Effect of Stapling on the Thermodynamics of Protein-Peptide Binding

Protein-protein interaction (PPI) is one of the key regulatory features to drive biomolecular processes and hence is targeted for designing therapeutics against diseases. Small peptides are a new and emerging class of therapeutics owing to their high specificity and low toxicity. For achieving efficient targeting of the PPI, amino acid side chains are often stapled together resulting in the rigidification of these peptides. Exploring the scope of these peptides demands a comprehensive understanding of their working principle. In this work, two stapled p53 peptides have been considered to delineate their binding mechanism with mdm2 using computational approaches. Addition of stapling protects the secondary structure of the peptides even in the case of thermal and chemical denaturation. Although the introduction of a stapling agent increases the hydrophobicity of the peptide surprisingly the enthalpic stabilization decreases. This is overcome by the lowering of the entropic penalty and the overall binding affinity improves. The mechanistic insights into the benefit of peptide stapling can be adopted for further improvement of peptide therapeutics.

Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation

Background:Proteogenomics is an emerging field at the intersection of genomics and proteomics. Many variant peptides corresponding to single nucleotide variations (SNVs) are associated with specific diseases. The aim of this study was to demonstrate the feasibility of proteogenomic-based variant peptide detection in disease models and clinical specimens.Methods:We sought to detect p53 single amino acid variant (SAAV) peptides in breast cancer tumor samples that have been previously subjected to sequencing analysis. Initially, two cancer cell lines having a cellular tumor antigen p53 (TP53) mutation and one wild type for TP53 were analyzed by selected reaction monitoring (SRM) assays as controls. One pool of wild type and one pool of mutated for TP53 cytosolic extracts were assayed with a shotgun proteogenomic workflow. Furthermore, 18 individual samples having a mutation in TP53 were assayed by SRM.Results:Two mutant p53 peptides were successfully detected in two cancer cell lines as expected from their DNA sequence. Wild type p53 peptides were detected in both cytosolic pools, however, none of the mutant p53 peptides were identified. Mutations at the protein level were detected in two cytosolic extracts and whole tumor lysates from the same patients by SRM analysis. Six thousand and six hundred and twenty eight non-redundant proteins were identified in the two cytosolic pools, thus greatly improving a previously reported cytosolic proteome.Conclusions:In the current study we show the great potential of using proteogenomics for the direct identification of cancer-associated mutations in clinical samples and we discuss current limitations and future perspectives.

Investigating peptide sequence variations for ‘double-click’ stapled p53 peptides

Evaluating the influence of staple position, azido amino acid side-chain length and point mutation on the activity of ‘double-click’ stapled p53 peptides.

Functionalised staple linkages for modulating the cellular activity of stapled peptides

A divergent synthetic strategy for generating helical p53 peptides bearing functionalised staple linkages, allowing for efficient optimisation of cellular activity.

Phase I adjuvant trial of multi-epitope p53 vaccine for patients with squamous cell carcinoma of the head and neck (SCCHN): A preliminary report

3012 Background: Alteration in the tumor suppressor protein, p53, is one of the most common events in human cancers. Since most mutations of p53 are associated with accumulation of p53, the nonmutated portions of protein are readily accessible to degradation into wild type (wt) sequence peptides for immune recognition by T lymphocytes. Methods: A phase I trial was conducted in stage I-IVa patients (pts) with SCCHN with no active disease using autologous dendritic cells (DC) loaded with two HLA-A*0201-restricted T cell-defined p53 peptides alone (Arm 1, 5 pts), plus either a wt p53 helper peptide (Arm 2, 5 pts) or nonspecific helper peptide (dervied from tetanus toxoid, Arm 3, 6 pts). The accrual goal is 24 patients, divided into the 3 arms. Each pt received 3 intranodal vaccinations at 2-week intervals. The primary endpoint was immunological response using ELISPOT, tetramer or delayed type hypersensitivity (DTH)reactions to p53 peptides. Results: 16 pts have been vaccinated so far. Erythema or hematoma were observed at vaccine injection sites in two pts. At 15-mo median follow-up, 11/16 pts are alive without evidence of disease, 2 developed disease recurrence, and 1 pt developed a second primary lung cancer. Two have died of unrelated causes. Immunological analyses of the SCCHN patients’ responses to immunization indicate responses to both HLA-A2 binding peptides. Tetramer frequencies for p53-specific CTL or IFN-gamma ELISPOT assays indicated a 5/16 responders. Ongoing correlative analyses include characterization of human papillomavirus (HPV) infection, HLA, antigen processing machinery, and staining with soluble T cell receptor for HLA-A2-p53 peptide complexes on tumor cells. Conclusions: Adjuvant p53 peptide-loaded DC-based vaccination is feasible and safe in pts with SCCHN. A phase II wt p53-based vaccine trial will be proposed based on the most efficacious regimen used in this trial. No significant financial relationships to disclose.