Publications by authors named "Paola Storici"

33 Publications

An anti-HER2 nanobody binds to its antigen HER2 via two independent paratopes.

Int J Biol Macromol 2021 Apr 14;182:502-511. Epub 2021 Apr 14.

Lab of Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000 Rožna Dolina, Nova Gorica, Slovenia. Electronic address:

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation. In parallel, the complex was cross-linked, digested and the resulting peptides were characterized by mass-spectrometry to define maximal distance restraints between HER2 and A10 amino acids in their complex. These independent datasets guided a docking process, refined by molecular dynamics simulations, to develop a model of the complex and estimate per-residue free-energy contributions. Such a model explains the experimental data and identifies a second, non-canonical paratope, located in the region opposite to the conventional nanobody paratope, formed by the hypervariable loop regions LH1 and LH3. Both paratopes contributed substantially to the overall affinity by binding to independent HER2 epitopes. Nanobody mutants with substitution of key interaction residues, as indicated by the model, possess significantly lower affinity for HER2. This is the first described case of a "natural" biparatopic nanobody, directly selected by in-vitro panning.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.04.032DOI Listing
April 2021

Community-wide experimental evaluation of the PROSS stability-design method.

J Mol Biol 2021 Mar 26:166964. Epub 2021 Mar 26.

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel. Electronic address:

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. Up to six designs were evaluated in each case for expression, stability, and, in some cases, activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.
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http://dx.doi.org/10.1016/j.jmb.2021.166964DOI Listing
March 2021

Known Drugs Identified by Structure-Based Virtual Screening Are Able to Bind Sigma-1 Receptor and Increase Growth of Huntington Disease Patient-Derived Cells.

Int J Mol Sci 2021 Jan 28;22(3). Epub 2021 Jan 28.

Institute of Molecular Biology and Pathology, National Research Council of Italy, 00185 Rome, Italy.

Huntington disease (HD) is a devastating and presently untreatable neurodegenerative disease characterized by progressively disabling motor and mental manifestations. The sigma-1 receptor (σ1R) is a protein expressed in the central nervous system, whose 3D structure has been recently determined by X-ray crystallography and whose agonists have been shown to have neuroprotective activity in neurodegenerative diseases. To identify therapeutic agents against HD, we have implemented a drug repositioning strategy consisting of: (i) Prediction of the ability of the FDA-approved drugs publicly available through the ZINC database to interact with σ1R by virtual screening, followed by computational docking and visual examination of the 20 highest scoring drugs; and (ii) Assessment of the ability of the six drugs selected by computational analyses to directly bind purified σ1R in vitro by Surface Plasmon Resonance and improve the growth of fibroblasts obtained from HD patients, which is significantly impaired with respect to control cells. All six of the selected drugs proved able to directly bind purified σ1R in vitro and improve the growth of HD cells from both or one HD patient. These results support the validity of the drug repositioning procedure implemented herein for the identification of new therapeutic tools against HD.
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http://dx.doi.org/10.3390/ijms22031293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865886PMC
January 2021

Investigational Studies on a Hit Compound Cyclopropane-Carboxylic Acid Derivative Targeting -Acetylserine Sulfhydrylase as a Colistin Adjuvant.

ACS Infect Dis 2021 02 29;7(2):281-292. Epub 2021 Jan 29.

P4T Group, Department of Food and Drugs, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an acetylserine sulfhydrylase (OASS) inhibitor can be used as a colistin adjuvant to treat infections caused by Gram-positive and Gram-negative pathogens. A compound that binds OASS with a nM dissociation constant was tested as an adjuvant of colistin against six critical pathogens responsible for infections spreading worldwide, , serovar Typhimurium, , , methicillin-resistant , and . The compound showed promising synergistic or additive activities against all of them. Knockout experiments confirmed the intracellular target engagement supporting the proposed mechanism of action. Moreover, compound toxicity was evaluated by means of its hemolytic activity against sheep defibrinated blood cells, showing a good safety profile. The 3D structure of the compound in complex with OASS was determined at 1.2 Å resolution by macromolecular crystallography, providing for the first time structural insights about the nature of the interaction between the enzyme and this class of competitive inhibitors. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants and the structural basis for further structure-activity relationship studies.
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http://dx.doi.org/10.1021/acsinfecdis.0c00378DOI Listing
February 2021

Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant.

