Periodic Reporting for period 1 - DPP9-TACDrug (DPP9 degradation-induced pyroptosis for treatment of acute myeloid leukemia)
Reporting period: 2023-04-01 to 2025-03-31
Acute myeloid leukemia (AML) is an aggressive malignancy of the blood and bone marrow, characterized by the rapid proliferation of abnormal myeloid cells. It is the most common type of acute leukemia in adults and remains challenging to treat due to its high relapse rate and resistance to conventional therapies. Despite advances in chemotherapy, targeted therapies, and hematopoietic stem cell transplantation, long-term remission remains elusive for many patients. Therefore, identifying novel therapeutic targets and innovative treatment strategies is crucial for improving clinical outcomes in AML.
Dipeptidyl-peptidase 9 (DPP9) is a proline-selective serine protease belonging to the peptidase S9 family. While initially recognized for its role in protein turnover and immune regulation, recent studies have highlighted its significance in AML. Notably, the inhibition of DPP9 has been shown to induce inflammatory cell death (pyroptosis) selectively in myeloid leukemia cells, positioning it as an attractive target for therapeutic intervention. Mechanistically, DPP9 regulates the activation of NLRP1, a key inflammasome sensor involved in pyroptosis. Inhibition of DPP9 disrupts its interaction with NLRP1, leading to inflammasome activation, caspase-1 activation, and subsequent cell death via pyroptosis. This targeted mechanism offers a promising approach for eradicating AML cells while sparing healthy tissues. However, to date, only small-molecule inhibitors of DPP9 have been reported, and their effects on the DPP9-NLRP1 interaction remain limited, showing only mild destabilization of the protein-protein interaction (PPI).
Targeted Protein Degradation (TPD) is an emerging and highly promising therapeutic modality in drug discovery, offering an alternative to traditional small-molecule inhibition. The first and most well-established TPD approach is PROTAC (PROteolysis Targeting Chimera) technology, which exploits the ubiquitin-proteasome system (UPS) to selectively degrade proteins of interest (POI).
Unlike conventional small-molecule inhibitors that require sustained occupancy to exert their effects, PROTACs function catalytically by inducing molecular proximity between the POI (in this case, DPP9) and an E3 ubiquitin ligase. This leads to the ubiquitination and subsequent proteasomal degradation of DPP9, effectively eliminating its cellular function. This catalytic mode of action allows PROTACs to achieve prolonged pharmacodynamic effects with lower dosages, reducing the likelihood of off-target toxicity and drug resistance.
Beyond PROTACs, other TPD strategies, such as AUTACs (Autophagy-Targeting Chimeras), offer additional avenues for targeted protein clearance through the autophagy-lysosome pathway. These approaches provide alternative mechanisms for degrading disease-associated proteins, broadening the potential for TPD-based therapies in AML.
The primary goal of this project was to design, synthesize, and validate small-molecule degraders targeting DPP9 for AML treatment. Successful implementation of this approach could revolutionize AML therapy by providing a highly selective, durable, and well-tolerated treatment option. By harnessing the power of targeted protein degradation, this strategy holds the potential to improve patient outcomes, reduce relapse rates, and pave the way for next-generation cancer therapeutics.
Dipeptidyl-peptidase 9 (DPP9) is a proline-selective serine protease belonging to the peptidase S9 family. While initially recognized for its role in protein turnover and immune regulation, recent studies have highlighted its significance in AML. Notably, the inhibition of DPP9 has been shown to induce inflammatory cell death (pyroptosis) selectively in myeloid leukemia cells, positioning it as an attractive target for therapeutic intervention. Mechanistically, DPP9 regulates the activation of NLRP1, a key inflammasome sensor involved in pyroptosis. Inhibition of DPP9 disrupts its interaction with NLRP1, leading to inflammasome activation, caspase-1 activation, and subsequent cell death via pyroptosis. This targeted mechanism offers a promising approach for eradicating AML cells while sparing healthy tissues. However, to date, only small-molecule inhibitors of DPP9 have been reported, and their effects on the DPP9-NLRP1 interaction remain limited, showing only mild destabilization of the protein-protein interaction (PPI).
Targeted Protein Degradation (TPD) is an emerging and highly promising therapeutic modality in drug discovery, offering an alternative to traditional small-molecule inhibition. The first and most well-established TPD approach is PROTAC (PROteolysis Targeting Chimera) technology, which exploits the ubiquitin-proteasome system (UPS) to selectively degrade proteins of interest (POI).
