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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
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.
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Nov 2019
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I02-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[11690]
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.
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Apr 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11690]
Open Access
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.
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Mar 2017
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[15832, 1949]
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.
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Sep 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[1358]
Open Access
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.
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Mar 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[11690]
Open Access
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.
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Aug 2016
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[8574]
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.
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Aug 2015
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|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[8574]
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
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Sep 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[8574]
Abstract: BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower Km values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties.
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Sep 2016
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Simon
Leiris
,
Alicia
Coelho
,
Jérôme
Castandet
,
Maëlle
Bayet
,
Clarisse
Lozano
,
Juliette
Bougnon
,
Justine
Bousquet
,
Martin John
Everett
,
Marc
Lemonnier
,
Nicolas
Sprynski
,
Magdalena
Zalacain
,
Thomas David
Pallin
,
Michael
Cramp
,
Neil
Jennings
,
Gilles
Raphy
,
Mark William
Jones
,
Ramesh
Pattipati
,
Battu
Shankar
,
Relangi
Sivasubrahmanyam
,
Ashok Kumar
Soodhagani
,
Ramakrishna Reddy
Juventhala
,
Narender
Pottabathini
,
Srinivasu
Pothukanuri
,
Manuela
Benvenuti
,
Cecilia
Pozzi
,
Stefano
Mangani
,
Filomena
De Luca
,
Giulia
Cerboni
,
Jean-denis
Docquier
,
David
Davies
Diamond Proposal Number(s):
[8574, 15832]
Abstract: The clinical effectiveness of carbapenem antibiotics such as meropenem is becoming increasingly compromised by the spread of both metallo β-lactamase (MBL) and serine β-lactamase (SBL) enzymes on mobile genetic elements, stimulating research to find new β-lactamase inhibitors to be used in conjunction with carbapenems and other β-lactam antibiotics. Herein we describe our initial exploration of a novel chemical series of metallo β-lactamase inhibitors, from concept through to efficacy in a survival model using an advanced tool compound (ANT431) in conjunction with meropenem.
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Nov 2018
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