J Transl Med 2020 04 22;18(1):179. Epub 2020 Apr 22.

Ulisse BioMed - Area Science Park, Trieste, Italy.

Background: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance.

Methods: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance.

Results: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001).

Conclusions: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.
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http://dx.doi.org/10.1186/s12967-020-02344-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174922PMC
April 2020

Insight into GEBR-32a: Chiral Resolution, Absolute Configuration and Enantiopreference in PDE4D Inhibition.

Molecules 2020 Feb 19;25(4). Epub 2020 Feb 19.

Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy.

Alzheimer's disease is the most common type of dementia, affecting millions of people worldwide. One of its main consequences is memory loss, which is related to downstream effectors of cyclic adenosine monophosphate (cAMP). A well-established strategy to avoid cAMP degradation is the inhibition of phosphodiesterase (PDE). In recent years, GEBR-32a has been shown to possess selective inhibitory properties against PDE type 4 family members, resulting in an improvement in spatial memory processes without the typical side effects that are usually correlated with this mechanism of action. In this work, we performed the HPLC chiral resolution and absolute configuration assignment of GEBR-32a. We developed an efficient analytical and semipreparative chromatographic method exploiting an amylose-based stationary phase, we studied the chiroptical properties of both enantiomers and we assigned their absolute configuration by H-NMR (nuclear magnetic resonance). Lastly, we measured the IC values of both enantiomers against both the PDE4D catalytic domain and the long PDE4D3 isoform. Results strongly support the notion that GEBR-32a inhibits the PDE4D enzyme by interacting with both the catalytic pocket and the regulatory domains.
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http://dx.doi.org/10.3390/molecules25040935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070305PMC
February 2020

A consensus protocol for the in silico optimisation of antibody fragments.

Chem Commun (Camb) 2019 Nov;55(93):14043-14046

International School for Advanced Studies (SISSA), Via Bonomea 265, 34136, Trieste, Italy.

We present an in silico mutagenetic protocol for improving the binding affinity of single domain antibodies (or nanobodies, VHHs). The method iteratively attempts random mutations in the interacting region of the protein and evaluates the resulting binding affinity towards the target by scoring, with a collection of scoring functions, short explicit solvent molecular dynamics trajectories of the binder-target complexes. The acceptance/rejection of each attempted mutation is carried out by a consensus decision-making algorithm, which considers all individual assessments derived from each scoring function. The method was benchmarked by evolving a single complementary determining region (CDR) of an anti-HER2 VHH hit obtained by direct panning of a phage display library. The optimised VHH mutant showed significantly enhanced experimental affinity with respect to the original VHH it matured from. The protocol can be employed as it is for the optimization of peptides, antibody fragments, and (given enough computational power) larger antibodies.
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http://dx.doi.org/10.1039/c9cc06182gDOI Listing
November 2019

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action.

Int J Mol Sci 2019 Oct 21;20(20). Epub 2019 Oct 21.

Dipartimento di Medicina e Chirurgia, Università di Parma, Via Gramsci 14, 43126 Parma, Italy.

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and -acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein-protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.
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http://dx.doi.org/10.3390/ijms20205219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829319PMC
October 2019

Comparative analysis of fusion tags used to functionalize recombinant antibodies.

Protein Expr Purif 2020 02 26;166:105505. Epub 2019 Sep 26.

Lab of Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Rožna Dolina, Nova Gorica, Slovenia. Electronic address:

Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody. Furthermore, we assessed the efficiency of the derivatization process. The constructs always bore a 6xHis tag plus either the controls (EGFP and C-tag) or CLIP, HALO, AviTag, the LEPTG sequence recognized by Sortase A (Sortase tag), or a free cysteine. The advantages and drawbacks of the different systems were analyzed and discussed.
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http://dx.doi.org/10.1016/j.pep.2019.105505DOI Listing
February 2020

Investigating Drug-Target Residence Time in Kinases through Enhanced Sampling Simulations.

