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Discovery and Characterization of Peptide Inhibitors for Calcium and Integrin Binding Protein 1
Ana C Puhl1, Jonathan W Bogart1, Victoria A Haberman1
1Center for Integrative Chemical Biology and Drug Discovery, Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
Abstract:
Calcium and integrin binding protein 1 (CIB1) is an EF-hand-containing, small intracellular protein that has recently been implicated in cancer cell survival and proliferation. In particular, CIB1 depletion significantly impairs tumor growth in triple-negative breast cancer (TNBC). Thus, CIB1 is a potentially attractive target for cancer chemotherapy that has yet to be validated by a chemical probe. To produce a probe molecule to the CIB1 helix 10 (H10) pocket and demonstrate that it is a viable target for molecular intervention, we employed random peptide phage display to screen and select CIB1-binding peptides. The top peptide sequence selected, UNC10245092, was produced synthetically, and binding to CIB1 was confirmed by isothermal titration calorimetry (ITC) and a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Both assays showed that the peptide bound to CIB1 with low nanomolar affinity. CIB1 was cocrystallized with UNC10245092, and the 2.1 Å resolution structure revealed that the peptide binds as an α-helix in the H10 pocket, displacing the CIB1 C-terminal H10 helix and causing conformational changes in H7 and H8. UNC10245092 was further derivatized with a C-terminal Tat-derived cell penetrating peptide (CPP) to demonstrate its effects on TNBC cells in culture, which are consistent with results of CIB1 depletion. These studies provide a first-in-class chemical tool for CIB1 inhibition in cell culture and validate the CIB1 H10 pocket for future probe and drug discovery efforts.
Insights
Researchers developed a novel peptide probe to target Calcium and Integrin Binding Protein 1 (CIB1) in triple-negative breast cancer (TNBC). This chemical tool validates CIB1 as a therapeutic target and aids future drug discovery for TNBC.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Calcium and Integrin Binding Protein 1 (CIB1) is an intracellular protein linked to cancer cell survival and proliferation.
- CIB1 depletion significantly hinders tumor growth, particularly in triple-negative breast cancer (TNBC).
- CIB1 represents a promising, yet unvalidated, target for cancer chemotherapy.
Purpose of the Study:
- To develop a chemical probe targeting the CIB1 helix 10 (H10) pocket.
- To validate CIB1 as a viable target for molecular intervention in cancer therapy.
- To create a first-in-class chemical tool for CIB1 inhibition.
Main Methods:
- Random peptide phage display was used to identify CIB1-binding peptides.
- Isothermal titration calorimetry (ITC) and time-resolved fluorescence resonance energy transfer (TR-FRET) assays confirmed binding affinity.
- X-ray crystallography elucidated the binding mode and conformational changes induced by the peptide.
Main Results:
- A high-affinity CIB1-binding peptide (UNC10245092) was identified and synthesized.
- The peptide binds to the CIB1 H10 pocket with low nanomolar affinity, acting as an alpha-helix.
- Structural analysis revealed displacement of the CIB1 C-terminal helix and conformational changes in other regions.
- A cell-penetrating peptide derivative showed effects on TNBC cells consistent with CIB1 depletion.
Conclusions:
- A novel chemical probe, UNC10245092, was developed for CIB1 inhibition in cell culture.
- The CIB1 H10 pocket is validated as a druggable target for future drug discovery efforts.
- This study provides a crucial tool for CIB1-targeted cancer therapy research, especially for TNBC.
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