast cancer. Using ChIP and luciferase assays, we also demonstrated that the cancer cells with wild-type but not mutant BRCA1 directly repress the expression of ß-hCG by binding to its promoter. Further, ß-hCG promotes migration and invasion predominantly in BRCA1 mutant breast cancer cells. Interestingly, stable overexpression of ß-hCG in BRCA1 mutant but not wild-type breast cancer cells results in the formation of spheres even on monolayer cultures. The cells of these spheres show high expression of both EMT and stem cell markers. Since ß-hCG belongs to a cysteine knot family of proteins like TGFß and TGFß signaling is deregulated in BRCA1 defective tumors, we checked whether ß-hCG can mediate signaling through TGFßRII in BRCA1 mutated cells. We found for the first time that ß-hCG can bind and phosphorylate TGFßRII, irrespective of LHCGR status and induce proliferation in BRCA1 defective cells. Our results confirmed that there exists a transcriptional regulation of BRCA1 on ß-hCG and BRCA1 mutation promotes ß-hCG mediated tumorigenesis through TGFßRII signaling. Thus inhibiting ß-hCG-TGFßRII could prove an effective treatment strategy for BRCA1 mutated tumors.

Data on overlapping brain disorders and emerging drug targets in human Dopamine Receptors Interaction Network.

Intercommunication of Dopamine Receptors (DRs) with their associate protein partners is crucial to maintain regular brain function in human. Majority of the brain disorders arise due to malfunctioning of such communication process. Hence, contributions of genetic factors, as well as phenotypic indications for various neurological and psychiatric disorders are often attributed as sharing in nature. In our earlier research article entitled "Human Dopamine Receptors Interaction Network (DRIN): a systems biology perspective on topology, stability and functionality of the network" (Podder et al., 2014) [1], we had depicted a holistic interaction map of human Dopamine Receptors. Given emphasis on the topological parameters, we had characterized the functionality along with the vulnerable properties of the network. In support of this, we hereby provide an additional data highlighting the genetic overlapping of various brain disorders in the network. The data indicates the sharing nature of disease genes for various neurological and psychiatric disorders in dopamine receptors connecting protein-protein interactions network. The data also indicates toward an alternative approach to prioritize proteins for overlapping brain disorders as valuable drug targets in the network.

Investigating the structural impact of S311C mutation in DRD2 receptor by molecular dynamics & docking studies.

Dopamine receptors (DR) are neuronal cell surface proteins that mediate the action of neurotransmitter dopamine in brain. Dopamine receptor D2 (DRD2) that belongs to G-protein coupled receptors (GPCR) family is a major therapeutic target for of various neurological and psychiatric disorders in human. The third inter cellular loop (ICL3) in DRD2 is essential for coupling G proteins and several signaling scaffold proteins. A mutation in ICL3 can interfere with this binding interface, thereby altering the DRD2 signaling. In this study we have examined the deleterious effect of serine to cysteine mutation at position 311 (S311C) in the ICL3 region that is implicated in diseases like schizophrenia and alcoholism. An in silico structure modeling approach was employed to determine the wild type (WT) and mutant S311C structures of DRD2, scaffold proteins - Gαi/o and NEB2. Protein-ligand docking protocol was exercised to predict the interactions of natural agonist dopamine with both the WT and mutant structures of DRD2. Besides, atomistic molecular dynamics (MD) simulations were performed to provide insights into essential dynamics of the systems-unbound and dopamine bound DRD2 (WT and mutant) and three independent simulations for Gαi, Gαo and NEB2 systems. To provide information on intra-molecular arrangement of the structures, a comprehensive residue interactions network of both dopamine bound WT and mutant DRD2 protein were studied. We also employed a protein-protein docking strategy to find the interactions of scaffold proteins - Gαi/o and NEB2 with both dopamine bound WT and mutant structures of DRD2. We observed a marginal effect of the mutation in dopamine binding mechanism on the trajectories analyzed. However, we noticed a significant structural alteration of the mutant receptor which affects Gαi/o and NEB2 binding that can be causal for malfunctioning in cAMP-dependent signaling and Ca(+) homeostasis in the brain dopaminergic system leading to neuropsychiatric disorders.

