Hitting "Undruggable" Targets: Determining the Properties of Cell Penetrant Stabilized Peptide Therapeutics for Intracellular Targets

Abstract

Nearly two-thirds of all disease-associated proteins are ‘undruggable’ by modern therapeutics, meaning they are inside cells, out of the reach of biologics, but lack small molecule binding pockets. Stabilized peptides have the potential to hit these targets, which would open a vast array of potential new therapies. One such target is the p53/MDM2 interaction—a protein-protein interaction central to many cancers. Several inhibitors have been developed against the MDM2 protein because this target degrades the “the guardian of the genome” protein, p53. However, few of these peptides demonstrate the serum-independent, on-target efficacy required for clinical translation. In addition to having a strong target binding affinity, these peptides must efficiently penetrate the cell and evade proteolytic degradation. The design criteria for developing agents that can meet all of these requirements are still poorly understood. This work focuses on identifying the most important physicochemical properties that promote overall in vitro efficacy, taking into account the relevant molecular and cellular parameters in order to aid in future design of stabilized peptide therapeutics. The research presented here begins with measuring the effects that lipophilicity and charge have on have on cellular uptake as these are two commonly tuned parameters for promoting stabilized peptide efficacy. Furthermore, cellular membrane penetration is largely thought to be a major limiting factor for this class of drugs. Results showed that incremental increases in charge caused significant increases in uptake and that although lipophilic peptides are more efficient at entering cells than hydrophilic peptides, there is a point at which increased lipophilicity begins to instead decrease uptake (logD>~3.5). After obtaining these results, I moved on to selecting peptides discovered via bacterial surface display and measuring their binding affinities and in vitro efficacies as a precursor to a full physicochemical property profile in order to identify what the biggest contributors to efficacy are. After selecting those peptides, I measured their lipophilicities, cellular penetration rates, membrane interactions, and proteolytic stability. Ultimately, results showed that the cellular potency of this series of compounds appears to be driven by intracellular stability, which correlated with efficacy, rather than permeability, which did not at all correlate with efficacy. This was demonstrated by ATSP-7041, a promising MDM2/p53 inhibiting peptide and the only p53-based peptide that has led to a clinical lead compound, as well as pepC, a novel peptide with efficacy close to that of ATSP-7041. These two peptides showed the highest resistance to proteolytic degradation as well as the highest cellular potency, although ATSP-7041 had the slowest cellular uptake (~3-fold slower than pepC). Characterization of the molecules demonstrated they all had high affinity and modest membrane permeability, leading to stability as the differentiating factor. These results exhibit the need for a wholistic assessment of peptide properties to help inform efficacy outcomes and serve as a basis for future peptide development.