Final Report Summary - HYPOXPROBE (The development of hypoxia-activated probes for imaging and therapy)
a. Regions of low oxygen (hypoxia) occur in a diverse range of biological contexts including diseases, bacterial infections and some healthy organs. During tumour growth and development, new blood vessels are formed, which demand more cellular oxygen than is available supply, leading to an oxygen deficient state. Regions of hypoxia occur in most of the solid tumours, and predict poor patient prognosis irrespective of the treatment modality. Many bacterial infections lead to biofilm formation, where the oxygen tension varies from hypoxia to anoxia (at the core) and are relevant in many situations including opportunistic infections resulting from cystic fibrosis (CF).
Hypoxia-activated prodrugs function by attaching a bioreductive group to a biologically activated molecule so as to substantially reduce the primary biological activity of the compound. These compounds potentially offer the ability to deliver compounds selectively to regions of hypoxia, and could find application in the treatment of diseases including cancers and CF. To determine when the use of hypoxia-activated prodrugs is useful, an effective imaging of hypoxia is essential. Although there is significant amount of research been conducted into imaging hypoxia, there is currently no simple method of quantitatively imaging gradients of oxygen concentration (oxygen tension). Development of such imaging method would find wide applications, ranging from understanding the fundamental biology of bacterial infections, to determining the applicability of hypoxia-activated prodrugs to solid tumours or hypoxic bacterial infections in the lungs of CF patients.
To develop a system that allows imaging of hypoxia gradients in bacteria and transfected mammalian cells, we put the effort towards developing novel hypoxia-activated small molecule inducers of gene expression. The specific aim of this project was to develop a protocol that places a fluorescent reporter gene under the control of a hypoxia-activated small molecule inducer (see Figure 1). In this system, the fluorescent protein will only be expressed when the small molecule inducer is released by removal of the hypoxia-activated group.
Towards this endeavor, we used green fluorescent protein (GFP) as the reporter gene under the control of the lac operator (lacO). The lacO sequence binds to the lac operon repressor protein (LOR) that inhibits RNA polymerase from performing gene transcription. Upon binding of a sugar molecule, β-D-1-thiogalactopyranoside (IPTG), a conformational change occurs in LOR, which releases it from binding to lacO and allows gene transcription.
Conceptually, upon understanding the interactions of the sugar molecule with LOR, if those interacting functional groups of the sugar can be protected with a hypoxia activated moiety, IPTG will not bind under normal condition, but only in hypoxic condition after the removal of the protecting group. In order to test this hypothesis, we have prepared benzylidine protected IPTG derivatives (compound 1 and 2, Figure 2) C2, C-3 and C-6 protected (as 4-nitrobenzyl ether) IPTG derivatives (compound 3, 4 and 5 Figure 2). A sequential protection-deprotection maneuver was needed to prepare these derivatives from IPTG. We also prepared C-6 protected derivative of 4-nitrobenzyl ether with a carbonate linker (compound 6 Figure 2). In order to evaluate the bio-reduction of IPTG derivatives, the first step was to identify the E. coli cell lines transformed with the appropriate plasmid, which could efficiently express GFP under hypoxia in presence of IPTG. We found that the Rosetta and BL21 cells transformed with sfGFP, could induce the GFP expression under hypoxia (level of GFP expression was analyzed by confocal microscopy and flow cytometry).
Based on preliminary experiments, a subset of the IPTG analogues (compound 3, 5, 6 and 7, Figure 2) were subjected to incubation under hypoxia and normixia with Rosetta and BL21 cells transformed with sfGFP. Level of GFP expression was analyzed by flow cytometry (see Figure 3). The preliminary experiment showed that, IPTG derivatives 5 (JS-64-1) and 6 (JS-66-1) are capable of inducing GFP expression similar to IPTG itself under normoxia. This behavior is consistent in both Rosetta and BL21 cell lines (see Figure 3). However, the level of GFP expression in hypoxia using these IPTG derivatives is weak. Further investigations to rationalise their behaviour under normoxia and hypoxia is currently underway.
b. Nitro-imidazole group have found wide applications in developing bioreductive prodrugs and in the context of imaging hypoxia. Among many existing nitroimidazole containing drug candidates, Metronidazole, an anti-parasitic drug candidate and Evofosfamide (TH-302), an anti-cancer therapeutic, which is currently being tested in advanced Phase II clinical trial are notable. We found that, despite of such frequent use of nitroimidazole derivatives in the development of HAP, the existing literature methods for their preparation is often low yielding, time intensive and capricious. With an aim to develop hypoxia-activated prodrugs based on Chk1 inhibitor SAR020106, we simultaneously put our effort to devise a robust and repeatable synthesis of 2-nitroimidazole (in a scale of 1-2 g) compound and to synthesize the bioreductive prodrug by conjugation of the nitroimidazole moiety to SAR020106. We have accomplished an improved and repeatable synthesis of 2-aminoimidazole (see Figure 4), a key intermediate for the 2-nitroimidazoyl alcohol /chloride synthesis and attached them to SAR020106 to prepare the corresponding HAPs (see Figure 5). These compounds constitute an important class of hypoxia activated prodrugs (HAP) based on Chk1 inhibitor. Their applications in hypoxic tumour cells are currently underway.
