Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo.

Uropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70-90 % of community or hospital-acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECs fluorescent probe (NO-AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO-AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on the outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within the periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

Activity Sensing of Coagulation and Fibrinolytic Proteases.

The blood coagulation cascade is a complex physiological process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.

Synthesis of Nε-acetyl-L-homolysine by the Lossen rearrangement and its application for probing deacetylases and binding modules of acetyl-lysine.

Lysine acetylation is a posttranslational protein modification mediating protein-protein interactions by recruitment of bromodomains. Investigations of bromodomains have focused so far on the sequence context of the modification site and acyl-modifications installed at lysine side chains. In contrast, there is only little information about the impact of the lysine residue that carries the modification on bromodomain binding. Here, we report a synthesis strategy for L-acetyl-homolysine from L-2-aminosuberic acid by the Lossen rearrangement. Peptide probes containing acetylated homolysine, lysine, and ornithine were generated and used for probing the binding preferences of four bromodomains from three different families. Tested bromodomains showed distinct binding patterns, and one of them bound acetylated homolysine with similar efficiency as the native substrate containing acetyl-lysine. Deacetylation assays with a bacterial sirtuin showed a strong preference for acetylated lysine, despite a broad specificity for N-acyl modifications.

Computer-Assisted Design of Peptide-Based Radiotracers.

In medical imaging, techniques such as magnetic resonance imaging, contrast-enhanced computerized tomography, positron emission tomography (PET), and single-photon emission computed tomography (SPECT) are extensively available and routinely used for disease diagnosis. PET probes with peptide-based targeting are typically composed of small peptides especially developed to have high affinity and specificity for a range of cellular and tissue targets. These probes' key benefits include being less expensive than traditional antibody-based PET tracers and having an effective chemical modification process that allows them to be radiolabeled with almost any radionuclide, making them highly appealing for clinical usage. Currently, as with every pharmaceutical design, the use of in silico strategies is steadily growing in this field, even though it is not part of the standard toolkit used during radiopharmaceutical design. This review describes the recent applications of computational design approaches in the design of novel peptide-based radiopharmaceuticals.

Update on Disease-Specific Biomarkers in Transthyretin Cardiac Amyloidosis.

PURPOSE OF REVIEW: Transthyretin cardiac amyloidosis (ATTR-CM) is an infiltrative cardiomyopathy and an increasingly recognized cause of morbidity and mortality. There remains substantial delay between initial symptoms and diagnosis. With the recent emergence of various targeted therapies proven to reduce morbidity and mortality, there is an imperative to diagnose subclinical disease. Biomarkers may be well-suited for this role. RECENT FINDINGS: Conventional markers of heart failure, such as natriuretic peptides and cardiac troponins, and estimated glomerular filtration rate are associated with risk in ATTR-CM. Circulating transthyretin (TTR) levels parallel TTR kinetic stability, correlate with disease severity, and may serve as indirect markers of ATTR-CM disease activity and response to targeted treatment. There is also growing evidence for the correlation of TTR to retinol-binding protein 4, a biomarker which independently associates with this disease. The rate-limiting step for ATTR pathogenesis is dissociation of the TTR homotetramer, which may be quantified using subunit exchange to allow for early risk assessment, prognostication, and assessment of treatment response. The protein species that result from the dissociation and misfolding of TTR are known as nonnative transthyretin (NNTTR). NNTTR is quantifiable via peptide probes and is a specific biomarker whose reduction is positively correlated with improvement in neuropathic ATTR amyloidosis. Neurofilament light chain (NfL) is released into the blood after axonal damage and correlates with neuropathic ATTR amyloidosis, but its clinical use in ATTR-CM is uncertain. Conventional markers of heart failure, transthyretin, retinol-binding protein 4, transthyretin kinetic stability, nonnative transthyretin, peptide probes, and neurofilament light chain have potential as biomarkers to enable early, subclinical diagnosis in patients with transthyretin cardiac amyloidosis.

An eco-friendly solvent-free reaction based on peptide probes: design an extraction-free method for analysis of acrylamide under microliter volume.

