Final Report Summary - MOLECULARDOCTORS (RNA-codified release of cytotoxic peptides from PNA prodrugs as a new therapeutic approach to cancer)
The study of DNA has progressed over the last two decades from the initial structural and functional characterization in living organisms, to what it is now known as DNA technology, encompassing the development of DNA as a tool in biological sciences, and more recently as a structural and nanotechnological scaffold. However, despite that the development of DNA-based functional and dynamic processes it is still in its infancy, the manipulation, control and the use of oligonucleotides might open promising and still unexplored opportunities for the development of DNA-encoded molecular therapies for specific diseases such as cancer. The current ultrafast sequencing technologies uncover and ever increase the amount of data that links diseases such as cancer to personal genetic information. One of the central questions is: Can we take advantage of this genetic information in the development of new therapeutic principles?
Conventional cytotoxic cancer therapies, such as radiation or chemotherapy, have little selectivity and originate therefore a broad spectrum of severe side effects. This fact, together with the observed complications resulting from individual genetic differences, make more urgent than ever to develop innovative chemical approaches based on the concept of tailored personalized medicine. In this context, the in situ generation of drugs on the basis of nucleic acid-templated reactions should provide a unique strategy to develop cell-specific responses. DNA and RNA template-directed reactions have previously been used mainly in the area of organic synthesis, or as diagnostic, or imaging tools. Inspired by the successful former DNA-triggered transfer of our group, where the DNA catalyzes the transfer of only one amino acid residue to form a peptide-PNA-conjugates with biological activity, now in our Molecular Doctor project we are exploring the possibilities of the use highly specific gene expression-specific chemotherapies based on mRNA-templated activation of PNA prodrugs by peptidyl transfer reactions. In our strategy two short PNA-oligomers equipped with a fragment of the cytotoxic peptide (non-toxic) hybridize adjacently to a complemetary RNA-template, which must be an overexpressed biomarker of malignant cells. One of the PNA probes presented the peptidyl sequence as donating group covalently bound as thioester, while the other bears an N-terminal cysteine peptidyl sequence (accepting group). This adjacent alignment triggers a templated native chemical ligation, which could allow the transfer of one part of the peptide to reconstitute the full cytotoxic peptide sequence (figure 1). After the transfer reaction the PNA with the full cytotoxic peptide could dissociate of the template, therefore the turnover and the catalytic production of more peptides could be ensured.
To accomplish the project's objectives we attained the following the milestones in brackets. As a cytotoxic peptide we chose the mitochondrial peptide KLAK (KLAKLAKKLAKLAK), which shows powerful cytotoxic properties in vitro as well as in vivo by disrupting the negatively charged mitochondrial membrane and releasing the cytochrome c that triggers apoptosis. Since the cytotoxic peptides will be synthesized by templated-catalyzed acyl transfer reactions, the strategy will require the mutation of a residue in the peptide sequence for a cysteine that is required for the native chemical ligation reaction. Consequently, we first synthesized the entire collection of cysteine full length peptides and their correspondent fragments (milestone a), and identified the best peptide candidate pairs to perform the transfer reaction (milestone b). The best pair is that in which the toxicity of the full length peptide has to be significant higher than the correspondent fragment. Based on the toxicity assays we selected two peptide pairs as the most promising ones, which entails the transfer of four (KLAK) or six (KLAKLA) residues in each case. Once the best peptide candidate pairs were discovered, the researcher synthesized the accepting and donating PNA-peptide conjugates by standard Fmoc/Boc-solid phase methodology (milestone c). In the next step the researcher selected the RNA XIAP as a template since it has been observed in many tumour cells, like in the case of prostate, pancreatic, gastric, ovarian, lung, breast cancers, and in certain patients XIAP overexpression is associated with poor clinical outcome. Initially, the transfer reaction was studied and optimized by using a 26 base pair DNA, analoge of XIAP mRNA, as a template for the four selected pairs (milestone d). Afterwards the concept was extrapolated to the in vitro assays of mRNA target-catalyzed transfer reaction (milestone e). The accomplishment of the catalytic transfer reaction with RNA demanded an optimization of the length of both PNA probes. Besides in our case the peptidyl transfer reactions involve two large peptide segments and are, thus, expected to occur at slower rates than the former acyl transfer reaction reported. However, the rate of the transfer reaction could be modulated by the appropriate selection of different thioester structures and linkers between the peptide and the PNA, which provided a different reactivity. The RNA-programmed chemistry was successfully developed and it was demonstrated to be remarkably robust, achieving even excellent turnovers with 0.1 eq of RNA. The results obtained in these studies were already very relevant by themselves being even suitable as starting point to develop parallel projects. Furthermore the toxicity study of the PNA-peptide conjugates alone and in presence of external RNA revealed some crucial aspects for the implementation of the strategy in vivo. The conjugation between the PNA and the peptide can affect the toxicity. In fact, in our particularly case the toxicity decreased four fold, which entails the need of the scale up the reaction. Besides the researcher also demonstrated that the RNA that triggers the reaction could influence on the biological activity of the peptide formed. Here, the project has tough us two important lessons: 1) it is difficult to use this method for the construction of positively charged peptides; 2) because of this drawback, dissociation of the product from the RNA template would be desirable. Considering these discoveries we seek for alternative means and since the use of intracellular mRNA XIAP as a template in vivo might entails difficulties impossible to overcome. As a proof of concept of this initial attempt, we decided to synthesize the accepting probe with a cell penetrating peptide (R8) and performed a catalytic template reaction with an extracellular RNA (milestone f). After the transfer reaction the full-length peptide-PNA conjugate would go inside the cells and trigger the death of the cancer cells. This stage was the most challenging. At the moment we are finishing touches to the project and at least two publications are expected in the near future.
