48 results found
Galderisi S, Rossi M, Cicaloni V, et al., 2022, Homogentisic acid induces autophagy alterations leading to chondroptosis in human chondrocytes: Implications in Alkaptonuria, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, Vol: 717, ISSN: 0003-9861
Pardo O, Chrysostomou S, Roy R, et al., 2021, Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer, Science Translational Medicine, Vol: 13, ISSN: 1946-6234
Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.
Cronin R, Brooke GN, Prischi F, 2021, The role of the p90 ribosomal S6 kinase family in prostate cancer progression and therapy resistance, ONCOGENE, Vol: 40, Pages: 3775-3785, ISSN: 0950-9232
Prischi F, Filippakopoulos P, 2021, Editorial: Structural Studies of Protein Complexes in Signaling Pathways, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 8
Obomighie I, Lapenas K, Murphy BE, et al., 2021, The Role of Ribosomal Protein S6 Kinases in Plant Homeostasis, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 8
Deganutti G, Prischi F, Reynolds CA, 2020, Supervised molecular dynamics for exploring the druggability of the SARS-CoV-2 spike protein, JOURNAL OF COMPUTER-AIDED MOLECULAR DESIGN, Vol: 35, Pages: 195-207, ISSN: 0920-654X
Brooke GN, Prischi F, 2020, Structural and functional modelling of SARS-CoV-2 entry in animal models, SCIENTIFIC REPORTS, Vol: 10, ISSN: 2045-2322
Deganutti G, Prischi F, Reynolds CA, 2020, Supervised molecular dynamics for exploring the druggability of the SARS-CoV-2 spike protein
<jats:title>Abstract</jats:title> <jats:p>The recent outbreak of the respiratory syndrome-related coronavirus (SARS-CoV-2) is stimulating an unprecedented scientific campaign to alleviate the burden of the coronavirus disease (COVID-19). One line of research has focused on targeting SARS-CoV-2 proteins fundamental for its replication by repurposing drugs approved for other diseases. The first interaction between the virus and the host cell is mediated by the spike protein on the virus surface and the human angiotensin-converting enzyme (ACE2). Small molecules able to bind the receptor-binding domain (RBD) of the spike protein and disrupt the binding to ACE2 would offer an important tool for slowing, or even preventing, the infection. Here, we screened 2421 approved small molecules<jats:italic> in silico</jats:italic> and validated the docking outcomes through extensive molecular dynamics simulations. Out of six drugs characterized as putative RBD binders, the cephalosporin antibiotic cefsulodin was further assessed for its effect on the binding between the RBD and ACE2, suggesting the importance of considering the dynamic formation of the heterodimer when judging any potential candidate.</jats:p>
Trezza A, Iovinelli D, Santucci A, et al., 2020, An integrated drug repurposing strategy for the rapid identification of potential SARS-CoV-2 viral inhibitors, SCIENTIFIC REPORTS, Vol: 10, ISSN: 2045-2322
Trezza A, Iovinelli D, Prischi F, et al., 2020, An integrated drug repurposing strategy for the rapid identification of potential SARS-CoV-2 viral inhibitors
<jats:title>Abstract</jats:title> <jats:p>The Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2). The virus has rapidly spread in humans, causing the ongoing Coronavirus pandemic. Recent studies have shown that, similarly to SARS-CoV, SARS-CoV-2 utilises the Spike glycoprotein on the envelope to recognise and bind the human receptor ACE2. This event initiates the fusion of viral and host cell membranes and then the viral entry into the host cell. Despite several ongoing clinical studies, there are currently no approved vaccines or drugs that specifically target SARS-CoV-2. Until an effective vaccine is available, repurposing FDA approved drugs could significantly shorten the time and reduce the cost compared to de novo drug discovery. In this study we attempted to overcome the limitation of in silico virtual screening by applying a robust <jats:italic>in silico</jats:italic> drug repurposing strategy. We combined and integrated docking simulations, with molecular dynamics (MD), Supervised MD (SuMD) and Steered MD (SMD) simulations to identify a Spike protein – ACE2 interaction inhibitor. Our data showed that Simeprevir and Lumacaftor bind the receptor-binding domain of the Spike protein with high affinity and prevent ACE2 interaction.Authors Alfonso Trezza and Daniele Iovinelli contributed equally to this work.</jats:p>
Brooke GN, Prischi F, 2020, Structural and functional modelling of SARS-CoV-2 entry in animal models
