I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
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.
|
Jul 2024
|
|
I03-Macromolecular Crystallography
|
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.
|
Nov 2023
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
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.
|
Jun 2022
|
|
I03-Macromolecular Crystallography
|
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.
|
Jun 2021
|
|
I02-Macromolecular Crystallography
|
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.
|
Jun 2020
|
|
I02-Macromolecular Crystallography
|
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.
|
Apr 2019
|
|
I04-Macromolecular Crystallography
|
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.
|
Sep 2017
|
|
I04-Macromolecular Crystallography
|
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.
|
Mar 2017
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Aug 2016
|
|