1,721,059 research outputs found
The anti-apoptotic proteins DJ-1 and Mcl-1: molecular basis of different protein-ligand interactions leading to apoptosis.
Full text linkDJ-1 and Mcl-1 are two anti-apoptotic proteins involved in distinct severe diseases such as Parkinson disease (PD) and Cancer. Even though they belong to different protein families, both genes take part in the apoptotic process; also, DJ-1 lies upstream in a pathway that affects cell death through members of the Mcl-1 protein family.
The first part of this thesis regards the effects of the conjugation of dopamine-derived quinones on DJ-1. Various functions have been ascribed to DJ-1 and many of these are linked with PD, such as a protective role against oxidative stress, either as a redox sensor or as an antioxidant protein. Oxidative stress is considered the major cause in neuronal death in PD and, in detail, the cause seems to involve the excessive oxidative stress generated by auto- and enzymatic oxidation of dopamine, which leads to the formation of highly reactive quinones (DAQs). The reactivity of DAQs toward DJ-1 in cells is reported in the literature, but the molecular basis and the related structural effects are not yet studied. DAQ are reactive toward nucleophilic atoms such as the thiolic sulfur atom in cysteine. The three cysteine residues seem to have very different roles in DJ-1: Cys106 is implicated in the oxidative activation of chaperone activity. The other two cysteines, Cys53 and Cys46, are located at the homodimer interface. The main objective of the thesis is the understanding of the structural modifications induced by the conjugation of DA onto the cysteine residues of DJ-1 using Molecular Dynamics, NMR, and Circular Dichroism (CD). The presence of different amounts of protein modified by one (+150 Da) or two (+300 Da) DAQs was confirmed by SDS-PAGE, radioactivity assay and mass spectroscopy. Interestingly, the same sample also showed the presence of a seemingly covalent dimer. To clarify which cysteine was involved in the dimerization, the same reaction was performed on two mutants, C106A and C53A. Covalent dimer formation was not detected in the C53A mutant. To characterize the structural modifications, we acquired several 1H-15N heteronuclear single quantum coherence (HSQC) spectra on the wt, C53A and C106A samples before and after DAQs treatment. Numerous modifications in the spectrum caused by the conjugation with DAQs were observed. Specifically, a notable number of peaks show a decrease in intensity, indicating a dynamic perturbation induced by DAQ conjugation. A molecular dynamics simulation study was used to explore the molecular basis of the covalent modification. We observed a different residue-fluctuation profile caused by the conjugation on Cys106, in agreement with NMR studies. We also provided a possible explanation of the molecular basis leading to dimerization. To clarify if covalent modification by dopamine may carry implications on the stability of DJ-1, we performed a thermal stability assay monitored by CD, analyzing wt DJ-1 and its C53A and C106A mutants, before and after the reaction with DAQs. For each pair, we observed different profiles, observing a lower thermal stability when Cys106 is involved.
The second part of this thesis regards the myeloid cell leukemia-1 (Mcl-1) protein and its role in apoptosis. One of the major anti-apoptotic mechanisms involves the alteration in the expression of B-cell lymphoma-2 (Bcl-2) family members, which consists of 25 genes divided in pro- and anti-apoptotic members. The balance between the levels of pro- and anti-apoptotic proteins is the key aspect, leading the cell to death or survival. Mcl-1 is a member of the anti-apoptotic family, and is a highly expressed pro-survival protein in several cancer cell lines. Even though the mechanism is still under discussion, Mcl-1 plays its anti-apoptotic role interacting with BAK and BAX, pro-apoptotic members of the Bcl-2 family, and the inhibition of this interaction promotes cell death in cancer cells. To identify new small peptides able to bind the BH3 cleft of Mcl-1 and to displace the pro-apoptotic binder, we performed a screening of a 109 different 12-mer peptides using the phage display technique. NMR was used as the technique of choice to validate the binding while Isothermal Titration Calorimetry (ITC) and fluorescence polarization assays (FPA) were used to measure the affinity. Three peptides with affinity in the low micromolar range were identified. The binding mode of these peptides was investigated in silico mixing the information harvested during the NMR studies. BLAST analysis of the identified sequences against the human genome identifies this characteristic pattern in a selection of interesting proteins including glucokinase, hexokinase, and a number of tumor suppressors among others. A short peptide sequence derived from glucokinase exhibits binding to Mcl-1 comparable to that seen for a 12-residue endogenous peptide. The sequence likely binds in a reverse orientation to that of the canonical BH3 helix, thereby placing the conserved glutamic acid residue in the location of the conserved aspartic acid residue of the BH3 sequence. These peptides are the shortest sequences ever observed to bind Mcl-1 and they may warrant development into improved Mcl-1 specific small molecules and peptide-based therapeutics. Further, their identification may provide the basis for increased understanding of possible cross-talk taking place between a number of divergent cellular signaling and homeostatic processes and the regulation of apoptosis.DJ-1 e Mcl-1 sono due proteine anti-apoptotiche coinvolte in diverse gravi patologie come il morbo di Parkinson (PD) e il cancro. Pur appartenendo a famiglie di proteine differenti, entrambi i geni sono coinvolti nel processo apoptotico, inoltre, DJ-1 si trova a monte in un percorso che interessa la morte cellulare attraverso i membri della famiglia della proteina Mcl-1.
