Two sigma receptor subtypes (SR) have been identified to date, the sigma 1 receptor (S1R) and the sigma 2 receptor (S2R), differentiable by pharmacological profile, size, subcellular location and function. In recent decades, SRs have been proposed as innovative therapeutic targets for the treatment of tumors, being involved in mechanisms of cancer cell proliferation and survival. This has strengthened the interest of the pharmaceutical chemistry field for the identification and study of molecules related to both receptor subtypes as potential anticancer agents. This PhD project fits into this scenario and has two main objectives: (i) to develop and validate a new approach for cancer treatment based on pan-SRs modulators, using RC-106, a previously identified new pan-SRs modulator, as a pharmacological tool; (ii) to develop and characterize new RC-106 analogs with good affinity for both receptor subtypes and with antitumor activity. To achieve objective (i) RC-106 was the subject of a large biological study conducted on a panel of pancreatic cancer cell lines. On this panel, the antiproliferative and pro-apoptotic activity of RC-106 was studied, showing effectiveness at micromolar concentrations (IC50). In parallel, the biodistribution profile of RC-106 in mice was investigated. The compound was found to be 25 times more concentrated in the pancreas than in the plasma, reaching a concentration in the target organ at least equal to that effective in all performed in vitro experiments. Furthermore, the ability of RC-106 to overcome the blood brain barrier suggests the potential use of the molecule also for the treatment of brain tumors. Once the anticancer activity of RC-106 had been validated, the work focused on the characterization of the molecular mechanisms underlying this activity. Since SRs are mainly localized at the interface between the endoplasmic reticulum (ER) and mitochondria and due to their current re-classification as ligand-activated chaperones, we hypothesized their potential role in regulating the response of cells to ER stress. To validate this hypothesis, we focused on a particular adaptive mechanism, known as Unfolded Protein Response (UPR). This mechanism, in conditions of chronic stress, switches from a cell survival signal to a signal of death and takes the name of terminal UPR, triggering programmed cell death. We hypothesized that, the modulation of SRs activity in tumor cells could induce the activation of terminal UPR, thus causing cell death. The data obtained showed that the anticancer activity of RC-106 is related to the activation of the terminalUPR and to the inhibition of the proteasome. From a more general point of view, our data support the hypothesis of pan-SRs modulators as a valid tool for pharmacological studies aimed at a better knowledge of this class of receptors. Finally, to further define the role of SRs in tumors, we planned confocal microscopy imaging studies aimed at localizing and tracing SRs at the intracellular level and obtaining information on the mechanism of internalization, uptake and retention of RC-106. First, the fluorescence spectrum of RC-106 was studied, showing a fluorescence emission very similar to that of the endogenous fluorophores present in the cells. Therefore, RC-106 was not found to be usable for imaging studies. We then designed two hydroxylated derivatives of RC-106, RC-172 and RC-174, suitable for the subsequent introduction of a fluorescent tag. Relative to the objective (ii), we designed and prepared RC-106 analogs characterized by the presence of a variously functionalized piperazine ring, using a combinatorial approach and finally evaluated their cytotoxic activity in multiple myeloma (MM) cell lines and in glioblastoma (GB). The results obtained led to the identification of two compounds with an interesting antitumor potential useful for the treatment of MM, and one worthy of further investigations for the treatment of GB.

