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Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor’s resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

It is now evident that the M1 family of aminopeptidases play important roles in many pathophysiological processes. Among them, the enzymatic properties of arginyl aminopeptidase-like 1 (RNPEPL1) are characterized only by its truncated form. No peptide substrate has been identified. To characterize the enzymatic properties of RNPEPL1 in more detail, the full-length protein was expressed in Escherichia coli and purified to homogeneity. The full-length RNPEPL1 showed rather restricted substrate specificity and basic amino acid preference towards synthetic substrates, which was different from the previously reported specificity characterized by the truncated form. Searching for peptide substrates, we found that several peptides, such as Met-enkephalin and kallidin, were cleaved. RNPEPL1 cleaved bradykinin to de-[Arg]-bradykinin despite the presence of proline at the P2'-position. The enzyme cleaved Met-enkephalin but not dynorphin A1–17. Similar to aminopeptidase B, the full-length RNPEPL1 showed basic amino acid preference towards both synthetic and peptide substrates. In addition to the unusual cleavage of bradykinin, this enzyme shows chain length-dependent cleavage of peptide substrates sharing N-terminal amino acid sequence. This is the first study to report the enzymatic properties of the full-length human RNPEPL1 as an aminopeptidase enzyme. [ABSTRACT FROM AUTHOR]

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Several attempts have been made over the past decade to explore the concept of prodrug strategies that exploit PSA as a molecular target for the release of anticancer drugs in prostate tumors using various prostate specific antigen (PSA)-cleavable peptide linkers, but the desired antitumor and antimetastatic efficacy has not yet been fully achieved. We set out to look for new PSA-cleavable peptide substrates that could be cleaved more rapidly and efficiently than the previously used peptides. To look for the most susceptible PSA-cleavable peptide substrates, we used the so-called spot technology. With the following general formula, we designed 25 different fluorogenic heptapeptides; Cellulose-P5-P4-P3-P2-P1-P1′-P2’ (Fluorophore). The increase of the fluorescence in the supernatant of the reaction mixture was monitored using a 96-well fluorometric plate reader with excitation of λex 485 nm and λem 535 nm. Three sequences showed a high fluorogenic liberation after incubation with PSA, i.e., Arg-Arg-Leu-His-Tyr-Ser-Leu (7), Arg-Arg-Leu-Asn-Tyr-Ser-Leu (8) and Arg-Ser-Ser-Tyr-Arg-Ser-Leu (23). Future incorporation of these optimized substrates in the PSA-cleavable prodrug formulations could further optimize the cleavage pattern and so the release characteristics of these prodrugs to rapidly and efficiently liberate the free cytotoxic agents inside the tumor tissues.

The G protein-coupled receptor kinase (GRK) family consists of seven cytosolic serine/threonine (Ser/Thr) protein kinases, and among them, GRK2 is involved in the regulation of an enormous range of both G protein-coupled receptors (GPCRs) and non-GPCR substrates that participate in or regulate many critical cellular processes. GRK2 dysfunction is associated with multiple diseases, including cancers, brain diseases, cardiovascular and metabolic diseases, and therefore GRK2-specific substrates/inhibitors are needed not only for studies of GRK2-mediated cellular functions but also for GRK2-targeted drug development. Here, we first review the structure, regulation and functions of GRK2, and its synthetic substrates and inhibitors. We then highlight recent work on synthetic peptide substrates/inhibitors as promising tools for fundamental studies of the physiological functions of GRK2, and as candidates for applications in clinical diagnostics. [ABSTRACT FROM AUTHOR]

Efficient deposition of biomolecules on the surface, maintaining their full activity and stability, is a most significant factor in biosensor construction. For this reason, more and more research is focused on the development of electrochemical biosensors that have the ability to electrically detect adsorbed molecules on electrode surface with high selectivity and sensitivity. The presented research aims to develop an efficient methodology that allows quantification of processes related to the evaluation of enzyme activity (proprotein convertase) using electrochemical methods. In this study we used impedance spectroscopy to investigate the immobilization of peptide substrate (Arg-Val-Arg-Arg) modified with 11-mercaptoundecanoic acid on the surface of gold electrode. Both the synthesis of the peptide substrate as well as the full electrochemical characteristics of the obtained electrode materials have been described. Experimental conditions, including concentration of peptide substrate immobilization, modification time, linker, and the presence of additional blocking groups have been optimized. The main advantages of the described method is that it makes it possible to observe the peptide substrate–enzyme interaction without the need to use fluorescent labels. This also allows observation of this interaction at a very low concentration. Both of these factors make this new technique competitive with the standard spectrofluorimetric method.