La prima parte di questa tesi riguarda gli effetti della coniugazione subita da DJ-1 da parte di derivati chinonici della dopamina. Svariate funzioni sono state attribuite a DJ-1 e molti di queste sono collegati con PD, come ad esempio un ruolo protettivo contro lo stress ossidativo, sia come un sensore redox e come antiossidante. Lo stress ossidativo è considerato la principale causa di morte neuronale nel PD e, in particolare, la causa sembra coinvolgere lo stress ossidativo eccessivo generato da auto-ossidazione ed enzimatica della dopamina, che porta alla formazione di chinoni altamente reattivi (DAQ). La reattività del DAQ verso DJ-1 nelle cellule è riportata in letteratura, ma le basi molecolari e strutturali non sono ancora state studiate. I DAQ sono reattivi verso gli atomi nucleofili come l'atomo di zolfo tiolico tipico della cisteina. I tre residui di cisteina sembrano avere molto in ruoli diversi DJ-1: Cys106 è implicato l'attivazione di attività ossidativa e di chaperone. Le altre due cisteine, Cys53 e Cys46, si trovano nell’interfaccia dimerica. L'obiettivo principale della tesi è la comprensione delle modificazioni strutturali indotte dalla coniugazione di DA sui residui di cisteina mediante Dinamica Molecolare, NMR e dicroismo circolare (CD). La presenza di diverse quantità di proteina modificata da uno (150 Da) o due (300 Da) DAQ è stata confermata da SDS-PAGE, da analisi basate sulla radioattività 14C e dalla spettroscopia di massa. È interessante notare che lo stesso campione ha anche mostrato la presenza di una frazione dimerizzata covalente. Per chiarire quale cisteina e’ coinvolta nella dimerizzazione covalente, la stessa reazione è stata effettuata su due mutanti, C106A e C53A. formazione del dimero covalente non è stata rilevata nel mutante C53A. Per caratterizzare le modifiche strutturali, abbiamo acquisito molti 1H-15N heteronuclear single quantum coherence (HSQC) spettri sul WT, i campioni C53A e C106A prima e dopo trattamento con iDAQ. Numerose modifiche nello spettro causati dalla coniugazione con DAQ sono stati osservati. In particolare, un notevole numero di picchi mostra una diminuzione d’intensità, che indica una perturbazione dinamica indotta dalla coniugazione con i DAQ. Una simulazione di dinamica molecolare studio è stato utilizzata per esplorare le basi molecolari della modificazione covalente. Abbiamo osservato un profilo diverso residui fluttuazione causata dalla coniugazione in Cys106, in accordo con gli studi NMR. Abbiamo anche fornito una possibile spiegazione delle basi molecolari che porta alla dimerizzazione covalente. Per chiarire se la modifica covalente di dopamina possa portare ripercussioni sulla stabilità del DJ-1, abbiamo effettuato un test di stabilità termica controllata da CD, analizzando WT DJ-1 e la sua C53A e mutanti C106A, prima e dopo la reazione con DAQ. Per ogni coppia abbiamo trovato diversi profili, osservando una stabilità termica inferiore Cys106 quando è coinvolto.
