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Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues
Published on: July 14, 2015
Optimization of the PROTAC linker region of the proteasome substrate receptor hRpn13 rationalized by structural
Xiuxiu Lu1, Venkata R Sabbasani2, Bakar Hassan1
1Protein Processing Section, Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.
Abstract:
Proteasome substrate receptor hRpn13 is a promising target for cancer therapy. hRpn13 proteolysis-targeting chimera (PROTACs) induce apoptosis by targeting the hRpn13 proteolytic product hRpn13Pru, which contains an intact ubiquitin- and proteasome-binding Pru domain. We generated a PROTAC series based on hRpn13Pru-targeting XL5 by varying the linker that connects it to a warhead against the VHL-based ubiquitin E3 ligase machinery. Among eight tested derivatives, XL5-VHL-7 with a -(CH2)5- alkyl linker promoted hRpn13Pru degradation and induced cellular apoptosis with 2-fold improved potency compared to the original PROTAC. By using this PROTAC series with slight chemical modifications in the linker region, we were able to evaluate the efficacy of structural modeling with molecular dynamics for refining PROTACs. Overall, we found that the experimental data correlated with efficacy predictions based on molecular dynamics and structural modeling. Moreover, we could observe hRpn13:PROTAC:VHL complexes by 2D NMR that support the structural modeling and stronger affinity of XL5-VHL-7 compared to the original hRpn13 PROTAC. Our NMR data further indicate that hRpn13 Pru affinity for XL5-VHL-7 is higher within the VHL complex present than with XL5-VHL-7 alone. Altogether, we develop an hRpn13 PROTAC with 2-fold increased potency by optimizing the linker and demonstrate the current benefit and limitations for including modeling with molecular dynamics to aid PROTAC optimization.
Insights
Researchers developed a more potent proteolysis-targeting chimera (PROTAC) by optimizing the linker, leading to a 2-fold increase in efficacy for targeting hRpn13 in cancer therapy. This advancement aids in cancer treatment strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapeutics
Background:
- The proteasome substrate receptor hRpn13 is a validated target for cancer therapy.
- hRpn13 proteolysis-targeting chimeras (PROTACs) induce apoptosis by targeting the hRpn13 proteolytic product (hRpn13Pru).
Purpose of the Study:
- To optimize hRpn13-targeting PROTACs by modifying the linker region.
- To evaluate the utility of structural modeling and molecular dynamics in refining PROTAC design.
Main Methods:
- Synthesis of a PROTAC series based on hRpn13Pru-targeting XL5 with varied linkers.
- Assessment of PROTAC-induced hRpn13Pru degradation and cellular apoptosis.
- Application of molecular dynamics and structural modeling for efficacy prediction.
- 2D Nuclear Magnetic Resonance (NMR) spectroscopy to characterize PROTAC:protein complexes.
Main Results:
- XL5-VHL-7, featuring a -(CH2)5- alkyl linker, demonstrated a 2-fold improvement in potency for hRpn13Pru degradation and apoptosis induction.
- Experimental data correlated well with predictions from molecular dynamics and structural modeling.
- NMR confirmed the formation of hRpn13:PROTAC:VHL complexes, supporting structural models and indicating higher affinity for XL5-VHL-7.
Conclusions:
- Optimizing the linker in hRpn13 PROTACs significantly enhances potency and therapeutic efficacy.
- Structural modeling and molecular dynamics are valuable tools for guiding PROTAC optimization, despite current limitations.
- The developed PROTAC, XL5-VHL-7, represents a promising advancement in targeting hRpn13 for cancer therapy.
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