Two sigma receptor subtypes (SR) have been identified to date, the sigma 1 receptor (S1R) and the sigma 2 receptor (S2R), differentiable by pharmacological profile, size, subcellular location and function. In recent decades, SRs have been proposed as innovative therapeutic targets for the treatment of tumors, being involved in mechanisms of cancer cell proliferation and survival. This has strengthened the interest of the pharmaceutical chemistry field for the identification and study of molecules related to both receptor subtypes as potential anticancer agents. This PhD project fits into this scenario and has two main objectives: (i) to develop and validate a new approach for cancer treatment based on pan-SRs modulators, using RC-106, a previously identified new pan-SRs modulator, as a pharmacological tool; (ii) to develop and characterize new RC-106 analogs with good affinity for both receptor subtypes and with antitumor activity. To achieve objective (i) RC-106 was the subject of a large biological study conducted on a panel of pancreatic cancer cell lines. On this panel, the antiproliferative and pro-apoptotic activity of RC-106 was studied, showing effectiveness at micromolar concentrations (IC50). In parallel, the biodistribution profile of RC-106 in mice was investigated. The compound was found to be 25 times more concentrated in the pancreas than in the plasma, reaching a concentration in the target organ at least equal to that effective in all performed in vitro experiments. Furthermore, the ability of RC-106 to overcome the blood brain barrier suggests the potential use of the molecule also for the treatment of brain tumors. Once the anticancer activity of RC-106 had been validated, the work focused on the characterization of the molecular mechanisms underlying this activity. Since SRs are mainly localized at the interface between the endoplasmic reticulum (ER) and mitochondria and due to their current re-classification as ligand-activated chaperones, we hypothesized their potential role in regulating the response of cells to ER stress. To validate this hypothesis, we focused on a particular adaptive mechanism, known as Unfolded Protein Response (UPR). This mechanism, in conditions of chronic stress, switches from a cell survival signal to a signal of death and takes the name of terminal UPR, triggering programmed cell death. We hypothesized that, the modulation of SRs activity in tumor cells could induce the activation of terminal UPR, thus causing cell death. The data obtained showed that the anticancer activity of RC-106 is related to the activation of the terminalUPR and to the inhibition of the proteasome. From a more general point of view, our data support the hypothesis of pan-SRs modulators as a valid tool for pharmacological studies aimed at a better knowledge of this class of receptors. Finally, to further define the role of SRs in tumors, we planned confocal microscopy imaging studies aimed at localizing and tracing SRs at the intracellular level and obtaining information on the mechanism of internalization, uptake and retention of RC-106. First, the fluorescence spectrum of RC-106 was studied, showing a fluorescence emission very similar to that of the endogenous fluorophores present in the cells. Therefore, RC-106 was not found to be usable for imaging studies. We then designed two hydroxylated derivatives of RC-106, RC-172 and RC-174, suitable for the subsequent introduction of a fluorescent tag. Relative to the objective (ii), we designed and prepared RC-106 analogs characterized by the presence of a variously functionalized piperazine ring, using a combinatorial approach and finally evaluated their cytotoxic activity in multiple myeloma (MM) cell lines and in glioblastoma (GB). The results obtained led to the identification of two compounds with an interesting antitumor potential useful for the treatment of MM, and one worthy of further investigations for the treatment of GB.

DEVELOPMENT AND CHARACTERIZATION OF NEW pan-SIGMA RECEPTOR MODULATORS: CHEMICAL, BIOLOGICAL AND PHARMACOLOGICAL STUDIES FOR PRECLINICAL VALIDATION OF NEW THERAPEUTIC TARGETS

