Investigating dynamic and energetic determinants of protein nucleic acid recognition: analysis of the zinc finger zif268-DNA complexes

<p>Abstract</p> <p>Background</p> <p>Protein-DNA recognition underlies fundamental biological processes ranging from transcription to replication and modification. Herein, we present a computational study of the sequence modulation of internal dynamic properties and of intraprotein networks of aminoacid interactions that determine the stability and specificity of protein-DNA complexes.</p> <p>Results</p> <p>To this aim, we apply novel theoretical approaches to analyze the dynamics and energetics of biological systems starting from MD trajectories. As model system, we chose different sequences of Zinc Fingers (ZF) of the Zif268 family bound with different sequences of DNA. The complexes differ for their experimental stability properties, but share the same overall 3 D structure and do not undergo structural modifications during the simulations. The results of our analysis suggest that the energy landscape for DNA binding may be populated by dynamically different states, even in the absence of major conformational changes. Energetic couplings between residues change in response to protein and/or DNA sequence variations thus modulating the selectivity of recognition and the relative importance of different regions for binding.</p> <p>Conclusions</p> <p>The results show differences in the organization of the intra-protein energy-networks responsible for the stabilization of the protein conformations recognizing and binding DNA. These, in turn, are reflected into different modulation of the ZF's internal dynamics. The results also show a correlation between energetic and dynamic properties of the different proteins and their specificity/selectivity for DNA sequences. Finally, a dynamic and energetic model for the recognition of DNA by Zinc Fingers is proposed.</p

Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site

congenital tuberculosis after in vitro fertilization presenting with endobronchial granuloma

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Surveillance of acute flaccid paralysis in the Marches region (Italy): 1997–2007

BACKGROUND: The last case of poliomyelitis due to transmission of indigenous wild poliovirus occurred in Italy in 1982, however, it is important to guarantee a high quality surveillance as there is a risk of importation of cases from areas where polio is endemic. Stopping poliovirus transmission is pursued through a combination of high infant immunization coverage and surveillance for wild poliovirus through reporting and laboratory testing of all cases of acute flaccid paralysis (AFP) among children under fifteen years of age. The aim of this study was to describe and to evaluate 11 years of active surveillance in the Marches (Italy) in terms of: incidence, aetiology and clinical manifestation of AFP cases. METHODS: The active Acute Flaccid Paralysis surveillance has been carried out in the Marches region since February 1997 by the Chair of Hygiene which established a regional hospital network. Active surveillance involves 15 hospital centres. RESULTS: In the considered period, 0-15 years population varied between 187,051 in 1997 to 201,625 in 2007, so the number of AFP expected cases is 2 per year. From February 1997 to October 2007, 27 cases were found with rates of 1.0/100,000 in 1997; 2.0/100,000 in 1998; 1.0/100,000 in 1999; 0.5/100,000 in 2000; 2.5/100,000 in 2001; 1.0/100,000 in 2002; 0 in 2003; 0.5/100,000 in 2004; 1.5/100,000 in 2005; 2.0/100,000 in 2006; 1.5/100,000 in 2007. In 29.6% of cases two stool samples were collected in 14 days from the symptoms onset. The 60-days follow-up is available for 23 out of 27 cases reported. In 44.5% of cases the definite diagnosis was Guillain Barrè syndrome. CONCLUSION: In general, the surveillance activity is satisfactory even if in presence of some criticalities in biological samples collection. The continuation of surveillance, in addition to the maintenance of current levels of performance, will tend to a further and more detailed sensitization of all workers involved, in order to obtain spontaneous and prompt reporting, and to achieve the optimal standards recommended by the WHO both in the collection of biological samples and the availability of 60 days follow-up, with the goal of eradicating polio from all countries

Glutamate Receptor-Mediated Neurotoxicity in a Model of Ethanol Dependence and Withdrawal in Rat Organotypic Hippocampal Slice Cultures

Long-term alcohol use can lead to alterations in brain structure and functions and, in some cases, to neurodegeneration. Several mechanisms have been proposed to explain ethanol (EtOH)-related brain injury. One of the most relevant mechanisms of alcohol-induced neurodegeneration involves glutamatergic transmission, but their exact role is not yet fully understood. We investigated the neurochemical mechanisms underlying the toxicity induced by EtOH dependence and/or withdrawal by exposing rat organotypic hippocampal slices to EtOH (100–300 mM) for 7 days and then incubating the slices in EtOH-free medium for the subsequent 24 h. EtOH withdrawal led to a dose-dependent CA1 pyramidal cell injury, as detected with propidium iodide fluorescence. Electron microscopy of hippocampal slices revealed that not only EtOH withdrawal but also 7 days chronic EtOH exposure elicited signs of apoptotic cell death in CA1 pyramidal cells. These data were supported by electrophysiological recordings of spontaneus Excitatory Post Synaptic Currents (sEPSCs) from CA1 pyramidal cells. The average amplitude of sEPSCs in slices treated with EtOH for 7 days was significantly increased, and even more so during the first 30 min of EtOH withdrawal, suggesting that the initial phase of the neurodegenerative process could be due to an excitotoxic mechanism. We then analyzed the expression levels of presynaptic (vGlut1, vGlut2, CB1 receptor, synaptophysin) and postsynaptic (PSD95, GluN1, GluN2A, GluN2B, GluA1, GluA2, mGluR1 and mGluR5) proteins after 7 days EtOH incubation or after EtOH withdrawal. We found that only GluA1 and mGluR5 expression levels were significantly increased after EtOH withdrawal and, in neuroprotection experiments, we observed that AMPA and mGluR5 antagonists attenuated EtOH withdrawal-induced toxicity. These data suggest that chronic EtOH treatment promotes abnormal synaptic transmission that may lead to CA1 pyramidal cell death after EtOH withdrawal through glutamate receptors and increased excitotoxicity

