Protein and Peptide Letters - Volume 25, Issue 12, 2018

Peptide-derived drugs constitute a significant fraction of therapeutic agents. In 2013, The global market of peptide therapeutics was ca. $19 billion; this value does not include revenue from insulin derivatives of $28 million. The combined sales of insulin and non-insulin peptide drugs is estimated to exceed $70 billion by 2019. A significant fraction of peptide-derived drugs is composed of an amino acid sequence and additional chemical functionalities that improve biological and pharmacological properties of the drug. In this review, we focus on synthetic cross-linkers that we refer to as “linchpins”, which are commonly used to constrain the secondary structure of peptides and equip them with added benefits such as resistance to proteolytic degradation and conformational stability. The latter property leads to an increase in binding potency and increased bioavailability due to increased permeation through biological membranes. Some linchpins can even introduce properties not found in natural peptides such as light-responsiveness. Peptides cyclized by linchpins can be viewed as a sub-class of a larger family of peptide-derived drugs with desired pharmacological performance in vivo. To understand how chemical modifications by linchpins improve drug discovery, this review also briefly summarizes canonical examples of chemical modification used in modern peptide therapeutics.

Background: Interactions between proteins play a key role in nearly all cellular process, and therefore, its dysregulation may lead to many different types of cellular dysfunctions. Hence, pathologic Protein-Protein Interactions (PPIs) constitute highly attractive drug targets and hold great potential for developing novel therapeutic agents for the treatment of incurable human diseases. Unfortunately, the identification of PPI inhibitors is an extremely challenging task, since traditionally used small molecules ligands are mostly unable to cover and anchor on the extensive and flat surfaces that define those binary protein complexes. In contrast, large biomolecules such as proteins or peptides are ideal fits for this so-called “undruggable” sites. However, their poor pharmacokinetic properties have also limited their applications as therapeutics. In this context, peptidomimetic molecules have emerged as an alternative and viable solution to this problem, since they conserve the architectural and structural features of peptides and also exhibit substantially improved pharmacokinetic profiles. Conclusion: In the last decades, a wide array of chemical approaches granting access to conformationally constrained peptides with substantially improved pharmacokinetic profiles have been described, with a special focus on those affording stapled peptides and allowing large-scale macrocyclizations. These peptidomimetic molecules have been successfully applied to target a plethora of biological hosts, which highlights their promising future as novel therapeutics for the treatment of incurable human diseases.

Background: Inducible Nitric Oxide Synthase (iNOS or NOS2) produces Nitric Oxide (NO) and related reactive nitrogen species, which are critical effectors of the host innate response and play key roles in the intracellular killing of bacterial and parasitic pathogens. The SPRY domain- containing SOCS box proteins SPSB1 and SPSB2 are key physiological regulators of this important enzyme. Disrupting the endogenous SPSB-iNOS interaction should prolong the intracellular lifetime of iNOS and enhance the production of NO, and therefore be beneficial in treating chronic and persistent infections such as tuberculosis. By using structure-based design, potent peptide inhibitors of this interaction have been developed. Conclusion: Inhibitors of the SPSB-iNOS interaction have therapeutic potential as a novel class of anti-infective agents. Various strategies are being pursued to target these peptide inhibitors to macrophages and deliver them to the cytoplasm of these cells. It will then be possible to assess the efficacy of such inhibitors in boosting the capacity of macrophages to destroy infectious pathogens.

Background: Mitochondria are the primary source of energy in most tissues. Mitochondrial dysfunction results in cellular energy deficiency, triggers the production of reactive oxygen species, and initiates various cell death and inflammatory pathways. Several cell-permeable peptides (SS peptides) have been described that selectively target cardiolipin on the inner mitochondrial membrane and promote efficiency of the electron transport chain to produce more ATP. Conclusion: In preclinical disease models, these peptides have been shown to repair damaged mitochondria and promote cellular repair and restore function. By mitigating cell injury, these peptides prevent inflammatory responses that can result in chronic inflammation and tissue remodeling. This peptide technology platform represents a paradigm shift in targeting the fundamental cause of cellular energy failure for age-related degenerative diseases.

Background: Tyrosine sulfation is an important post-translational modification of secreted and membrane proteins in multi-cellular organisms. This modification is catalyzed by tyrosylprotein sulfotransferases that often modify tyrosine residues in their target substrates in a heterogeneous manner. Chemokine receptors such as CCR5, which play roles in inflammation, immunity and viral infection, are sulfated on tyrosine residues in their extracellular N-termini. The heterogeneity of the sulfation has made it difficult to obtain atomic-resolution information on this region of CCR5. Homogeneously sulfated peptide surrogates can be efficiently synthesized by chemical and biochemical approaches. This communication reviews current chemical and biochemical methods for peptide tyrosine sulfation and the use of N-terminal CCR5 peptide surrogates in biochemical and structural analyses. Conclusion: Using solid phase peptide synthesis and synthons containing sulfotyrosine or sulfotyrosine neopentyl esters peptides containing up to 30 residues with multiple sulfotyrosines can be synthesized and purified in high (>50-70%) yield. Such peptides can be isotopically labeled at selected positions and used in detailed NMR investigations to investigate the interactions of sulfotyrosine residues with receptors. The application of transferred NOE studies to investigate CCL5/CCR5 interactions has led to the determination of pairwise interactions between the chemokine and its receptor.

Background: Venom peptides are a proven resource for identifying novel drugs, however the process of identifying bioactive venom peptides is currently labor intensive, costly, and rarely results in pharmaceutical success. As venom peptides are modulators of ion channels and receptors their potential for manipulating cell signals in diseased states are unique and offer an untapped resource for finding new medicines. Recent advances in -omic technologies, and microfluidic biosensing systems have transformed how venom peptides are discovered and characterized. Conclusion: This review will cover past, present and future approaches for screening venom peptides for drug discovery and development. Specifically, we will highlight online high-resolution microfluidic biosensing systems and new fluorescence detection methods that can be adapted to expand the discovery and characterization of venom peptide drugs.

Background: Research has been directed at the optimization of insulin for medicinal purposes. An insulin analog that could be reversibly activated might provide more precise pharmacokinetic control and broaden the inherent therapeutic index of the hormone. The prospect of using intramolecular structural constraint to reversibly inactive insulin might constitute the first step to achieving such an optimized analog. Chemically crosslinked insulin analogs have been reported where two amines are covalently linked by reaction with symmetrical bifunctional active esters. There is little selectivity in this synthetic approach to molecular constraint with multiple derivatives being formed. Objective: To systematically evaluate the synthesis of covalently crosslinked insulin analogs by asymmetric methods and the biological consequences. Method: We report synthesis of amine crosslinked insulin analogs via a two-step procedure. The stepwise approach was initiated by amide bond formation and followed by second site alkylation to produce site-specific, cross-linked insulin analogs. Results: A set of unique insulin analogs crosslinked at the two of the three native amines were synthesized. They were chemical characterized and assessed by in vitro bioanalysis to result in a significant and reasonably consistent reduction in biological potency. Conclusion: We achieved an unambiguous two-step synthesis of several crosslinked insulin analogs differing in location of the chemical tether. Bioanalysis demonstrated the ability of the molecular constraint to reduce bioactivity. The results set the stage for in vivo assessment of whether such a reduction in potency can be used pharmacologically to establish a constrained hormone upon which reversible tethering might be subsequently introduced.