Exploring the Functional Consequences of Protein Backbone Alteration in Ubiquitin through Native Chemical Ligation.

Ubiquitin (Ub) plays critical roles in myriad protein degradation and signaling networks in the cell. We report herein Ub mimetics based on backbones that blend natural and artificial amino acid units. The variants were prepared by a modular route based on native chemical ligation. Biological assays show that some are enzymatically polymerized onto protein substrates, and that the resulting Ub tags are recognized for downstream pathways. These results advance the size and complexity of folded proteins mimicked by artificial backbones and expand the functional scope of such agents.

Thermodynamic and Structural Impact of α,α-Dialkylated Residue Incorporation in a ß-Hairpin Peptide.

Peptides containing α,α-dialkylated α-amino acids, owing to their ability to disrupt aggregation of ß-amyloid proteins, have therapeutic potential in the treatment of neurodegenerative diseases. Thermodynamic and structural analyses are reported for a series of ß-hairpin peptides containing α,α-dialkylated α-amino acids with varying side-chain lengths. The results of these experiments show that α,α-dialkylated α-amino acids with side-chain lengths longer than one carbon unit are tolerated in a ß-hairpin, although at a moderate cost to folded stability.

Thermodynamic origin of α-helix stabilization by side-chain cross-links in a small protein.

Peptide cross-linking has been widely explored as a means of constraining short sequences into stable folded conformations, most commonly α-helices. The prevailing hypothesis for the origin of helix stabilization is an entropic effect resulting from backbone pre-organization; however, obtaining direct evidence bearing on this hypothesis is challenging. Here, we compare the folding thermodynamics of a small helix-rich protein domain and analogues containing one of three common cross-linking motifs. Analysis of the folding free energy landscapes of linear vs. cyclized species reveal consistent trends in the effect of cyclization on folding energetics, as well as subtle differences based on the chemistry of the cross link. Stabilization in all three systems arises entirely from a reduction in the entropic penalty of folding that more than compensates for an enthalpic destabilization of the folded state.

Peptide Backbone Composition and Protease Susceptibility: Impact of Modification Type, Position, and Tandem Substitution.

The clinical utility of peptides is limited by their rapid degradation by endogenous proteases. Modification of the peptide backbone can generate functional analogues with enhanced proteolytic stability. Existing principles for the design of such oligomers have focused primarily on effective structural mimicry. A more robust strategy would incorporate a rational approach for engineering maximal proteolytic stability with minimal unnatural residue content. We report here the systematic comparison of the proteolytic resistance imparted by four backbone modifications commonly employed in the design of protease-stable analogues of peptides with complex folding patterns. The degree of protection was quantified as a function of modification type, position, and tandem substitution in the context of a long, unstructured host sequence and a canonical serine protease. These results promise to inform ongoing work to develop biostable mimics of increasingly complex peptides and proteins.

Folding and function in α/ß-peptides: targets and therapeutic applications.

Combining natural α-amino acid residues and unnatural ß-amino acid residues in a single chain leads to heterogeneous-backbone oligomers called α/ß-peptides. Despite their unnatural backbones, α/ß-peptides can manifest a variety of folding patterns and biological functions reminiscent of natural peptides and proteins. Moreover, incorporation of ß-residues can impart useful properties to the oligomer such as improved stability to degradation by protease enzymes. α/ß-Peptides have been developed that engage diverse biological targets, including proteins involved in apoptotic signalling, HIV-cell fusion, hormone signalling, and angiogenesis. For some systems, promising results obtained in vitro have paved the way for demonstrated activity in vivo, where α/ß-peptides show equal potency and improved duration of effect compared to α-peptide counterparts.

Amphimedosides A-C: synthesis, chemoselective glycosylation, and biological evaluation.

The amphimedosides, discovered in 2006, are the first examples of naturally occurring glycosylated alkoxyamines. We report syntheses of amphimedosides A-C that feature a stereoselective oxyamine neoglycosylation and found that these alkaloids display modest cytotoxicity toward seven diverse human cancer cell lines, exhibiting IC(50) values ranging from 3.0 µM to greater than 100 µM.

Synthesis and biological evaluation of RON-neoglycosides as tumor cytotoxins.

Cardenolides such as digitoxin have been shown to inhibit cancer cell growth, to reduce cancer metastasis, and to induce apoptosis in tumor cells. Among the most potent digitoxin-based cytotoxins identified to date are MeON-neoglycosides generated via oxyamine neoglycosylation. Here, we report our studies of oxyamine neoglycosylation aimed at facilitating the elucidation of linkage-diversified digitoxin neoglycoside structure-activity relationships. We identified conditions suitable for the convenient synthesis of digitoxin neoglycosides and found that sugar structure, rather than RON-glycosidic linkage, exerts the strongest influence on neoglycoside yield and stereochemistry. We synthesized a library of digitoxin neoglycosides and assessed their cytotoxicity against eight human cancer cell lines. Consistent with previous findings, our data show that the structure of RON-neoglycosidic linkages influences both the potency and selectivity of digitoxin neoglycosides.