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19 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
	Discovery of taniborbactam (VNRX-5133): A broad-spectrum serine- and metallo-β-lactamase inhibitor for carbapenem-resistant bacterial infections Bin Liu , Robert E. Lee Trout , Guo-hua Chu , Daniel Mcgarry , Randy W Jackson , Jodie Hamrick , Denis Daigle , Susan Cusick , Cecilia Pozzi , Filomena De Luca , Manuela Benvenuti , Stefano Mangani , Jean-denis Docquier , William J. Weiss , Daniel C. Pevear , Luigi Xerri , Christopher J. Burns Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b01518 Show Abstract ▼ Abstract: A major resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. Originally recognized for their ability to hydrolyze penicillins, emergent β-lactamases can now confer resistance to other β-lactam drugs, including both cephalosporins and carbapenems. The emergence and global spread of β-lactamase-producing multi-drug-resistant "superbugs" has caused increased alarm within the medical community due to the high mortality rate associated with these difficult-to-treat bacterial infections. To address this unmet medical need, we initiated an iterative program combining medicinal chemistry, structural biology, biochemical testing and microbiological profiling to identify broad-spectrum inhibitors of both serine- and metallo-β-lactamase enzymes. Lead optimization, beginning with narrower-spectrum, weakly active compounds provided 20 (VNRX-5133, taniborbactam), a boronic acid-containing pan-spectrum β-lactamase inhibitor. In vitro and in vivo studies demonstrated that 20 restored the activity of β-lactam antibiotics against carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Enterobacteriaceae. Taniborbactam is the first pan-spectrum β-lactamase inhibitor to enter clinical development.	Nov 2019
I02-Macromolecular Crystallography	Structural insights into the development of cycloguanil derivatives as Trypanosoma brucei pteridine reductase 1 inhibitors Giacomo Landi , Pasquale Linciano , Chiara Borsari , Claudia P. Bertolacini , Carolina Borsoi Moraes , Anabela Cordeiro-da-silva , Sheraz Gul , Gesa Witt , Maria Kuzikov , Maria Paola Costi , Cecilia Pozzi , Stefano Mangani Acs Infectious Diseases DOI: 10.1021/acsinfecdis.8b00358 Diamond Proposal Number(s): [11690] Show Abstract ▼ Abstract: Cycloguanil is a known dihydrofolate reductase (DHFR) inhibitor, but there is no evidence of its activity on pteridine reductase (PTR), the main metabolic bypass to DHFR inhibition in trypanosomatid parasites. Here, we provide experimental evidence of cycloguanil as an inhibitor of Trypanosoma brucei PTR1 (TbPTR1). A small library of cycloguanil derivatives was develop, resulting in 1 and 2a having IC50 of 692 and 186 nM, respectively, towards TbPTR1. Structural analysis revealed that the increased potency of 1 and 2a is due to the combined contributions of hydrophobic interactions, H-bonds and halogen bonds. Moreover, in vitro cell growth inhibition tests indicated that 2a is also effective on T. brucei. The simultaneous inhibition of DHFR and PTR1 activity in T. brucei is a new promising strategy for the treatment of human African Trypanosomiasis. On this purpose, 1,6-dihydrotriazines represent new molecular tools to develop potent dual PTR and DHFR inhibitors.	Apr 2019
I02-Macromolecular Crystallography	High-resolution crystal structure of Trypanosoma brucei pteridine reductase 1 in complex with an innovative tricyclic-based inhibitor Giacomo Landi , Pasquale Linciano , Giusy Tassone , Maria Paola Costi , Stefano Mangani , Cecilia Pozzi Acta Crystallographica Section D Structural Biology 76, 558 - 564 DOI: 10.1107/S2059798320004891 Diamond Proposal Number(s): [11690] Show Abstract ▼ Abstract: The protozoan parasite Trypanosoma brucei is the etiological agent of human African trypanosomiasis (HAT). HAT, together with other neglected tropical diseases, causes serious health and economic issues, especially in tropical and subtropical areas. The classical antifolates targeting dihydrofolate reductase (DHFR) are ineffective towards trypanosomatid parasites owing to a metabolic bypass by the expression of pteridine reductase 1 (PTR1). The combined inhibition of PTR1 and DHFR activities in Trypanosoma parasites represents a promising strategy for the development of new effective treatments for HAT. To date, only monocyclic and bicyclic aromatic systems have been proposed as inhibitors of T. brucei PTR1 (TbPTR1); nevertheless, the size of the catalytic cavity allows the accommodation of expanded molecular cores. Here, an innovative tricyclic-based compound has been explored as a TbPTR1-targeting molecule and its potential application for the development of a new class of PTR1 inhibitors has been evaluated. 