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A–G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD50 toxin present in buffer solution. [ABSTRACT FROM AUTHOR]

We designed a multiple signal sensing probe based on highly luminescent nanosized Cu-PTC metal-organic framework (MOF). The as synthesized nano-MOF was characterized by various techniques to confirm its morphology, crystallinity and elemental composition. A positively charged matrix metalloproteinase-2 (MMP-2) substrate (a polypeptide) was designed and prepared to interact electrostatically with the negatively charged Cu-PTC MOF probe, resulting in decrease in fluorescence response of the probe. Subsequently, MMP-2 was specifically recognized by the MMP-2 substrate and cleavage of the MMP-2 substrate resulted in "turn-on" fluorescence response from 1 to 175 ng/mL of MMP-2 concentrations with a limit of detection (LOD) of 0.8 ng/mL. In addition, the developed Cu-PTC MOF also exhibited peroxidase-like activity, which was exploited in the electrochemical detection of MMP-2 subjected to the catalysis of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H 2 O 2. The large surface area of the probe and unique catalytic activity toward TMB facilitated fabrication of an excellent sensing platform to detect MMP-2 with a linear range of 0.5–200 ng/mL with a LOD of 0.25 ng/mL. Various potential interfering biomolecules were evaluated and the stability of the constructed biosensor was assessed in optical and electrochemical modes. The reliability of the electrochemical detection mode was further executed by monitoring MMP-2 in biological samples (human serum and cell samples) and good recoveries were recorded. [Display omitted] • Designed a multiple signal sensing probe based on Cu-PTC metal organic framework (MOF). • Developed Cu-PTC MOF also exhibited peroxidase-like activity. • Excellent electrochemical sensing platform to detect MMP-2. • Proposed opto-electrochemical sensor was verified by quantitative measurement of MMP-2 in human serum. [ABSTRACT FROM AUTHOR]

BACKGROUND: Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeated sequences of high molecular weight glutenin subunits were synthesized. RESULTS: Hydrolysis of peptides by pest protease was determined by high‐performance liquid chromatography. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer. The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the assay developed was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants, which are proportional to the protease content of the flour, were determined. CONCLUSION: The total analysis time for the assay developed is estimated as 15 min. The assay developed permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) is generated by proteolytic processing of amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Amyloid-β is responsible for the formation of senile plaques in Alzheimer's disease (AD). Consequently, inhibition of β-secretase (BACE-1), a rate-limiting enzyme in the production of Aβ, constitutes an attractive therapeutic approach to the treatment of AD. This paper reports an inkjet printing-based fluorescence assay for high throughput screening of β-secretase inhibitors achieved by employing a BACE-1 FRET substrate (Rh-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-Quencher). This peptide substrate is known to be a readily available and suitable substrate for proteolytic activity, and it has high affinity to BACE-1. The BACE-1 peptide substrate printed on parchment paper was effectively cleaved by BACE-1, which was printed on the same spot. The amount of enzyme and substrate required for this inkjet printing-based BACE-1 assay can be less than 1.4ⅹ10 3 , permitting the evaluation of inhibitor activity with femtomolar potency. The inkjet-printing-based BACE-1 inhibitory assay revealed inhibitory effects of inhibitor IV and STA on BACE-1 with an IM 50 of 1.00 × 10 −15  mol and 1.01 × 10 −14  mol, respectively. These data confirm that both BACE-1 inhibitors (inhibitor IV and STA) actively inhibited the BACE-1 proteolysis of BACE-1 substrate on parchment paper. It important to note that the number of mole of BACE-1-substrate and enzyme utilized in the printing-based enzymatic assay are 1.4ⅹ10 3 smaller than the amount used in the conventional well-plate assay. The inkjet printing-based inhibitory assay constitutes a versatile high throughput technique and the IM 50 values of the inhibitors were obtained with satisfactory reproducibility, suggesting that this inkjet-printing BACE-1 inhibitory assay could be quite suitable for the screening of new potential BACE-1 inhibitors for AD. [ABSTRACT FROM AUTHOR]

