10 research outputs found
Glutatione s-trasferasi p1-1 come possibile bersaglio per lo sviluppo di nuovi chemioterapici: un approccio bivalente
Nella presente tesi si è studiato il meccanismo d' azione di molecole che hanno come bersaglio intracellulare la Glutatione S-trasferasi (GST).
Le GST sono una famiglia di enzimi di detossificazione. La reazione generale catalizzata da tutte le isoforme è la coniugazione del glutatione (GSH) ad una molecola contenente un gruppo elettrofilo. Tale reazione permette lâ eliminazione della molecola in quanto il coniugato è più solubile ed atto a essere escreto. Inoltre, le GST sono dei modulatori endogeni della proteine che regolano la morte cellulare programmata (apoptosi). Dal secolo scorso, è noto che l' iperespressione delle GST è associata all' insorgenza dei tumori e porta alla resistenza ai farmaci chemioterapici.
Lo studio è stato condotto su due fronti. Sono stati sintetizzati e caratterizzati degli analoghi del 6-(7-nitro-2,1,3-benzossadiazol-4-yltio)esanolo (NBDHEX), un promettente inibitore di alcune classi di GST(GSTP1-1, GSTM2-2), con lo scopo di individuare delle molecole più idrosolubili e con una maggiore selettività verso l' isoforma GSTP1-1, iperespressa nei tumori (parte 1). Inoltre è stato caratterizzato il meccanismo d' azione della brostallicina, una molecola che sfrutta l' iperespressione del sistema GSH/GST per la produzione di una specie alchilante del DNA (parte 2).
1. L' analisi cristallografica del sito attivo della GSTM2-2 e della GSTP1-1 ha messo in evidenza che lâ anello benzossadiazolico è ben stabilizzato dai residui aminoacidici presenti nel sito H di entrambe le isoforme. Nel sito attivo della GSTP1-1, invece, si possono migliorare le interazioni tra il nitro gruppo, carico negativamente, e alcuni residui carichi positivamente. Inoltre la catena alcolica dell'NBDHEX interagisce solo debolmente con i residui aminoacidici della sito attivo. Basandosi su queste evidenze, si sono valutati gli effetti della sostituzione del gruppo nitro con un gruppo solfonico e la variazione della catena alcolica con un gruppo benzene variamente sostituito. I composti che ne sono derivati sono: il 6-(7-sulfo-2,1,3-benzossadiazol-4-yltio)esanolo (SBDHEX), e derivati del nitrobenzofurazano funzionalizzati in catena laterale, a cui sono state date le sigle 2759, 2766, 2760, 2868, 2871, 2872, 2874, 2875 e 2876. Questi composti, sebbene non si siano dimostrati più efficaci dellâ NBDHEX hanno fornito informazioni utili per la progettazione di nuovi inibitori con scheletro nitro-benzossafurazanico. Dagli studi condotti è emerso che il gruppo-nitro è fondamentale per l' interazione con l' enzima poiché l' SBDHEX non sembra interagire con la GSTP1-1 e non è in grado di formare un complesso-Ï . La presenza di un anello aromatico, come sostituente della catena laterale dell' anello benzossadiazolico, aumenta la selettività delle molecole nei confronti dell' isoforma GSTP1-1. Infine, le molecole estremamente solubili (SBDHEX, 2760, 2875) hanno un basso effetto citotossico imputabile alla loro difficoltà di diffondere nel doppio strato fosfolipidico.