Unlike conventional small-molecule inhibitors that require sustained occupancy to exert their effects, PROTACs function catalytically by inducing molecular proximity between the POI (in this case, DPP9) and an E3 ubiquitin ligase. This leads to the ubiquitination and subsequent proteasomal degradation of DPP9, effectively eliminating its cellular function. This catalytic mode of action allows PROTACs to achieve prolonged pharmacodynamic effects with lower dosages, reducing the likelihood of off-target toxicity and drug resistance.
Beyond PROTACs, other TPD strategies, such as AUTACs (Autophagy-Targeting Chimeras), offer additional avenues for targeted protein clearance through the autophagy-lysosome pathway. These approaches provide alternative mechanisms for degrading disease-associated proteins, broadening the potential for TPD-based therapies in AML.
The primary goal of this project was to design, synthesize, and validate small-molecule degraders targeting DPP9 for AML treatment. Successful implementation of this approach could revolutionize AML therapy by providing a highly selective, durable, and well-tolerated treatment option. By harnessing the power of targeted protein degradation, this strategy holds the potential to improve patient outcomes, reduce relapse rates, and pave the way for next-generation cancer therapeutics.
During the fellowship, several activities were carried out to ensure the successful achievement of the proposal’s main objectives.
1. Organic Synthesis of PROTACs, AUTACs, and CLIPTACs
The initial phase focused on the design and synthesis of various Targeted Protein Degradation (TPD) modalities, including PROTACs, AUTACs, and CLIPTACs. While CLIPTACs were not part of the original plan, they were incorporated to address the permeability challenges encountered with PROTACs.
The rational design of these molecules was based on an in-house developed DPP9 inhibitor, with systematic variations in linker lengths and proteasome/autophagy-targeting motifs. In total, 15 PROTACs, 10 AUTACs, and 4 CLIPTACs were successfully synthesized and fully characterized.
2. Evaluation of DPP9 Clearance by PROTACs, AUTACs, and CLIPTACs
Following synthesis, the next phase focused on assessing the ability of the newly developed compounds to degrade DPP9. A series of biological assays were conducted to evaluate their efficacy:
- Inhibitory Potency and Selectivity: Compounds were tested against DPP9 and compared with related peptidases (DPP8, DPP4, DPP2, FAP, and PREP) to determine their selectivity.
- Cellular Permeability: An in-house assay was employed to predict the permeability of the synthesized molecules.
- DPP9 Clearance Potency: The efficiency of PROTACs, AUTACs, and CLIPTACs in degrading DPP9 was assessed in human cell lines.
IC₅₀ values for all synthesized compounds indicated high inhibitory potency against DPP9, with activity in the low nanomolar range. As anticipated, both PROTACs and AUTACs exhibited low to moderate cellular permeability due to their high molecular weight. PROTACs showed no significant DPP9 degradation, while AUTACs did not induce measurable DPP9 degradation at the tested conditions. CLIPTACs demonstrated a superior DPP9 degradation profile compared to PROTACs, making them a promising alternative.
3. Comparison of Pyroptosis Signatures: PROTACs, CLIPTACs, and DPP9 Inhibitors
To assess the pyroptotic potential of the synthesized compounds, their effects were compared with conventional DPP9 small-molecule inhibitors. Lactate dehydrogenase (LDH) release assays were performed in cell lines as an indicator of pyroptosis induction. AUTACs were excluded from this phase due to their lack of DPP9 degradation in earlier studies.
PROTACs exhibited only a modest effect on LDH release, suggesting a weaker pyroptosis signature compared to conventional DPP9 inhibitors. DPP9 small-molecule inhibitors induced a stronger pyroptotic response, reinforcing their superior ability to trigger inflammatory cell death. The evaluation of CLIPTACs is still ongoing.
1. Organic Synthesis of PROTACs, AUTACs, and CLIPTACs
The initial phase focused on the design and synthesis of various Targeted Protein Degradation (TPD) modalities, including PROTACs, AUTACs, and CLIPTACs. While CLIPTACs were not part of the original plan, they were incorporated to address the permeability challenges encountered with PROTACs.
The rational design of these molecules was based on an in-house developed DPP9 inhibitor, with systematic variations in linker lengths and proteasome/autophagy-targeting motifs. In total, 15 PROTACs, 10 AUTACs, and 4 CLIPTACs were successfully synthesized and fully characterized.
