Designing Superhydrophilic, Disordered Peptides to Improve the Stability and Efficacy of Protein Therapeutics

Abstract

The therapeutic potential of protein drugs (biologics) has been hindered by difficulties with long-term storage and rapid clearance from the body. Recombinant fusion proteins provide a scalable synthesis platform for engineered biologics, whereby a polypeptide domain can be appended to alter the physical characteristics of a therapeutic protein and enhance its pharmaceutical viability. Two simple design principles, based on the physical properties of the polypeptide domain, have been separately applied to address issues with the storage and delivery of biologics. Superhydrophilic peptides, exemplified by the alternating-charge peptide poly(EK), have been shown to increase the thermostability of proteins in vitro. “Conformationally disordered” peptides, exemplified by the homo amino-acid peptide polyG, have been shown to increase the circulation half-life and bioactivity of protein therapeutics in vivo. The combination of superhydrophilicity and conformational disorder in a single fusion peptide may be necessary to simultaneously address concerns regarding the storage and therapeutic lifetime of biologics. In the current work, we use enhanced sampling molecular dynamics (MD) simulations to investigate the conformational ensemble of poly(EK) and glycine-substituted poly(EK) variants, and validate our structural predictions with circular dichroism (CD). We find the (EK)15 peptide exhibits a high propensity for forming anti-parallel beta strand secondary structures, which are stabilized by extensive salt-bridging of the positive and negative sidechains. MD simulations predict that limited glycine substitutions effectively disrupt the secondary structure and promote disordered conformations at physiologically relevant temperatures. We conclude that conformational disorder should be rationally programmed into alternating-charge peptides to improve their suitability for drug delivery applications. We also contribute a computational approach to quantify conformational disorder in polypeptides, which should facilitate the de novo design of more effective fusion proteins.