title: Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.
abstract: The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.
authors.affiliation: Dipartimento di Biologia e Biotecnologie "Charles Darwin", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.affiliation: Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Piazzale Aldo Moro 5, 00185 Roma, Italy; Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Tramonti
authors.affiliation: Department of Chemistry, Goshen College, 1700 S Main St, Goshen, IN 46526, United States.
authors.forename: Doug
authors.initials: D
authors.lastname: Schirch
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Guiducci
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Martino Luigi
authors.initials: ML
authors.lastname: di Salvo
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Fiascarelli
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Giorgi
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Maras
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy. Electronic address: francesca.cutruzzola@uniroma1.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Cutruzzolà
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy. Electronic address: roberto.contestabile@uniroma1.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Contestabile
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D012696:Glycine Hydroxymethyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011773:Pyruvates / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000970:Antineoplastic Agents; D011773:Pyruvates; D011994:Recombinant Proteins; D012694:Serine; C017092:bromopyruvate; D012696:Glycine Hydroxymethyltransferase; C520541:SHMT protein, human; D003545:Cysteine
keywords: 3-Bromopyruvate; Cancer metabolic reprogramming; Enzyme inhibition; Isozymes; Serine hydroxymethyltransferase