Design of a serum stability tag for bioactive peptides.

Serum has a high intrinsic proteolytic activity that leads to continuous processing of peptides and proteins. Strategies to protect bioactive peptides from serum proteolytic degradation include incorporation of unnatural amino acids, conformational constraints, large polymeric tags, or other synthetic manipulations such as amide bond replacements. Here we explored a possibility of designing a serum stability tag made of natural amino acids. We observed that a diproline motif (-Pro-Pro-) shows remarkable stability against serum endopeptidases. Accordingly, we designed close to 50 peptides to identify natural amino acids flanking the -Pro-Pro- sequence that can enhance the serum stability of this motif. As a result, a tetrapeptide with the sequence Asp-Pro-Pro-Glu (DPPE) was identified that remains intact in human serum for more than 24 h. at 37°C.

Design of a peptide inhibitor of tyrosine kinase 2.

Tyrosine kinase inhibitors show great promise as clinical therapies, but small molecule inhibitors that are available in the clinic and under development bind to the adenosine triphosphate binding domain of the kinase, potentially limiting efficacy and selectivity. The development of antisense peptide inhibitors is a relatively unexplored area of research, and here we investigate inhibitory peptides specific for the Janus-associated kinase (JAK) family member, tyrosine kinase 2 (TYK2). We have developed peptides that are 2-3 times more selective for TYK2 than other JAK family members, with a TYK2 IC50 of 1.2 µM. In addition, TYK2 inhibitory peptides show specificity for TYK2-mediated functions over JAK1 functions in cell-based assays. These peptides provide a new tool for the development of specific peptide inhibitors for closely related tyrosine kinases.

Modulation of the cannabinoid receptors by hemopressin peptides.

Changes in the endocannabinoid system are implicated in numerous diseases, making it an attractive target for pharmaceutical development. The endocannabinoid receptors have traditionally been thought to act through the effects of lipophilic messengers called cannabinoids. The exciting finding of endocannabinoid system modulation by the nonapeptide hemopressin and its N-terminal extensions has highlighted the complexity of cannabinoid biology and pharmacology and sparked interest for therapeutic purposes. However, many questions surrounding the generation and regulation of the hemopressin peptides, the self-assembly of hemopressin and the potential for drug development based on hemopressin remain and are discussed in this review.

Proteolytic fingerprinting of complex biological samples using combinatorial libraries of fluorogenic probes.

Proteases have garnered interest as candidate biomarkers and therapeutic targets for many human diseases. A key challenge is the identification and characterization of disease-relevant proteases in the complex milieu of biological fluids such as serum, plasma, and bronchoalveolar lavage, in which a multitude of hydrolases act in concert. This unit describes a protocol to map the global proteolytic substrate specificities of complex biological samples using a concise combinatorial library of internally quenched fluorogenic peptide probes (IQFPs). This substrate profiling approach provides a global and quantitative comparison of protease specificities between different biological samples. Such a comparative analysis can lead to the identification of disease-specific 'fingerprints' of proteolytic activities with potential utility in diagnosis and therapy.

Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α.

Post-proline cleaving peptidases are promising therapeutic targets for neurodegenerative diseases, psychiatric conditions, metabolic disorders, and many cancers. Prolyl oligopeptidase (POP; E.C. 3.4.21.26) and fibroblast activation protein α (FAP; E.C. 3.4.24.B28) are two post-proline cleaving endopeptidases with very similar substrate specificities. Both enzymes are implicated in numerous human diseases, but their study is impeded by the lack of specific substrate probes. We interrogated a combinatorial library of proteolytic substrates and identified novel and selective substrates of POP and FAP. These new sequences will be useful as probes for fundamental biochemical study, scaffolds for inhibitor design, and triggers for controlled drug delivery.

The ST Pinch: A side chain-to-side chain hydrogen-bonded motif.

The ST Pinch is a 12-membered hydrogen-bonded motif (Ser/Thr-Xaa-Ser/Thr) involving the side chain oxygen atoms of two Ser/Thr residues. We identified the ST Pinch in 104 proteins in a database containing high-resolution crystal structures. Conformational analysis of the ST Pinch in these proteins points to specific preferences for the Xaa residue and a high propensity of this residue to adopt positive φ angles. Our results suggest that this motif serves as a linker of secondary structural elements within proteins and is a new addition to the existing list of short hydrogen bond-stabilized motifs in proteins.

Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases.

Proteases are candidate biomarkers and therapeutic targets for many diseases. Sensitive and robust techniques are needed to quantify proteolytic activities within the complex biological milieu. We hypothesized that a combinatorial protease substrate library could be used effectively to identify similarities and differences between serum and bronchoalveolar lavage fluid (BALF), two body fluids that are clinically important for developing targeted therapies and diagnostics. We used a concise library of fluorogenic probes to map the protease substrate specificities of serum and BALF from guinea pigs. Differences in the proteolytic fingerprints of the two fluids were striking: serum proteases cleaved substrates containing cationic residues and proline, whereas BALF proteases cleaved substrates containing aliphatic and aromatic residues. Notably, cleavage of proline-containing substrates dominated all other protease activities in both human and guinea pig serum. This substrate profiling approach provides a foundation for quantitative comparisons of protease specificities between complex biological samples.

Design of short linear peptides that show hydrogen bonding constraints in water.

Using a combination of an aromatic amino acid, a homoserine side chain, and a d-amino acid, a series of linear tetrapeptides were designed that adopt an "Hse turn" in water. The conformation was stabilized by intramolecular hydrogen bonds even in the presence of surrounding water molecules. In particular, the peptide with sequence H-Abz-Homoser-Ser-d-Gln-NH(2) showed significant through-space interactions and its free energy of folding is estimated to be on the order of -4 kcal/mol. We report the design of the tetrapeptides using a novel mimicry approach and their characterization based on NMR spectroscopy and MD simulations.

Fluorescence probe with a pH-sensitive trigger.

Acid-catalyzed hydrolysis was used as the mechanism to design a new type of environmentally sensitive fluorescence probe. A mild and selective periodate oxidation of the 2-amino alcohol of serine in the presence of a disulfide bond was developed to prepare dialdehyde peptides. Two identical fluorochrome hydrazide derivatives were then linked to the dialdehyde peptide forming an acid-labile hydrazone linkage. This self-quenched probe is weakly fluorescent at a physiological pH of 7.4 but shows more than 3-fold fluorescence enhancement at pH 4.5.

Potent inhibitors of LXXLL-based protein-protein interactions.

Protein-protein interactions between estrogen receptors, ERalpha and ERbeta, and their coactivators (CoAs) are an attractive target for drug intervention. This interaction is mediated by a small pentapeptide motif (LXXLL), termed the NR box. Based on this motif, a variety of cyclic and linear peptides were synthesized in order to gain a better understanding of the association of CoA proteins with the ER isoforms. Utilizing a time-resolved florescence-based coactivator interaction assay, we determined the abilities of these peptides to inhibit this interaction. Using molecular modeling and CD spectroscopy, we have examined the structural basis of their bioactivities with both hormone receptor isoforms. Either homocysteine or penicillamine was utilized as a substitute for cysteine in the disulfide-bridged peptides, while tertiary leucine and neopentyl glycine were used as the surrogates for the NR box leucines. The most potent disufide-bridged peptide (K(i)= 70 pM, with ERalpha) incorporates neopentyl glycine in the NR box, while the most active peptide in this series with ERbeta (K(i)=350 pM) incorporates tertiary leucine. Surprisingly, several linear peptides containing a single cysteine residue showed activities with low nanomolar K(i) values. Collectively, our results suggest a synthetic approach for designing potent and selective peptidomimetics for ERalpha and ERbeta interactions with CoA proteins effecting estrogen action.

Understanding base-assisted desulfurization using a variety of disulfide-bridged peptides.

A recently rediscovered reaction of base-assisted lanthionine formation has been applied to several systems of disulfide-bridged peptides. In addition to previously described nonapeptides consisting of i, i+3 cystine linkages, the reaction has now been extended to systems consisting of shorter (i, i+2) and longer (i, i+4) disulfide bridges. The desulfurization reaction is also compatible with disulfide bridges formed through homocysteines and penicillamines, yielding unusual amino acids such as cystathionine and beta,beta-dimethyl lanthionine (referred to as "penthionine") in a peptide chain, respectively. Systematic study of this transformation has provided several new insights into its mechanism. We have observed formation of dehydroalanine and dehydrovaline residues resulting from i, i+2-bridged cysteines and i, i+3-bridged cysteine/penicillamine peptides, respectively, thereby supporting a beta-elimination/Michael-addition mechanism for this transformation. Amino acid analysis and NMR data from total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum correlation (HSQC) experiments show three diastereomeric lanthionine-bridged peptides in the product mixture. But in the case of desulfurization of a cysteine/homocysteine containing disulfide-bridged peptide, Michael addition appears to be stereoselective, yielding a single stereoisomer of cystathionine within the peptide. According to molecular modeling and CD spectroscopy, constrained peptides such as those containing penicillamine are less likely to undergo facile desulfurization. Flexibility of the torsional angles (C(alpha)H-C(alpha)-C(beta)-S) corresponding to the cysteine residues and temperature appear to be contributing factors determining the extent of desulfurization.

Solid-phase synthesis of disulfide heterodimers of peptides.

[structure: see text] The synthesis of the orthogonal disulfide template 1 and its use to synthesize unsymmetrical intermolecular disulfide bond peptides on a solid support are described. Application of template 1 to synthesize bioconjugates of cell permeable moieties based on the disulfide bond is demonstrated.