2,4-Diaminopyrimido[4,5-b]indol-6-ol (1) was designed and synthesized, and was found to be effective in blocking TbPTR1 activity, with a Ki in the low-micromolar range. The binding mode of 1 was clarified through the structural characterization of its ternary complex with TbPTR1 and the cofactor NADP(H), which was determined to 1.30 Å resolution. The compound adopts a substrate-like orientation inside the cavity that maximizes the binding contributions of hydrophobic and hydrogen-bond interactions. The binding mode of 1 was compared with those of previously reported bicyclic inhibitors, providing new insights for the design of innovative tricyclic-based molecules targeting TbPTR1.	Jun 2020
I03-Macromolecular Crystallography	Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ 3 -oxo)Tris[(μ 2 -peroxo)] triiron(III) cluster Cecilia Pozzi , Silvia Ciambellotti , Caterina Bernacchioni , Flavio Di Pisa , Stefano Mangani , Paola Turano Proceedings Of The National Academy Of Sciences 114, 2580 - 2585 DOI: 10.1073/pnas.1614302114 Diamond Proposal Number(s): [11690] Open Access Show Abstract ▼ Abstract: X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (μ3-oxo)Tris[(μ2-peroxo)(μ2-glutamato-κO:κO′)](glutamato-κO)(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin.	Mar 2017
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Probing the role of Arg97 in Heat shock protein 90 N-terminal domain from the parasite Leishmania braziliensis through site-directed mutagenesis on the human counterpart Giusy Tassone , Stefano Mangani , Maurizio Botta , Cecilia Pozzi Biochimica Et Biophysica Acta (bba) - Proteins And Proteomics DOI: 10.1016/j.bbapap.2018.09.005 Diamond Proposal Number(s): [15832, 1949] Show Abstract ▼ Abstract: In Brazil, the mucocutaneous form of leishmaniasis, caused by the parasite Leishmania braziliensis, is a widespread and very challenging disease responsible for disfiguration and, in the most severe cases, death. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone playing a pivotal role in the folding process of client proteins, and therefore its activity is fundamental for cell survival and proliferation. Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP binding pocket in the protein N-terminal domain (NTD) act as Hsp90 inhibitors. The development of selective molecules targeting the ATPase site of protozoan Hsp90 is tricky for the high homology with the human Hsp90 NTD (hNTD). Notably, only the human Lys112 is replaced by Arg97 in the L. braziliensis enzyme. Recently, this difference has been probed to design selective inhibitors targeting parasite Hsp90s. Here, a reliable protocol for expression and purification of LbHsp90-NTD (LbNTD) was developed but its structural characterization was unsuccessful. The role of Arg97 in LbNTD was hence probed by means of the “leishmanized” K112R variant of hNTDα. To deeply investigate the role of this residue, also the hNTDα K112A variant was generated. Structural studies performed on hNTDα and its variants using various ADP and ATP analogues and cAMP revealed that this residue is not crucial for nucleotide binding. This finding strongly suggests that Arg97 in LbNTD and more generally the conserved arginine residue in parasite Hsp90s are not exploitable for the development of selective inhibitors.	Sep 2018
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Iron biomineral growth from the initial nucleation seed in L‐ferritin Silvia Ciambellotti , Cecilia Pozzi , Stefano Mangani , Paola Turano Chemistry – A European Journal DOI: 10.1002/chem.202000064 Diamond Proposal Number(s): [15832, 21741] Show Abstract ▼ Abstract: X‐ray structures of homopolymeric human L‐ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa‐nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (μ 3 ‐oxo)tris[(μ 2 ‐glutamato‐κO:κO′)](glutamato‐κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the level of the protein surface, capable of contributing coordination bonds and electrostatic guidance.	Feb 2020
