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]
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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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin.
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Aug 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11690]
Open Access
Abstract: Trypanosoma and Leishmania parasites are the etiological agents of various threatening neglected tropical diseases (NTDs), including human African trypanosomiasis (HAT), Chagas disease, and various types of leishmaniasis. Recently, meaningful progresses in the treatment of HAT, due to Trypanosoma brucei (Tb), have been achieved by the introduction of fexinidazole and the combination therapy eflornithine–nifurtimox. Nevertheless, due to drug resistance issues and the exitance of animal reservoirs, the development of new NTD treatments is still required. For this purpose, we explored the combined targeting of two key folate enzymes, dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1). We formerly showed that the TbDHFR inhibitor cycloguanil (CYC) also targets TbPTR1, although with reduced affinity. Here, we explored a small library of CYC analogues to understand how their substitution pattern affects the inhibition of both TbPTR1 and TbDHFR. Some novel structural features responsible for an improved, but preferential, ability of CYC analogues to target TbPTR1 were disclosed. Furthermore, we showed that the known drug pyrimethamine (PYR) effectively targets both enzymes, also unveiling its binding mode to TbPTR1. The structural comparison between PYR and CYC binding modes to TbPTR1 and TbDHFR provided key insights for the future design of dual inhibitors for HAT therapy.
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Jun 2021
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I04-Macromolecular Crystallography
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David T.
Davies
,
Simon
Leiris
,
Magdalena
Zalacain
,
Nicolas
Sprynski
,
Jérôme
Castandet
,
Justine
Bousquet
,
Clarisse
Lozano
,
Agustina
Llanos
,
Laethitia
Alibaud
,
Srinivas
Vasa
,
Ramesh
Pattipati
,
Ravindar
Valige
,
Bhaskar
Kummari
,
Srinivasu
Pothukanuri
,
Cyntia
De Piano
,
Ian
Morrissey
,
Kirsty
Holden
,
Peter
Warn
,
Francesca
Marcoccia
,
Manuela
Benvenuti
,
Cecilia
Pozzi
,
Giusy
Tassone
,
Stefano
Mangani
,
Jean-Denis
Docquier
,
David
Pallin
,
Richard
Elliot
,
Marc
Lemonnier
,
Martin
Everett
Diamond Proposal Number(s):
[21741]
Abstract: The diazabicyclooctanes (DBOs) are a class of serine β-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of β-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of β-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO “critical priority pathogen” producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure–activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.
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Dec 2020
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