Regarding the relevance of this project, there is no need to emphasise the importance of inventing new therapeutic strategies for cancer. It is clear that cancer is a modern day pandemic. In fact, nowadays cancer kills more people than tuberculosis, malaria and HIV put together. Moreover, diagnosis and treatment for most tumours are becoming sophisticated and very expensive. This increase in cost could have a dramatic impact in countries in which the health expenditure per capita is only a few dollars yearly; some health systems, even in the most affluent countries, have increasing difficulties in being able to provide the most expensive new drugs. The dramatic situation prevailing in the treatment of HIV-infected people, whereby most countries are unable to afford modern therapies, could soon also become a reality in oncology. Consequently improving, expanding and developing oncologic treatments will undoubtedly have profound socio-economic impact worldwide. In this context our project contributes by combining challenging and pioneering objectives from a basic point of view with important applicability possibilities. Relying on chemistry: synthesis and designed reactions, we approached objectives in the areas of chemical and cell biology that might lead to new advances in medicinal chemistry. Indeed, to our knowledge this is the first time that a cytotoxic drug is formed by this type of reactions in a gene-encoded approach, we demonstrated that the RNA-programmed chemistry works and even using prodrugs, which is also of great importance in terms of selectivity and side effects. Therefore, we are opening the possibility to reach the challenging goal of specific mRNA-directed release of bioactive species inside the cytoplasm in the near future. This implies new potential therapeutics that could be used in the war against cancer and also for other drug developments.
Conventional cytotoxic cancer therapies, such as radiation or chemotherapy, have little selectivity and originate therefore a broad spectrum of severe side effects. This fact, together with the observed complications resulting from individual genetic differences, make more urgent than ever to develop innovative chemical approaches based on the concept of tailored personalized medicine. In this context, the in situ generation of drugs on the basis of nucleic acid-templated reactions should provide a unique strategy to develop cell-specific responses. DNA and RNA template-directed reactions have previously been used mainly in the area of organic synthesis, or as diagnostic, or imaging tools. Inspired by the successful former DNA-triggered transfer of our group, where the DNA catalyzes the transfer of only one amino acid residue to form a peptide-PNA-conjugates with biological activity, now in our Molecular Doctor project we are exploring the possibilities of the use highly specific gene expression-specific chemotherapies based on mRNA-templated activation of PNA prodrugs by peptidyl transfer reactions. In our strategy two short PNA-oligomers equipped with a fragment of the cytotoxic peptide (non-toxic) hybridize adjacently to a complemetary RNA-template, which must be an overexpressed biomarker of malignant cells. One of the PNA probes presented the peptidyl sequence as donating group covalently bound as thioester, while the other bears an N-terminal cysteine peptidyl sequence (accepting group). This adjacent alignment triggers a templated native chemical ligation, which could allow the transfer of one part of the peptide to reconstitute the full cytotoxic peptide sequence (figure 1). After the transfer reaction the PNA with the full cytotoxic peptide could dissociate of the template, therefore the turnover and the catalytic production of more peptides could be ensured.