La seconda parte di questa tesi riguarda la proteina myeloid cell leukemia-1 (Mcl-1) e il suo ruolo nell’ apoptosi. Uno dei principali meccanismi anti-apoptotici comporta l'alterazione dell'espressione dei membri della famiglia Bcl-2, che è composta da 25 membri suddivisi in geni pro-e anti-apoptotici. L'equilibrio tra i livelli di proteine pro-ed anti-apoptotico è l'aspetto chiave, conducendo la cellula alla morte o alla sopravvivenza. Mcl-1 è un membro della classe anti-apoptotica, ed è una proteina altamente espressa in diverse linee cellulari tumorali. Anche se il meccanismo è ancora in discussione, Mcl-1 svolge il suo ruolo interagendo con BAK e BAX, membri pro-apoptotici della famiglia Bcl-2, e l'inibizione di questa interazione promuove la morte delle cellule in cellule tumorali. Per identificare nuovi piccoli peptidi in grado di impegnare la cavita-BH3 di Mcl-1 e per inibire l’interazione con i membri pro-apoptotici, abbiamo effettuato uno screening di 109 diversi peptidi 12-mer con la tecnica del phage display. NMR è stata utilizzata come tecnica di scelta per convalidare l’interazione, mentre la Calorimetria isotermica di titolazione (ITC) e le analisi a fluorescenza polarizzata (FPA) sono stati utilizzati per misurare l'affinità. Tre peptidi con affinità nella gamma bassa micromolare sono stati identificati. La modalità di legame di questi peptidi è stata studiata in silico unitamente con le informazioni raccolte nel corso degli studi NMR. BLAST analisi delle sequenze individuate, identifica queste sequenze in una selezione di proteine interessanti tra cui glucochinasi, esochinasi, e una serie di soppressori tumorali. Una breve sequenza del peptide derivato da mostre glucochinasi dimostra un’affinità per Mcl paragonabile a quella osservata per un peptide endogeno di 12 residui. La sequenza si lega probabilmente con un orientamento contrario a quello tipico dell’elica BH3, ponendo in tal modo i residui conservati acido glutammico in luogo del residuo di acido aspartico conservate della sequenza BH3. Questi peptidi rappresentano la più corte sequenza mai osservata inibire Mcl-1, e possono giustificare lo sviluppo di composti di sintesi specifici e terapie a basati su peptidi. Inoltre, la loro identificazione può fornire la base per una maggiore comprensione delle possibili interazioni che si svolgono tra un numero di differenti proteine nella regolazione dell'apoptosi
A Novel NMR-Based Protocol to Screen Ultralow Molecular Weight Fragments
No full textFragment-based lead discovery has emerged as one of the most efficient screening strategies for finding hit molecules in drug discovery. Recently, a novel strategy based on a class of fragments characterized by an ultralow molecular weight (ULMW) has been proposed. These fragments bind to the target with a very low affinity, requiring reliable biophysical methods for detection. The most notable application of ULMW used a set of 81 fragments, named MiniFrags, and screened them by X-ray crystallography. We extended the utilization of this novel class of fragments to another gold standard technique for fragment-based screening: nuclear magnetic resonance (NMR). Here, we present a novel NMR protocol to detect and analyze such weak interactions in a challenging real-world scenario: a flexible target with a flat, water-exposed binding site. We identified a subset of 69 highly water-soluble MiniFrags that were screened against the antiapoptotic protein human Bfl-1
Synthesis and preliminary structure-activity relationship study of 2-aryl-2H-pyrazolo[4,3-c]quinolin-3-ones as potential checkpoint kinase 1 (Chk1) inhibitors
Full text linkThe serine-threonine checkpoint kinase 1 (Chk1) plays a critical role in the cell cycle arrest in response to DNA damage. In the last decade, Chk1 inhibitors have emerged as a novel therapeutic strategy to potentiate the anti-tumour efficacy of cytotoxic chemotherapeutic agents. In the search for new Chk1 inhibitors, a congeneric series of 2-aryl-2 H-pyrazolo[4,3-c]quinolin-3-one (PQ) was evaluated by in-vitro and in-silico approaches for the first time. A total of 30 PQ structures were synthesised in good to excellent yields using conventional or microwave heating, highlighting that 14 of them are new chemical entities. Noteworthy, in this preliminary study two compounds 4e 2 and 4h 2 have shown a modest but significant reduction in the basal activity of the Chk1 kinase. Starting from these preliminary results, we have designed the second generation of analogous in this class and further studies are in progress in our laboratories.Fil: Malvacio, Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Cuzzolin, Alberto. Università di Padova; ItaliaFil: Sturlese, Mattia. Università di Padova; ItaliaFil: Vera, Domingo Mariano Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; ArgentinaFil: Moyano, Elizabeth Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Moro, Stefano. Università di Padova; Itali
G-quadruplexes formation within the promoter of TEAD4 oncogene and their interaction with Vimentin
