I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21741, 29907]
Open Access
Abstract: Glutaminyl-peptide cyclotransferases (QCs) convert the N-terminal glutamine or glutamate residues of protein and peptide substrates into pyroglutamate (pE) by releasing ammonia or a water molecule. The N-terminal pE modification protects peptides/proteins against proteolytic degradation by amino- or exopeptidases, increasing their stability. Mammalian QC is abundant in the brain and a large amount of evidence indicates that pE peptides are involved in the onset of neural human pathologies such as Alzheimer’s and Huntington’s disease and synucleinopathies. Hence, human QC (hQC) has become an intensively studied target for drug development against these diseases. Soon after its characterization, hQC was identified as a Zn-dependent enzyme, but a partial restoration of the enzyme activity in the presence of the Co(II) ion was also reported, suggesting a possible role of this metal ion in catalysis. The present work aims to investigate the structure of demetallated hQC and of the reconstituted enzyme with Zn(II) and Co(II) and their behavior in the presence of known inhibitors. Furthermore, our structural determinations provide a possible explanation for the presence of the mononuclear metal binding site of hQC, despite the presence of the same conserved metal binding motifs present in distantly related dinuclear aminopeptidase enzymes.
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Jul 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Valeria
Francesconi
,
Marco
Rizzo
,
Cecilia
Pozzi
,
Lorenzo
Tagliazucchi
,
Claude U.
Konchie Simo
,
Giulia
Saporito
,
Giacomo
Landi
,
Stefano
Mangani
,
Anna
Carbone
,
Silvia
Schenone
,
Nuno
Santarém
,
Joana
Tavares
,
Anabela
Cordeiro-Da-Silva
,
Maria Paola
Costi
,
Michele
Tonelli
Diamond Proposal Number(s):
[29907]
Abstract: Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.
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Jul 2024
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I03-Macromolecular Crystallography
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Pasquale
Linciano
,
Cecilia
Pozzi
,
Giusy
Tassone
,
Giacomo
Landi
,
Stefano
Mangani
,
Matteo
Santucci
,
Rosaria
Luciani
,
Stefania
Ferrari
,
Nuno
Santarem
,
Lorenzo
Tagliazucchi
,
Anabela
Cordeiro-Da-Silva
,
Michele
Tonelli
,
Donatella
Tondi
,
Laura
Bertarini
,
Sheraz
Gul
,
Gesa
Witt
,
Carolina B.
Moraes
,
Luca
Costantino
,
Maria Paola
Costi
Diamond Proposal Number(s):
[21741]
Abstract: Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diammino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class phenyl-, heteroaryl- and benzyloxy-nitro derivatives of the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the x-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective T. brucei inhibitors were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-β-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Finally, compound 1p, selected for the best pharmacodynamic and pharmacokinetic properties, showed promising activity in a mouse model of T. brucei infection. Compound 1p can be considered a good candidate for further bioavailability and efficacy studies.
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Nov 2023
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I04-Macromolecular Crystallography
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Alice
Legru
,
Federica
Verdirosa
,
Yen
Vo-Hoang
,
Giusy
Tassone
,
Filippo
Vascon
,
Caitlyn A.
Thomas
,
Filomena
Sannio
,
Giuseppina
Corsica
,
Manuela
Benvenuti
,
Georges
Feller
,
Rémi
Coulon
,
Francesca
Marcoccia
,
Savannah Rowane
Devente
,
Ezeddine
Bouajila
,
Catherine
Piveteau
,
Fabrice
Leroux
,
Rebecca
Deprez-Poulain
,
Benoît
Deprez
,
Patricia
Licznar-Fajardo
,
Michael W.
Crowder
,
Laura
Cendron
,
Cecilia
Pozzi
,
Stefano
Mangani
,
Jean-Denis
Docquier
,
Jean-François
Hernandez
,
Laurent
Gavara
Diamond Proposal Number(s):
[21741]
Abstract: Metallo-β-lactamases (MBLs) contribute to the resistance of Gram-negative bacteria to carbapenems, last-resort antibiotics at hospital, and MBL inhibitors are urgently needed to preserve these important antibacterial drugs. Here, we describe a series of 1,2,4-triazole-3-thione-based inhibitors displaying an α-amino acid substituent, which amine was mono- or disubstituted by (hetero)aryl groups. Compounds disubstituted by certain nitrogen-containing heterocycles showed submicromolar activities against VIM-type enzymes and strong NDM-1 inhibition (Ki = 10–30 nM). Equilibrium dialysis, native mass spectrometry, isothermal calorimetry (ITC), and X-ray crystallography showed that the compounds inhibited both VIM-2 and NDM-1 at least partially by stripping the catalytic zinc ions. These inhibitors also displayed a very potent synergistic activity with meropenem (16- to 1000-fold minimum inhibitory concentration (MIC) reduction) against VIM-type- and NDM-1-producing ultraresistant clinical isolates, including Enterobacterales and Pseudomonas aeruginosa. Furthermore, selected compounds exhibited no or moderate toxicity toward HeLa cells, favorable absorption, distribution, metabolism, excretion (ADME) properties, and no or modest inhibition of several mammalian metalloenzymes.