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Structural comparison of enterococcus faecalis and human thymidylate synthase complexes with the substrate dUMP and its analogue FdUMP provides hints about enzyme conformational variabilities Cecilia Pozzi , Stefania Ferrari , Rosaria Luciani , Giusy Tassone , Maria Costi , Stefano Mangani Molecules 24 DOI: 10.3390/molecules24071257 Diamond Proposal Number(s): [1358] Open Access Show Abstract ▼ Abstract: Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) using N5,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the half-site reactivity of Enterococcus faecalis TS and the mechanisms underlying the conformational changes occurring in the two enzymes. We also identify relevant differences in cofactor and inhibitor binding between Enterococcus faecalis and human TS that can guide the design of selective inhibitors against bacterial TSs.	Mar 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs Chiara Borsari , Rosaria Luciani , Cecilia Pozzi , Ina Poehner , Stefan Henrich , Matteo Trande , Anabela Cordeiro-da-silva , Nuno Santarem , Catarina Baptista , Annalisa Tait , Flavio Di Pisa , Lucia Dello Iacono , Giacomo Landi , Sheraz Gul , Markus Wolf , Maria Kuzikov , Bernhard Ellinger , Jeanette Reinshagen , Gesa Witt , Philip Gribbon , Manfred Kohler , Oliver Keminer , Birte Behrens , Luca Costantino , Paloma Tejera Nevado , Eugenia Bifeld , Julia Eick , Joachim Clos , Juan Torrado , María D. Jiménez-antón , María J. Corral , José M Alunda , Federica Pellati , Rebecca C. Wade , Stefania Ferrari , Stefano Mangani , Maria Paola Costi Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.6b00698 Diamond Proposal Number(s): [11690] Open Access Show Abstract ▼ Abstract: Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure−activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4- methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.	Aug 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	The soluble Y115E–Y117E variant of human glutaminyl cyclase is a valid target for X-ray and NMR screening of inhibitors against Alzheimer disease Flavio Dipisa , Cecilia Pozzi , Manuela Benvenuti , Matteo Andreini , Guido Marconi , Stefano Mangani Acta Crystallographica Section F Structural Biology Communications 71 (8), 986 - 992 DOI: 10.1107/S2053230X15010389 PMID: 26249687 Diamond Proposal Number(s): [8574] Show Abstract ▼ Abstract: Recent developments in molecular pathology and genetics have allowed the identification of human glutaminyl cyclase (hQC) among the abnormal proteins involved in many neurodegenerative disorders. Difficulties in obtaining large quantities of pure protein may limit the use of crystallographic screening for drug development on this target. Site-directed mutagenesis experiments have led to the identification of some solvent-exposed residues that are absolutely critical to achieve increased solubility and to avoid precipitation of the enzyme in inclusion bodies when expressed in Escherichia coli. The designed variant Y115E–Y117E has been found to be able to provide large amounts of monodisperse, pure hQC from an E. coli expression system. To validate the use of the artificial construct as a target for large-scale X-ray and NMR screening campaigns in the search for new inhibitors of hQC, the X-ray crystal structures of the hQC Y115E–Y117E variant and of its adduct with the inhibitor PBD-150 were determined.	Aug 2015
I04-1-Macromolecular Crystallography (fixed wavelength)	Iron binding to human heavy-chain ferritin Cecilia Pozzi , Flavio Di Pisa , Caterina Bernacchioni , Silvia Ciambellotti , Paola Turano , Stefano Mangani Acta Crystallographica Section D Biological Crystallography 71, 1909 - 1920 DOI: 10.1107/S1399004715013073 PMID: 2632738 Diamond Proposal Number(s): [8574] Show Abstract ▼ Abstract: Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein from Rana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with its R. catesbeiana counterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species	Sep 2015

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