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Article ; Online: Development of a Liquid Chromatography and High-Resolution and -Accuracy Mass Spectrometry Method to Evaluate New Biotherapeutic Entity Processing in Human Liver Lysosomes.

Colangelo, Gabriele Sergio / Di Ianni, Andrea / Cowan, Kyra / Riccardi Sirtori, Federico / Barbero, Luca Maria

ImmunoHorizons

2023 Volume 7, Issue 6, Page(s) 467–479

Abstract: Biotherapeutic immunogenicity remains a great challenge for researchers because multiple factors trigger immune responses. Predicting and assessing the potential human immune response against biological drugs could represent an impressive breakthrough ... ...

Abstract	Biotherapeutic immunogenicity remains a great challenge for researchers because multiple factors trigger immune responses. Predicting and assessing the potential human immune response against biological drugs could represent an impressive breakthrough toward generating potentially safer and more efficacious therapeutic proteins. This article describes an in vitro assay that can contribute to evaluating the potential immunogenicity of biotherapeutics by focusing on lysosomal proteolysis. We selected human liver lysosomes (hLLs) from four different donors as a surrogate in vitro model instead of APC lysosomes because they are a ready-to-use lysosomal source. To assess the biological comparability of this surrogate to APC lysosomal extract, we compared the proteome content of hLLs with literature data of lysosomal fractions extracted from murine bone marrow and human blood-derived dendritic cells. Then we tested infliximab (IFX; Remicade) under different proteolytic conditions using liquid chromatography and high-resolution and -accuracy mass spectrometry to better define the degradation kinetics inside the lysosomes. hLLs revealed similar enzymatic content compared with human and murine dendritic cell lysosomes. Degradation assays demonstrated that our liquid chromatography and high-resolution and -accuracy mass spectrometry method could identify both the intact protein and the peptides resulting from proteolysis with high specificity and resolution. The rapid and easy assay described in this article can be extremely useful for evaluating the immunogenic risk associated with therapeutic proteins. In addition, this method can complement information from MHC class II-associated peptide proteomics assays and other in vitro and in silico techniques.
MeSH term(s)	Humans ; Mice ; Animals ; Histocompatibility Antigens Class II/metabolism ; Liver/metabolism ; Mass Spectrometry ; Infliximab/therapeutic use ; Infliximab/metabolism ; Proteome/metabolism ; Lysosomes/metabolism ; Chromatography, Liquid
Chemical Substances	Histocompatibility Antigens Class II ; Infliximab (B72HH48FLU) ; Proteome
Language	English
Publishing date	2023-06-16
Publishing country	United States
Document type	Journal Article
ISSN	2573-7732
ISSN (online)	2573-7732
DOI	10.4049/immunohorizons.2300035
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Assessing MAPPs assay as a tool to predict the immunogenicity potential of protein therapeutics.

Di Ianni, Andrea / Fraone, Tiziana / Balestra, Piercesare / Cowan, Kyra / Riccardi Sirtori, Federico / Barbero, Luca

Life science alliance

2023 Volume 7, Issue 1

Abstract: MHC-II-associated peptide proteomics (MAPPs) is a mass spectrometry-based (MS) method to identify naturally presented MHC-II-associated peptides that could elicit CD4+T cell activation. MAPPs assay is considered one of the assays that better characterize ...

Abstract	MHC-II-associated peptide proteomics (MAPPs) is a mass spectrometry-based (MS) method to identify naturally presented MHC-II-associated peptides that could elicit CD4+T cell activation. MAPPs assay is considered one of the assays that better characterize the safety of biotherapeutics by driving the selection of the best candidates concerning their immunogenicity risk. However, there is little knowledge about the impact of bead material on the recovery of MHC-II MS-eluted ligands in MAPPs assays. Here, we firstly describe a robust MAPPs protocol by implementing streptavidin magnetic beads for the isolation of these peptides instead of commonly used NHS-activated beads. Moreover, we assessed the impact of the cell medium used for cell cultures on the morphology and recovery of the in vitro-generated APCs, and its potential implications in the amount of MHC-II isolated peptides. We also described an example of a MAPPs assay application to investigate drug-induced immunogenicity of two bispecific antibodies and compared them with monospecific trastuzumab IgG1 control. This work highlighted the importance of MAPPs in the preclinical in vitro strategy to mitigate the immunogenicity risk of biotherapeutics.
MeSH term(s)	Histocompatibility Antigens Class II/metabolism ; CD4-Positive T-Lymphocytes/metabolism ; Peptides/chemistry ; Immunoglobulin G ; Lymphocyte Activation
Chemical Substances	Histocompatibility Antigens Class II ; Peptides ; Immunoglobulin G
Language	English
Publishing date	2023-10-13
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2575-1077
ISSN (online)	2575-1077
DOI	10.26508/lsa.202302095
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Article: MS methods to study macromolecule-ligand interaction: Applications in drug discovery

Riccardi Sirtori, Federico / Alessandra Altomare / Giancarlo Aldini / Luca Regazzoni / Marina Carini

Methods. 2018 July 15, v. 144

2018

Abstract: The interaction of small compounds (i.e. ligands) with macromolecules or macromolecule assemblies (i.e. targets) is the mechanism of action of most of the drugs available today. Mass spectrometry is a popular technique for the interrogation of ... ...

Abstract	The interaction of small compounds (i.e. ligands) with macromolecules or macromolecule assemblies (i.e. targets) is the mechanism of action of most of the drugs available today. Mass spectrometry is a popular technique for the interrogation of macromolecule-ligand interactions and therefore is also widely used in drug discovery and development. Thanks to its versatility, mass spectrometry is used for multiple purposes such as biomarker screening, identification of the mechanism of action, ligand structure optimization or toxicity assessment. The evolution and automation of the instruments now allows the development of high throughput methods with high sensitivity and a minimized false discovery rate. Herein, all these approaches are described with a focus on the methods for studying macromolecule-ligand interaction aimed at defining the structure–activity relationships of drug candidates, along with their mechanism of action, metabolism and toxicity.
Keywords	automation ; biomarkers ; drugs ; ligands ; mass spectrometry ; mechanism of action ; metabolism ; screening ; structure-activity relationships ; toxicity
Language	English
Dates of publication	2018-0715
Size	p. 152-174.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2018.06.005
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Article ; Online: MS methods to study macromolecule-ligand interaction: Applications in drug discovery.

Riccardi Sirtori, Federico / Altomare, Alessandra / Carini, Marina / Aldini, Giancarlo / Regazzoni, Luca

Methods (San Diego, Calif.)

2018 Volume 144, Page(s) 152–174

Abstract	The interaction of small compounds (i.e. ligands) with macromolecules or macromolecule assemblies (i.e. targets) is the mechanism of action of most of the drugs available today. Mass spectrometry is a popular technique for the interrogation of macromolecule-ligand interactions and therefore is also widely used in drug discovery and development. Thanks to its versatility, mass spectrometry is used for multiple purposes such as biomarker screening, identification of the mechanism of action, ligand structure optimization or toxicity assessment. The evolution and automation of the instruments now allows the development of high throughput methods with high sensitivity and a minimized false discovery rate. Herein, all these approaches are described with a focus on the methods for studying macromolecule-ligand interaction aimed at defining the structure-activity relationships of drug candidates, along with their mechanism of action, metabolism and toxicity.
MeSH term(s)	DNA/chemistry ; DNA/metabolism ; Drug Discovery/methods ; Ligands ; Macromolecular Substances/chemistry ; Macromolecular Substances/metabolism ; Mass Spectrometry/methods ; Proteins/chemistry ; Proteins/metabolism ; RNA/chemistry ; RNA/metabolism ; Structure-Activity Relationship
Chemical Substances	Ligands ; Macromolecular Substances ; Proteins ; RNA (63231-63-0) ; DNA (9007-49-2)
Language	English
Publishing date	2018-06-22
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2018.06.005
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Article ; Online: Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

Scalabrin, Matteo / Quintieri, Luigi / Palumbo, Manlio / Riccardi Sirtori, Federico / Gatto, Barbara

Chemical research in toxicology

2017 Volume 30, Issue 2, Page(s) 614–624

Abstract: The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded ... ...

Abstract	The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.
MeSH term(s)	Chromatography, High Pressure Liquid ; DNA/chemistry ; DNA Adducts/chemistry ; Doxorubicin/analogs & derivatives ; Doxorubicin/chemistry ; Kinetics ; Mass Spectrometry ; Spectrophotometry, Ultraviolet
Chemical Substances	3'-deamino-3'',4'-anhydro-(2''-methoxy-3''-oxy-4''-morpholinyl)doxorubicin ; DNA Adducts ; Doxorubicin (80168379AG) ; DNA (9007-49-2)
Language	English
Publishing date	2017-01-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	639353-6
ISSN	1520-5010 ; 0893-228X
ISSN (online)	1520-5010
ISSN	0893-228X
DOI	10.1021/acs.chemrestox.6b00362
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Article ; Online: Identification of unprecedented ATP-competitive choline kinase inhibitors.

Bioorganic & medicinal chemistry letters

2021 Volume 51, Page(s) 128310

Abstract: In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS- ... ...

Abstract	In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.
MeSH term(s)	Adenosine Triphosphate/antagonists & inhibitors ; Adenosine Triphosphate/metabolism ; Choline Kinase/antagonists & inhibitors ; Choline Kinase/metabolism ; Cyclohexanes/chemical synthesis ; Cyclohexanes/chemistry ; Cyclohexanes/pharmacology ; Dose-Response Relationship, Drug ; Humans ; Molecular Structure ; Protein Kinase Inhibitors/chemical synthesis ; Protein Kinase Inhibitors/chemistry ; Protein Kinase Inhibitors/pharmacology ; Structure-Activity Relationship
Chemical Substances	Cyclohexanes ; NMS-P830 ; Protein Kinase Inhibitors ; Adenosine Triphosphate (8L70Q75FXE) ; CHKA protein, human (EC 2.7.1.32) ; Choline Kinase (EC 2.7.1.32)
Language	English
Publishing date	2021-08-18
Publishing country	England
Document type	Journal Article
ZDB-ID	1063195-1
ISSN	1464-3405 ; 0960-894X
ISSN (online)	1464-3405
ISSN	0960-894X
DOI	10.1016/j.bmcl.2021.128310
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Article ; Online: Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90.

Riccardi Sirtori, Federico / Caronni, Dannica / Colombo, Maristella / Dalvit, Claudio / Paolucci, Mauro / Regazzoni, Luca / Visco, Carlo / Fogliatto, Gianpaolo

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

2015 Volume 76, Page(s) 83–94

Abstract: ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. ...

Abstract	ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer. In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100μM, were tested to assess the reliability of ESI-MS for the study of protein-ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry. After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90. In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments. These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with unpredicted or allosteric sites, without the need of any binding probes.
MeSH term(s)	Automation, Laboratory ; Binding Sites ; Binding, Competitive ; Crystallography, X-Ray ; Drug Discovery/methods ; Flow Injection Analysis ; Fluorescence Polarization ; HSP90 Heat-Shock Proteins/antagonists & inhibitors ; HSP90 Heat-Shock Proteins/chemistry ; HSP90 Heat-Shock Proteins/metabolism ; High-Throughput Screening Assays ; Ligands ; Magnetic Resonance Spectroscopy ; Protein Binding ; Reproducibility of Results ; Small Molecule Libraries ; Spectrometry, Mass, Electrospray Ionization
Chemical Substances	HSP90 Heat-Shock Proteins ; Ligands ; Small Molecule Libraries
Language	English
Publishing date	2015-08-30
Publishing country	Netherlands
Document type	Comparative Study ; Journal Article ; Validation Studies
ZDB-ID	1154366-8
ISSN	1879-0720 ; 0928-0987
ISSN (online)	1879-0720
ISSN	0928-0987
DOI	10.1016/j.ejps.2015.05.001
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Article: Discovery of Stereospecific PARP-1 Inhibitor Isoindolinone NMS-P515.

Papeo, Gianluca / Orsini, Paolo / Avanzi, Nilla R / Borghi, Daniela / Casale, Elena / Ciomei, Marina / Cirla, Alessandra / Desperati, Viviana / Donati, Daniele / Felder, Eduard R / Galvani, Arturo / Guanci, Marco / Isacchi, Antonella / Posteri, Helena / Rainoldi, Sonia / Riccardi-Sirtori, Federico / Scolaro, Alessandra / Montagnoli, Alessia

ACS medicinal chemistry letters

2019 Volume 10, Issue 4, Page(s) 534–538

Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs ... ...

Abstract	Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD
Language	English
Publishing date	2019-03-13
Publishing country	United States
Document type	Journal Article
ISSN	1948-5875
ISSN	1948-5875
DOI	10.1021/acsmedchemlett.8b00569
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Article ; Online: Molecular recognition of T:G mismatched base pairs in DNA as studied by electrospray ionization mass spectrometry.

Riccardi Sirtori, Federico / Aldini, Giancarlo / Colombo, Maristella / Colombo, Nicoletta / Malyszko, Jan / Vistoli, Giulio / D'Alessio, Roberto

ChemMedChem

2012 Volume 7, Issue 6, Page(s) 1112–1122

Abstract: Postreplicative mismatch repair (MMR) is a cellular system involved in the recognition and correction of DNA polymerase errors that escape detection in proofreading. Of the various mismatched bases, T:G pairing in DNA is one of the more common mutations ... ...

Abstract	Postreplicative mismatch repair (MMR) is a cellular system involved in the recognition and correction of DNA polymerase errors that escape detection in proofreading. Of the various mismatched bases, T:G pairing in DNA is one of the more common mutations leading to the formation of tumors in humans. In addition, the absence of the MMR system can generate resistance to several chemotherapeutic agents, particularly DNA-damaging substances. The main purpose of this study was the setup and validation of an electrospray ionization (ESI) mass spectrometry method for the identification of small molecules that are able to recognize T:G mismatches in DNA targets. These findings could be useful for the discovery of new antitumor drugs. The analytical method is based on the ability of electrospray to preserve the noncovalent adducts present in solution and transfer them to the gas phase. Lexitropsin derivatives (polyimidazole compounds) have been previously described as selective for T:G mismatch binding by NMR and ITC studies. We synthesized and tested various polyimidazole derivatives, one of which in particular (NMS-057) showed a higher affinity for an oligonucleotide DNA sequence containing a T:G mismatched base pair. To rationalize these findings, molecular docking studies were performed using available NMR structures. Moreover, ESI-MS experiments, performed on an orbitrap mass spectrometer, highlighted the formation of heterodimeric complexes between DNA sequences, distamycin A, and polyimidazole compounds. Our results confirm that this ESI method could be a valuable tool for the identification of new molecules able to specifically recognize T:G mismatched base pairs.
MeSH term(s)	Base Pair Mismatch ; DNA/chemistry ; Guanine/chemistry ; Netropsin/analogs & derivatives ; Netropsin/chemistry ; Spectrometry, Mass, Electrospray Ionization ; Thymine/chemistry
Chemical Substances	lexitropsin (110124-49-7) ; Guanine (5Z93L87A1R) ; Netropsin (64B3O0RD7N) ; DNA (9007-49-2) ; Thymine (QR26YLT7LT)
Language	English
Publishing date	2012-06
Publishing country	Germany
Document type	Journal Article
ZDB-ID	2218496-X
ISSN	1860-7187 ; 1860-7179
ISSN (online)	1860-7187
ISSN	1860-7179
DOI	10.1002/cmdc.201100526
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Article ; Online: Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

Pellegatta, Serena / Valletta, Lorella / Corbetta, Cristina / Patanè, Monica / Zucca, Ileana / Riccardi Sirtori, Federico / Bruzzone, Maria Grazia / Fogliatto, Gianpaolo / Isacchi, Antonella / Pollo, Bianca / Finocchiaro, Gaetano

Acta neuropathologica communications

2015 Volume 3, Page(s) 4

Abstract: The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than ... ...

Abstract	The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.
MeSH term(s)	Animals ; Biomarkers, Tumor/genetics ; Biomarkers, Tumor/immunology ; Brain Neoplasms/genetics ; Brain Neoplasms/immunology ; Cell Line, Tumor ; Disease Models, Animal ; Down-Regulation ; Gas Chromatography-Mass Spectrometry ; Glioma/genetics ; Glioma/immunology ; Glioma/therapy ; Glutarates/metabolism ; Granzymes/metabolism ; Immunotherapy/methods ; Interferon-gamma/metabolism ; Interleukin-10/metabolism ; Isocitrate Dehydrogenase/genetics ; Isocitrate Dehydrogenase/immunology ; Mice ; Mice, Inbred C57BL ; Mutation ; Perforin/metabolism ; Transforming Growth Factor beta2/metabolism
Chemical Substances	Biomarkers, Tumor ; Glutarates ; Transforming Growth Factor beta2 ; Perforin (126465-35-8) ; Interleukin-10 (130068-27-8) ; alpha-hydroxyglutarate (2889-31-8) ; Interferon-gamma (82115-62-6) ; Isocitrate Dehydrogenase (EC 1.1.1.41) ; IDH1 protein, human (EC 1.1.1.42.) ; Granzymes (EC 3.4.21.-)
Language	English
Publishing date	2015-01-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2715589-4
ISSN	2051-5960 ; 2051-5960
ISSN (online)	2051-5960
ISSN	2051-5960
DOI	10.1186/s40478-014-0180-0
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