J Chem Theory Comput 2019 Aug 12;15(8):4646-4659. Epub 2019 Jul 12.

Computational and Chemical Biology , Fondazione Istituto Italiano di Tecnologia , via Morego 30 , 16163 Genova , Italy.

It is widely accepted that drug-target association and dissociation rates directly affect drug efficacy and safety. To rationally optimize drug binding kinetics, one must know the atomic arrangement of the protein-ligand complex during the binding/unbinding process in order to detect stable and metastable states. Whereas experimental approaches can determine kinetic constants with fairly good accuracy, computational approaches based on molecular dynamics (MD) simulations can deliver the atomistic details of the unbinding process. Furthermore, they can also be utilized prospectively to predict residence time (i.e., the inverse of unbinding kinetics constant, ) with an acceptable level of accuracy. Here, we report a novel method based on adiabatic bias MD with an electrostatics-like collective variable (dubbed elABMD) for sampling protein-ligand dissociation events in two kinases. elABMD correctly ranked a ligand series on glucokinase, in agreement with experimental data and previous calculations. Subsequently, we applied the new method prospectively to a congeneric series of GSK-3β inhibitors. For this series, new crystal structures were generated and the residence time was experimentally measured with surface plasmon resonance (SPR). There was good agreement between computational predictions and experimental measures, suggesting that elABMD is an innovative and efficient tool for calculating residence times.
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http://dx.doi.org/10.1021/acs.jctc.9b00104DOI Listing
August 2019

A Triazolotriazine-Based Dual GSK-3β/CK-1δ Ligand as a Potential Neuroprotective Agent Presenting Two Different Mechanisms of Enzymatic Inhibition.

ChemMedChem 2019 02 15;14(3):310-314. Epub 2019 Jan 15.

Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Licio Giorgeri 1, 34127, Trieste, Italy.

Glycogen synthase kinase 3β (GSK-3β) and casein kinase 1δ (CK-1δ) are emerging targets for the treatment of neuroinflammatory disorders, including Parkinson's disease. An inhibitor able to target these two kinases was developed by docking-based design. Compound 12, 3-(7-amino-5-(cyclohexylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)-2-cyanoacrylamide, showed combined inhibitory activity against GSK-3β and CK-1δ [IC (GSK-3β)=0.17 μm; IC (CK-1δ)=0.68 μm]. In particular, classical ATP competition was observed against CK-1δ, and a co-crystal of compound 12 inside GSK-3β confirmed a covalent interaction between the cyanoacrylamide warhead and Cys199, which could help in the development of more potent covalent inhibitors of GSK-3β. Preliminary studies on in vitro models of Parkinson's disease revealed that compound 12 is not cytotoxic and shows neuroprotective activity. These results encourage further investigations to validate GSK-3β/CK-1δ inhibition as a possible new strategy to treat neuroinflammatory/degenerative diseases.
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http://dx.doi.org/10.1002/cmdc.201800778DOI Listing
February 2019

Structure-Activity Relationships of Hexahydrocyclopenta[c]quinoline Derivatives as Allosteric Inhibitors of CDK2 and EGFR.

ChemMedChem 2018 12 20;13(24):2627-2634. Epub 2018 Nov 20.

Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy.

Following the discovery of a type III allosteric modulator of cyclin-dependent kinase 2 (CDK2) characterized by a hexahydrocyclopenta[c]quinolone scaffold, three different series of its derivatives were synthesized and biologically evaluated. Docking of the synthesized compounds into the allosteric pocket of CDK2 allowed the elucidation of structure-activity relationships (SARs). Moreover, the compounds were tested on the wild-type epidermal growth factor receptor (EGFR) kinase domain (KD) and its clinically relevant T790M/L858R mutant form. Herein we describe the first SAR investigation of allosteric ligands that bind to the type III inhibitor pocket of CDK2 and EGFR-KD. Although the activity of the synthesized inhibitors needs to be improved, the obtained results provide clear-cut indications about pharmacophore requirements and selectivity determinants. Remarkably, this study led to the identification of a selective T790M/L858R EGFR allosteric inhibitor that is inactive toward both wild-type EGFR and CDK2. Finally, docking into the T790M/L858R EGFR-KD led us to hypothesize that the compounds bind to the double-mutant EGFR-KD by adopting a binding mode different from that in CDK2, thus rationalizing the observed selectivity profile.
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http://dx.doi.org/10.1002/cmdc.201800687DOI Listing
December 2018

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity.

Biochim Biophys Acta Proteins Proteom 2018 12 27;1866(12):1260-1270. Epub 2018 Sep 27.

Department of Food and Drug, University of Parma, Parma, Italy; Institute of Biophysics, National Research Council, Pisa, Italy; National Institute of Biostructures and Biosystems, Rome, Italy. Electronic address:

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in K, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.
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http://dx.doi.org/10.1016/j.bbapap.2018.09.011DOI Listing
December 2018

Identification of small-molecule EGFR allosteric inhibitors by high-throughput docking.

Future Med Chem 2018 07 16;10(13):1545-1553. Epub 2018 May 16.

Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125, Modena, Italy.

Aim: The EGFR inhibitors represent the first-line treatment of non-small-cell lung cancer. However, the emergence of resistance urgently requires the development of new inhibitors targeting drug-resistant mutants.

Methodology: A recently released structure of an EGFR kinase domain in complex with an allosteric inhibitor and a mutant protein model derived from it were used to set up a low-cost high-throughput docking protocol for the fast identification of EGFR allosteric inhibitors.

Conclusion: The virtual screening of commercially available compounds led to the identification of interesting new hit compounds. The most promising hit was confirmed to be a new allosteric inhibitor of wild-type and T790M/L858R double mutant EGFR which was able to inhibit the growth of  non-small-cell lung cancer cell lines.
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http://dx.doi.org/10.4155/fmc-2018-0063DOI Listing
July 2018

Molecular Bases of PDE4D Inhibition by Memory-Enhancing GEBR Library Compounds.

Biochemistry 2018 05 1;57(19):2876-2888. Epub 2018 May 1.

Center for Nano Science and Technology @ PoliMi , Istituto Italiano di Tecnologia , via Giovanni Pascoli 70/3 , 20133 Milano , Italy.

Selected members of the large rolipram-related GEBR family of type 4 phosphodiesterase (PDE4) inhibitors have been shown to facilitate long-term potentiation and to improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, few if any structure-activity relationship studies have been performed to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degree of selectivity between the different PDE4 isoforms.
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http://dx.doi.org/10.1021/acs.biochem.8b00288DOI Listing
May 2018

Baculovirus-driven protein expression in insect cells: A benchmarking study.

J Struct Biol 2018 08 12;203(2):71-80. Epub 2018 Mar 12.

Max-Planck Institute of Biochemistry, Biochemistry Core Facility, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address:

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
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http://dx.doi.org/10.1016/j.jsb.2018.03.004DOI Listing
August 2018

A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action.

Nat Commun 2017 06 9;8:15772. Epub 2017 Jun 9.

National Laboratory CIB (LNCIB), Area Science Park Padriciano, Trieste 34149, Italy.

The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed in the majority of cancers and its activity strongly contributes to tumour initiation and progression. Inactivation of PIN1 function conversely curbs tumour growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening, we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinone-mimicking drug that generates reactive oxygen species and DNA damage, inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.
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http://dx.doi.org/10.1038/ncomms15772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472749PMC
June 2017

Structural basis of the specificity of USP18 toward ISG15.

Nat Struct Mol Biol 2017 03 6;24(3):270-278. Epub 2017 Feb 6.

Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany, Breisacherstraße 64, 79106 Freiburg, Germany.

Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin proteases and Ubl proteases, collectively termed DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates the interferon-induced Ubl ISG15. To identify the molecular determinants of this specificity, we solved the crystal structures of mouse USP18 alone and in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation, thus revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15, thus providing evidence that USP18's ISG15 specificity is mediated by a small interaction interface. Our results may provide a structural basis for the development of new drugs modulating ISG15 linkage.
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http://dx.doi.org/10.1038/nsmb.3371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405867PMC
March 2017

Topoisomerase IIβ mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs.

Cancer Cell Int 2016 26;16:58. Epub 2016 Jul 26.

Structural Biology Laboratory, Elettra-Sincrotrone Trieste, Strada Statale 14-km 163, 5, Basovizza, 34149 Trieste, Italy ; Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO USA.

Background: Glioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation.

Methods: In this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage.

Results: We found several differentially expressed genes and we identified topoisomerase IIβ (Top2β) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2β was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2β expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2β increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2β depletion in NCH421k cells.

Conclusion: We suggest that Top2β may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics.
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http://dx.doi.org/10.1186/s12935-016-0339-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960855PMC
July 2016

Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell differentiation.

Biophys Chem 2015 Dec 25;207:90-6. Epub 2015 Sep 25.

SISSI beamline, Elettra-Sincrotrone Trieste, Italy.

According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem cells - a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection. Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative, labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics. The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol in clinical practice to estimate the content of stem cells in a tumor sample.
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http://dx.doi.org/10.1016/j.bpc.2015.09.005DOI Listing
December 2015

From protein structure to function via single crystal optical spectroscopy.

Front Mol Biosci 2015 28;2:12. Epub 2015 Apr 28.

Department of Pharmacy, University of Parma Parma, Italy ; National Institute of Biostructures and Biosystems Rome, Italy ; Institute of Biophysics, Consiglio Nazionale delle Ricerche Pisa, Italy.

The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic "artifacts," including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5'-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms.
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http://dx.doi.org/10.3389/fmolb.2015.00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428442PMC
May 2015

Synthesis, characterization, and optimization for in vivo delivery of a nonselective isopeptidase inhibitor as new antineoplastic agent.

J Med Chem 2015 Feb 12;58(4):1691-704. Epub 2015 Feb 12.

Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste , Via Giorgieri 1, 34127 Trieste, Italy.

Bis-arylidenecycloalkanones structurally related to the nonselective isopeptidase inhibitor G5 were synthesized and tested for cytotoxic activity against glioblastoma cells. Cytotoxicities correlate well with Hammett σ constants for substituted arylidene groups, confirming the proposed inhibition mechanism. A new inhibitor (2c) based on the 4-hydroxycyclohexanone scaffold, which favors apoptosis over necrosis, was selected for further development. 2c inhibited representative deubiquitinases with micromolar IC50, and its proapoptotic activity was studied on several cancer cell lines. Inhibitor 2c was conjugated to PEG via dicarbamate and diester linkers. While the dicarbamate was inactive, the diester (2cPE) behaves like a prodrug and is converted into the active species 2c by secreted esterase activities. Finally, 2cPE was also tested in vivo on A549 lung carcinoma xenografts generated in mice. Intravenous treatment with 2cPE led to a significant reduction in primary tumor growth, without appreciable toxicity to mice.
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http://dx.doi.org/10.1021/jm501336hDOI Listing
February 2015

Thieno[3,2-c]pyrazoles: a novel class of Aurora inhibitors with favorable antitumor activity.

Bioorg Med Chem 2010 Oct 25;18(19):7113-20. Epub 2010 Jul 25.

Nerviano Medical Sciences-Oncology, via Pasteur 10, 20014 Nerviano, Milan, Italy.

A novel series of 3-amino-1H-thieno[3,2-c]pyrazole derivatives demonstrating high potency in inhibiting Aurora kinases was developed. Here we describe the synthesis and a preliminary structure-activity relationship, which led to the discovery of a representative compound (38), which showed low nanomolar inhibitory activity in the anti-proliferation assay and was able to block the cell cycle in HCT-116 cell line. This compound demonstrated favorable pharmacokinetic properties and good efficacy in the HL-60 xenograft tumor model.
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http://dx.doi.org/10.1016/j.bmc.2010.07.048DOI Listing
October 2010

Identification of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as a new class of orally and selective Polo-like kinase 1 inhibitors.

J Med Chem 2010 May;53(9):3532-51

Nerviano Medical Sciences Srl, Oncology, Viale Pasteur 10, 20014 Nerviano, (Mi), Italy.

Polo-like kinase 1 (Plk1) is a fundamental regulator of mitotic progression whose overexpression is often associated with oncogenesis and therefore is recognized as an attractive therapeutic target in the treatment of proliferative diseases. Here we discuss the structure-activity relationship of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline class of compounds that emerged from a high throughput screening (HTS) campaign as potent inhibitors of Plk1 kinase. Furthermore, we describe the discovery of 49, 8-{[2-methoxy-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide, as a highly potent and specific ATP mimetic inhibitor of Plk1 (IC(50) = 0.007 microM) as well as its crystal structure in complex with the methylated Plk1(36-345) construct. Compound 49 was active in cell proliferation against different tumor cell lines with IC(50) values in the submicromolar range and active in vivo in the HCT116 xenograft model where it showed 82% tumor growth inhibition after repeated oral administration.
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http://dx.doi.org/10.1021/jm901713nDOI Listing
May 2010

PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer.

Mol Cancer Ther 2007 Dec;6(12 Pt 1):3158-68

Nerviano Medical Sciences S.r.l.-Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

PHA-739358 is a small-molecule 3-aminopyrazole derivative with strong activity against Aurora kinases and cross-reactivities with some receptor tyrosine kinases relevant for cancer. PHA-739358 inhibits all Aurora kinase family members and shows a dominant Aurora B kinase inhibition-related cellular phenotype and mechanism of action in cells in vitro and in vivo. p53 status-dependent endoreduplication is observed upon treatment of cells with PHA-739358, and phosphorylation of histone H3 in Ser(10) is inhibited. The compound has significant antitumor activity in different xenografts and spontaneous and transgenic animal tumor models and shows a favorable pharmacokinetic and safety profile. In vivo target modulation is observed as assessed by the inhibition of the phosphorylation of histone H3, which has been validated preclinically as a candidate biomarker for the clinical phase. Pharmacokinetics/pharmacodynamics modeling was used to define drug potency and to support the prediction of active clinical doses and schedules. We conclude that PHA-739358, which is currently tested in clinical trials, has great therapeutic potential in anticancer therapy in a wide range of cancers.
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http://dx.doi.org/10.1158/1535-7163.MCT-07-0444DOI Listing
December 2007

1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles: identification of a potent Aurora kinase inhibitor with a favorable antitumor kinase inhibition profile.

J Med Chem 2006 Nov;49(24):7247-51

Nerviano Medical Sciences S.r.l. viale Pasteur 10, 20014 Nerviano, Milan, Italy.

The optimization of a series of 5-phenylacetyl 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivatives toward the inhibition of Aurora kinases led to the identification of compound 9d. This is a potent inhibitor of Aurora kinases that also shows low nanomolar potency against additional anticancer kinase targets. Based on its high antiproliferative activity on different cancer cell lines, favorable chemico-physical and pharmacokinetic properties, and high efficacy in in vivo tumor models, compound 9d was ultimately selected for further development.
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http://dx.doi.org/10.1021/jm060897wDOI Listing
November 2006

Crystal structure of osmoporin OmpC from E. coli at 2.0 A.

J Mol Biol 2006 Oct 3;362(5):933-42. Epub 2006 Aug 3.

Division of Structural Biology, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

Porins form transmembrane pores in the outer membrane of Gram-negative bacteria with matrix porin OmpF and osmoporin OmpC from Escherichia coli being differentially expressed depending on environmental conditions. The three-dimensional structure of OmpC has been determined to 2.0 A resolution by X-ray crystallography. As expected from the high sequence similarity, OmpC adopts the OmpF-like 16-stranded hollow beta-barrel fold with three beta-barrels associated to form a tight trimer. Unlike in OmpF, the extracellular loops form a continuous wall at the perimeter of the vestibule common to the three pores, due to a 14-residues insertion in loop L4. The pore constriction and the periplasmic outlet are very similar to OmpF with 74% of the pore lining residues being conserved. Overall, only few ionizable residues are exchanged at the pore lining. The OmpC structure suggests that not pore size, but electrostatic pore potential and particular atomic details of the pore linings are the critical parameters that physiologically distinguish OmpC from OmpF.
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http://dx.doi.org/10.1016/j.jmb.2006.08.002DOI Listing
October 2006

PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity.

Clin Cancer Res 2006 Jul;12(13):4080-9

Nerviano Medical Sciences S.r.l.-Oncology, Milan, Italy.

Purpose: Aurora kinases play critical roles during mitosis in chromosome segregation and cell division. The aim of this study was to determine the preclinical profile of a novel, highly selective Aurora kinase inhibitor, PHA-680632, as a candidate for anticancer therapy.

Experimental Design: The activity of PHA-680632 was assayed in a biochemical ATP competitive kinase assay. A wide panel of cell lines was evaluated for antiproliferative activity. Cell cycle analysis. Immunohistochemistry, Western blotting, and Array Scan were used to follow mechanism of action and biomarker modulation. Specific knockdown of the targets by small interfering RNA was followed to validate the observed phenotypes. Efficacy was determined in different xenograft models and in a transgenic animal model of breast cancer.

Results: PHA-680632 is active on a wide range of cancer cell lines and shows significant tumor growth inhibition in different animal tumor models at well-tolerated doses. The mechanism of action of PHA-680632 is in agreement with inhibition of Aurora kinases. Histone H3 phosphorylation in Ser10 is mediated by Aurora B kinase, and our kinetic studies on its inhibition by PHA-680632 in vitro and in vivo show that phosphorylation of histone H3 is a good biomarker to follow activity of PHA-680632.

Conclusions: PHA-680632 is the first representative of a new class of Aurora inhibitors with a high potential for further development as an anticancer therapeutic. On treatment, different cell lines respond differentially, suggesting the absence of critical cell cycle checkpoints that could be the basis for a favorable therapeutic window.
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http://dx.doi.org/10.1158/1078-0432.CCR-05-1964DOI Listing
July 2006

Potent and selective Aurora inhibitors identified by the expansion of a novel scaffold for protein kinase inhibition.

J Med Chem 2005 Apr;48(8):3080-4

Nerviano Medical Sciences - Oncology, via Pasteur 10, 20014 Nerviano, Milan, Italy.

Potent and selective Aurora kinase inhibitors were identified from the combinatorial expansion of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a novel and versatile scaffold designed to target the ATP pocket of protein kinases. The most potent compound reported in this study had an IC(50) of 0.027 microM in the enzymatic assay for Aur-A inhibition and IC(50)s between 0.05 microM and 0.5 microM for the inhibition of proliferation of different tumor cell lines.
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http://dx.doi.org/10.1021/jm049076mDOI Listing
April 2005

Mechanistic crystallography. Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid as elucidated by crystallography.

Biochemistry 2004 Nov;43(44):14057-63

Division of Structural Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

(1R,3S,4S)-3-Amino-4-fluorocyclopentane-1-carboxylic acid (7) was previously shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) [Qiu, J. and Silverman, R. B. (2000) J. Med. Chem. 43, 706-720]. Two mechanisms were considered as reasonable possibilities, a Michael addition mechanism and an enamine mechanism. On the basis of a variety of chemical studies, including tedious radiolabeling experiments, it was concluded that inactivation by 7 proceeds by a Michael addition mechanism. Here, a crystal structure of 7 bound to pig liver GABA-AT is reported, which clearly demonstrates that the adduct formed is derived from an enamine mechanism. This represents another example of how crystallography is an important tool for elucidation of inactivation mechanisms.
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http://dx.doi.org/10.1021/bi0487185DOI Listing
November 2004