Periodic Reporting for period 1 - ABPP-PROTDRUG (An Activity-based Protein Profiling-supported Protein Therapeutic Drug discovery platform for the development of innovative anti-cancer therapies based on Lin28 inhibition)
Berichtszeitraum: 2021-09-01 bis 2023-08-31
Cancer remains one of the most prevalent diseases worldwide and new therapeutic targets are highly sought. Many proteins or protein-associated molecular pathways have relevant roles in many cancers, making research in cancer-associated protein-based therapies a popular area. In this project, we aim to take advantage of cancer-associated protein-protein interactions and develop innovative therapeutics for cancer treatment.
Certain proteins in our cells interact amongst each other, often only temporarily, and this interactions can result in both anti-cancer effects or cancer potentiating effects. We aimed to perform small chemical modifications in proteins that will allow them to form irreversible chemical connections with their targets, stabilizing their interaction and potentiating these effects. This process will translate into more potent anti-cancer effects due the irreversible nature of the new bond. The successful development of these agents will effectively contribute to the field of protein therapeutics, or protein drugs, a novel class of therapeutic strategy.
Our approach focused on the PD-1/PD-L1 interaction. PD-L1 is frequently overexpressed in tumors and PD-1 is a protein that interacts with it, with the interaction between both proteins leading to inhibition of immune system cells, release of cytokines and general cellular toxicity, leading to increased cell death of important immune cells. Interrupting this interaction via the use of protein drugs has been shown to lead to reduction in tumor growth in mice. We focused our project in creating protein drugs that interact with PD-L1 in an irreversible manner, keeping PD-1 and PD-L1 from interacting and overall leading to an anti-cancer effect.
The success of our project will translate into better therapeutics for cancer patients and better quality of life for people suffering from cancer. The targeting of unusual proteins with innovative tools might be advantageous for cancers where conventional therapies fail. Innovative cancer therapies will also alleviate the economic burden of cancer, with the potential of improving patient care by allowing better allocating of medical resources. Despite focusing solely on cancer in this project, the concept of interfering with protein-protein interactions is present virtually in all cell processes both in heathy states and diseased states. We believe the popularization of these techniques will allow its application in other diseases, creating further impact at a society level and generating numerous other projects both in the host lab and in other labs.
Our main project objectives were:
(i) Create a library of protein drugs targeting PD-L1 using a variety of PD-L1 interacting proteins and chemistries developed in the host lab;
(ii) Evaluate the binding of these proteins to PD-L1 and the stability of their interaction;
(iii) Study the crosslinking between the protein drugs and PD-L1 using proteomics;
(iv) Evaluate the biological effects of the protein drugs in cancer cells.
In addition to these objectives we also developed other anti-cancer therapeutics like PROTACs, molecular condensate modulators and proteomics platforms to study anti-cancer agents.
Certain proteins in our cells interact amongst each other, often only temporarily, and this interactions can result in both anti-cancer effects or cancer potentiating effects. We aimed to perform small chemical modifications in proteins that will allow them to form irreversible chemical connections with their targets, stabilizing their interaction and potentiating these effects. This process will translate into more potent anti-cancer effects due the irreversible nature of the new bond. The successful development of these agents will effectively contribute to the field of protein therapeutics, or protein drugs, a novel class of therapeutic strategy.
Our approach focused on the PD-1/PD-L1 interaction. PD-L1 is frequently overexpressed in tumors and PD-1 is a protein that interacts with it, with the interaction between both proteins leading to inhibition of immune system cells, release of cytokines and general cellular toxicity, leading to increased cell death of important immune cells. Interrupting this interaction via the use of protein drugs has been shown to lead to reduction in tumor growth in mice. We focused our project in creating protein drugs that interact with PD-L1 in an irreversible manner, keeping PD-1 and PD-L1 from interacting and overall leading to an anti-cancer effect.
The success of our project will translate into better therapeutics for cancer patients and better quality of life for people suffering from cancer. The targeting of unusual proteins with innovative tools might be advantageous for cancers where conventional therapies fail. Innovative cancer therapies will also alleviate the economic burden of cancer, with the potential of improving patient care by allowing better allocating of medical resources. Despite focusing solely on cancer in this project, the concept of interfering with protein-protein interactions is present virtually in all cell processes both in heathy states and diseased states. We believe the popularization of these techniques will allow its application in other diseases, creating further impact at a society level and generating numerous other projects both in the host lab and in other labs.
Our main project objectives were:
(i) Create a library of protein drugs targeting PD-L1 using a variety of PD-L1 interacting proteins and chemistries developed in the host lab;
(ii) Evaluate the binding of these proteins to PD-L1 and the stability of their interaction;
(iii) Study the crosslinking between the protein drugs and PD-L1 using proteomics;
(iv) Evaluate the biological effects of the protein drugs in cancer cells.
In addition to these objectives we also developed other anti-cancer therapeutics like PROTACs, molecular condensate modulators and proteomics platforms to study anti-cancer agents.
We used synthetic miniproteins with very high affinity for PDL-1 as a starting point. A library of miniproteins and a library of small molecule probes with dual reactivity were combined to produce a library of protein drugs targeting PDL-1. These protein drugs were characterized by a combination of LC-MS, BLI, gel-based approaches and proteomics to fully characterize the protein drugs and their interaction with PDL-1.
The combination of these techniques allowed us to evaluate how efficiently the protein drugs bound to PD-L1, how stable this reaction was, if the irreversible covalent bond had formed after proximity between both proteins, and if the reaction was somehow reversible. Notably, crosslinking proteomics allowed us to observe the exact aminoacid residues participating in the covalent crosslink formed between both proteins.
Excitingly, this strategy can be easily adapted to other protein-protein interactions, making it a project with high potential for a strong scientific and societal impact. Variations in the probes used will allow the established methodology to be adapted to other entities like serine hydrolases, well known players in cancer and metastasis, or sulfenic acids, oxidized cysteines known to take part in redox control of the cellular environment.
Briefly, regarding the additional projects developed in the same period we achieved:
(i) A library of serine hydrolase-targeting PROTACs was developed with the help of the computational tool PRosettaC. The compounds were shown to efficiently degrade their model target APT1 with excellent selectivity. Targeting APT1 is a potential anti-cancer strategy.
(ii) Small cysteine reactive molecules were used to modify G3BP1, a protein known to be a part of molecular condensates. Modulation of molecular condensates is a recent approach that is believed to have potential as an anti-cancer strategy.
(iii) Novel proteomics platforms for evaluation of small cysteine-reactive molecules, specifically carbonylacrylic compounds, uncovered the targeting of NPM1 which could potentially explain their anti-leukemia effects.
(iv) Innovative dual-reactivity probes were developed that recognize specific aminoacid signatures on the surface of proteins.
The combination of these techniques allowed us to evaluate how efficiently the protein drugs bound to PD-L1, how stable this reaction was, if the irreversible covalent bond had formed after proximity between both proteins, and if the reaction was somehow reversible. Notably, crosslinking proteomics allowed us to observe the exact aminoacid residues participating in the covalent crosslink formed between both proteins.
Excitingly, this strategy can be easily adapted to other protein-protein interactions, making it a project with high potential for a strong scientific and societal impact. Variations in the probes used will allow the established methodology to be adapted to other entities like serine hydrolases, well known players in cancer and metastasis, or sulfenic acids, oxidized cysteines known to take part in redox control of the cellular environment.
Briefly, regarding the additional projects developed in the same period we achieved:
(i) A library of serine hydrolase-targeting PROTACs was developed with the help of the computational tool PRosettaC. The compounds were shown to efficiently degrade their model target APT1 with excellent selectivity. Targeting APT1 is a potential anti-cancer strategy.
(ii) Small cysteine reactive molecules were used to modify G3BP1, a protein known to be a part of molecular condensates. Modulation of molecular condensates is a recent approach that is believed to have potential as an anti-cancer strategy.
(iii) Novel proteomics platforms for evaluation of small cysteine-reactive molecules, specifically carbonylacrylic compounds, uncovered the targeting of NPM1 which could potentially explain their anti-leukemia effects.
(iv) Innovative dual-reactivity probes were developed that recognize specific aminoacid signatures on the surface of proteins.
The global outcome of this project will establish powerful platforms to weaponize transient non-covalent interactions as innovative therapeutic modulators, as well as additional innovative therapies targeting cancer.
At the moment we have in our hands a considerable library of protein drugs, made from different PD-L1 targeting proteins and different chemistries. These have been shown to perform well in terms of binding to PD-L1 and mechanistically, with demonstrated stable crosslinks forming between both proteins. We have evaluated these constructs extensively and are in the process of applying these protein drugs in biological settings, with good preliminary results.
At the same time, we’ve delivered a large library of PROTAC compounds targeting APT1 with potential applications in cancer. This work is currently submitted for publishing.
A new strategy for modulation of molecular condensates via small reactive molecules was demonstrated. It’s direct effect in biological systems remains to be proven.
Carbonylacrylic compounds, which had previously been impossible to study by proteomics, were able to be studied using modified innovative proteomics platforms developed in this study. Targets of these compounds were found, including NPM1, which was shown to be directly modified by them and could explain most of the anticancer effects observed upon treatment with these compounds.
New probes that recognize specific protein surface signatures were developed. These can be finetuned to recognize different signatures and to tether different families of proteins.
Overall, the current results are very promising, despite not having immediate measurable societal implications at the moment. We believe that further development of protein drugs using small molecule probes and the proper establishment of the field could have a tremendous impact in the field of cancer therapies and also therapies for other diseases, which in turn could evolve into extraordinary societal value.
At the moment we have in our hands a considerable library of protein drugs, made from different PD-L1 targeting proteins and different chemistries. These have been shown to perform well in terms of binding to PD-L1 and mechanistically, with demonstrated stable crosslinks forming between both proteins. We have evaluated these constructs extensively and are in the process of applying these protein drugs in biological settings, with good preliminary results.
At the same time, we’ve delivered a large library of PROTAC compounds targeting APT1 with potential applications in cancer. This work is currently submitted for publishing.
A new strategy for modulation of molecular condensates via small reactive molecules was demonstrated. It’s direct effect in biological systems remains to be proven.
Carbonylacrylic compounds, which had previously been impossible to study by proteomics, were able to be studied using modified innovative proteomics platforms developed in this study. Targets of these compounds were found, including NPM1, which was shown to be directly modified by them and could explain most of the anticancer effects observed upon treatment with these compounds.
New probes that recognize specific protein surface signatures were developed. These can be finetuned to recognize different signatures and to tether different families of proteins.
Overall, the current results are very promising, despite not having immediate measurable societal implications at the moment. We believe that further development of protein drugs using small molecule probes and the proper establishment of the field could have a tremendous impact in the field of cancer therapies and also therapies for other diseases, which in turn could evolve into extraordinary societal value.