To accomplish the project's objectives we attained the following the milestones in brackets. As a cytotoxic peptide we chose the mitochondrial peptide KLAK (KLAKLAKKLAKLAK), which shows powerful cytotoxic properties in vitro as well as in vivo by disrupting the negatively charged mitochondrial membrane and releasing the cytochrome c that triggers apoptosis. Since the cytotoxic peptides will be synthesized by templated-catalyzed acyl transfer reactions, the strategy will require the mutation of a residue in the peptide sequence for a cysteine that is required for the native chemical ligation reaction. Consequently, we first synthesized the entire collection of cysteine full length peptides and their correspondent fragments (milestone a), and identified the best peptide candidate pairs to perform the transfer reaction (milestone b). The best pair is that in which the toxicity of the full length peptide has to be significant higher than the correspondent fragment. Based on the toxicity assays we selected two peptide pairs as the most promising ones, which entails the transfer of four (KLAK) or six (KLAKLA) residues in each case. Once the best peptide candidate pairs were discovered, the researcher synthesized the accepting and donating PNA-peptide conjugates by standard Fmoc/Boc-solid phase methodology (milestone c). In the next step the researcher selected the RNA XIAP as a template since it has been observed in many tumour cells, like in the case of prostate, pancreatic, gastric, ovarian, lung, breast cancers, and in certain patients XIAP overexpression is associated with poor clinical outcome. Initially, the transfer reaction was studied and optimized by using a 26 base pair DNA, analoge of XIAP mRNA, as a template for the four selected pairs (milestone d). Afterwards the concept was extrapolated to the in vitro assays of mRNA target-catalyzed transfer reaction (milestone e). The accomplishment of the catalytic transfer reaction with RNA demanded an optimization of the length of both PNA probes. Besides in our case the peptidyl transfer reactions involve two large peptide segments and are, thus, expected to occur at slower rates than the former acyl transfer reaction reported. However, the rate of the transfer reaction could be modulated by the appropriate selection of different thioester structures and linkers between the peptide and the PNA, which provided a different reactivity. The RNA-programmed chemistry was successfully developed and it was demonstrated to be remarkably robust, achieving even excellent turnovers with 0.1 eq of RNA. The results obtained in these studies were already very relevant by themselves being even suitable as starting point to develop parallel projects. Furthermore the toxicity study of the PNA-peptide conjugates alone and in presence of external RNA revealed some crucial aspects for the implementation of the strategy in vivo. The conjugation between the PNA and the peptide can affect the toxicity. In fact, in our particularly case the toxicity decreased four fold, which entails the need of the scale up the reaction. Besides the researcher also demonstrated that the RNA that triggers the reaction could influence on the biological activity of the peptide formed. Here, the project has tough us two important lessons: 1) it is difficult to use this method for the construction of positively charged peptides; 2) because of this drawback, dissociation of the product from the RNA template would be desirable. Considering these discoveries we seek for alternative means and since the use of intracellular mRNA XIAP as a template in vivo might entails difficulties impossible to overcome. As a proof of concept of this initial attempt, we decided to synthesize the accepting probe with a cell penetrating peptide (R8) and performed a catalytic template reaction with an extracellular RNA (milestone f). After the transfer reaction the full-length peptide-PNA conjugate would go inside the cells and trigger the death of the cancer cells. This stage was the most challenging. At the moment we are finishing touches to the project and at least two publications are expected in the near future.
Regarding the relevance of this project, there is no need to emphasise the importance of inventing new therapeutic strategies for cancer. It is clear that cancer is a modern day pandemic. In fact, nowadays cancer kills more people than tuberculosis, malaria and HIV put together. Moreover, diagnosis and treatment for most tumours are becoming sophisticated and very expensive. This increase in cost could have a dramatic impact in countries in which the health expenditure per capita is only a few dollars yearly; some health systems, even in the most affluent countries, have increasing difficulties in being able to provide the most expensive new drugs. The dramatic situation prevailing in the treatment of HIV-infected people, whereby most countries are unable to afford modern therapies, could soon also become a reality in oncology. Consequently improving, expanding and developing oncologic treatments will undoubtedly have profound socio-economic impact worldwide. In this context our project contributes by combining challenging and pioneering objectives from a basic point of view with important applicability possibilities. Relying on chemistry: synthesis and designed reactions, we approached objectives in the areas of chemical and cell biology that might lead to new advances in medicinal chemistry. Indeed, to our knowledge this is the first time that a cytotoxic drug is formed by this type of reactions in a gene-encoded approach, we demonstrated that the RNA-programmed chemistry works and even using prodrugs, which is also of great importance in terms of selectivity and side effects. Therefore, we are opening the possibility to reach the challenging goal of specific mRNA-directed release of bioactive species inside the cytoplasm in the near future. This implies new potential therapeutics that could be used in the war against cancer and also for other drug developments.