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Nov 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29907]
Open Access
Abstract: Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that stabilizes client proteins in a folded and functional state. It is composed of two identical and symmetrical subunits and each monomer consists of three domains, the N-terminal (NTD), the middle (MD), and the C-terminal domain (CTD). Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP-binding pocket in the NTD act as Hsp90 inhibitors, leading to client protein degradation and cell death. Therefore, human Hsp90 represents a validated target for developing new anticancer drugs. Since protozoan parasites use their Hsp90 to trigger important transitions between different stages of their life cycle, this protein also represents a profitable target in anti-parasite drug discovery. Nevertheless, the development of molecules able to selectively target the ATP-binding site of protozoan Hsp90 is challenging due to the high homology with the human Hsp90 NTD (hHsp90-NTD). In a previous work, a series of potent Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold was developed. The most promising inhibitor of the series, JMC31, showed potent Hsp90 binding and antiproliferative activity in NCI-H460 cells in the low-nanomolar range. In this work, we present the structural characterization of hHsp90-NTD in complex with JMC31 through X-ray crystallography. In addition, to elucidate the role of residue 112 on the ligand binding and its exploitability for the development of selective inhibitors, we investigated the crystal structures of hHsp90-NTD variants (K112R and K112A) in complex with JMC31.
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Aug 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Open Access
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.
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Jun 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11690]
Open Access
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.
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Apr 2022
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I04-Macromolecular Crystallography
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Federica
Verdirosa
,
Laurent
Gavara
,
Laurent
Sevaille
,
Giusy
Tassone
,
Giuseppina
Corsica
,
Alice
Legru
,
Georges
Feller
,
Giulia
Chelini
,
Paola S.
Mercuri
,
Silvia
Tanfoni
,
Filomena
Sannio
,
Manuela
Benvenuti
,
Giulia
Cerboni
,
Filomena
De Luca
,
Ezeddine
Bouajila
,
Yen
Vo Hoang
,
Patricia
Licznar-Fajardo
,
Moreno
Galleni
,
Cecilia
Pozzi
,
Stefano
Mangani
,
Jean-Denis
Docquier
,
Jean-François
Hernandez
Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Metallo-β-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need . We previously reported several series of compounds based on the 1,2,4-triazole-3-thione scaffold. In particular, Schiff bases formed between diversely 5-substituted-4-amino compounds and 2-carboxybenzaldehyde were broad-spectrum inhibitors of VIM-type, NDM-1 and IMP-1 MBLs. Unfortunately, they were unable to restore antibiotic susceptibility of MBL-producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we developed compounds where the hydrazone-like bond of the Schiff bases was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM-1 and IMP-1, but some showed a significantly better activity on VIM-type enzymes, with K i values in the μM to sub-μM range. The resolution of the crystallographic structure of VIM-2 in complex with one inhibitor yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the β-lactam susceptibility of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cells at high concentration, thus showing promising safety.
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Jan 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Giusy
Tassone
,
Marco
Paolino
,
Cecilia
Pozzi
,
Annalisa
Reale
,
Laura
Salvini
,
Gianluca
Giorgi
,
Maurizio
Orlandini
,
Federico
Galvagni
,
Stefano
Mangani
,
Xuchun
Yang
,
Benedetta
Carlotti
,
Fausto
Ortica
,
Loredana
Latterini
,
Massimo
Olivucci
,
Andrea
Cappelli
Diamond Proposal Number(s):
[15832, 21741]
Abstract: The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the in vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such library, made of variable unnatural chromophores inserted in an easy to mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.
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Oct 2021
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I04-Macromolecular Crystallography
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Alice
Legru
,
Federica
Verdirosa
,
Jean-François
Hernandez
,
Giusi
Tassone
,
Filomena
Sannio
,
Manuela
Benvenuti
,
Pierre-Alexis
Conde
,
Guillaume
Bossis
,
Caitlyn A.
Thomas
,
Michael W.
Crowder
,
Melissa
Dillenberger
,
Katja
Becker
,
Cecilia
Pozzi
,
Stefano
Mangani
,
Jean-Denis
Docquier
,
Laurent
Gavara
Diamond Proposal Number(s):
[21741]
Abstract: Metallo-β-lactamases (MBLs) are important contributors of Gram-negative bacteria resistance to β-lactam antibiotics. MBLs are highly worrying because of their carbapenemase activity, their rapid spread in major human opportunistic pathogens while no clinically useful inhibitor is available yet. In this context, we are exploring the potential of compounds based on the 1,2,4-triazole-3-thione scaffold as an original ligand of the di-zinc active sites of MBLs, and diversely substituted at its positions 4 and 5. Here, we present a new series of compounds substituted at the 4-position by a thioether-containing alkyl chain with a carboxylic and/or an aryl group at its extremity. Several compounds showed broad-spectrum inhibition with Ki values in the μM to sub-μM range against VIM-type enzymes, NDM-1 and IMP-1. The presence of the sulfur and of the aryl group was important for the inhibitory activity and the binding mode of a few compounds in VIM-2 was revealed by X-ray crystallography. Importantly, in vitro antibacterial susceptibility assays showed that several inhibitors were able to potentiate the activity of meropenem on Klebsiella pneumoniae clinical isolates producing VIM-1 or VIM-4, with a potentiation effect of up to 16-fold. Finally, a selected compound was found to only moderately inhibit the di-zinc human glyoxalase II, and several showed no or only moderate toxicity toward several human cells, thus favourably completing a promising behaviour.
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Oct 2021
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