2. La brostallicina un pro-farmaco che sfrutta l' iperespressione del sistema GSH/GST. Partendo dalle evidenze indirette, è stato chiarito e caratterizzato il meccanismo citotossico di questa molecola che è in fase di sperimentazione clinica. Dagli studi condotti con differenti tecniche sperimentali è stato possibile concludere che sia la GSTP1-1 che la GSTM2-2 catalizzano la reazione tra la brostallicina e il GSH. In particolare, l' isoforma GSTM2-2 ha un' affinità maggiore verso la molecola e ne permette l' impiego in quei tumori che la iperesprimono. Entrambe le isoforme catalizzano la formazione di un intermedio di reazione, l' addotto brostallicina-GSH, che presenta un cloro come sostituente del Cα. Questo intermedio è convertito lentamente nei prodotti di reazione in cui il cloro viene sostituito da un gruppo fosfato o un ossidrile. Basandosi sui dati cinetici, è stato proposto un meccanismo per spiegare in modo soddisfacente la reazione tra brostallicina e GSH catalizzata dalla GSTP1-1. Infine l' ipotesi che il composto con il cloro potesse rappresentare la forma attiva del farmaco, è stata validata utilizzando la tecnica dell' interruzione della polimerasi. Infatti questa molecola, in assenza di GSH/GST, è in grado di alchilare il DNA con la stessa specificità di sequenza della miscela contenete brostallicina, GSH e GSTP1-1. Questi studi hanno permesso di chiarire il ruolo svolto dalle GST nel meccanismo d' azione della brostallicina e il motivo per cui questo chemioterapico sia avvantaggiato dallâ iperespressione del sistema GSH/GST.In this experimental work the mechanism of action of drugs, which have as
intracellular target the Glutathion S-Transferases (GST), is investigated.
GST are a family of enzymes involved in the detoxification pathway. These
enzymes catalyze the conjugation of glutathione (GSH) to a wide variety of
endogenous and exogenous electrophilic compounds, leading to a more
soluble species which can be easily eliminated. Furthermore, GST are
endogenous modulators of proteins involved in the apoptotic pathway. From
the last century, it is known that the aberrant expression of GST isozymes is
correlate with tumor onset and implicated in the development of resistance
toward chemotherapy agents.
We used two different approaches studying GST as target for the development
of new anticancer compounds. We synthesized new molecules starting from
the 6-(7-nitro-2,1,3-benzossadiazol-4-ylthio)exanol (NBDHEX), a promising
inhibitor of Pi and Mu classes of GSTs (GSTP1-1 and GSTM2-2), with the
aim to produce compounds more soluble and specific for the GSTP1-1
(section 1). Moreover, we characterize the mechanism of action of brostallicin,
which take advantage from the overexpression of GSH/GST system to lead a
molecules able to bind DNA (section 2).
1. The crystallographic analysis of the active site of GSTM2-2 and
GSTP1-1 reveals that the residues in the H site of both isoenzymes well
stabilize the benzossadiazole ring. However in the active site of
GSTP1-1 the interactions can be improved. In particular, the interaction
among the nitro group, which has a negative charge when forming Ï -
complex, and some residues which have a positive charges, and also
between alcoholic chain of NBDHEX with the active site. Based on
these evidences we will assess the effects of the substitution of the nitro
group with a sulfonic one and the changing of the alcoholic chain with
aromatic group variously substituted. The resulting molecules are: 6-(7-
sulfo-2,1,3-benzossadiazol-4-ylthio)exanol (SBDHEX), and derivatives
of the nitrobenzofurazane ring, functionalized on the lateral chain:
2759, 2766, 2760, 2868, 2871, 2872, 2874, 2875 e 2876. These
compounds are less effective than NBDHEX, however we collect
important information to better design new inhibitor with
bezoxadiazole bone. From our studies we clarified the important of the
nitro group. Infact, the nitro group make possible the interaction with
the enzyme because SBDHEX does not interact with GSTP1-1 and the
molecule is not able to form the Ï -complex. The benzene ring improve
the selectivity towards the GSTP1-1. Finally, compounds which are
more soluble (SBDHEX, 2760, 2875) have less cytotoxic effect
because of they are unable to diffuse cell membrane.