Hypoxia-activated prodrugs function by attaching a bioreductive group to a biologically activated molecule so as to substantially reduce the primary biological activity of the compound. These compounds potentially offer the ability to deliver compounds selectively to regions of hypoxia, and could find application in the treatment of diseases including cancers and CF. To determine when the use of hypoxia-activated prodrugs is useful, an effective imaging of hypoxia is essential. Although there is significant amount of research been conducted into imaging hypoxia, there is currently no simple method of quantitatively imaging gradients of oxygen concentration (oxygen tension). Development of such imaging method would find wide applications, ranging from understanding the fundamental biology of bacterial infections, to determining the applicability of hypoxia-activated prodrugs to solid tumours or hypoxic bacterial infections in the lungs of CF patients.
To develop a system that allows imaging of hypoxia gradients in bacteria and transfected mammalian cells, we put the effort towards developing novel hypoxia-activated small molecule inducers of gene expression. The specific aim of this project was to develop a protocol that places a fluorescent reporter gene under the control of a hypoxia-activated small molecule inducer (see Figure 1). In this system, the fluorescent protein will only be expressed when the small molecule inducer is released by removal of the hypoxia-activated group.
Towards this endeavor, we used green fluorescent protein (GFP) as the reporter gene under the control of the lac operator (lacO). The lacO sequence binds to the lac operon repressor protein (LOR) that inhibits RNA polymerase from performing gene transcription. Upon binding of a sugar molecule, β-D-1-thiogalactopyranoside (IPTG), a conformational change occurs in LOR, which releases it from binding to lacO and allows gene transcription.
Conceptually, upon understanding the interactions of the sugar molecule with LOR, if those interacting functional groups of the sugar can be protected with a hypoxia activated moiety, IPTG will not bind under normal condition, but only in hypoxic condition after the removal of the protecting group. In order to test this hypothesis, we have prepared benzylidine protected IPTG derivatives (compound 1 and 2, Figure 2) C2, C-3 and C-6 protected (as 4-nitrobenzyl ether) IPTG derivatives (compound 3, 4 and 5 Figure 2). A sequential protection-deprotection maneuver was needed to prepare these derivatives from IPTG. We also prepared C-6 protected derivative of 4-nitrobenzyl ether with a carbonate linker (compound 6 Figure 2). In order to evaluate the bio-reduction of IPTG derivatives, the first step was to identify the E. coli cell lines transformed with the appropriate plasmid, which could efficiently express GFP under hypoxia in presence of IPTG. We found that the Rosetta and BL21 cells transformed with sfGFP, could induce the GFP expression under hypoxia (level of GFP expression was analyzed by confocal microscopy and flow cytometry).
Based on preliminary experiments, a subset of the IPTG analogues (compound 3, 5, 6 and 7, Figure 2) were subjected to incubation under hypoxia and normixia with Rosetta and BL21 cells transformed with sfGFP. Level of GFP expression was analyzed by flow cytometry (see Figure 3). The preliminary experiment showed that, IPTG derivatives 5 (JS-64-1) and 6 (JS-66-1) are capable of inducing GFP expression similar to IPTG itself under normoxia. This behavior is consistent in both Rosetta and BL21 cell lines (see Figure 3). However, the level of GFP expression in hypoxia using these IPTG derivatives is weak. Further investigations to rationalise their behaviour under normoxia and hypoxia is currently underway.
b. Nitro-imidazole group have found wide applications in developing bioreductive prodrugs and in the context of imaging hypoxia. Among many existing nitroimidazole containing drug candidates, Metronidazole, an anti-parasitic drug candidate and Evofosfamide (TH-302), an anti-cancer therapeutic, which is currently being tested in advanced Phase II clinical trial are notable. We found that, despite of such frequent use of nitroimidazole derivatives in the development of HAP, the existing literature methods for their preparation is often low yielding, time intensive and capricious. With an aim to develop hypoxia-activated prodrugs based on Chk1 inhibitor SAR020106, we simultaneously put our effort to devise a robust and repeatable synthesis of 2-nitroimidazole (in a scale of 1-2 g) compound and to synthesize the bioreductive prodrug by conjugation of the nitroimidazole moiety to SAR020106. We have accomplished an improved and repeatable synthesis of 2-aminoimidazole (see Figure 4), a key intermediate for the 2-nitroimidazoyl alcohol /chloride synthesis and attached them to SAR020106 to prepare the corresponding HAPs (see Figure 5). These compounds constitute an important class of hypoxia activated prodrugs (HAP) based on Chk1 inhibitor. Their applications in hypoxic tumour cells are currently underway.