<jats:title>Abstract</jats:title> <jats:p>SARS-CoV-2 is the novel <jats:italic>coronavirus </jats:italic>responsible for the outbreak of COVID-19, a disease that has spread to over 100 countries and, as of the 13 May 2020, has infected over 4 million people. Despite the urgent need to find effective therapeutics, research on SARS-CoV-2 has been affected by a lack of suitable animal models. To facilitate the development of medical approaches and novel treatments, we compared the ACE2 receptor and TMPRSS2 protease usage of the SARS-CoV-2 Spike glycoprotein in human and in a panel of animal models, i.e. guinea pig, dog, cat, rat, rabbit, ferret and mouse. Here we showed that ACE2, but not TMPRSS2, has a higher level of sequence variability in the Spike protein interaction surface, which greatly influences Spike protein binding mode. Comparison of SARS-CoV and SARS-CoV-2 S proteins bound the ACE2 receptors showed that the SARS-CoV-2 Spike glycoprotein has adapted to bind the human, but not rodents, ACE2 with high affinity. In contrast, we did not detect species-specific adaptation for TMPRSS2. Analysis of binding modes and protein contacts indicates that ferrets are the most suitable model for the study of inhibitory antibodies and small molecules targeting the SARS-CoV-2 Spike protein interaction with ACE2. Since TMPRSS2 is similar across species, our data also suggest that transgenic animal models expressing human ACE2, such as the K18-hACE2 mouse, are also likely to be useful models for studies investigating viral entry.</jats:p>
Spiga O, Cicaloni V, Fiorini C, et al., 2020, Machine learning application for development of a data-driven predictive model able to investigate quality of life scores in a rare disease, ORPHANET JOURNAL OF RARE DISEASES, Vol: 15
Chrysostomou S, Roy R, Prischi F, et al., 2019, Abstract 1775: Targeting RSK4 prevents both chemoresistance and metastasis in lung cancer, AACR Annual Meeting on Bioinformatics, Convergence Science, and Systems Biology, Publisher: American Association for Cancer Research, Pages: 1-2, ISSN: 0008-5472
Lung cancer is the commonest cause of cancer death worldwide with a five-year survival rate of less than five percent for metastatic tumors. Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer cases of which adenocarcinoma prevails. Patients almost invariably develop metastatic drug-resistant disease and this is responsible for our failure to provide curative therapy. Hence, a better understanding of the mechanisms underlying these biological processes is urgently required to improve clinical outcome.The 90-kDa ribosomal S6 kinases (RSKs) are downstream effectors of the RAS/MAPK cascade. RSKs are highly conserved serine/threonine protein kinases implicated in diverse cellular processes, including cell survival, proliferation, migration and invasion. Four isoforms exist in humans (RSK1-4) and are uniquely characterized by the presence of two non-identical N- and C-terminal kinase domains. RSK isoforms are 73-80% identical at protein level and this has been thought to suggest overlapping functions.However, through functional genomic kinome screens, we show that RSK4, contrary to RSK1, promotes both drug resistance and metastasis in lung cancer. This kinase is overexpressed in the majority (57%) of NSCLC biopsies and this correlates with poor overall survival in lung adenocarcinoma patients. Genetic silencing of RSK4 sensitizes lung cancer cells to chemotherapy and prevents their migration and invasiveness in vitro and in vivo. RSK4 downregulation decreases the anti-apoptotic proteins Bcl2 and cIAP1/2 which correlates with increased apoptotic signalling, whilst it also induces mesenchymal-epithelial transition (MET) through inhibition of NFκB activity. A small-molecule inhibitor screen identified several floxacins, including trovafloxacin, as potent allosteric inhibitors of RSK4 activation. Trovafloxacin reproduced all biological and molecular effects of RSK4 silencing in vitro and in vivo, and is predicted to bind a novel allosteric site revealed
Prischi F, Chrysostomou S, Roy R, et al., 2019, Targeting RSK4 prevents both chemoresistance and metastasis in lung and bladder cancer, FEBS Open Bio, Publisher: WILEY, Pages: 330-330, ISSN: 2211-5463
Prischi F, Ali MM, 2019, Phosphorylated RSK4 N-terminal Kinase Domain in complex with AMP-PNP
Prischi F, Ali MM, 2019, RSK4 N-terminal Kinase Domain S232E in complex with AMP-PNP
Prischi F, Ali MM, 2019, RSK4 N-terminal Kinase Domain in complex with AMP-PNP
Trampari E, Holden ER, Wickham GJ, et al., 2019, Antibiotics select for novel pathways of resistance in biofilms
<jats:title>Abstract</jats:title><jats:p>Most bacteria in nature exist in aggregated communities known as biofilms. Bacteria within biofilms are inherently highly resistant to antibiotics. Current understanding of the evolution and mechanisms of antibiotic resistance is largely derived from work from cells in liquid culture and it is unclear whether biofilms adapt and evolve in response to sub-inhibitory concentrations of drugs. Here we used a biofilm evolution model to show that biofilms of a model food borne pathogen,<jats:italic>Salmonella</jats:italic>Typhimurium rapidly evolve in response to exposure to three clinically important antibiotics. Whilst the model strongly selected for improved biofilm formation in the absence of any drug, once antibiotics were introduced the need to adapt to the drug was more important than the selection for improved biofilm formation. Adaptation to antibiotic stress imposed a marked cost in biofilm formation, particularly evident for populations exposed to cefotaxime and azithromycin. We identified distinct resistance phenotypes in biofilms compared to corresponding planktonic control cultures and characterised new mechanisms of resistance to cefotaxime and azithromycin. Novel substitutions within the multidrug efflux transporter, AcrB were identified and validated as impacting drug export as well as changes in regulators of this efflux system. There were clear fitness costs identified and associated with different evolutionary trajectories. Our results demonstrate that biofilms adapt rapidly to low concentrations of antibiotics and the mechanisms of adaptation are novel. This work will be a starting point for studies to further examine biofilm specific pathways of adaptation which inform future antibiotic use.</jats:p>
Zabet NR, Catoni M, Prischi F, et al., 2017, Cytosine methylation at CpCpG sites triggers accumulation of non-CpG methylation in gene bodies, Nucleic Acids Research, Vol: 45, Pages: 3777-3784, ISSN: 1362-4962
Methylation of cytosine is an epigenetic mark involved in the regulation of transcription, usually associated with transcriptional repression. In mammals, methylated cytosines are found predominantly in CpGs but in plants non-CpG methylation (in the CpHpG or CpHpH contexts, where H is A, C or T) is also present and is associated with the transcriptional silencing of transposable elements. In addition, CpG methylation is found in coding regions of active genes. In the absence of the demethylase of lysine 9 of histone 3 (IBM1), a subset of body-methylated genes acquires non-CpG methylation. This was shown to alter their expression and affect plant development. It is not clear why only certain body-methylated genes gain non-CpG methylation in the absence of IBM1 and others do not. Here we describe a link between CpG methylation and the establishment of methylation in the CpHpG context that explains the two classes of body-methylated genes. We provide evidence that external cytosines of CpCpG sites can only be methylated when internal cytosines are methylated. CpCpG sites methylated in both cytosines promote spreading of methylation in the CpHpG context in genes protected by IBM1. In contrast, CpCpG sites remain unmethylated in IBM1-independent genes and do not promote spread of CpHpG methylation.
Prischi F, Pastore A, 2017, Hybrid Methods in Iron-Sulfur Cluster Biogenesis, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 4
Prischi F, Pastore A, 2016, Application of Nuclear Magnetic Resonance and Hybrid Methods to Structure Determination of Complex Systems, Advanced Technologies for Protein Complex Production and Characterization. Advances in Experimental Medicine and Biology, Editors: Vega, Publisher: SPRINGER INT PUBLISHING AG, Pages: 351-368, ISBN: 978-3-319-27214-6
The current main challenge of Structural Biology is to undertake the structure determination of increasingly complex systems in the attempt to better understand their biological function. As systems become more challenging, however, there is an increasing demand for the parallel use of more than one independent technique to allow pushing the frontiers of structure determination and, at the same time, obtaining independent structural validation. The combination of different Structural Biology methods has been named hybrid approaches. The aim of this review is to critically discuss the most recent examples and new developments that have allowed structure determination or experimentally-based modelling of various molecular complexes selecting them among those that combine the use of nuclear magnetic resonance and small angle scattering techniques. We provide a selective but focused account of some of the most exciting recent approaches and discuss their possible further developments.
Carrara M, Prischi F, Ali MMU, 2015, Crystal structures reveal transient PERK luminal domain tetramerization in ER stress signaling
Carrara M, Prischi F, Ali MMU, 2015, Crystal structures reveal transient PERK luminal domain tetramerization in ER stress signaling
Carrara M, Prischi F, Nowak P, et al., 2015, Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling, EMBO Journal, Vol: 34, Pages: 1589-1600, ISSN: 0261-4189
Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein‐folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X‐ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation.
Popovic M, Sanfelice D, Pastore C, et al., 2015, Selective observation of the disordered import signal of a globular protein by in-cell NMR: The example of frataxins, Protein Science, Vol: 24, Pages: 996-1003, ISSN: 0961-8368
We have exploited the capability of in-cell NMR to selectively observe flexible regions withinfolded proteins to carry out a comparative study of two members of the highly conserved frataxin fam-ily which are found both in prokaryotes and in eukaryotes. They all contain a globular domain whichshares more than 50% identity, which in eukaryotes is preceded by an N-terminal tail containing themitochondrial import signal. We demonstrate that the NMR spectrum of the bacterial ortholog CyaYcannot be observed in the homologous E. coli system, although it becomes fully observable as soonas the cells are lysed. This behavior has been observed for several other compact globular proteins asseems to be the rule rather than the exception. The NMR spectrum of the yeast ortholog Yfh1 containsinstead visible signals from the protein. We demonstrate that they correspond to the flexible N-terminal tail indicating that this is flexible and unfolded. This flexibility of the N-terminus agrees withprevious studies of human frataxin, despite the extensive sequence diversity of this region in the twoproteins. Interestingly, the residues that we observe in in-cell experiments are not visible in the crystalstructure of a Yfh1 mutant design ed to destabilize the first helix. More importantly, our results showthat, in cell, the protein is predominantly present not as an aggregate but as a monomeric species.
Carrara M, Prischi F, Nowak PR, et al., 2015, Noncanonical binding of BiP ATPase domain to Ire1 and Perk is dissociated by unfolded protein C(H)1 to initiate ER stress signaling, eLife, Vol: 4, Pages: 1-16, ISSN: 2050-084X
The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response in order to maintain homeostasis. How cells detect the accumulation of misfolded proteins remains unclear. In this study, we identify a noncanonical interaction between the ATPase domain of the ER chaperone BiP and the luminal domains of the UPR sensors Ire1 and Perk that dissociates when authentic ER unfolded protein CH1 binds to the canonical substrate binding domain of BiP. Unlike the interaction between chaperone and substrates, we found that the interaction between BiP and UPR sensors was unaffected by nucleotides. Thus, we discover that BiP is dual functional UPR sensor, sensing unfolded proteins by canonical binding to substrates and transducing this event to noncanonical, signaling interaction to Ire1 and Perk. Our observations implicate BiP as the key component for detecting ER stress and suggest an allosteric mechanism for UPR induction.
Prischi F, Nowak PR, Carrara M, et al., 2014, Phosphoregulation of Ire1 RNase splicing activity (vol 5, pg 3554, 2014), NATURE COMMUNICATIONS, Vol: 5, ISSN: 2041-1723
Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation.
Carrara M, Prischi F, Ali MMU, 2013, UPR Signal Activation by Luminal Sensor Domains, Int. j. Mol. Sci., Vol: 14
Bernini A, Spiga O, Venditti V, et al., 2012, The use of a ditopic Gd(III) paramagnetic probe for investigating alpha-bungarotoxin surface accessibility, JOURNAL OF INORGANIC BIOCHEMISTRY, Vol: 112, Pages: 25-31, ISSN: 0162-0134
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