Rational Design of Allosteric and Selective Inhibitors of the Molecular Chaperone TRAP1

Summary: TRAP1 is the mitochondrial paralog of the heat shock protein 90 (HSP90) chaperone family. Its activity as an energy metabolism regulator has important implications in cancer, neurodegeneration, and ischemia. Selective inhibitors of TRAP1 could inform on its mechanisms of action and set the stage for targeted drug development, but their identification was hampered by the similarity among active sites in HSP90 homologs. We use a dynamics-based approach to identify a TRAP1 allosteric pocket distal to its active site that can host drug-like molecules, and we select small molecules with optimal stereochemical features to target the pocket. These leads inhibit TRAP1, but not HSP90, ATPase activity and revert TRAP1-dependent downregulation of succinate dehydrogenase activity in cancer cells and in zebrafish larvae. TRAP1 inhibitors are not toxic per se, but they abolish tumorigenic growth of neoplastic cells. Our results indicate that exploiting conformational dynamics can expand the chemical space of chaperone antagonists to TRAP1-specific inhibitors with wide therapeutic opportunities. : The molecular chaperone TRAP1 regulates energy metabolism, and its activity is relevant in cancer and degenerative diseases. Here, Sanchez-Martin et al. identify highly selective allosteric inhibitors of TRAP1. These compounds revert biochemical and pro-neoplastic effects of TRAP1 and could both enlighten its mode of action and disclose novel therapeutic strategies. Keywords: chaperone inhibitors, anticancer compound, molecular dynamics, allosteric ligands, TRAP1, HSP90, mitochondria, mitochondrial biology, zebrafish, cancer cells, neurofibrom

Design, Synthesis and Biological Evaluation of Biphenylamide Derivatives as Hsp90 C-terminal Inhibitors

Modulation of Hsp90 C-terminal function represents a promising therapeutic approach for the treatment of cancer and neurodegenerative diseases. Current drug discovery efforts toward Hsp90 C-terminal inhibition focus on novobiocin, an antibiotic that was transformed into an Hsp90 inhibitor. Based on structural information obtained during the development of novobiocin derivatives and molecular docking studies, scaffolds containing a biphenyl moiety in lieu of the coumarin ring present in novobiocin were identified as new Hsp90 C-terminal inhibitors. Structure-activity relationship studies produced new derivatives that inhibit the proliferation of breast cancer cell lines at nanomolar concentrations, which corresponded directly with Hsp90 inhibition

A Fe2+-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes

Multifunctional human collagen lysyl hydroxylase (LH/PLOD) enzymes catalyze post-translational hydroxylation and subsequent glycosylation of collagens, enabling their maturation and supramolecular organization in the extracellular matrix (ECM). Recently, the overexpression of LH/PLODs in the tumor microenvironment results in abnormal accumulation of these collagen post-translational modifications, which has been correlated with increased metastatic progression of a wide variety of solid tumors. These observations make LH/PLODs excellent candidates for prospective treatment of aggressive cancers. The recent years have witnessed significant research efforts to facilitate drug discovery on LH/PLODs, including molecular structure characterizations and development of reliable high-throughput enzymatic assays. Using a combination of biochemistry and in silico studies, we characterized the dual role of Fe2+ as simultaneous cofactor and inhibitor of lysyl hydroxylase activity and studied the effect of a promiscuous Fe2+ chelating agent, 2,2'-bipyridil, broadly considered a lysyl hydroxylase inhibitor. We found that at low concentrations, 2,2'-bipyridil unexpectedly enhances the LH enzymatic activity by reducing the inhibitory effect of excess Fe2+. Together, our results show a fine balance between Fe2+-dependent enzymatic activity and Fe2+-induced self-inhibited states, highlighting exquisite differences between LH/PLODs and related Fe2+, 2-oxoglutarate dioxygenases and suggesting that conventional structure-based approaches may not be suited for successful inhibitor development. These insights address outstanding questions regarding druggability of LH/PLOD lysyl hydroxylase catalytic site and provide a solid ground for upcoming drug discovery and screening campaigns

Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 æ from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, cochaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary