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
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I03-Macromolecular Crystallography
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Rosaria
Luciani
,
Puneet
Saxena
,
Sachin
Surade
,
Matteo
Santucci
,
Alberto
Venturelli
,
Chiara
Borsari
,
Gaetano
Marverti
,
Glauco
Ponterini
,
Stefania
Ferrari
,
Tom L.
Blundell
,
Maria Paola
Costi
Diamond Proposal Number(s):
[9537, 9007]
Abstract: Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. Herein, we selected 16 compounds through a virtual screening approach. We solved the first X-ray crystal structure of Thermatoga maritima (Tm) ThyX in complex with a nonsubstrate analog inhibitor. Given the active site similarities between Mycobacterium tuberculosis ThyX (Mtb-ThyX) and Tm-ThyX, our crystal structure paves the way for a structure-based design of novel antimycobacterial compounds. The 1H-imidazo[4,5-d]pyridazine was identified as scaffold for the development of Mtb-ThyX inhibitors.
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Oct 2016
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I04-Macromolecular Crystallography
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Flavio
Di Pisa
,
Giacomo
Landi
,
Lucia
Dello Iacono
,
Cecilia
Pozzi
,
Chiara
Borsari
,
Stefania
Ferrari
,
Matteo
Santucci
,
Nuno
Santarem
,
Anabela
Cordeiro-Da-Silva
,
Carolina
Moraes
,
Laura
Alcantara
,
Vanessa
Fontana
,
Lucio
Freitas-Junior
,
Sheraz
Gul
,
Maria
Kuzikov
,
Birte
Behrens
,
Ina
Pöhner
,
Rebecca
Wade
,
Maria
Costi
,
Stefano
Mangani
Diamond Proposal Number(s):
[8574]
Open Access
Abstract: Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1–3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1–TbPTR1 and Leishmania major–LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.
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Mar 2017
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[8574]
Open Access
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.
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Sep 2017
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I24-Microfocus Macromolecular Crystallography
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Joanna
Panecka-Hofman
,
Ina
Pöhner
,
Francesca
Spyrakis
,
Talia
Zeppelin
,
Flavio Di
Pisa
,
Lucia
Dello Iacono
,
Alessio
Bonucci
,
Antonio
Quotadamo
,
Alberto
Venturelli
,
Stefano
Mangani
,
Maria Paola
Costi
,
Rebecca C.
Wade
Diamond Proposal Number(s):
[11690]
Abstract: Background
Multi-target approaches are necessary to properly analyze or modify the function of a biochemical pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of experimentally validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: (i) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR–TS), (ii) the primary off-targets: human DHFR and TS, and (iii) the secondary on-target: human folate receptor β, a folate/antifolate transporter.
Methods
We computationally compared the structural, dynamic and physico-chemical properties of the targets. We based our analysis on available inhibitory activity and crystallographic data, including a crystal structure of the bifunctional T. cruzi DHFR–TS with tetrahydrofolate bound determined in this work. Due to the low sequence and structural similarity of the targets analyzed, we employed a mapping of binding pockets based on the known common ligands, folate and methotrexate.
Results
Our analysis provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallographic structures.
Conclusions
The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosmatid agents.
General significance
Apart from the target–based discovery of selective compounds, our approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families.
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Sep 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[11690, 15832]
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.
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Mar 2019
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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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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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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11690]
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.
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Jun 2020
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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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