WO2016064343A1 - Conjugués de détection de protéase membranaire et leur utilisation - Google Patents

Conjugués de détection de protéase membranaire et leur utilisation Download PDF

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WO2016064343A1
WO2016064343A1 PCT/SG2015/050347 SG2015050347W WO2016064343A1 WO 2016064343 A1 WO2016064343 A1 WO 2016064343A1 SG 2015050347 W SG2015050347 W SG 2015050347W WO 2016064343 A1 WO2016064343 A1 WO 2016064343A1
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protease
compound
moiety
cleavage site
amino acid
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PCT/SG2015/050347
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English (en)
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Bengang Xing
Mu JING
Fang Liu
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Nanyang Technological University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention lies in the field of biochemistry and relates to protease detection conjugates. Further, the present invention relates to manufacturing methods of the conjugates of the present invention, their use to monitor target protease activity, methods of screening a compound library and kits comprising the conjugate.
  • Cell surface proteolytic enzymes play critical roles in physiological and pathological processes ranging from extracellular matrix processing, growth factors and receptors' activation to microbial invasion, etc. These proteolytic events not only have been implicated in the site specific cleavage of bioactive proteins or peptide substrates within transmembrane domains, thus performing various biological functions, they are also involved in the progression of various degenerative diseases including cancer, atherosclerosis and neurological disorder. Therefore, diagnostic targeting and regulation of cell surface proteolytic enzymes becomes one promising approach to understand the basic pathological pathways for cancer and other diseases and to establish new treatment methods.
  • Furin a membrane localized proteolytic processing enzyme that belongs to the pro-protein convertase (PC) family, is ubiquitously expressed and functions within the secretory and endocytic pathways and at the cell surface.
  • the furin-like convertases can activate a variety of protein precursors and pro-proteins in the intracellular membrane and on the cell surface by processing them into biologically functional peptides and proteins.
  • furin has been well-known to be involved in the intra-membrane processing of several kinds of matrix metalloproteinases (MMPs), especially the MMP-2 enzyme which was found elevated in several types of human cancers.
  • MMPs matrix metalloproteinases
  • a-secretase and ⁇ -secretase which are two key enzymes in the processing of toxic amyloidal peptide generation during the development of Alzheimer's disease (AD)
  • AD Alzheimer's disease
  • the processing of cell-surface associated furin or furin-like proprotein convertases is highly relevant for the maturation of bacterial toxins and the propagation of many viral pathogens. These processes are prerequisites for the bacterial or viral invasion into the host cell.
  • fluorescent bio-reporters such as green fluorescence protein (GFP) labelling or genetically encoded fluorescent protein variants have been used to image the extracellular furin activities.
  • GFP green fluorescence protein
  • Similar non-invasive investigation of cell-surface proteolytic furin activity was also achieved in living cells on the basis of reengineered fusions of the anthrax toxin-P-lactamase and mutants of furin with the altered protease cleavage specificity.
  • these genetic manipulations may potentially perturb the cell's physiology and cause unexpected biological responses.
  • small-molecule-based probes which are easy to prepare and manipulate do not cause such physiological perturbations.
  • most of the current small-molecule-based reporters for furin-like assays e.g. Boc-RVRR-AMC, etc.
  • the protease detection conjugates of the present invention can be bound to cellular membranes without further chemical or genetic assistance and that the membrane-bound conjugate can be processed by a given target protease to release a previously retained fluorescence signal.
  • the fluorescence signal is directly correlated with the activity of the protease of interest. This direct correlation allows the monitoring of protease activity towards membrane-bound substrates in living cells that are not chemically fixed to their substrate or genetically modified.
  • the conjugates of the present invention allow the detection of signals of single substrates. And due to their minimal cytotoxicity to cells even after long time incubation, the conjugates of the present invention can be used for real-time visualization of protease activities without affecting physiological processes.
  • the conjugates of the invention comprise four parts: a lipophilic moiety that tethers the conjugate to a membrane-of-interest; an amino acid sequence which comprises a cleavage site of the protease of interest; a fluorescent reporter moiety and a modular moiety that absorbs the fluorescence signal of the reporter moiety in a distance-dependent manner.
  • the amino acid sequence comprising the cleavage site is arranged between the fluorescent reporter moiety and the modular moiety, they are separated from each other by catalytic cleavage of the protease cleavage site.
  • the influence of the modulator moiety on the fluorescent reporter is lost, as the modulation is distance-dependent.
  • the fluorescence signal of the reporter moiety can be measured by techniques well-known in the art.
  • the present invention is directed to a protease detection conjugate comprising or consisting of a lipophilic moiety that can insert into a membrane and tether the detection conjugate to said membrane, an amino acid sequence comprising a protease cleavage site, a fluorescent reporter moiety and a modulator moiety that can modulate the fluorescence of the reporter moiety, wherein the amino acid sequence is arranged between the fluorescent reporter moiety and the modulator moiety such that upon cleavage of the amino acid sequence by the target protease the modulation of the modulator moiety on the fluorescent reporter moiety is terminated.
  • the present invention relates to a method of manufacturing a compound according to the present invention, wherein the amino acid sequence comprising the protease cleavage site comprises an N-terminal lysine residue and a C-terminal lysine residue, comprising: (a) synthesizing the amino acid sequence comprising the protease cleavage site by solid phase peptide synthesis; (b) reacting the compound of step (a) with an unbranched carboxylic acid comprising at least seven carbon atoms to conjugate the carboxylic acid to the ⁇ -amino group of the N-terminal lysine residue; (c) reacting the compound of step (b) with the fluorescent reporter moiety to conjugate the fluorescent reporter moiety to the terminal amino group of the amino acid sequence comprising the protease cleavage site and (d) reacting the compound of step (c) with the fluorescent modulator moiety to conjugate the fluorescent modulator moiety to the ⁇ -amino group of the C
  • the present invention relates to a method of manufacturing a compound according to present invention, wherein the amino acid sequence comprising the protease cleavage site comprises a cysteine residue and a C-terminal lysine residue, comprising: (a) synthesizing the amino acid sequence comprising the protease cleavage site by solid phase peptide synthesis; (b) reacting the compound of step (a) with the fluorescent reporter moiety to conjugate the fluorescent reporter moiety to the terminal amino group of the amino acid sequence comprising the protease cleavage site; (c) reacting the compound of step (b) with the fluorescent modulator moiety to conjugate the fluorescent modulator moiety to the ⁇ -amino group of the C-terminal lysine residue of the sequence comprising the protease cleavage site and (d) reacting the compound of step (c) with l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-2-[
  • the present invention is directed to the use of the compound of the present invention to monitor the activity of a target protease.
  • the invention is directed to a method of screening a compound library, comprising: contacting cells or cell lysate with the compound according to the present invention; contacting said cells or cell lysate with at least one compound from a compound library; detecting the target protease activity of the cells or cell lysate treated with the compound from the compound library; comparing the detected protease activity with the protease activity of a control sample that has not been contacted with the compound from the compound library and determining based on the comparison of the protease activity if the compound of the compound library is a target protease inhibitor, a target protease activator or does not influence target protease activity.
  • the invention is relates to a kit for monitoring protease activity, comprising the compound according to the present invention and instructions to monitor protease activity.
  • Figure 1 shows an illustration of the structure of a membrane- anchored and furin-responsive probe (MFP) and the surface associated furin cleavage on the cell membranes.
  • MFP membrane- anchored and furin-responsive probe
  • FIG. 2 shows the synthesis of a membrane-anchored and furin-responsive probe (MFP).
  • MFP membrane-anchored and furin-responsive probe
  • Figure 3 shows a) UV absorbance spectra of FITC (in black) and MFP (in grey); b) Fluorescence Intensity of FITC (in black) and probe (in grey).
  • Figure 5 shows fluorescence imaging of U251 cells detecting MFP.
  • Living cells were incubated with MFP alone (130 nM), MFP and inhibitor (40 ⁇ ) or control probe FP (130 nM) for 15 min at 37 °C.
  • Cells stained with CellMask Deep Red (2.5 ⁇ / ⁇ ) were used as standard to image the membrane. The relative fluorescence intensity was plotted as a function of distance along the indicated arrows. Lovo cells incubated with MFP as control.
  • Figure 6 shows time-lapse imaging of U251 cells after 2 min incubation with MFP (260 nM) and subsequent washing procedure. Bottom was the normalized fluorescence intensity values plotted over time.
  • Figure 8 shows fluorescence imaging of Lovo cells and U251 cells after incubation with high concentrations of MFP (1 ⁇ ) for 30 min at 37 °C.
  • Figure 9 shows A) fluorescence signals of MFP (10 ⁇ ) for quantification of the furin activity in U251 and Lovo cell lysates (10 ⁇ g total protein was incubated with MFP at 37 °C). The inhibitor Dec-RVKR-cmk (100 ⁇ ) was added in U251 cell lysates for 30 min prior to the probe addition; B) Analysis of furin expression by western blot in U251 and Lovo cells and ⁇ -tubulin was included as internal control.
  • Figure 11 shows fluorescence live-cell imaging of HEK 293 cells after incubation with higher concentration of MFP (2.5 ⁇ ) for 2 hrs at 37 °C. (FITC: green; Lysotracker, red; H33258, blue).
  • Figure 12 shows two-photon fluorescence imaging in U251 cell lines and mouse ear tissue after incubation with MFP.
  • Figure 14 shows fluorescence signal of Atto-MFP (1 nM) in HEK293 cells by using wide field microscopy set-up from 10 s to 200s, each image was taken at 10s intervals.
  • FIG 15 shows an illustration of human serum albumin (HAS) coated FRET reporters for real-time imaging of furin activity in the Golgi apparatus of cancer cells.
  • HAS human serum albumin
  • Figure 16 shows an illustration of the synthesis of a lipid-probe of the invention.
  • conjugates as described herein allow the robust and efficient detection of protease activity.
  • conjugates of the present invention are integrated into cellular membranes without the use of further chemical or physical support, they can be used as a mimetic for membrane -bound substrates of proteases ( Figure 1).
  • the conjugates of the present invention allow the measurement of a protease-of-interest activity in living, non-fixed cells under physiological conditions and without genetic manipulation of the investigated cells.
  • the conjugates of the present invention can be used for live-cell imaging and to monitor single substrate turnover.
  • the conjugates of the present invention comprise four parts, wherein a lipophilic moiety tethers the conjugate to the membrane.
  • An amino acid sequence comprises a protease cleavage site and is located between a fluorescence reporter moiety and a modulator moiety that absorbs the fluorescence signal of the reporter moiety in a distance-dependent manner.
  • the fluorescence reporter moiety and the modulator moiety are separated and the modulation is terminated.
  • the fluorescence signal of the reporter can be detected. This signal correlates with the activity of the protease- of-interest and can thus be used to determine catalytic parameters, such as K cat and K m , of the protease-of-interest.
  • the present invention is directed to a protease detection conjugate comprising or consisting of a lipophilic moiety that can insert into a membrane and tether the detection conjugate to said membrane, an amino acid sequence comprising a protease cleavage site, a fluorescent reporter moiety and a modulator moiety that can modulate the fluorescence of the reporter moiety, wherein the amino acid sequence is arranged between the fluorescent reporter moiety and the modulator moiety such that upon cleavage of the amino acid sequence by the target protease the modulation of the modulator moiety on the fluorescent reporter moiety is terminated.
  • protease refers to an agent that cleaves a peptide bond between sequential amino acids in a polypeptide chain.
  • the protease catalyzes the hydrolysis of peptide bonds.
  • the protease is an enzyme
  • the protease is a protein (i.e. a protein enzyme) comprising one or more polypeptide chains.
  • Proteases are generally identified by their catalytic type, e.g., aspartic acid peptidases, cysteine (thiol) peptidases, metallopeptidases, serine peptidases, threonine peptidases, alkaline or semi-alkaline proteases, neutral and peptidases of unknown catalytic mechanism.
  • Non-limiting examples of proteases suitable as proteases-of-interest in the present invention include serine proteases, threonine proteases, cysteine proteases, aspartic acid proteases, metalloproteases and glutamic acid proteases.
  • proteases suitable for use in the present invention include, but are not limited to animal proteases, bacterial proteases, fungal proteases, plant proteases, recombinant proteases (e.g., produced via recombinant DNA technology by a suitable host cell, selected from any one of bacteria, yeast, fungi, plant, insect or mammalian host cells in culture, or recombinant proteases, which include an amino acid sequence that is homologous or substantially identical to a naturally occurring sequence, proteases encoded by a nucleic acid that is homologous or substantially identical to a naturally occurring protease-encoding nucleic acid, etc.), chemically modified proteases, and mixtures thereof.
  • suitable host cell selected from any one of bacteria, yeast, fungi, plant, insect or mammalian host cells in culture, or recombinant proteases, which include an amino acid sequence that is homologous or substantially identical to a naturally occurring sequence, proteases encoded by a nucleic acid that is
  • the protease cleavage site is the cleavage site of a membrane -bound protease.
  • Membrane-bound protease broadly refers to a target protease being present on or in a cell membrane or the membrane of intracellular organelles (for example, endoplasmic reticulum and Golgi apparatus).
  • Proteases that are membrane -bound include, but are not limited to, disintegrin and metalloproteinase (ADAM), matrix metalloproteinases (MMPs), type II transmembrane serine proteases (TTSPs) and furin.
  • the protease cleavage site is a proprotein convertase cleavage site.
  • Proprotein convertases are a family of proteins that activate other proteins.
  • proprotein convertases remove those chains and activate the protein.
  • the prototypical proprotein convertase is furin.
  • Proprotein convertases have medical significance, because they are involved in many important biological processes, such as cholesterol synthesis.
  • Many proprotein convertases, especially furin and PACE4 are involved in pathological processes such as viral infection, inflammation, hypercholesterolemia, and cancer, and have been postulated as therapeutic targets for some of these diseases.
  • Other proprotein convertases, aside furin, include proprotein contervase PCSK
  • furin refers to a protein that in humans is encoded by the FURIN gene.
  • the reference sequence numbers of human furin are RefSeq (mRNA) NM_001289823 for mRNA and
  • RefSeq protein NP_001276752 for the protein.
  • the reference sequence numbers are RefSeq (mRNA) NM_001081454 for mRNA and RefSeq (protein) NP_001074923 for the protein.
  • Some proteins are inactive when they are first synthesized, and must have sections deleted in order to become active. Furin deletes these sections and activates the proteins. It was named furin because it was in the upstream region of an oncogene known as FES. The gene was known as FUR (FES Upstream Region) and therefore the protein was named furin. Furin is also known as PACE (Paired basic Amino acid Cleaving Enzyme).
  • Furin cleavage sequences that can be incorporated in the amino acid comprising the protease cleavage site comprise the sequence of (K/R)(X) n (K/R), wherein K is lysine, R is arginine, X is any amino acid and n is 0, 2, 4 or 6; the sequence of (R)(X)(K/R)(R), wherein K is lysine, R is arginine and X is any amino acid and the sequence of (R)(V)(R)(R), wherein R is arginine and V is valine.
  • the protease of interest has at least one membrane -bound substrate.
  • lipophilic moiety refers to the ability of said chemical moiety to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. Further, it refers to a moiety that can tether the detection conjugate of the present invention to a membrane.
  • the lipophilic moiety tethers the protease detection conjugate to the cell membrane, nuclear membrane, Golgi membrane, endoplasmatic reticulum membrane or mitochondrial membrane.
  • the lipophilic moiety includes functional groups which associate with or bind to a cell membrane. Thus, the lipophilic moiety brings the substance to which this moiety is attached in close proximity to the membrane of a target cell.
  • the cell membrane is eukaryotic or prokaryotic.
  • the lipophilic moiety is desirably a hydrophobic moiety. This moiety can include a mixed sequence peptide or a homopolymer peptide such as poly leucine or poly arginine less than 10 amino acids long.
  • the lipophilic moiety may be attached to the C-terminal amino acid, the N- terminal amino acid, or to an amino acid between the N- terminal and C-terminal amino acid in the peptide.
  • the lipophilic moieties also include cholesterol, phospholipids, steroids, sphingosine, ceramide, octyl- glycine, 2-cyclohexylalanine, or benzolylphenylalanine.
  • Other lipophilic moieties include CI or C2 acyl groups, or a C3-C20 branched and non-branched fatty acid moiety such as propanoic acid (C3); butanoic acid (C4); pentanoic acid (C5); hexanoic acid
  • C6 heptanoic acid
  • C7 heptanoic acid
  • C8 nonanoic acid
  • decanoic acid CIO
  • undecanoic acid Cl l
  • dodecanoic acid CI 2
  • tridecanoic acid CI 3
  • the lipophilic moiety comprises at least 7 carbon atoms.
  • the fatty acid has 7 to 17 carbon atoms.
  • the lipophilic moiety is dodecanoyl, octadecanoyl or 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-2-[monomethoxy poly(ethylene glycol)] .
  • membrane refers to an enclosing or separating membrane that acts as a selectively permeable barrier within living organisms.
  • Biological membranes for example in the form of cell membranes, often consist of a phospholipid bilayer with embedded, integral and peripheral proteins used in communication and transportation of chemicals and ions. Bulk lipid in membrane provides a fluid matrix for proteins to rotate and laterally diffuse for physiological functioning.
  • fluorescent reporter moiety any reagent which possesses fluorescent properties, or which is conjugated to a fluorescent dye.
  • fluorescent dyes may be conjugated to different functional groups. Suitable functional groups for conjugation include, but are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups being particularly preferred.
  • fluorescent dyes containing amino groups can be attached to detection reagents containing amino groups using cross-linkers as are known in the art; for example, homo-or hetero-bifunctional cross- linkers as are well known.
  • Fluorophores of the fluorescent reporter moiety can be either "small molecule" fluors, or proteinaceous fluors (e.g. green fluorescent proteins and all variants thereof). Suitable fluorophores include, but are not limited to, l,l '-diethyl-2,2'-cyanine iodide, 1,2- diphenylacetylene, 1,4-diphenylbutadiene, 1,6-Diphenylhexatriene, 2-Methylbenzoxazole, 2,5-Diphenyloxazole (PPO), 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H- pyran (DCM), 4-Dimethylamino-4'-nitrostilbene, 4',6-Diamidino-2-phenylindole (DAPI), 5- ROX, 7-AAD, 7-Benzylamino-4-nitrobenz-2-oxa-l,3-diazole, 7-Methoxy
  • the fluorescent dye may be an Alexa Fluor® dye, including Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, and Alexa Fluor® 750 (Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, Calif. 92008).
  • Alexa Fluor® 350 Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alex
  • the fluorescent dye may be a tandem fluorophore conjugate, including Cy5-PE, Cy5.5-PE, Cy7-PE, Cy5.5-APC, Cy7-APC, Cy5.5-PerCP, Alexa Fluor® 610-PE, Alexa Fluor® 700- APC, and Texas Red-PE. Tandem conjugates are less stable than monomeric fluorophores, so comparing a detection reagent labeled with a tandem conjugate to reference solutions may yield MESF calibration constants with less precision than if a monomeric fluorophore had been used.
  • the fluorescent dye may be a fluorescent protein such as green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech— Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6: 178-182 (1996)), cyan fluorescent protein (CFP), and enhanced yellow fluorescent protein (EYFP; 1.
  • GFP green fluorescent protein
  • EGFP blue fluorescent protein
  • CFP cyan fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • the fluorescent dye is dTomato, FlAsH, mBanana, mCherry, mHoneydew, mOrange, mPlum, mStrawberry, mTangerine, ReAsH, Sapphire, mKO, mCitrine, Cerulean, Ypet, tdTomato, Emerald, or T- Sapphire (Shaner et al., Nature Methods, 2(12):905-9. (2005)).
  • the fluorescent dye may be a fluorescent semiconductor nanocrystal particle, or quantum dot, including Qdot® 525 nanocrystals, Qdot® 565 nanocrystals, Qdot® 585 nanocrystals, Qdot® 605 nanocrystals, Qdot® 655 nanocrystals, Qdot® 705 nanocrystals, Qdot® 800 nanocrystals (Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, Calif. 92008).
  • the fluorescent dye may be an upconversion nanocrystal, as described in Wang et al., Chem. Soc. Rev., 38:976-989 (2009).
  • the fluorescent dye may be an ATTO 390 dye, ATTO 425 dye, ATTO 465 dye, ATTO 488 dye, ATTO 495 dye, ATTO 520 dye, ATTO 532 dye, ATTO 550 dye, ATTO 565 dye, ATTO 590 dye, ATTO 594 dye, ATTO 610 dye, ATTO 61 IX dye, ATTO 620 dye, ATTO 633 dye, ATTO 635 dye, ATTO 637 dye, ATTO 647 dye, ATTO 647N dye, ATTO 655 dye, ATTO 665 dye, ATTO 680 dye, ATTO 700 dye, ATTO 725 dye and ATTO 740 dye manufactured by ATTO-TEC GmbH (Siegen, Germany).
  • modulator moiety refers to a chemical group that can absorb emission energy of the fluorescent reporter moiety. As the modulator moiety absorbs emission energy of the fluorescent reporter moiety both chemical groups are connected by Forster resonance energy transfer or fluorescence resonance energy transfer (FRET), a mechanism describing energy transfer between two light-sensitive molecules (chromophores). Therefore, the fluorescent reporter moiety and the modulator moiety form a FRET pair.
  • FRET fluorescence resonance energy transfer
  • a donor chromophore or FRET donor initially in its electronic excited state, transfers energy to an acceptor chromophore or FRET acceptor through non-radiative dipole- dipole coupling.
  • the efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other.
  • modulation is terminated or modulation is lost refers to separation of the fluorescent reporter molecule and the modulator moiety (by cleavage of the protease cleavage site) and the "release" of the previously absorbed fluorescence signal of the reporter moiety. This allows the detection of the fluorescence signal of the reporter by methods well- known in the art.
  • the fluorescent reporter moiety is conjugated to the N- terminus of the amino acid sequence comprising the protease cleavage site or, when the N- terminal amino acid of the sequence comprising the protease cleavage site is a lysine, to the ⁇ - amino group of said lysine.
  • N-terminus refers to the amino acid residue which is the last residue in N-terminal direction of a peptide.
  • C- terminus refers to the amino acid residue which is the last residue in the C-terminal direction of a peptide.
  • the ⁇ -amino group of lysine is attached to the fifth carbon from the a-carbon, which is attached to the carboxyl group of the lysine.
  • the modulator moiety absorbs emission energy of the fluorescent reporter moiety. This means that fluorescent reporter moiety, in its electronic excited state, transfers energy to the modulator moiety through non- radiative dipole-dipole coupling.
  • the modulator moiety is a quencher.
  • quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on pressure and temperature. Molecular oxygen, iodide ions and acrylamide are common chemical quenchers.
  • the modulator moiety and the fluorescent reporter moiety form a FRET pair, wherein the modulator moiety fluorescent reporter moiety is the FRET donor and the modulator moiety is the FRET acceptor.
  • FRET is Forster resonance energy transfer or fluorescence resonance energy transfer.
  • the FRET donor is fluorescein isothiocyanate (FITC) or Atto647N.
  • FITC fluorescein isothiocyanate
  • Atto647N In various embodiments of the invention, the FRET acceptor is Dabcyl or BHQ3.
  • the conjugate of the invention has the structure of
  • R is arginine
  • V is valine
  • S is serine
  • FITC fluorescein
  • the conjugate of the invention has the structure of
  • R is arginine
  • V is valine
  • S is serine
  • the conjugate of the invention has the structure of
  • the present invention relates to a method of manufacturing a compound according to the present invention, wherein the amino acid sequence comprising the protease cleavage site comprises an N-terminal lysine residue and a C-terminal lysine residue, comprising: (a) synthesizing the amino acid sequence comprising the protease cleavage site by solid phase peptide synthesis; (b) reacting the compound of step (a) with an unbranched carboxylic acid comprising at least seven carbon atoms to conjugate the carboxylic acid to the ⁇ -amino group of the N-terminal lysine residue; (c) reacting the compound of step (b) with the fluorescent reporter moiety to conjugate the fluorescent reporter moiety to the terminal amino group of the amino acid sequence comprising
  • the above method uses in step (a) diisopropylethylamin and dichloromethane, in step (b) N,N,N',N'-Tetramethyl-0-(lH- benzotriazol-l-yl)uronium hexafluorophosphate/ diisopropylethylamin and dichloromethane, in step (c) 2% hydrazine, dichloromethane, fluorescein isothiocyanate and diisopropylethylamin, and in step (d) diisopropylethylamin, 4-((4- (dimethylamino)phenyl)azo)benzoic acid succinimidyl ester, trifluoroacetic acid, water and triisopropylsilyl.
  • the present invention relates to a method of manufacturing a compound according to present invention, wherein the amino acid sequence comprising the protease cleavage site comprises a cysteine residue and a C-terminal lysine residue, comprising: (a) synthesizing the amino acid sequence comprising the protease cleavage site by solid phase peptide synthesis; (b) reacting the compound of step (a) with the fluorescent reporter moiety to conjugate the fluorescent reporter moiety to the terminal amino group of the amino acid sequence comprising the protease cleavage site; (c) reacting the compound of step (b) with the fluorescent modulator moiety to conjugate the fluorescent modulator moiety to the ⁇ -amino group of the C-terminal lysine residue of the sequence comprising the protease cleavage site and (d) reacting the compound of step (c) with l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-2-[
  • the above method uses in step (a) diisopropylethylamin and dichloromethane, in step (b) fluorescein isothiocyanate, diisopropylethylamin, 2% hydrazine and 4-((4-(dimethylamino)phenyl)azo)benzoic acid succinimidyl ester, in step (c) trifluoroacetic acid and in step (d) l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-2-[monomethoxy poly(ethylene glycol)] 2-maleimidoethyl ether and dimethylformamide/dichloromethane.
  • the term “synthesizing”, as used herein, refers to a purposeful execution of chemical reactions to obtain a product, or several products. This happens by physical and chemical manipulations usually involving one or more reactions.
  • the term “reacting” as used with regard to the method of manufacturing the conjugate of the invention refers to contacting the educts under conditions that allow formation of the product. Exemplary reaction conditions are described herein.
  • solid phase peptide synthesis refers to a method of generating peptides or proteins, wherein small porous beads are treated with functional units (“linkers") on which peptide chains can be built.
  • the peptide will remain covalently attached to the bead until cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid.
  • a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid.
  • the peptide is thus "immobilized" on the solid-phase and can be retained during a filtration process while liquid-phase reagents and byproducts of synthesis are flushed away.
  • conjugate refers to a molecule comprising two or more chemical groups (e.g., peptides, carbohydrates, nanoparticles, small molecules, or nucleic acid molecules) that are linked.
  • the chemical groups are covalently coupled.
  • the two or more groups are chemically linked using any suitable chemical bond (e.g., covalent bond).
  • suitable chemical bonds are well known in the art and include disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds (e.g. peptide bonds), thioether, and esterase labile bonds.
  • the present invention is directed to the use of the compound of the present invention to monitor the activity of a target protease.
  • Monitoring protease activity or “detecting protease activity”, as interchangeably used herein, relate to quantitatively or qualitatively identifying the catalytic turn-over of a protease-of-interest.
  • protease activity refers to the ability of a given protease to act as a catalyst in a process, such as the conversion of one compound to another compound.
  • desired activity can include the activity of one or more enzymes being actively expressed in one cell.
  • target protease or “protease-of-interest”, as interchangeably used herein, is furin.
  • the compound of the invention is coated by human serum albumin (HSA).
  • HSA human serum albumin
  • the components are human serum albumin and the conjugates of the invention. This includes also the situation where the individual polypeptide complex components, e.g. human serum albumin, are synthesized or recombinantly expressed and subsequently isolated and combined with other components to form a complex, in vitro and prior to administration to a subject.
  • the polypeptide-conjugate complexes are added to nanoparticles to yield nanoparticles with adsorbed or coupled polypeptide complexes having a ratio of number of molecules:number of nanoparticle ratios from about, at least about or at most about 0.1, 0.5, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500 or more to: l, more typically 0.1: 1, 1: 1 to 50: 1 or 300: 1.
  • the ratio of the number of human serum albumin/conjugate molecules to the number of nanoparticles is from about 10: 1 to about 1000: 1.
  • human serum albumin refers to the protein having the reference sequence number RefSeq NM_000477.
  • the protease activity is monitored in a sample from a patient.
  • the sample is a biological sample, for example a body fluid, cell or tissue sample.
  • Body fluids comprise, but are not limited to blood, blood plasma, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), endolymph and perilymph, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, tears, vaginal secretion, nipple aspirate fluid, vomit and urine.
  • the cell or tissue sample may comprise material originated from any part of the body such as connective tissue, muscle tissue, nervous tissue, and epithelial tissue.
  • obtaining a sample relates to different methods known in the art that comprise, but not limited to, biopsy, sentinel node biopsy or removal of blood, bone marrow, sputum or bronchial fluids.
  • patient as used herein, is to be interpreted broadly to include elderly persons, persons actively being treated or monitored for specific medical ailments, as well as persons who wish to have their general medical condition monitored by health practitioners, or animals. [00061]
  • the patient is a cancer patient, atherosclerosis patient or neurological disorder patient.
  • the neurological disorder patient is a Alzheimer's disease (AD) patient.
  • AD Alzheimer's disease
  • neurological disorder refers to a disease or disorder of the nervous system, sometimes involving the brain, that manifests with symptoms characteristic of brain or nerve dysfunction, e.g., short-term or long-term memory lapse or defects, dementia, cognition defects, balance and coordination problems, and emotional and behavioral deficiencies and tremor.
  • Non-limiting examples of neurological disorders include as Parkinson's disease, Essential tremor, Huntington's disease, Alzheimer's disease, Multiple sclerosis and organic psychosis.
  • Alzheimer's disease is a chronic neurodegenerative disease that usually starts slowly and gets worse over time. The most common early symptom is difficulty in remembering recent events (short-term memory loss).
  • symptoms can include problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, not managing self-care, and behavioral issues. Gradually, bodily functions are lost, ultimately leading to death. Although the speed of progression can vary, the average life expectancy following diagnosis is three to nine years.
  • arteriosclerosis refers to a specific form of arteriosclerosis in which an artery wall thickens as a result of invasion and accumulation of white blood cells (WBCs)(foam cell) and proliferation of intimal smooth muscle cell creating a fibrofatty plaque.
  • WBCs white blood cells
  • cancer refers to diseases caused by uncontrolled cell division and the ability of cells to metastasize, or to establish new growth in additional sites.
  • malignant refers to cancerous cells or groups of cancerous cells. Exemplary cancers include: carcinoma, melanoma, sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma.
  • cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases.
  • lung cancer including small-cell lung cancer, non- small cell lung cancer, adenocarcino
  • Carcinoma includes all carcinomas and adenocarcinomas present in humans.
  • Carcinoma is a type of cancer that develops from epithelial cells.
  • a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that generally arises from cells originating in the endodermal or ectodermal germ layer during embryogenesis.
  • the term "sarcoma”, as used herein, is a cancer that arises from transformed cells in one of a number of tissues that develop from embryonic mesoderm.
  • sarcomas include tumors of bone, cartilage, fat, muscle, vascular, and hematopoietic tissues.
  • osteosarcoma arises from bone
  • chondrosarcoma arises from cartilage
  • liposarcoma arises from fat
  • leiomyosarcoma arises from smooth muscle.
  • the protease activity is monitored in real-time. In still various embodiments, the protease activity is monitored in living cells. This means that the investigated cells are not fixed or permeabilized and still proceed with metabolic processes, such as the cell cycle.
  • the fluorescence signal of the conjugate of the invention can be monitored directly without time delay on a read-out device, such as fluorescence microscopy.
  • the invention relates to a method of screening a compound library, comprising: contacting cells or cell lysate with the compound according to claims 1- 19; contacting said cells or cell lysate with at least one compound from a compound library; detecting the target protease activity of the cells or cell lysate treated with the compound from the compound library; comparing the detected protease activity with the protease activity of a control sample that has not been contacted with the compound from the compound library and determining based on the comparison of the protease activity if the compound of the compound library is a target protease inhibitor, a target protease activator or does not influence target protease activity.
  • screening refers to means finding out a certain property, including susceptibility to or activity on a certain compound, such as an enzyme, etc. In various embodiments, this property is the ability of a given compound to inhibit or to activate the catalytic activity of a given target protease. In various preferred embodiments, the target protease is furin.
  • compound library refers to any collection of agents that includes a plurality of molecular structures. Compound libraries can include, for example, combinatorial chemical libraries, natural products libraries, peptide libraries, and aptamer libraries. In certain embodiments, peptide and aptamer libraries can be generated by transcription and translation from nucleic acid sequences included within a genetic library.
  • contacting refers generally to providing access of one component, reagent, analyte or sample to another.
  • contacting can involve mixing a solution comprising a small-molecule compound or another member of a compound library with living cells.
  • This solution comprising one component, reagent, analyte or sample may also comprise another component or reagent, such as dimethyl sulfoxide (DMSO), a detergent or human serum albumin, which facilitates mixing, interaction, uptake, or other physical or chemical phenomenon advantageous to the contact between components, reagents, analytes and/or samples.
  • DMSO dimethyl sulfoxide
  • a detergent or human serum albumin a detergent or human serum albumin
  • cell refers to eukaryotic or prokaryotic cells. In preferred embodiments the cells are eukaryotic cells.
  • eukaryotic cell includes nucleated cells and naturally occurring membrane-enclosed ATP-containing bodies of eukaryotic origin without nuclei, such as platelets, that are suspected to be contained in a fluid.
  • Cell lysate refers to a fluid containing the contents of lysed cells. Cell ly sates may be whole cell lysates and/or crude (unpurified) cell ly sates. In some embodiments, cell lysates may be partially purified (e.g.
  • cell lysates are prepared by high pressure lysis, thereby forming intracellular membrane vesicles that enable oxidative phosphorylation in the cell lysates.
  • Other methods of preparing cell lysate include, without limitation, sonication, homogenization, enzymatic lysis using lysozyme, and freezing and grinding.
  • the term "detecting”, as used herein, refers to a quantitative rather than a merely qualitative analysis.
  • comparing encompasses comparing the amount of a target protease activity comprised by the sample to be analyzed with an amount of a suitable reference source specified below in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample.
  • control sample or “control”, as interchangeably used herein, means that any treatment with chemicals from a compound library or symptoms and/or complications that may relate to a specific disease that are investigated by the methods of the present application are absent in said control cells or control cell lysate.
  • the health status of the control cells or individuals may have been determined by tests known in the art.
  • inhibitor refers to any small molecule, peptide or aptamer that binds one or more subunit of a protease of interest, thereby inhibiting the ability of said protease to hydrolyze the cleavage site(s) in at least one of its substrates.
  • activator refers to any small molecule, peptide or aptamer that binds one or more subunit of a protease of interest, thereby enhancing the ability of said protease to hydrolyze the cleavage site(s) in at least one of its substrates.
  • kits for monitoring protease activity comprising the compound according to any one of claims 1-19 and instructions to monitor protease activity.
  • the kit further comprises human serum albumin (HSA).
  • HSA human serum albumin
  • kits of the invention comprise the conjugates of the present invention.
  • such a kit may comprise instructions for use as well as typical reagents known to those skilled in the art. For example, the exact number of reaction tubes, their holders, etc. can be determined by the skilled person.
  • covalently coupled relates to a chemical bond that involves the sharing of electron pairs between atoms.
  • the stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding.
  • At least seven relates to seven or more, in particular 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.
  • Fluorescence spectroscopy is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light.
  • a complementary technique is absorption spectroscopy.
  • Devices that measure fluorescence are called fluorometers or fluorimeters.
  • a fluoro meter generates the wavelength of light required to excite the analyte of interest; it selectively transmits the wavelength of light emitted, then it measures the intensity of the emitted light.
  • Fluorometers employ monochromators (a spectrofluorometer), optical filters (a filter fluorometer), or narrow band light sources like LED's or lasers to select excitation and emission wavelengths.
  • monochromators a spectrofluorometer
  • optical filters a filter fluorometer
  • narrow band light sources like LED's or lasers to select excitation and emission wavelengths.
  • Well-known manufacturer of fluorometers include PerkinElmer, Horiba Scientific, Jenway, Berthold Technologies, Thermo Fisher Scientific, Ocean Optics, Promega, Walchem, Eppendorf and Agilent Scientific.
  • Furin was purchased from Biolabs (2000 U mL -1 ) wherein one unit releases 1 pmol of AMC from the fluorogenic peptide Boc-RVRR-MCA( ITS Science & Medical Pte Ltd) per minute at 30°C.
  • the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-cmk) was purchased from Bachem.
  • Mass spectra were measured with a Thermo LCQ Deca XP Max for ESI.
  • Analytical reverse-phase HPLC analysis was performed on a Shimadzu HPLC system with an Alltima C-18 (250 x 10 mm) column at a flow rate of 3.0 mL / min.
  • UV-Vis absorption spectra were recorded in a 10-mm path quartz cell on Cary 60 UV-Vis Spectrophotometer. Fluorescence emission spectra were performed on a Varian Cary eclipse fluorescence spectrometer.
  • Two-photon images were acquired on A TriM Scope II single- beam two-photon microscope (La Vision BioTec) with a laser (Coherent Chameleon Ultra II One Box Ti:sapphire) and water-dipping objectives (20x, Olympus).
  • the fluorescent signals were detected with a filter at 525/50 BP (Chroma, cat. no. ET525/50m).
  • the peptide sequence was synthesized by Fmoc based solid phase peptide synthesis (SPPS) on a 2-Chlorotrityl Chloride.
  • SPPS Fmoc based solid phase peptide synthesis
  • the coupling reaction of two amino acids lasted 2-4 hours at room temperature in the presence of HBTU as a coupling reagent and N,
  • DIPEA N-Diisopropylethylamine
  • the reaction was allowed to last for 4 hours for coupling with arginine and 2 hours for other amino acids.
  • the deprotection of Fmoc group was performed within 20% peperidine in Dimethyformamide (DMF) for 20 min.
  • the dodecanoic acid was coupled to the N-terminus of the peptide using the same SPPS protocol. Then the Dde protecting group was removed in 2% hydrazine in DMF for 20 min. Then FITC was reacted with the amino group of the lysine for 4h. Then the product was cleaved from resin using 2% trifluoroacetic acid TFA in Dichloromethane (DCM) for 10 minutes. The Mtt protecting group was also removed during this process. After removing DCM, the diethyl ether was added to obtain the precipitate.
  • DCM dichloromethane
  • control probe FP was synthesized following the synthesis of the furin- responsive probe MFP.
  • the prepared MFP (100 ⁇ ) was incubated in 0.15 mL of buffer or serum solution (Fetal Bovine Serum, Standard Quality solution, EU approved from GE Healthcare) for different time intervals at room temperature. At different time points, the mixture was re- suspended in MeOH (0.3 mL) and then centrifuged at 16,000 g for 2 min. The supernatant was analyzed by HPLC to evaluate the stability. There was no obvious MFP probe degradation observed in PBS (for more than one week) and serum (for 24 hours) indicating the sufficient stability of MFP in vitro and in living cells.
  • buffer or serum solution Fetal Bovine Serum, Standard Quality solution, EU approved from GE Healthcare
  • U251 and Lovo cells were seeded at 35-mm diameter ⁇ -dish plastic-bottom (ibidi GmbH, Germany) in complete medium for 24 h incubation at 5% C0 2 and 37 °C. Then cells were incubated with the probe (130 nM) or probe together with the inhibitor Dec- RVKR-cmk (40 ⁇ ) for 15 min incubation. Subsequently, cells were incubated with CellMask Deep Red (2.5 ⁇ ⁇ ) for 15 min and washed three times with PBS. Live cell imaging was performed under confocal laser scanning microscope (CLSM) (Nikon, Eclipse TE2000-E).
  • CLSM confocal laser scanning microscope
  • ROI region of interest
  • the MFP or FP probes (6.5 ⁇ ) were incubated with pure furin enzyme for 120 min at 37°C in 0.1 mL assay buffer.
  • the assay buffer contains 100 mM HEPES, pH 7.4, 0.5 % Triton X-100, 1 mM CaCl 2 , and ImM 2-mercaptoethanol.
  • the emission spectrum ⁇ ⁇ 450 nm) was recorded over time.
  • HPLC condition for the analysis of products and enzyme hydrolysis is a linear 20 min gradient from 30% to 95% acetonitrile in water with 0.1% TFA.
  • HEK293 cells were seeded on 15 ⁇ -8 ⁇ 4 wells (ibidi GmbH, Germany) in complete medium for 24 h incubation at 5% C0 2 and 37°C. Then the medium was changed to fresh serum- free medium in the presence of 1 ⁇ C6-F, C12-F, and C18-F.
  • the quantum yield of MFP in PBS IX is about 44%.
  • the dye reagent was diluted for 5 times with distilled water before use. 20 ⁇ ⁇ of the tested sample was mixed with 200 ⁇ ⁇ of the diluted reagent in 96-well plates. After incubation at room temperature for 5 minutes, the absorbance at 595 nm was recorded by a Tecan' s Infinite M200 microplate reader. The 10 ⁇ g total protein was incubated with probe (10 ⁇ ) for lh. For the control experiment, the U251 supernatant was pre-incubated with the inhibitor Dec- RVKR-cmk (100 ⁇ ) for 30 min before the addition of the probe. Then the fluorescence was measured at 523 nm using the microplate reader.
  • the membrane was washed 3 times with PBST, then incubated with secondary antibody (Goat Anti-Mouse IgG (H + L)-HRP Conjugate #170-6516) (1: 10000 dilutions) for lh at RT and washed 3 times with PBST.
  • the samples were detected with LAS-4000 machine using ECL exposure buffer. After that, another 20 (the same amount as the above) of each sample was analyzed in 10% SDS-PAGE and transferred to 0.22 ⁇ PVDF membrane (100V, 110 min, 4°C).
  • the membrane was incubated with mouse ⁇ -tubulin antibody (1: 1000 dilutions) lh at RT, washed 3 times with PBST, then incubated with goat anti-mouse secondary antibody (1: 10000 dilutions).
  • Glioblastoma U251 Cells and Lovo cells were seeded in 96-well plates with a density of 10 5 cells per well. After 24 hours' incubation at 5% C0 2 and 37°C, the medium was replaced with fresh medium containing different concentrations of MFP. After 24 hours' incubation, the cytotoxicity was evaluated by the standard MTT assay. The absorbance at 570 nm was measured by a Tecan's Infinite M200 microplate reader.
  • Example 1 Synthesis and characterization of a membrane-anchored and fluorescence probe (MFP)
  • Example 2 In vitro furin enzyme assays [00093] Firstly, the enzyme hydrolysis of the developed MFP probe was analyzed (6.5 ⁇ ) by measuring the changes in fluorescence emission upon the addition of furin in HEPES buffer (0.1 M, pH 7.4). As shown in Figure 4A, the MFP probe itself was almost non- fluorescent due to efficient FRET quenching. After incubation with enzyme for 2 hours, intense fluorescent enhancement (-12 folds) was observed at a maximum wavelength of 525 nm, corresponding to the connected FITC. Similar enzyme hydrolysis with standard inhibitor (Dec-RVKR-cmk) demonstrated limited fluorescence enhancement after 2 hours incubation, suggesting that the specific furin interaction splits the FRET pair by releasing the quencher Dabcyl.
  • Example 3 Imaging and quantification of furin-like activity in live cells
  • the chemiluminescence signals shown in Figure 9B confirmed the highly expressed furin in U251 cell lines, whereas the same enzyme expression was greatly reduced in Lovo cell lines.
  • the different levels of furin expression in U251 and Lovo cells verified by immunoblotting further confirmed that the MFP probe could be used to reliably evaluate furin activities in different cell lines.
  • the potential cytotoxicity of the MFP probe was evaluated by using standard MTT assay. The results demonstrate that the MFP probe is not cytotoxic for both, U251 and Lovo cells, even after long incubation. This suggests that the MFP probe could be used in real-time visualization of furin activities without affecting physiological processes of the investigated cells.
  • C6-F caproic acid, lauric acid and stearic acid, respectively
  • This molecule was not conjugated to the quencher Dabcyl.
  • the FITC-peptide fatty acid conjugates abbreviated as C6-F, C12-F and C18-F (1 ⁇ ), respectively, were incubated with HEK 293 cells at 37 °C for different time intervals. As shown in Figure 10, there was almost no fluorescence observed in C6-F incubated HEK 293 cells compared to the cells treated with C12-F and C18-F. The results confirmed that the increased length of carbon chain enhances the immobilization of the probe onto the cell surface.
  • the MFP containing the dodecanoyl moiety is a better tracer to specifically localize the probe onto the cell membrane, and thus significantly facilitate long-time trackable visualization of surface associated furin in living cells.
  • Example 6 Two photon live cell and tissue imaging
  • Example 7 Single molecule reporters measure cell surface-associated furin activies
  • this system was modified to use a precise wide field microscopy set-up.
  • FITC was replaced by Atto647N in the MFP probe.
  • Atto647N is well-known for its strong absorption and high photostablity.
  • the quencher Dabcyl was replaced by BHQ3 ( Figure 13).
  • real time imaging of U251 cells by normal confocal microscopy was carried out as described above. As shown before, the fluorescence of the Atto-MFP probe was quenched before incubation with cells. However, after incubation with cells within a short time, fluorescence was recovered gradually. This turn-on fluorescence was mostly observed on the cell surface, which is consistent with the previous imaging results.
  • Example 8 Novel FRET reporters for real-time imaging of furin activity in the Golgi apparatus of cancer cells
  • Furin is located in several processing compartments and mostly localized to the Golgi apparatus.
  • Golgi apparatus is a central station that receives biomolecules from the endoplasmic reticulum (ER) and is involved in the protein processing and trafficking.
  • Dysregulated furin substrate processing in the Golgi apparatus concerning substrates like matrix metalloproteases and viral glycopeptides, is closely related to many human disorders such as cancer and neuronal diseases. Therefore, organelle-localized fluorescent probes offer a direct imaging tool for elucidating more details about furin activities in the Golgi compartment.

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Abstract

La présente invention concerne des conjugués de détection de protéase. En outre, la présente invention concerne des procédés de production des conjugués de la présente invention, leur utilisation pour surveiller l'activité des protéases cible, des procédés de criblage d'une bibliothèque de composés et des kits comprenant le conjugué.
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CN111492241A (zh) * 2017-09-29 2020-08-04 由联邦材料研究和检测机构主席所代表的经济与能源部长所代表的德意志联邦共和国 检测土壤和水中的烃污染
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CN111492241A (zh) * 2017-09-29 2020-08-04 由联邦材料研究和检测机构主席所代表的经济与能源部长所代表的德意志联邦共和国 检测土壤和水中的烃污染
CN109608635A (zh) * 2018-12-03 2019-04-12 淮海工学院 一种新型高分子发光材料及其制备方法
CN109608635B (zh) * 2018-12-03 2021-03-02 淮海工学院 一种高分子发光材料及其制备方法
WO2022008720A1 (fr) * 2020-07-10 2022-01-13 Life & Brain Gmbh Capteurs de tension optiques hybrides novateurs

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