A peptide probe-based MMP-9 inhibitory assay was developed to assess anticancer activity of compounds on parchment paper using FRET-based inkjet printing. The fluorescent peptide containing 5-FAM (donor) and QXL520 (acceptor) was used as a substrate with the sequence of Pro-Leu-Gly-Cys-His-Ala-Arg-Lys specific to MMP-9. The suitability of the peptide as a substrate of MMP-9 was confirmed by HPLC. When both MMP-9 and the peptide were printed on the identical reaction spot of parchment paper, the peptide was cleaved and fragmented by MMP-9. The fluorescence was then recovered by 5-FAM and detected at Ex/Em = 490 ± 20 nm/520 ± 20 nm. Based on the successful performance of FRET on the parchment paper without any surface treatment, the assay platform was applied to test compounds, and a successful anticancer assay could be accomplished. This methodology holds notable strengths in versatile applications to the MMP enzyme family, as well as control of ejection volume, reduction of consumed enzyme amount, and rapid reaction time. Hence, we suggest the FRET peptide-based assay using inkjet printing as a viable replacement for sensitive and high-throughput evaluation of anticancer compounds. [ABSTRACT FROM AUTHOR]

Rapid and sensitive detection of botulinum neurotoxins (BoNTs), which cause botulism, is essential in a public health emergency or bioterrorism event. We have previously developed a mass spectrometry (MS)-based functional method, Endopep-MS assay, for the fast detection and differentiation of all BoNT serotypes by affinity enriching the toxin and detecting the serotype-specific cleavage products of peptide substrates derived from the in vivo targets. To improve the performance of the Endopep-MS assay, we report here the further optimization of the peptide substrate for the detection of serotype A botulinum neurotoxins. An increased substrate cleavage was achieved by extending the original peptide N-terminus with optimized amino acid sequence, increasing the detection sensitivity of the method. In addition, the resistance of the substrate to nonspecific hydrolysis was dramatically improved by selectively substituting amino acids at the scissile bond and various other positions of the extended peptide. Moreover, incorporating the N-terminal hydrophobic residues dramatically improved the relative intensity of the cleavage products in the mass spectra. This allowed easy detection of the cleavage products, further enhancing the performance of the assay. The limit of detection for spiked serum sample was enhanced from 0.5 to 0.1 mouseLD and from 0.5 to 0.2 mouseLD for spiked stool. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Endopeptidase-mass-spectrometry (Endopep-MS) is used as a specific and rapid in - vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific-peptide-substrate, and the cleavage products are then detected by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type A (BoNT/A). Our strategy was based on reported BoNT/A-SNAP-25 interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate led to a 10-fold increase in the assay sensitivity both in buffer and in clinically relevant samples. [ABSTRACT FROM AUTHOR]

This manuscript reports on a novel substrate for the amperometric detection of cellular apoptosis, Asp-Glu-Val-Asp- p-methoxyaniline (DEVD- pMA). This substrate showed electrochemical inertia from −0.2 to 0.7 V versus Ag/AgCl in phosphate-buffered saline (PBS), but was cleaved by caspase-3, releasing pMA, which was detected with cyclic voltammetry and chronoamperometry. Compared with p-aminophenol ( pAP), pMA showed lower toxicity to the human hepatocellular carcinoma cell line (HepG2) and higher stability in PBS. By using this substrate, we successfully detected caspase-3 in the range of 0.31-2.5×10−2 units/mL and cellular apoptosis in HepG2 cells by using simple amperometry without cell lysis. This work will help to develop a simple method for the electrochemical monitoring of apoptosis, which can be applied in biological research and drug development. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase-9 (MMP-9) is a core protease of cancer invasion and metastasis. In this paper, we report an electrochemical biosensor integrated with a redox reporter labeled peptide for the detection of MMP-9. The peptide substrate conjugated with methylene blue (MB) specific to MMP-9 was immobilized on Au electrodes using a thiol group in cysteine. The peptide cleavage by MMP-9 caused the release of electroactive MB from the electrodes and resulted in the decrease of the electrical tunneling current. The concentric electrode device consisted of an island electrode and an enclosing electrode which is ca. 1000 folds larger than the island electrode. The self-gating effect from the concentric electrode device was able to provide a reliable electrical measurement without a reference electrode. From the electrochemical biosensor, a linear correlation between the concentration of MMP-9 and the electrical signal reduction was found in a range from 1 pM to 1 nM and the limit of detection was determined at 7 pM. [ABSTRACT FROM AUTHOR]

