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

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Multitarget, selective compound design yields potent inhibitors of a kinetoplastid pteridine reductase 1 Ina Pöhner , Antonio Quotadamo , Joanna Panecka-Hofman , Rosaria Luciani , Matteo Santucci , Pasquale Linciano , Giacomo Landi , Flavio Di Pisa , Lucia Dello Iacono , Cecilia Pozzi , Stefano Mangani , Sheraz Gul , Gesa Witt , Bernhard Ellinger , Maria Kuzikov , Nuno Santarem , Anabela Cordeiro-Da-Silva , Maria P. Costi , Alberto Venturelli , Rebecca C. Wade Journal Of Medicinal Chemistry 51 DOI: 10.1021/acs.jmedchem.2c00232 Open Access Show Abstract ▼ Abstract: The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure–activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.	Jun 2022
I04-Macromolecular Crystallography	Crystal structure of the ternary complex of Leishmania major pteridine reductase 1 with the cofactor NADP+/NADPH and the substrate folic acid Lucia Dello Iacono , Flavio Di Pisa , Stefano Mangani Acta Crystallographica Section F Structural Biology Communications 78 DOI: 10.1107/S2053230X22002795 Diamond Proposal Number(s): [11690] Open Access Show Abstract ▼ Abstract: Pteridine reductase 1 (PTR1) is a key enzyme of the folate pathway in protozoan parasites of the genera Leishmania and Trypanosoma and is a valuable drug target for tropical diseases. This enzyme is able to catalyze the NADPH-dependent reduction of both conjugated (folate) and unconjugated (biopterin) pterins to their tetrahydro forms, starting from oxidized- or dihydro-state substrates. The currently available X-ray structures of Leishmania major PTR1 (LmPTR1) show the enzyme in its unbound, unconjugated substrate-bound (with biopterin derivatives) and inhibitor-bound forms. However, no structure has yet been determined of LmPTR1 bound to a conjugated substrate. Here, the high-resolution crystal structure of LmPTR1 in complex with folic acid is presented and the intermolecular forces that drive the binding of the substrate in the catalytic pocket are described. By expanding the collection of LmPTR1 structures in complex with process intermediates, additional insights into the active-site rearrangements that occur during the catalytic process are provided. In contrast to previous structures with biopterin derivatives, a small but significant difference in the orientation of Asp181 and Tyr194 of the catalytic triad is found. This feature is shared by PTR1 from T. brucei (TbPTR1) in complex with the same substrate molecule and may be informative in deciphering the importance of such residues at the beginning of the catalytic process.	Apr 2022
I04-Macromolecular Crystallography	Elucidating the 3D structure of a surface membrane antigen from Trypanosoma cruzi as a serodiagnostic biomarker of Chagas disease Flavio Di Pisa , Stefano De Benedetti , Enrico Mario Alessandro Fassi , Mauro Bombaci , Renata Grifantini , Angelo Musicò , Roberto Frigerio , Angela Pontillo , Cinzia Rigo , Sandra Abelli , Romualdo Grande , Nadia Zanchetta , Davide Mileto , Alessandro Mancon , Alberto Rizzo , Alessandro Gori , Marina Cretich , Giorgio Colombo , Martino Bolognesi , Louise Jane Gourlay Vaccines 10 DOI: 10.3390/vaccines10010071 Diamond Proposal Number(s): [20221] Open Access Show Abstract ▼ Abstract: Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 Å) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles.	Jan 2022
I24-Microfocus Macromolecular Crystallography	Structure, immunoreactivity, and in silico epitope determination of SmSPI S. mansoni serpin for immunodiagnostic application Stefano De Benedetti , Flavio Di Pisa , Enrico Mario Alessandro Fassi , Marina Cretich , Angelo Musicò , Roberto Frigerio , Alessandro Mussida , Mauro Bombaci , Renata Grifantini , Giorgio Colombo , Martino Bolognesi , Romualdo Grande , Nadia Zanchetta , Maria Rita Gismondo , Davide Mileto , Alessandro Mancon , Louise Jane Gourlay Vaccines 9 DOI: 10.3390/vaccines9040322 Diamond Proposal Number(s): [5912] Open Access Show Abstract ▼ Abstract: The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.	Apr 2021
I04-Macromolecular Crystallography	Release of soybean isoflavones by using a β‐glucosidase from Alicyclobacillus herbarius Lidia Delgado , Christian M. Heckmann , Flavio Di Pisa , Louise Gourlay , Francesca Paradisi Chembiochem 47 DOI: 10.1002/cbic.202000688 Diamond Proposal Number(s): [20221] Open Access Show Abstract ▼ Abstract: β‐Glucosidases are used in the food industry to hydrolyse glycosidic bonds in complex sugars, with enzymes sourced from extremophiles better able to tolerate the process conditions. In this work, a novel β‐glycosidase from the acidophilic organism Alicyclobacillus herbarius was cloned and heterologously expressed in Escherichia coli BL21(DE3). AheGH1 was stable over a broad range of pH values (5–11) and temperatures (4–55 °C). The enzyme exhibited excellent tolerance to fructose and good tolerance to glucose, retaining 65 % activity in the presence of 10 % (w/v) glucose. It also tolerated organic solvents, some of which appeared to have a stimulating effect, in particular ethanol with a 1.7‐fold increase in activity at 10 % (v/v). The enzyme was then applied for the cleavage of isoflavone from isoflavone glucosides in an ethanolic extract of soy flour, to produce soy isoflavones, which constitute a valuable food supplement, full conversion was achieved within 15 min at 30 °C.	Nov 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Two novel fish paralogs provide insights into the Rid family of imine deaminases active in pre-empting enamine/imine metabolic damage Stefania Digiovanni , Cristina Visentin , Genny Degani , Alberto Barbiroli , Matteo Chiara , Luca Regazzoni , Flavio Di Pisa , Andrew J. Borchert , Diana M. Downs , Stefano Ricagno , Maria Antonietta Vanoni , Laura Popolo Scientific Reports 10 DOI: 10.1038/s41598-020-66663-w Diamond Proposal Number(s): [20221] Open Access Show Abstract ▼ Abstract: Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.	Jun 2020