Acrylamide is a group 2A carcinogen and potential endocrine disruptor that can enter the ecosystem by various routes and has recently become a dangerous pollutant. This widely used chemical can enter the human body via air inhalation, food or water consumption, or skin contact. In this study, we developed a peptide probe for the detection of acrylamide by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) after its micro-tagging with a peptide. Direct detection of acrylamide by MALDI-TOF MS is not feasible due to its poor ionization in the MALDI interface, which hinders its analysis by the technique. After microwave irradiation for 2 min, the formed acrylamide-peptide derivative was detected easily by MALDI-TOF MS without the need for extraction procedures. The procedure does not involve organic solvents and a water-soluble peptide that allows detection of acrylamide in small sample volumes with a limit of detection (LOD) of 0.05 ng/µL. The relative standard deviation (RSD) and relative error (RE) of the measurements were < 6.7% for intra- and inter-day assays. Gel-washing solutions from a polyacrylamide gel experiment were used as a model to study the efficiency of the developed method. Finally, we used the proposed method for the detection of free acrylamide in small volumes of lung epithelial cells (a model to test the air inhalation of acrylamide under a tiny volume of sample) and human urine. The developed method will enable rapid acrylamide detection in environmental and biological samples via a green approach based on microwave-assisted derivatization in water alongside the use of a less toxic derivatization reagent, reusable target plate, and miniaturization protocols.

Simultaneous fingerprinting of multiplex collagen biomarkers in connective tissues by multicolor quantum dots-based peptide probes.

The accurate detection of multiplex collagen biomarkers is vital for diagnosing and treating various critical diseases such as tumors and fibrosis. Despite the attractive optical properties of quantum dots (QDs), it remains technically challenging to create stable and specific QDs-based probes for multiplex biological imaging. We report for the first time the construction of multi-color QDs-based peptide probes for the simultaneous fingerprinting of multiplex collagen biomarkers in connective tissues. A bipeptide system composed of a glutathione (GSH) host peptide and a collagen-targeting guest peptide (CTP) has been developed, yielding CTP-QDs probes that exhibit exceptional luminescence stability when exposed to ultraviolet irradiation and mildly acidic conditions. The versatile bipeptide system allows for facile one-pot synthesis of high-quality multicolor CTP-QDs probes, exhibiting superior selectivity in targeting critical collagen biomarkers including denatured collagen, type I collagen, type II collagen, and type IV collagen. The multicolor CTP-QDs probes have demonstrated remarkable efficacy in simultaneously fingerprinting multiple collagen types in diverse connective tissues, irrespective of their status, whether affected by injury, diseases, or undergoing remodeling processes. The innovative multicolor CTP-QDs probes offer a robust toolkit for the multiplex fingerprinting of the collagen suprafamily, demonstrating significant potential in the diagnosis and treatment of collagen-related diseases.

Peptide-based diagnostic and therapeutic agents: Where we are and where we are heading?

Peptides are increasingly present in all branches of medicine as innovative drugs, imaging agents, theragnostic, and constituent moieties of other sophisticated drugs such as peptide-drug conjugates. Due to new developments in chemical synthesis strategies, computational biology, recombinant technology, and chemical biology, peptide drug development has made a great progress in the last decade. Numerous natural peptides and peptide mimics have been obtained and studied, covering multiple therapeutic areas. Even though peptides have been investigated across the wide therapeutic spectrum, oncology, metabolism, and endocrinology are the most frequent medical indications of them. This review summarizes the current use of and the emerging new opportunities of peptides for diagnosis and treatment of various diseases.

Rational design, synthesis and structural characterization of peptides and peptidomimetics to target Hsp90/Cdc37 interaction for treating hepatocellular carcinoma.

Heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) work together as a molecular chaperone complex to regulate the activity of a multitude of client protein kinases. These kinases belong to a wide array of intracellular signaling networks that mediate multiple cellular processes including proliferation. As a result, Hsp90 and Cdc37 represent innovative therapeutic targets in various cancers (such as leukemia, multiple myeloma, and hepatocellular carcinoma (HCC)) in which their expression levels are elevated. Conventional small molecule Hsp90 inhibitors act by blocking the conserved adenosine triphosphate (ATP) binding site. However, by targeting less conserved sites in a more specific manner, peptides and peptidomimetics (modified peptides) hold potential as more efficacious and less toxic alternatives to the conventional small molecule inhibitors. Using a rational approach, we herein developed bioactive peptides targeting Hsp90/Cdc37 interaction. A six amino acid linear peptide derived from Cdc37, KTGDEK, was designed to target Hsp90. We used in silico computational docking to first define its mode of interaction, and binding orientation, and then conjugated the peptide with a cell penetrating peptide, TAT, and a fluorescent dye to confirm its ability to colocalize with Hsp90 in HCC cells. Based on the parent linear sequence, we developed a peptidomimetics library of pre-cyclic and cyclic derivatives. These peptidomimetics were evaluated for their binding affinity to Hsp90, and bioactivity in HCC cell lines. Among them, a pre-cyclic peptidomimetic demonstrates high binding affinity and bioactivity in HCC cells, causing reduced cell proliferation that is associated with induction of cell apoptosis, and down-regulation of phosphorylated MEK1/2. Overall, this generalized approach of rational design, structural optimization, and cellular validation of 'drug-like' peptidomimetics against Hsp90/Cdc37 offers a feasible and promising way to design novel therapeutic agents for malignancies and other diseases that are dependent on this molecular chaperone complex.