In this work we report the detection of plasmin protease by means of the thickness shear mode (TSM) acoustic method. The biorecognition element consists of a peptide substrate (PS) specific to plasmin immobilized on a piezoelectric quartz crystal electrode. After enzymatic reaction with plasmin, it cleaves a short fragment of the peptide causing increase in the resonance frequency of the piezo crystal. Plasmin was detected in the range of concentrations 1–20 nM, a target interval in which its presence presumably affects the quality of milk. The PS exhibited negligible response against to similar protease trypsin. This has been confirmed also by electrochemical detection method. Limit of detection of this acoustic transducer was found to be 0.65 nM. Formation of the sensing surface and kinetic effect of plasmin on the peptide substrate was studied by atomic force microscopy (AFM). The PS response was also validated in pretreated milk samples spiked by known concentrations of plasmin achieving an average recovery of 63 ± 0.6%. [ABSTRACT FROM AUTHOR]

We presented a strategy for fabricating graphene oxide (GO)-based fluorescent biosensors to monitor the change of phosphorylation state and detect phosphatase activity. By regulating the interaction between the negatively charged phosphate group and the positively charged amino residue, we found that GO showed different quenching efficiency toward the phosphorylated and dephosphorylated dye-labeled peptides. To demonstrate the application of our method, alkaline phosphatase (ALP) was tested as a model enzyme with phosphorylated fluorescein isothiocyanate (FITC)-labeled short peptide FITC-Gly-Gly-Gly-Tyr(PO32-)-Arg as the probe. When the negatively charged phosphate group in the Tyr residue was removed from the peptide substrate by enzymatic hydrolysis, the resulting FITC-Gly-Gly-Gly-Tyr-Arg was readily adsorbed onto the GO surface through electrostatic interaction. As a result, fluorescence quenching was observed. Furthermore, the method was applied for the screening of phosphatase inhibitors. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase–mass spectrometry (Endopep–MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep–MS assay. Our strategy was based on reported BoNT/B–substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum. [ABSTRACT FROM AUTHOR]

We report a comparative analysis of plasmin detection by means of electrochemical biosensor and photometric assays. The biosensor has been based on ferrocene-modified peptide (Fc-P) specific to plasmin. Fc-P substrate is enzymatically cleaved by plasmin and the short fragment of peptide contained Fc is released causing decrease of redox current measured by cyclic voltammetry. The biosensor was tested in the range 1-20 nM of freshly prepared plasmin and it was highly selective ( LOD=0.56±0.03 nM) demonstrating lower reaction to other proteins such as thrombin and β-casein. The concentration of plasmin has been determined also by a photometric analysis based on UV-vis spectroscopy ( LOD=3.68±0.04 nM). The assay has been validated in milk samples. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD 50 /ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples. [ABSTRACT FROM AUTHOR]

Highlights: [•] GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions. [•] The consensus phosphorylation site motifs for GRK2 are (D/E)X1–3(S/T), (D/E)X1–3(S/T)(D/E), or (D/E)X0–2(D/E)(S/T). [•] Among 17 peptide substrates, a peptide fragment of β-tubulin (DEMEFTEAESNMN) shows the highest affinity for GRK2. [Copyright &y& Elsevier]

Highlights: [•] Myristoylated peptides derived from amino acid residues 2–19 of the preS1 domain selectively recognized human hepatocellular carcinoma cells. [•] Non-myristoylated peptides showed much lower affinity for human hepatocellular carcinoma cells than myristoylated peptides. [•] A modified peptide exhibited the highest affinity for human hepatocellular carcinoma cells. [ABSTRACT FROM AUTHOR]