Serum biomarkers reveal long-term cardiac injury in isoproterenol-treated African green monkeys.

The assessment of cardiac toxicity is a major challenge in both drug development and clinical trials, and numerous marketed pharmaceuticals have been removed from the market due to unpredicted cardiac effects. Serum troponins are widely used indicators of cardiac injury; however, they are short-lived and have not been validated in preclinical animal models. In this study, we have used filter-aided sample preparation (FASP) and tandem mass tag (TMT) labeling to investigate serum protein alterations in isoproterenol-treated African green monkeys. Our results showed that the combination of FASP and TMT labeling provided highly reproducible and efficient sample preparation, which enables us to identify and quantify serum proteins with high confidence. We focused on the proteins that exhibit long-term alteration upon isoproterenol injection and discovered nine proteins exhibiting significant changes at 48 and 72 h postdosing. We further chose three proteins, serum amyloid A (SAA), frutose biphosphate aldolase A (FBAA), and fetuin A, for validation using enzyme-linked immunosorbent assay (ELISA). The serum concentration of SAA showed a ∼ 50 fold increase, while concentration of FBAA and fetuin A exhibited a significant decrease accompanying isoproterenol-induced cardiotoxicity. This work provides valuable insights for multimarker evaluation of long-term cardiac injury.

Prolyl endopeptidase activity in bronchoalveolar lavage fluid: a novel diagnostic biomarker in a guinea pig model of invasive pulmonary aspergillosis.

Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.

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.

Hemopressin forms self-assembled fibrillar nanostructures under physiologically relevant conditions.

The nonapeptide hemopressin, which is derived from the α chain of hemoglobin, has been reported to exhibit inverse agonist activity against the CB1 receptor. Administration of this peptide in animal models led to decreased food intake and elicited hypotensive and antinociceptive effects. On the basis of hemopressin's potential in therapeutic applications and the lack of a structure-activity relationship study in literature, we aimed to determine the conformational features of hemopressin under physiological conditions. We conducted transmission electron microscopy experiments of hemopressin, revealing that it self-assembles into fibrils under aqueous conditions at pH 7.4. Circular dichroism and nuclear magnetic resonance experiments indicate that the peptide adopts a mostly extended ß-like structure, which may contribute to its self-assembly and fibril formation.

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.

Repurposing FDA-approved drugs to combat drug-resistant Acinetobacter baumannii.

OBJECTIVE: The rising occurrence of drug-resistant pathogens accentuates the need to identify novel antibiotics. We wanted to identify new scaffolds for drug discovery by repurposing FDA-approved drugs against Acinetobacter baumannii, an emerging Gram-negative nosocomial drug-resistant pathogen. MATERIALS AND METHODS: In this study, we screened 1040 FDA-approved drugs against drug-susceptible A. baumannii ATCC 17978 and drug-resistant A. baumannii BAA-1605. RESULTS AND DISCUSSION: Twenty compounds exhibited significant antimicrobial activity (MIC ≤8 mg/L) against ATCC 17978 while only five compounds showed such activity against BAA-1605. Among the most notable results, tyrothricin, a bactericidal antibiotic typically active only against Gram-positive bacteria, exhibited equipotent activity against both strains. CONCLUSION: The paucity of identified compounds active against drug-resistant A. baumannii exemplifies its ability to resist antimicrobials as well as the resilience of drug-resistant Gram-negative pathogens. Repurposing of approved drugs is a viable alternative to de novo drug discovery and development.

Enzyme-targeted fluorescent imaging probes on a multiple antigenic peptide core.

Peptide dendrimers have a variety of applications in biology such as the vehicles for drug and gene delivery, molecular inhibitors, protein mimics, and synthetic vaccines. The multiple antigenic peptide (MAP) system is a well-known example of a discrete, dendrimeric scaffold. We explored a novel application of the MAP-based scaffold by designing molecular probes that fluoresce only after enzymatic treatment. The probes, which were synthesized on solid support, incorporate a cathepsin S dipeptide substrate (Leu-Arg), and a poly(ethylene glycol) (PEG) chain in their dendritic arms. The fluorescence emission of the near-infrared fluorochromes attached to the N-termini of the dendritic arms was quenched. Mechanistic studies revealed formation of H-type dye aggregates within the tetravalent MAP system. By varying the length of the PEG chain, three probes were synthesized, CyPEG-1, CyPEG-2, and CyPEG-3 with 4, 8, and 12 ethylene oxide units, respectively. CyPEG-2 showed optimum aqueous solubility and quenching efficiency for imaging applications. Upon proteolytic activation with cathepsin S (EC 3.4.22.27), CyPEG-2 showed greater than 70-fold increase and more than 95% recovery in fluorescence emission.