CORTESI, MICHELA
2021-02-25T00:00:00+01:00

Abstract

Two sigma receptor subtypes (SR) have been identified to date, the sigma 1 receptor (S1R) and the sigma 2 receptor (S2R), differentiable by pharmacological profile, size, subcellular location and function. In recent decades, SRs have been proposed as innovative therapeutic targets for the treatment of tumors, being involved in mechanisms of cancer cell proliferation and survival. This has strengthened the interest of the pharmaceutical chemistry field for the identification and study of molecules related to both receptor subtypes as potential anticancer agents. This PhD project fits into this scenario and has two main objectives: (i) to develop and validate a new approach for cancer treatment based on pan-SRs modulators, using RC-106, a previously identified new pan-SRs modulator, as a pharmacological tool; (ii) to develop and characterize new RC-106 analogs with good affinity for both receptor subtypes and with antitumor activity. To achieve objective (i) RC-106 was the subject of a large biological study conducted on a panel of pancreatic cancer cell lines. On this panel, the antiproliferative and pro-apoptotic activity of RC-106 was studied, showing effectiveness at micromolar concentrations (IC50). In parallel, the biodistribution profile of RC-106 in mice was investigated. The compound was found to be 25 times more concentrated in the pancreas than in the plasma, reaching a concentration in the target organ at least equal to that effective in all performed in vitro experiments. Furthermore, the ability of RC-106 to overcome the blood brain barrier suggests the potential use of the molecule also for the treatment of brain tumors. Once the anticancer activity of RC-106 had been validated, the work focused on the characterization of the molecular mechanisms underlying this activity. Since SRs are mainly localized at the interface between the endoplasmic reticulum (ER) and mitochondria and due to their current re-classification as ligand-activated chaperones, we hypothesized their potential role in regulating the response of cells to ER stress. To validate this hypothesis, we focused on a particular adaptive mechanism, known as Unfolded Protein Response (UPR). This mechanism, in conditions of chronic stress, switches from a cell survival signal to a signal of death and takes the name of terminal UPR, triggering programmed cell death. We hypothesized that, the modulation of SRs activity in tumor cells could induce the activation of terminal UPR, thus causing cell death. The data obtained showed that the anticancer activity of RC-106 is related to the activation of the terminalUPR and to the inhibition of the proteasome. From a more general point of view, our data support the hypothesis of pan-SRs modulators as a valid tool for pharmacological studies aimed at a better knowledge of this class of receptors. Finally, to further define the role of SRs in tumors, we planned confocal microscopy imaging studies aimed at localizing and tracing SRs at the intracellular level and obtaining information on the mechanism of internalization, uptake and retention of RC-106. First, the fluorescence spectrum of RC-106 was studied, showing a fluorescence emission very similar to that of the endogenous fluorophores present in the cells. Therefore, RC-106 was not found to be usable for imaging studies. We then designed two hydroxylated derivatives of RC-106, RC-172 and RC-174, suitable for the subsequent introduction of a fluorescent tag. Relative to the objective (ii), we designed and prepared RC-106 analogs characterized by the presence of a variously functionalized piperazine ring, using a combinatorial approach and finally evaluated their cytotoxic activity in multiple myeloma (MM) cell lines and in glioblastoma (GB). The results obtained led to the identification of two compounds with an interesting antitumor potential useful for the treatment of MM, and one worthy of further investigations for the treatment of GB.
Two sigma receptor subtypes (SR) have been identified to date, the sigma 1 receptor (S1R) and the sigma 2 receptor (S2R), differentiable by pharmacological profile, size, subcellular location and function. In recent decades, SRs have been proposed as innovative therapeutic targets for the treatment of tumors, being involved in mechanisms of cancer cell proliferation and survival. This has strengthened the interest of the pharmaceutical chemistry field for the identification and study of molecules related to both receptor subtypes as potential anticancer agents. This PhD project fits into this scenario and has two main objectives: (i) to develop and validate a new approach for cancer treatment based on pan-SRs modulators, using RC-106, a previously identified new pan-SRs modulator, as a pharmacological tool; (ii) to develop and characterize new RC-106 analogs with good affinity for both receptor subtypes and with antitumor activity. To achieve objective (i) RC-106 was the subject of a large biological study conducted on a panel of pancreatic cancer cell lines. On this panel, the antiproliferative and pro-apoptotic activity of RC-106 was studied, showing effectiveness at micromolar concentrations (IC50). In parallel, the biodistribution profile of RC-106 in mice was investigated. The compound was found to be 25 times more concentrated in the pancreas than in the plasma, reaching a concentration in the target organ at least equal to that effective in all performed in vitro experiments. Furthermore, the ability of RC-106 to overcome the blood brain barrier suggests the potential use of the molecule also for the treatment of brain tumors. Once the anticancer activity of RC-106 had been validated, the work focused on the characterization of the molecular mechanisms underlying this activity. Since SRs are mainly localized at the interface between the endoplasmic reticulum (ER) and mitochondria and due to their current re-classification as ligand-activated chaperones, we hypothesized their potential role in regulating the response of cells to ER stress. To validate this hypothesis, we focused on a particular adaptive mechanism, known as Unfolded Protein Response (UPR). This mechanism, in conditions of chronic stress, switches from a cell survival signal to a signal of death and takes the name of terminal UPR, triggering programmed cell death. We hypothesized that, the modulation of SRs activity in tumor cells could induce the activation of terminal UPR, thus causing cell death. The data obtained showed that the anticancer activity of RC-106 is related to the activation of the terminalUPR and to the inhibition of the proteasome. From a more general point of view, our data support the hypothesis of pan-SRs modulators as a valid tool for pharmacological studies aimed at a better knowledge of this class of receptors. Finally, to further define the role of SRs in tumors, we planned confocal microscopy imaging studies aimed at localizing and tracing SRs at the intracellular level and obtaining information on the mechanism of internalization, uptake and retention of RC-106. First, the fluorescence spectrum of RC-106 was studied, showing a fluorescence emission very similar to that of the endogenous fluorophores present in the cells. Therefore, RC-106 was not found to be usable for imaging studies. We then designed two hydroxylated derivatives of RC-106, RC-172 and RC-174, suitable for the subsequent introduction of a fluorescent tag. Relative to the objective (ii), we designed and prepared RC-106 analogs characterized by the presence of a variously functionalized piperazine ring, using a combinatorial approach and finally evaluated their cytotoxic activity in multiple myeloma (MM) cell lines and in glioblastoma (GB). The results obtained led to the identification of two compounds with an interesting antitumor potential useful for the treatment of MM, and one worthy of further investigations for the treatment of GB.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11571/1399174
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