Structural signatures of DRD4 mutants revealed using molecular dynamics simulations: Implications for drug targeting.

Human Dopamine Receptor D4 (DRD4) orchestrates several neurological functions and represents a target for many psychological disorders. Here, we examined two rare variants in DRD4; V194G and R237L, which elicit functional alterations leading to disruption of ligand binding and G protein coupling, respectively. Using atomistic molecular dynamics (MD) simulations, we provide in-depth analysis to reveal structural signatures of wild and mutant complexes with their bound agonist and antagonist ligands. We constructed intra-protein network graphs to discriminate the global conformational changes induced by mutations. The simulations also allowed us to elucidate the local side-chain dynamical variations in ligand-bound mutant receptors. The data suggest that the mutation in transmembrane V (V194G) drastically disrupts the organization of ligand binding site and causes disorder in the native helical arrangement. Interestingly, the R237L mutation leads to significant rewiring of side-chain contacts in the intracellular loop 3 (site of mutation) and also affects the distant transmembrane topology. Additionally, these mutations lead to compact ICL3 region compared to the wild type, indicating that the receptor would be inaccessible for G protein coupling. Our findings thus reveal unreported structural determinants of the mutated DRD4 receptor and provide a robust framework for design of effective novel drugs.

Decentralization and decision space in the health sector: a case study from Karnataka, India.

Various attempts have been made in India with respect to decentralization, most significantly the 73rd Amendment to the Constitution of India (1993) which provided the necessary legal framework for decentralization to take place. However, the outcome has been mixed: an evaluation of the impact of decentralization in the health sector found virtually no change in health system performance and access to health services in terms of availability of health personnel or improvement in various health indicators, such as Infant Mortality Rates or Maternal Mortality Ratio. Subsequently, there has been a conscious effort under the National Rural Health Mission (NRHM)-launched in 2005-to promote decentralization of funds, functions and functionaries to lower levels of government; and Karnataka had a head-start since devolution of all 29 functions prescribed by the 73rd Amendment had already taken place in the state by the late 1990s. This study presents the findings of an on-going research effort to build empirical evidence on decentralization in the health sector and its impact on system performance. The focus here is on analyzing the responses of health personnel at the district level and below on their perceived 'Decision Space'-the range of choice or autonomy they see themselves as having along a series of functional dimensions. Overall, the data indicate that there is a substantial gap between the spirit of the NRHM guidelines on decentralization and the actual implementation on the ground. There is a need for substantial capacity building at all levels of the health system to genuinely empower functionaries, particularly at the district level, in order to translate the benefits of decentralization into reality.

Hydroxyethylamine Based Phthalimides as New Class of Plasmepsin Hits: Design, Synthesis and Antimalarial Evaluation.

A novel class of phthalimides functionalized with privileged scaffolds was designed, synthesized and evaluated as potential inhibitors of plasmepsin 2 (Ki: 0.99 ± 0.1 µM for 6u) and plasmepsin 4 (Ki: 3.3 ± 0.3 µM for 6t), enzymes found in the digestive vacuole of the plasmodium parasite and considered as crucial drug targets. Three compounds were identified as potential candidates for further development. The listed compounds were also assayed for their antimalarial efficacy against chloroquine (CQ) sensitive strain (3D7) of Plasmodium falciparum. Assay of twenty seven hydroxyethylamine derivatives revealed four (5e, 6j, 6o and 6s) as strongly active, which were further evaluated against CQ resistant strain (7GB) of P. falciparum. Compound 5e possessing the piperidinopiperidine moiety exhibited promising antimalarial activity with an IC50 of 1.16 ± 0.04 µM. Further, compounds 5e, 6j, 6o and 6s exhibited low cytotoxic effect on MCF-7 cell line. Compound 6s possessing C2 symmetry was identified as the least cytotoxic with significant antimalarial activity (IC50: 1.30 ± 0.03 µM). The combined presence of hydroxyethylamine and cyclic amines (piperazines and piperidines) was observed as crucial for the activity. The current studies suggest that hydroxyethylamine based molecules act as potent antimalarial agent and may be helpful in drug development.