Dielectrophoresis assisted rapid, selective and single cell detection of antibiotic resistant bacteria with G-FETs.

The rapid increase in antibiotic resistant pathogenic bacteria has become a global threat, which besides the development of new drugs, requires rapid, cheap, scalable, and accurate diagnostics. Label free biosensors relying on electrochemical, mechanical, and mass based detection of whole bacterial cells have attempted to meet these requirements. However, the trade-off between selectivity and sensitivity of such sensors remains a key challenge. In particular, point-of-care diagnostics that are able to reduce and/or prevent unneeded antibiotic prescriptions require highly specific probes with sensitive and accurate transducers that can be miniaturized and multiplexed, and that are easy to operate and cheap. Towards achieving this goal, we present a number of advances in the use of graphene field effect transistors (G-FET) including the first use of peptide probes to electrically detect antibiotic resistant bacteria in a highly specific manner. In addition, we dramatically reduce the needed concentration for detection by employing dielectrophoresis for the first time in a G-FET, allowing us to monitor changes in the Dirac point due to individual bacterial cells. Specifically, we realized rapid binding of bacterial cells to a G-FET by electrical field guiding to the device to realize an overall 3 orders of magnitude decrease in cell-concentration enabling a single-cell detection limit, and 9-fold reduction in needed time to 5 min. Utilizing our new biosensor and procedures, we demonstrate the first selective, electrical detection of the pathogenic bacterial species Staphylococcus aureus and antibiotic resistant Acinetobacter baumannii on a single platform.

Peptide Probes of Colistin Resistance Discovered via Chemically Enhanced Phage Display.

Colistin is an antibiotic of last resort used to treat infections caused by multidrug-resistant Gram-negative bacterial pathogens. The recent surge in reported cases of colistin-resistant infections urgently calls for fast and reliable diagnostic methods, which can be used for the facile detection and proper treatment of these challenging infections. A major mechanism of colistin resistance involves phosphoethanolamine (PE) modification of lipopolysaccharide (LPS), the molecular target of colistin. This LPS modification mechanism has been recently reported to be transferrable via a plasmid-carried mcr-1 gene, which is particularly concerning as it may readily confer colistin resistance to a wide array of bacterial pathogens. To develop molecular tools to allow facile detection of colistin resistance, we have herein enlisted a novel phage library that incorporates dynamic covalent warheads to recognize PE modifications on bacterial cells. Screening of this chemically modified phage library against colistin-resistant pathogens revealed a number of peptide probes that readily differentiate colistin-resistant bacterial strains from their colistin-susceptible counterparts. With a fluorophore label, these peptide probes selectively stain colistin-resistant bacteria at sub-to-low micromolar concentrations. The bacterial staining is minimally inhibited by the presence of serum proteins or even blood serum. Mechanistic studies indicate that our peptide probes bind colistin-resistant bacteria primarily by targeting PE-modified lipids. However, some species-specific features of the cell surface can also contribute to the peptides' association to bacterial cells. Further elucidation of such cell surface features may give molecular probes with improved species and strain specificity, which will enable bacterial infection diagnosis with high precision.

A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification.

The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarker. There is much evidence indicating that a panel of miRNAs, termed "miRNA fingerprints", is more specific and informative than an individual miRNA as biomarker. Thus, multiplex assays for simultaneous quantification of multiple miRNAs could be more potent in clinical practice. However, current available assays normally require pre-enrichment, amplification and labeling steps, and most of them are semi-quantitative or lack of multiplexing capability. In this study, we developed a quasi-targeted proteomics assay for multiplexed miRNA quantification by a combination of DNA-peptide probes and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, the signal of target miRNAs (i.e., miR-21, miR-let7a, miR-200c, miR-125a and miR-15b) was converted into the mass response of reporter peptides by hybridization of miRNAs with DNA-peptide probes and subsequent tryptic digestion to release the peptides. After a careful optimization of conditions related to binding, conjugation, hybridization and multiple reaction monitoring (MRM) detection, the assay was validated for each miRNA and the limit of quantification (LOQ) for all the miRNAs can achieve 1 pM. Moreover, crosstalk between DNA-peptide probes in multiplex assay was sophisticatedly evaluated. Using this quasi-targeted proteomics assay, the level of target miRNAs was determined in 3 human breast cell lines and 36 matched pairs of breast tissue samples. Finally, simplex assay and qRT-PCR were also performed for a comparison. This approach grafts the strategy of targeted proteomics into miRNA quantification and may offer a new way for multiplexed miRNA profiling.