Full text linkG-quadruplexes (G4s) are nucleic acid secondary structures detected within human chromosomes, that cluster at gene promoters and enhancers. This suggests that G4s may play specific roles in the regulation of gene expression. Within a distinct subgroup of G-rich domains, the formation of two or more adjacent G4 units (G4-repeats) is feasible. Recently it was shown that Vimentin, a protein highly expressed within mesenchymal cells, selectively recognizes these arrangements. Putative G4-repeats have been searched within the human gene proximal promoters by the bioinformatics tool QPARSE and they resulted to be enriched at genes related to epithelial-to-mesenchymal transition (EMT). This suggested that Vimentin binding at these sites might be relevant for the maintenance of the mesenchymal phenotype. Among all the identified sequences, in the present study we selected the one located within the promoter of the TEAD4 oncogene. TEAD4 codifies for a transcriptional enhancer factor, TEAD4, that actively promotes EMT, supporting, cell proliferation and migration. Moreover, in colorectal cancer cells TEAD4 directly enhances the expression of Vimentin. Thus, the possible interaction of Vimentin with TEAD4 promoter could highlight a positive feedback loop between these two factors, associated to important tumor metastasis related events. Here, we exploited spectroscopic and electrophoretic measurements under different conditions to address the folding behavior of the selected sequence. This allowed us to validate the folding of TEAD4 promoter into a G4-repeat able to interact with Vimentin
Synthesis and structural studies of new analogues of PTH(1-11) containing C-alpha-tetra-substituted amino acids in position 8
No full textThe N-terminal 1–34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recently, analogues of PTH(1–11) fragments with helicity-enhancing substitutions have been demonstrated to yield potent analogues of PTH(1–34). The work describes the synthesis, biological activity and structure of analogues of the best modified PTH sequence H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 (I). In particular, the effect of the Ala/Aib substitution at positions 1 and 3 as well as of the replacement of Nle in position 8 with D-Nle, L-(αMe)-Nle and D-(αMe)-Nle was studied. The resulting peptides were characterized structurally by CD spectroscopy, solution NMR and MD, and in vitro for activity with respect to the cognate receptor, parathyroid hormone receptor
Implementing a Scoring Function Based on Interaction Fingerprint for Autogrow4: Protein Kinase CK1δ as a Case Study
Full text linkIn the last 20 years, fragment-based drug discovery (FBDD) has become a popular and consolidated approach within the drug discovery pipeline, due to its ability to bring several drug candidates to clinical trials, some of them even being approved and introduced to the market. A class of targets that have proven to be particularly suitable for this method is represented by kinases, as demonstrated by the approval of BRAF inhibitor vemurafenib. Within this wide and diverse set of proteins, protein kinase CK1δ is a particularly interesting target for the treatment of several widespread neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Computational methodologies, such as molecular docking, are already routinely and successfully applied in FBDD campaigns alongside experimental techniques, both in the hit-discovery and in the hit-optimization stage. Concerning this, the open-source software Autogrow, developed by the Durrant lab, is a semi-automated computational protocol that exploits a combination between a genetic algorithm and a molecular docking software for de novo drug design and lead optimization. In the current work, we present and discuss a modified version of the Autogrow code that implements a custom scoring function based on the similarity between the interaction fingerprint of investigated compounds and a crystal reference. To validate its performance, we performed both a de novo and a lead-optimization run (as described in the original publication), evaluating the ability of our fingerprint-based protocol to generate compounds similar to known CK1δ inhibitors based on both the predicted binding mode and the electrostatic and shape similarity in comparison with the standard Autogrow protocol
In silico 3D modeling of binding activities
No full textIn silico three-dimensional (3D) molecular modeling tools based upon the receptor/enzyme-ligand docking simulation in protein crystal structures and/or homology modeling of receptors have been reliably used in pharmacological research and development for decades. Molecular docking methodologies are helpful for revealing facets of activation and inactivation, thus improving mechanistic understanding and predicting molecular ligand binding activity, and they can have a high level of accuracy, and have also been explored and applied in chemical risk assessment. This computational approach is, however, only applicable for chemical hazard identification situations where the specific target receptor for a given chemical is known and the crystal structure/homology model of the receptor is available
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