Design, synthesis and biological evaluation of functionalized phthalimides: a new class of antimalarials and inhibitors of falcipain-2, a major hemoglobinase of malaria parasite.

Phthalimides functionalized with cyclic amines were synthesized, characterized and screened for their in vitro antimalarial efficacy against Plasmodium falciparum (Pf3D7). Of all the listed phthalimides evaluated, 14 and 24 were identified as potent antimalarial agents as advocated by assessment of their ability to inhibit [(3)H] hypoxanthine incorporation in the nucleic acid of parasites. In addition, phthalimides 14 and 24 were incubated for 60 and 90h and an enhanced antimalarial effect was noticed with increase in time to great extent. A reduction in IC50 values was observed with increase in exposure time of the parasite to the compounds. A symmetric phthalimide, 24 possessing piperazine as linker unit was identified as the most potent antimalarial agent with IC50 values of 5.97±0.78, 2.0±1.09 and 1.1±0.75µM on incubation period of 42, 60 and 90h, respectively. The abnormal morphologies such as delay in developmental stages, growth arrest and condensed nuclei of parasite were observed with the aid of microscopic studies upon exposure with 14 and 24. The evaluation of 14 and 24 against chloroquine resistant strain, (Pf7GB) of P. falciparum afforded IC50 values, 13.29±1.20 and 7.21±0.98µM, respectively. The combination of 24 with artemisinin (ART) showed enhanced killing of parasite against Pf3D7. Further, all phthalimides were evaluated for their activity against falcipain-2 (FP2), a major hemoglobinase of malarial parasite. The enzymatic assay afforded 6 as most active member against FP2. To the best of our knowledge this is the initial study represents phthalimide protected amino acids functionalized with cyclic amines as potent antimalarial agents.

Structure and dynamics of DRD4 bound to an agonist and an antagonist using in silico approaches.

Human dopamine receptor D4 (DRD4), a member of G-protein coupled receptor (GPCR) family, plays a central role in cell signaling and trafficking. Dysfunctional activity of DRD4 can lead to several psychiatric conditions and, therefore, represents target for many neurological disorders. However, lack of atomic structure impairs our understanding of the mechanism regulating its activity. Here, we report the modeled structure of DRD4 alone and in complex with dopamine and spiperone, its natural agonist and antagonist, respectively. To assess the conformational dynamics induced upon ligand binding, all-atom explicit solvent molecular dynamics simulations in membrane environment were performed. Comprehensive analyses of simulations reveal that agonist binding triggers a series of conformational changes in the transmembrane region, including rearrangement of residues, characteristic of transmission and tyrosine toggle molecular switches. Further, the trajectories indicate that a loop region in the intracellular region--ICL3, is significantly dynamic in nature, mainly due to the side-chain movements of conserved proline residues involved in SH3 binding domains. Interestingly, in dopamine-bound receptor simulation, ICL3 represents an open conformation ideal for G protein binding. The structural and dynamical information presented here suggest a mode of activation of DRD4, upon ligand binding. Our study will help in further understanding of receptor activation, as acquiring structural information is crucial for the design of highly selective DRD4 ligands.

Human Dopamine Receptors Interaction Network (DRIN): a systems biology perspective on topology, stability and functionality of the network.

Dopamine receptors (DR) are one of the major neurotransmitter receptors present in human brain. Malfunctioning of these receptors is well established to trigger many neurological and psychiatric disorders. Taking into consideration that proteins function collectively in a network for most of the biological processes, the present study is aimed to depict the interactions between all dopamine receptors following a systems biology approach. To capture comprehensive interactions of candidate proteins associated with human dopamine receptors, we performed a protein-protein interaction network (PPIN) analysis of all five receptors and their protein partners by mapping them into human interactome and constructed a human Dopamine Receptors Interaction Network (DRIN). We explored the topology of dopamine receptors as molecular network, revealing their characteristics and the role of central network elements. More to the point, a sub-network analysis was done to determine major functional clusters in human DRIN that govern key neurological pathways. Besides, interacting proteins in a pathway were characterized and prioritized based on their affinity for utmost drug molecules. The vulnerability of different networks to the dysfunction of diverse combination of components was estimated under random and direct attack scenarios. To the best of our knowledge, the current study is unique to put all five dopamine receptors together in a common interaction network and to understand the functionality of interacting proteins collectively. Our study pinpointed distinctive topological and functional properties of human dopamine receptors that have helped in identifying potential therapeutic drug targets in the dopamine interaction network.

A synthetic chimeric peptide harboring human papillomavirus 16 cytotoxic T lymphocyte epitopes shows therapeutic potential in a murine model of cervical cancer.

Infection with human papillomavirus (HPV) such as HPV16 is known to be associated with cervical cancer. The E6 and E7 oncoproteins of this virus are attractive targets for T-cell-based immunotherapy to cervical cancer. In our study, software predicted, multiple H-2D(b) restricted HPV16 cytotoxic T lymphocytes (CTL) epitopes on a synthetic chimeric peptide, was used along with different immunopotentiating adjuvants such as alum, heat-killed Mycobacterium w (Mw) cells, and poly D,L-lactic-co-glycolide (PLGA) microspheres. We have shown that subcutaneous immunization with H-2D(b)-restricted HPV16 peptide was able to generate CTL-mediated cytolysis of HPV16 E6- and E7-expressing TC-1 tumor cells in vitro, as well as protect against in vivo challenge with TC-1 cells in C57BL/6 mice. In vitro, this chimeric peptide showed best efficacy with PLGA microspheres, moderate with alum, and least with Mw as adjuvant. This approach may thus provide a potential peptide-based therapeutic candidate vaccine for the control of HPV infection and hence cervical cancer.

Functionalized hydroxyethylamine based peptide nanostructures as potential inhibitors of falcipain-3, an essential proteases of Plasmodium falciparum.

Self-assembled peptide based nanostructures gained enough popularity due to their easy biocompatibility and numerous potential applications. An excellent model of self-assembly of hydroxyethylamine based peptide nanostructures was synthesized and characterized by DLS and TEM. Spherical nano structures of I and III were observed with particle size ∼50 and ∼80nm, respectively. Further, I and III were screened against anti-malarial target, falcipain-3 (FP3), a crucial cysteine protease involved as a major hemoglobinase of Plasmodium falciparum. Interestingly, compound III completely inhibited the activity of FP3. The effective concentration (1.5µM) of III found to be more potent than I. This biochemical result was substantiated by molecular-docking studies indicating III to be best inhibitor of FP3. This is the first report showing that bis hydroxethylamine based peptide nanostructures could be very effective inhibitor of malarial cysteine proteases.

Pharmacophore modeling and virtual screening studies to design potential COMT inhibitors as new leads.

Catechol-O-methyltransferase (COMT) catalyzes the methylation of catecholamines, including neurotransmitters like dopamine, epinephrine and norepinephrine, leading to their degradation. COMT has been a subject of study for its implications in numerous neurological disorders like Parkinson's disease (PD), schizophrenia, and depression. The COMT gene is associated with many allelic variants, the Val108Met polymorphism being the most clinically significant. Availability of crystal structure of both 108V and 108M forms of human soluble-COMT (S-COMT) facilitated us to use structure-based virtual screening approach to obtain new hits by screening a library of CNS permeable compounds from ZINC database. In this study, E-pharmacophore was also used to generate pharmacophore models based on a series of known COMT inhibitors. A five-point pharmacophore model consisting of one hydrogen-bond acceptor (A), two hydrogen bond donors (D), and two aromatic rings (R) was generated for both the polymorphic forms of COMT. These models were then used for filtering ZINC-CNS permeable library to obtain new hits. Physicochemical properties were also calculated for all the hits obtained from both the approaches for favorable ADME properties. These identified hits maybe of interest for further structural optimization and biological evaluation assays.

(1S,1'S,2'R,4a'S,9a'S,9b'R)-1'-Acet-yloxy-2,4'-dioxo-2',4',4a',7',8',9',9a',9b'-octa-hydro-1'H,2H-spiro-[ace-naphthyl-ene-1,5'-pyrano[4,3-a]pyrrolizin]-2'-ylmethyl acetate.

In the title compound C26H25NO7, the mean plane through the lactone-substituted ring of the pyrrolizidine moiety forms dihedral angles of 78.46 (6) and 58.28 (8)° with the ace-naphthyl-ene moiety and the sugar based-lactone ring, respectively. The sum of the angles at the the N atom of the pyrrolizidine ring (335.0°) is in accordance with sp (3) hybridization. Some atoms of the acetate group are disordered and were refined using a split model [occupancy ratio 0.673 (10):0.327 (10)].

(4'-Acet-yloxy-1,3,1'-trioxo-1,3,4,4a,4b,5,6,7,9,9a-deca-hydro-spiro-[indene-2,9'-pyrano[4,3-a]pyrrolizin]-3'-yl)methyl acetate.

In the title compound, C23H23NO8, the dihedral angle between the five- and six-membered rings of the indene-dione moiety is 3.09 (13)°. The mean plane of the five-membered ring (which has a flat envelope conformation with the spiro C atom as the flap) is inclined to the mean plane of the central five-membered ring of the pyrrolizine unit by 76.48 (12)°. This central ring has a twist conformation on the N-C(spiro) bond. The outer ring of the pyrrolizine unit has an envelope conformation with the N atom as the flap. The mean planes of these two fused rings are inclined to one another by 65.28 (15)°. The pyran ring has a screw-boat conformation and its mean plane makes a dihedral angle of 29.50 (11)° with the mean plane of the central five-membered ring of the pyrrolizine unit. In the crystal, mol-ecules are linked via C-H⋯O hydrogen bonds, forming two-dimensional networks lying parallel to the ab plane.

From drug target to leads--sketching a physicochemical pathway for lead molecule design in silico.

The discovery of new pharmaceuticals via computer modeling is one of the key challenges in modern medicine. The advent of global networks of genomic, proteomic and metabolomic endeavors is ushering in an increasing number of novel and clinically important targets for screening. Computational methods are anticipated to play a pivotal role in exploiting the structural and functional information to understand specific molecular recognition events of the target macromolecule with candidate hits leading ultimately to the design of improved leads for the target. In this review, we sketch a system independent, comprehensive physicochemical pathway for lead molecule design focusing on the emerging in silico trends and techniques. We survey strategies for the generation of candidate molecules, docking them with the target and ranking them based on binding affinities. We present a molecular level treatment for distinguishing affinity from specificity of a ligand for a given target. We also discuss the significant aspects of drug absorption, distribution, metabolism, excretion and toxicity (ADMET) and highlight improved protocols required for higher quality and throughput of in silico methods employed at early stages of discovery. We present a realization of the various stages in the pathway proposed with select examples from the literature and from our own research to demonstrate the way in which an iterative process of computer design and validation can aid in developing potent leads. The review thus summarizes recent advances and presents a viewpoint on improvements envisioned in the years to come for automated computer aided lead molecule discovery.

A free energy based computational pathway from chemical templates to lead compounds: a case study of COX-2 inhibitors.

Automation of lead compound design in silico given the structure of the protein target and a definition of its active site vies for the top of the wish list in any drug discovery programme. We present here an enumeration of steps starting from chemical templates and propose a solution at the state of the art, in the form of a system independent comprehensive computational pathway. This methodology is illustrated with cyclooxygenase-2 (COX-2) as a target. We built candidate molecules including a few Non Steroidal Anti-inflammatory Drugs (NSAIDs) from chemical templates, passed them through empirical filters to assess drug-like properties, optimized their geometries, derived partial atomic charges via quantum calculations, performed Monte Carlo docking, carried out molecular mechanics and developed free energy estimates with Molecular Mechanics Generalized Born Solvent Accessibility (MMGBSA) methodology for each of the candidate molecules. For the case of aspirin, we also conducted molecular dynamics on the enzyme, the drug and the complex with explicit solvent followed by binding free energy analysis. Collectively, the results obtained from the above studies viz. sorting of drugs from non-drugs, semi-quantitative estimates of binding free energies, amply demonstrate the viability of the strategy proposed for lead selection/design for biomolecular targets.