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Enhancement of benzothiazoles as Pteridine Reductase-1 (PTR1) inhibitors for the treatment of Trypanosomatidic infections Pasquale Linciano , Cecilia Pozzi , Lucia Dello Iacono , Flavio Di Pisa , Giacomo Landi , Alessio Bonucci , Sheraz Gul , Maria Kuzikov , Bernhard Ellinger , Gesa Witt , Nuno Santarem , Catarina Baptista , Caio Franco , Carolina Borsoi Moraes , Wolfgang Müller , Ulrike Wittig , Rosaria Luciani , Antony Sesenna , Antonio Quotadamo , Stefania Ferrari , Ina Pöhner , Anabela Cordeiro-Da-Silva , Stefano Mangani , Luca Costantino , Maria Paola Costi Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.8b02021 Diamond Proposal Number(s): [11690, 15832] Show Abstract ▼ Abstract: 2-amino-benzo[d]thiazole has been identified as new core moiety for the development of improved PTR1 inhibitors and anti-Trypanosomatidic agents. Through a molecular docking approach and the crystal structure of 6-(methylsulfonyl)benzo[d]thiazol-2-amine ternary complex with TbPTR1, 42 new compounds were designed, synthesized and evaluated for their ability to inhibit T. brucei and L. major PTR1 enzymes and in-vitro activity against Trypanosoma brucei and amastigote stage of Leishmania infantum. We identified several 2-amino-benzo[d]thiazole derivatives with improved activity against the enzymes (TbPTR1 IC50 = 0.35 µM; LmPTR1 IC50 = 1.9 µM) and anti-parasitic activity against T. brucei in the low µM range. Ten compounds, with low/sub micromolar inhibitor activity against TbPTR1, were able to potentiate the antiparasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, with a Potentiating Index (PI) ranging between 1.2 and 2.7. The compound library was profile for an early ADME-Toxicity profile and the compounds showing the best in vitro/enzymatic inhibition properties were selected for progression. 2-amino-N-benzylbenzo[d]thiazole-6-carboxamide (4c), was finally identified as a novel potent and selective anti-trypanocydal agent (EC50 = 7.0 µM) with an overall safe early ADME-Toxicity profile. The pharmacokinetic studies of 4c in BALB/c mice using a hydroxypropyl-β-cyclodextrin formulation yielded good oral bioavailability, confirming its suitability for progression to in-vivo anti-parasitic studies.	Mar 2019
I04-Macromolecular Crystallography	The structure of the human glutaminyl cyclase–SEN177 complex indicates routes for developing new potent inhibitors as possible agents for the treatment of neurological disorders Cecilia Pozzi , Flavio Di Pisa , Manuela Benvenuti , Stefano Mangani Jbic Journal Of Biological Inorganic Chemistry 84 DOI: 10.1007/s00775-018-1605-1 Diamond Proposal Number(s): [8574] Show Abstract ▼ Abstract: Recent evidence links the role of human glutaminyl cyclase (hQC) to the amyloidogenic process involved in Alzheimer’s disease (AD). hQC is a zinc enzyme present in neuronal tissue and its activity is responsible for the cyclization of N-terminal Gln or Glu β-amyloid peptides, leading to N-pyroglutamic acid peptides (pE-Aβ) that is probably a crucial event in the initiation and progress of the disease. Indeed, pE-containing peptides exhibit an elevated neurotoxicity and a tendency to aggregate. These observations render hQC inhibition an attractive strategy for developing new molecules active against AD. We present here the crystal structure of hQC in complex with SEN177, a newly designed molecule. The SEN177-binding mode to hQC differs from that of the known hQC inhibitors. SEN177 Ki on hQC is 20 nM, comparable or better than that of the most potent known hQC inhibitors PBD150 and PQ912. In addition, SEN177 already demonstrated relevant pharmacological properties in in vivo models of Huntington’s disease. All these properties make SEN177 an important scaffold for developing molecules acting on AD and related diseases.	Aug 2018
I04-Macromolecular Crystallography	Exploiting the 2-amino-1,3,4-thiadiazole scaffold to inhibit Trypanosoma brucei pteridine reductase in support of early-stage drug discovery Pasquale Linciano , Alice Dawson , Ina Pöhner , David M. Costa , Monica S. Sá , Anabela Cordeiro-Da-Silva , Rosaria Luciani , Sheraz Gul , Gesa Witt , Bernhard Ellinger , Maria Kuzikov , Philip Gribbon , Jeanette Reinshagen , Markus Wolf , Birte Behrens , Véronique Hannaert , Paul A. M. Michels , Erika Nerini , Cecilia Pozzi , Flavio Di Pisa , Giacomo Landi , Nuno Santarem , Stefania Ferrari , Puneet Saxena , Sandra Lazzari , Giuseppe Cannazza , Lucio H. Freitas-Junior , Carolina B. Moraes , Bruno S. Pascoalino , Laura M. Alcântara , Claudia P. Bertolacini , Vanessa Fontana , Ulrike Wittig , Wolfgang Müller , Rebecca C. Wade , William N. Hunter , Stefano Mangani , Luca Costantino , Maria P. Costi Acs Omega 2, 5666 - 5683 DOI: 10.1021/acsomega.7b00473 Diamond Proposal Number(s): [8574] Open Access Show Abstract ▼ Abstract: Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.	Sep 2017
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

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