Site-specific Substitutions Eliminate Aggregation Properties of Hemopressin.

Hemopressin is a naturally occurring and therapeutically relevant peptide with applications in hypertension, pain, addiction, and obesity. We had previously demonstrated that hemopressin converts into amyloid-like fibrils under aqueous conditions. However, the amino acid residues that modulate the aggregation propensity of hemopressin were not identified. In this study, we designed and synthesized 25 different analogs of hemopressin and analyzed their aggregation properties using the principle of dynamic light scattering. As a result, we were able to identify four conservative changes in the peptide sequence (Val(2) →DVal(2), Asn(3) →Gln(3) Leu(7) →Npg(7) and C-OH→C-NH2) that minimize aggregation propensity of hemopressin. The results indicate that hemopressin aggregation is cooperative in nature and involves contribution from multiple amino acids within the peptide chain. The analogs and the corresponding aggregation propensity data reported in this study would be useful for researchers investigating therapeutic properties of hemopressin, which have been hampered due to the tendency of hemopressin to aggregate in aqueous solutions.

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.

Sample collection in clinical proteomics--proteolytic activity profile of serum and plasma.

PURPOSE: Proteolytic enzymes are promising diagnostic targets since they play key roles in diverse physiological processes and have been implicated in numerous human diseases. Human blood is a relatively noninvasive source for disease-specific protease biomarker detection and subsequent translation into diagnostic tests. However, the choice of serum or plasma, and more specifically, which anticoagulant to choose in plasma preparation, is important to address in the sample preparation phase of biomarker discovery. EXPERIMENTAL DESIGN: We have previously utilized a combinatorial library of internally quenched fluorogenic probes to successfully map the global proteolytic profiles of various biological fluids. In this study, we utilized the platform to ascertain the impact of three commonly used anticoagulants (EDTA, heparin, and citrate) on the proteolytic activity profile of plasma and serum collected from a healthy Caucasian male. RESULTS: Serum and plasma citrate were observed to be most proteolytically active, followed by plasma heparin and then plasma EDTA. Detailed analysis of the amino acid distribution of motifs cleaved and not cleaved by the samples offered significant insights in to active proteolytic components within them. CONCLUSION AND CLINICAL RELEVANCE: Broad quantitative comparison of proteolytic profiles of these samples revealed several novel insights related to the differential substrate recognition of proteases present in these biological fluids.

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.

In silico systems biology approaches for the identification of antimicrobial targets.

Classical antibiotic discovery efforts have relied mainly on molecular library screening coupled with target-based lead optimization. The conventional approaches are unable to tackle the emergence of antibiotic resistance and are failing to provide understanding of multiple mechanisms behind drug actions and the off-target effects. These insufficiencies have prompted researchers to focus on a multidisciplinary approach of systems biology-based antibiotic discovery. Systems biology is capable of providing a big-picture view for therapeutic targets through interconnected networks of biochemical reactions derived from both experimental and computational techniques. In this chapter, we have compiled software tools and databases that are typically used for target identification through in silico analyses. We have also identified enzyme- and broad-spectrum metabolite-based drug targets that have emerged through in silico systems microbiology.

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

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.

The serine-proline turn: a novel hydrogen-bonded template for designing peptidomimetics.

Serine-Proline (SP) dipeptide motifs have been shown to form unique hydrogen-bonding patterns in protein crystal structures. Peptides were designed to mimic these patterns by forming the 6 + 10 and the 9 + 10 hydrogen-bonded rings. Factors that contribute to the formation of SP turns include controlling backbone flexibility and amino acid chirality along with creating a hydrophobic environment around the intramolecular hydrogen bonds.

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.

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.

An enhanced ß turn in water.

Aiming to design short linear peptides featuring strong intramolecular hydrogen bonds in water, a series of tetrapeptides based on the sequence Ac-Ala-Pro-Ala-Ala-NH(2) containing all possible combinations of L- and D-amino acids was synthesized. A regiospecific combination of heterochiral residues (DDLL or its mirror image LLDD) can be used to increase turn formation and stability within short peptides in water.