Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis (ML-IAP)
Matthew C Franklin1, Saloumeh Kadkhodayan, Heidi Ackerly
1Department of Protein Engineering, Genentech, Inc., One DNA Way, South San Francisco, California 94080, USA.
Abstract:
Melanoma inhibitor of apoptosis (ML-IAP) is a potent anti-apoptotic protein that is upregulated in a number of melanoma cell lines but not expressed in most normal adult tissues. Overexpression of IAP proteins, such as ML-IAP or the ubiquitously expressed X-chromosome-linked IAP (XIAP), in human cancers has been shown to suppress apoptosis induced by a variety of stimuli. Peptides based on the processed N-terminus of Smac/DIABLO can negate the ability of overexpressed ML-IAP or XIAP to suppress drug-induced apoptosis. Such peptides have been demonstrated to bind to the single baculovirus IAP repeat (BIR) of ML-IAP and the third BIR of XIAP with similar high affinities (approximately 0.5 microM). Herein, we use phage-display of naïve peptide libraries and synthetic peptides to investigate the peptide-binding properties of ML-IAP-BIR and XIAP-BIR3. X-ray crystal structures of ML-IAP-BIR in complex with Smac- and phage-derived peptides, together with peptide structure-activity-relationship data, indicate that the peptides can be modified to provide increased binding affinity and selectivity for ML-IAP-BIR relative to XIAP-BIR3. For instance, substitution of Pro3' in the Smac-based peptide (AVPIAQKSE) with (2S,3S)-3-methylpyrrolidine-2-carboxylic acid [(3S)-methyl-proline] results in a peptide with 7-fold greater affinity for ML-IAP-BIR and about 100-fold specificity for ML-IAP-BIR relative to XIAP-BIR3.
Insights
Researchers developed modified peptides that selectively bind to Melanoma inhibitor of apoptosis (ML-IAP) proteins, offering a targeted approach to overcome apoptosis suppression in melanoma. This enhances potential cancer therapies by improving drug efficacy.
Area of Science:
- Oncology
- Molecular Biology
- Structural Biology
Background:
- Melanoma inhibitor of apoptosis (ML-IAP) is upregulated in melanoma, promoting cancer cell survival by inhibiting apoptosis.
- Inhibitor of Apoptosis Proteins (IAPs), including ML-IAP and XIAP, are frequently overexpressed in cancers, conferring resistance to apoptosis.
- Smac/DIABLO-derived peptides can antagonize IAPs, but optimizing their binding and selectivity is crucial for therapeutic development.
Purpose of the Study:
- To investigate the peptide-binding properties of ML-IAP baculovirus IAP repeat (BIR) and XIAP-BIR3 using phage display and synthetic peptides.
- To understand the structural basis of peptide-ML-IAP-BIR interactions through X-ray crystallography.
- To design modified peptides with enhanced binding affinity and selectivity for ML-IAP-BIR over XIAP-BIR3.
Main Methods:
- Utilized phage display of peptide libraries to identify potential ML-IAP binders.
- Synthesized peptides based on Smac/DIABLO and phage-derived sequences.
- Determined X-ray crystal structures of ML-IAP-BIR in complex with various peptides.
- Performed structure-activity relationship studies to guide peptide modifications.
Main Results:
- Identified peptides that bind to ML-IAP-BIR and XIAP-BIR3 with high affinity.
- Structural analysis revealed key interactions between peptides and the ML-IAP-BIR domain.
- Peptide modifications, such as incorporating (3S)-methyl-proline, significantly increased binding affinity (7-fold) and selectivity (100-fold) for ML-IAP-BIR compared to XIAP-BIR3.
Conclusions:
- Peptide modifications can precisely tune binding affinity and selectivity for ML-IAP-BIR.
- Developed peptides demonstrate potential as targeted therapeutics to overcome ML-IAP-mediated apoptosis resistance in melanoma.
- The findings provide a foundation for designing novel anti-cancer agents targeting IAP proteins.