2. Evaluation of DPP9 Clearance by PROTACs, AUTACs, and CLIPTACs
Following synthesis, the next phase focused on assessing the ability of the newly developed compounds to degrade DPP9. A series of biological assays were conducted to evaluate their efficacy:
- Inhibitory Potency and Selectivity: Compounds were tested against DPP9 and compared with related peptidases (DPP8, DPP4, DPP2, FAP, and PREP) to determine their selectivity.
- Cellular Permeability: An in-house assay was employed to predict the permeability of the synthesized molecules.
- DPP9 Clearance Potency: The efficiency of PROTACs, AUTACs, and CLIPTACs in degrading DPP9 was assessed in human cell lines.
IC₅₀ values for all synthesized compounds indicated high inhibitory potency against DPP9, with activity in the low nanomolar range. As anticipated, both PROTACs and AUTACs exhibited low to moderate cellular permeability due to their high molecular weight. PROTACs showed no significant DPP9 degradation, while AUTACs did not induce measurable DPP9 degradation at the tested conditions. CLIPTACs demonstrated a superior DPP9 degradation profile compared to PROTACs, making them a promising alternative.
3. Comparison of Pyroptosis Signatures: PROTACs, CLIPTACs, and DPP9 Inhibitors
To assess the pyroptotic potential of the synthesized compounds, their effects were compared with conventional DPP9 small-molecule inhibitors. Lactate dehydrogenase (LDH) release assays were performed in cell lines as an indicator of pyroptosis induction. AUTACs were excluded from this phase due to their lack of DPP9 degradation in earlier studies.
PROTACs exhibited only a modest effect on LDH release, suggesting a weaker pyroptosis signature compared to conventional DPP9 inhibitors. DPP9 small-molecule inhibitors induced a stronger pyroptotic response, reinforcing their superior ability to trigger inflammatory cell death. The evaluation of CLIPTACs is still ongoing.
1 - PROTACs, AUTACs, and CLIPTACs represent the first targeted protein degradation (TPD) modalities developed for DPP9. This marks a significant advancement in the field of TPD, opening new therapeutic opportunities for challenging cancers such as AML.
2 - The synthesis of these bifunctional molecules required optimization to achieve the proposed structures. The newly developed synthetic strategy can be readily applied to other bifunctional molecules, expanding its utility beyond this study.
3 - To evaluate the ability of these molecules to induce DPP9 degradation, key assays were optimized, including an extensive selection of antibodies for precise DPP9 detection. This methodological refinement provides valuable insights for researchers studying DPP9 biology.
4 - The strong DPP9-binding affinity observed for these molecules confirms the validity of the rational design approach. This information is particularly relevant for researchers aiming to develop additional DPP9-targeting chemical tools.
5- Different TPD strategies were assessed for their potential to induce DPP9 degradation and pyroptosis in human cells. CLIPTACs have so far shown the most promising degradation results, with additional studies underway to evaluate their pyroptosis-inducing potential.
Overall, by leveraging cutting-edge approaches such as AUTACs, PROTACs, and CLIPTACs, this research provides novel insights into selective protein degradation strategies, paving the way for next-generation chemical tools and DPP9-targeted therapeutics.
2 - The synthesis of these bifunctional molecules required optimization to achieve the proposed structures. The newly developed synthetic strategy can be readily applied to other bifunctional molecules, expanding its utility beyond this study.
3 - To evaluate the ability of these molecules to induce DPP9 degradation, key assays were optimized, including an extensive selection of antibodies for precise DPP9 detection. This methodological refinement provides valuable insights for researchers studying DPP9 biology.
4 - The strong DPP9-binding affinity observed for these molecules confirms the validity of the rational design approach. This information is particularly relevant for researchers aiming to develop additional DPP9-targeting chemical tools.
5- Different TPD strategies were assessed for their potential to induce DPP9 degradation and pyroptosis in human cells. CLIPTACs have so far shown the most promising degradation results, with additional studies underway to evaluate their pyroptosis-inducing potential.
Overall, by leveraging cutting-edge approaches such as AUTACs, PROTACs, and CLIPTACs, this research provides novel insights into selective protein degradation strategies, paving the way for next-generation chemical tools and DPP9-targeted therapeutics.