2. Brostallicin is a pro-drug which take advantage of the overexpression
of GSH/GST system. Based on this evidence we clarify the mechanism
of action of this compound which is in clinical trial. Through different
experimental approaches we can conclude that both GSTP1-1 than
GSTM2-2 catalyze the reaction between brostallicin and GSH. In
particular, the affinity of brostallicin is higher for GSTM2-2 and the
drug is fully active at the concentration using in cancer treatment. Both
isoenzyme catalyze the formation of an intermediate reactive species,
which is slowly converted into the final products. This intermediate,
identified as the alpha-chloroamido derivative of the GSH-brostallicin
adduct, is able to alkylate DNA in a sequence-specific manner and
appears to be the active form of the drug. The kinetic behavior of the
reaction between brostallicin and GSH, catalyzed by GSTP1-1, has
been studied in detail, and a minimum kinetic scheme that suitably
describes the experimental data is provided. Overall, these data fully
support and extend the findings that brostallicin could be indicated for
the treatment of tumor overexpressing the pi or mu class GST
15+ MILLION TOP 1% MOST CITED SCIENTIST 12.2% AUTHORS AND EDITORS FROM TOP 500 UNIVERSITIES 13 Advances in MicroRNAs and Alzheimer's Disease Research
KuQuinones equilibria assessment for biomedical applications
A small library of pentacyclic quinoid compounds, called KuQuinones (KuQs), has been prepared through a one-pot reaction. KuQuinones complex structure is made up by two naphthoquinone units connected by a five-membered ring. Due to KuQs structural features, keto− enol tautomerization in solution likely occurs, leading to the generation of four different species, i.e., the enol, the enolate, the external enol and the diquinoid species. The interchange among KuQ tautomers leads to substantial spectral variations of the dye depending on the experimental conditions used. The comprehension of tautomeric equilibria of this new class of quinoid compounds is strongly required in order to explain their behavior in solution and in biological environment. UV−vis, 1H NMR spectroscopies, and DFT calculations resulted appropriate tools to understand the nature of the prevalent KuQuinone species in solution. Moreover, due to the structural similarity of KuQuinones with camptothecin (CPT), a largely used anticancer agent, KuQs have been tested against Cisplatin-resistant SKOV3 and SW480 cancer cell lines. Results highlighted that KuQs are highly active toward the analyzed cell lines and almost nontoxic for healthy cell, indicating a high specific activityA small library of pentacyclic quinoid compounds, called KuQuinones (KuQs), has been prepared through a one-pot reaction. KuQuinones complex structure is made up by two naphthoquinone units connected by a five-membered ring. Due to KuQs structural features, keto− enol tautomerization in solution likely occurs, leading to the generation of four different species, i.e., the enol, the enolate, the external enol and the diquinoid species. The interchange among KuQ tautomers leads to substantial spectral variations of the dye depending on the experimental conditions used. The comprehension of tautomeric equilibria of this new class of quinoid compounds is strongly required in order to explain their behavior in solution and in biological environment. UV−vis, 1H NMR spectroscopies, and DFT calculations resulted appropriate tools to understand the nature of the prevalent KuQuinone species in solution. Moreover due to the structural similarity of KuQuinones with camptothecin (CPT), a largely used anticancer agent, KuQs have been tested against Cisplatin-resistant SKOV3 and SW480 cancer cell lines. Results highlighted that KuQs are highly active toward the analyzed cell lines and almost nontoxic for healthy cell, indicating a high specific activity
Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S
Role of glutathione transferases in the mechanism of brostallicin activation
Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-π or GST-μ. Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here indicate that brostallicin binds reversibly to both isoenzymes with Kd values in the micromolar range (the affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is slowly converted into the final products. This intermediate, identified as the R-chloroamido derivative of the GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1, has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the treatment of tumor overexpressing the pi or mu class GST
Immuno-Contexture and Immune Checkpoint Molecule Expression in Mismatch Repair Proficient Colorectal Carcinoma
Colorectal carcinoma (CRC) represents a lethal disease with heterogeneous outcomes. Only patients with mismatch repair (MMR) deficient CRC showing microsatellite instability and hyper-mutated tumors can obtain clinical benefits from current immune checkpoint blockades; on the other hand, immune- or target-based therapeutic strategies are very limited for subjects with mismatch repair proficient CRC (CRCpMMR). Here, we report a comprehensive typing of immune infiltrating cells in CRCpMMR. We also tested the expression and interferon-γ-modulation of PD-L1/CD274. Relevant findings were subsequently validated by immunohistochemistry on fixed materials. CRCpMMR contain a significantly increased fraction of CD163+ macrophages (TAMs) expressing TREM2 and CD66+ neutrophils (TANs) together with decrease in CD4−CD8−CD3+ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of conventional and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells displays an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRCpMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion, and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRCpMMR