Targeted disruption of key disease-linked protein-protein interactions with highly selective cell-penetrating peptide disruptors

Cell-penetrating peptide disruptors represent an exciting area of drug discovery, allowing for the development of novel therapeutics capable of targeting the inhibition of intracellular protein-protein interactions (PPIs). Key advantages of peptides as disruptors of PPIs, compared with small molecules and large biologics, is their unique ability to cross the cell membrane and target the large, flat, and featureless surface of intracellular proteins. Peptide disruptors are typically derived from a native protein sequence, binding their target with high selectivity due to comprising of multiple key binding residues. Such peptide disruptors act as dominant negative decoys (lacking functionality) that directly compete with the endogenous protein for its respective partner. Selective binding of a peptide disruptor to a target protein thus inhibits the formation of the endogenous PPI, attenuating related downstream signalling/protein function. As there are >500,000 unique PPIs estimated to exist within the human interactome, many of which drive aberrant signalling and disease pathogenesis, peptide disruptors represent an emerging approach to the development of novel and effective therapeutics, with many showing promise in a clinical setting. As such, my thesis focused on (1) the development of novel cell-penetrating peptide disruptors of the pro-senescence FOXO4 - p53 PPI, and (2) investigated the therapeutic efficacy of PPL-008 (an already existing cell-penetrating peptide disruptor of the PDE8A - c-Raf protein complex) in the context of BRAF inhibitor resistant melanoma and KRAS-induced pancreatic ductal adenocarcinoma (PDAC). The formation of the FOXO4 - p53 protein complex has been highlighted as a key driver of cellular senescence, with selective inhibition resulting in targeted apoptosis of senescent cells and subsequently demonstrating clear therapeutic efficacy in the context of ageing. As such, the initial section of my thesis focused on the development of cell-penetrating peptide disruptors of the FOXO4 - p53 PPI; aimed at establishing novel starting points for the design of senolytic compounds. Utilising the well-established high-throughput peptide array screening platform, I initially mapped the interaction between full-length human FOXO4 and p53 proteins. Fine mapping indicated that FOXO4 bound p53 at multiple regions within the DNA binding domain, oligomerisation domain and C-terminal negative regulatory domain. In-depth analysis of these binding regions led to the rapid generation of a novel short sequence p53-based peptide library. Through synthesising a C-terminal stearic acid, said p53 peptides were made cell-permeable, a step found to facilitate intracellular delivery and improve peptide stability. Although robust analysis of p53 peptide stability and biophysical binding kinetics remains to be carried out, all p53 peptides demonstrated a clear ability to inhibit complex formation between p53 and FOXO4 in an overexpressed human cell line. Resultingly, my findings indicate the successful development of novel short sequence cell-penetrating peptide disruptors of the pro-senescence FOXO4 - p53 PPI and represent promising start points for the development of associated senolytic compounds. Future investigations will focus on evaluating these peptide disruptors in the relevant models of cellular senescence, aimed at the eventual development of pre-clinical LEAD senolytic compounds for the treatment of senescence-associated disease and ageing. The second section of my thesis focused on the development and therapeutic evaluation of PPL-008 - Cell Porter®, a PDE8A-based cell-penetrating peptide disruptor of the PDE8A - c-Raf protein complex. First generation stearylated PPL-008 was previously shown to modulate crosstalk between the cAMP and MAPK signalling systems, attenuating MAPK signalling through c-Raf kinase inhibition. As such, the therapeutic efficacy of second-generation Cell Porter® conjugated PPL-008 was assessed in the context of BRAF inhibitor resistant malignant melanoma (both mutant NRASQ61L and BRAFV600E), where-by c-Raf kinase is known to drive aberrant paradoxical MAPK signalling. Unlike original stearylated PPL-008, all PPL-008 - Cell Porter® analogues attenuated ERK activity, suppressed BRAF inhibitor (Vemurafenib) induced paradoxical ERK activation and inhibited cell growth in an NRASQ61L MM415 human malignant melanoma cell line. PPL-008C (Cell Porter® conjugated to the C-terminus via a thioester linker) was considered the in vitro LEAD due to its robust consistency as a mono-agent and in combination with Vemurafenib. Anti-proliferative actions of PPL-008C were recapitulated in a Vemurafenib-resistant 3D spheroid model of malignant melanoma (BRAFV600E), halting spheroid growth in combination with Vemurafenib. Furthermore, PPL-008C demonstrated early pre-clinical translation in MM415 (NRASQ61L) xenograft mice, significantly inhibiting ERK activation as a monotherapy. ERK inhibition was not attributed to increased PKA-mediated phosphorylation of c-RafS259, previously hypothesised to be associated with PPL-008's mechanism of action. My findings demonstrate for the first time a therapeutic role for targeting the disruption of PDE8A - c-Raf protein complex in clinically relevant models of malignant melanoma. Future investigations will assess combining BRAF inhibition and PPL-008C as a novel synergistic therapeutic approach to overcoming BRAF inhibitor resistant melanoma. Novel inhibitors of c-Raf activity are of emerging interest in the hunt for effective therapies aimed at treating KRAS/c-Raf addicted cancers, including PDAC. As PPL-008C is believed to allosterically inhibit c-Raf activity through targeted disruption of the PDE8A - c-Raf PPI, the third section of my thesis investigated the therapeutic efficacy of PPL-008C in the context of KRAS-induced PDAC. Promisingly, PPL-008C robustly attenuated cell survival in a small, but heterogenous panel of patient-derived PDAC cell lines. Cell lines harbouring multiple KRAS/TP53 mutations appeared less sensitive to PPL-008C therapy than single KRAS or TP53 mutants. Protein expression levels of ERK (T202/Y204) or c-Raf (S338) kinase activity did not directly correlate with PPL-008C sensitivity, suggesting PPL-008C may induce anti-proliferative activity through c-Raf kinase independent mechanisms. Moreover, PPL-008C appeared to modestly reduce protein markers of AKT activity (phosphorylated T308/S473), suggesting PPL-008C does not induce acquired resistance through PI3K/AKT associated pathway reactivation. Interestingly, PPL-008C induced marked upregulation of inhibitory c-Raf phosphorylation markers S43 and S233, but not S259. These findings further contradict the original hypothesis that PPL-008C-mediated inhibition of c-Raf activity is a result of PKA phosphorylation of the inhibitory S259 residue. Instead, my findings suggest PKA phosphorylation of inhibitory S43/S233 residues are responsible for c-Raf inhibition. Thus, through targeting the disruption of PDE8A - c-Raf, PPL-008C represents a novel therapeutic approach to treating KRAS-induced PDAC. Future investigations aim to identify molecular characteristics that can predict PPL-008C sensitivity and evaluate potential synergistic combination therapies that can be assessed in appropriate pre-clinical models of PDAC. In summary, these findings embolden peptide array screening technology as a powerful and robust approach to developing novel cell-penetrating peptide disruptors, and reinforces the therapeutic value of targeting the disruption of disease-associated protein complexes with peptide disruptor therapeutics.