key: cord-1037559-gj9v83mx
authors: Breidenbach, Julian; Bartz, Ulrike; Gütschow, Michael
title: Coumarin as a structural component of substrates and probes for serine and cysteine proteases
date: 2020-05-13
journal: Biochim Biophys Acta Proteins Proteom
DOI: 10.1016/j.bbapap.2020.140445
sha: 5f620fc799356ceb155582612d3d147215b49c03
doc_id: 1037559
cord_uid: gj9v83mx

Coumarins represent well-established structures to introduce fluorescence into tool compounds for biochemical investigations. They are valued for their small size, chemical stability and accessibility as well as their tunable photochemical properties. As components of fluorophore/quencher pairs or FRET donor/acceptor pairs, coumarins have frequently been applied in substrate mapping approaches for serine and cysteine proteases. This review also focuses on the incorporation of coumarins into the side chain of amino acids and the exploitation of the resulting fluorescent amino acids for the positional profiling of protease substrates. The protease-inhibiting properties of certain coumarin derivatives and the utilization of coumarin moieties to assemble activity-based probes for serine and cysteine protease are discussed as well.

Coumarin derivatives have manifold pharmacological properties showing, for example, antidiabetic [1] , antiviral [2] , anti-inflammatory [3] , as well as anticancer and antileukemia activities [4, 5] and acting as adenosine receptor antagonists [6] and monoamine oxidase inhibitors [7, 8] . Beside the identification of coumarin derivatives as lead structures against different targets, coumarins are frequently used because of their fluorescent properties to label substrates or inhibitors of several enzymes [9] . Through some minor modifications at the coumarin scaffold, it is possible to obtain small molecules with excellent stability, good fluorescence, high quantum yield and sufficient water solubility [10, 11] .

This review will focus on coumarins as fluorescence dyes to generate substrates and probes for serine and cysteine proteases. These two classes of proteolytic enzymes share an acyl transfer mechanism of the peptide bond cleavage involving the nucleophilic attack of the active site serine hydroxyl and cysteine thiolate nucleophile, respectively, at the carbonyl carbon of the scissile bond (Fig. 1) . Specificity of proteases is generally achieved through defined interactions of amino acid residues and certain subsites at the active site. The small size of fluorescent coumarins suggests their incorporation into the side chain of amino acids and the generation of fluorescent peptides. Internally quenched fluorescent (IQF) peptide substrates make use of this option. They consist of a fluorophore, e.g. a coumarin, and a quencher moiety, separated by several amino acids. Proteolytic cleavage of any peptide bond within this portion leads to a loss of quenching and the concomitant generation of fluorescence. Such IQF peptides have frequently been employed for the positional profiling of protease substrates to perform an active-site mapping and to elucidate substrate specificity [9] . covering P2 to P3´ residues and the corresponding subsites comprising S2 to S3´ in the nomenclature of Schechter and Berger [12] .

In addition to the utilization of coumarins for the reconstruction of specificity profiles for serine and cysteine proteases, this review will also highlight the role of coumarins as structural components of activity-based probes (ABPs). Serine and cysteine proteases, both of which are characterized by a covalent mode of catalysis, are particularly suitable to be targeted by ABPs. These probes elicit an irreversible inhibition of the protease because the nucleophilic attack is directed to an electrophilic moiety of the probe, the so called warhead.

The sulfur of a cysteine protease represents a soft nucleophile and thus prefers soft electrophiles such as acyloxymethyl ketones, epoxysuccinates and Michael acceptors. In contrast, the serine proteases favor harder electrophiles like phosphonates or isocoumarins [13] . Next to the warhead, ABPs contain two further parts, a spacer and a tag (Fig. 2) . The highest impact on the specificity of an ABP is caused by the spacer, which can consist of a well-accepted peptidic recognition unit. The tag permits the detection of the inhibited protease, either through biotin or fluorescent reporters, among which cyanines, fluoresceins, boron-dipyrromethene (BODIPY) derivatives and coumarins have been frequently employed [14] . The introduction of the latter fluorophore into ABPs for serine and cysteine proteases will be considered in this review. We will not discuss the detection and quantification of inorganic ions or low-molecular weight thiols by means of coumarin-containing probes, which has comprehensively reviewed elsewhere [11] . 

Coumarins are aromatic lactones. The aromatic character of coumarin was confirmed by topological resonance energy and nucleus independent chemical shift values and calorimetric measurement [15, 16] . Thiocoumarin, in which the ring oxygen is replaced by sulfur, is somewhat more stable [15] , similar to the comparison of 4H-3,1-benzoxazin-4-ones and 4H-3,1-benzothiazin-4-ones [17] . Hydrolysis of coumarins at high pH produced ciscoumarinic acids, and lactonization of this species occurred at low pH. Coumarin (1, Fig. 3) had a half-life of 160 min at pH 10 and 25 °C [18] . A 7-methoxy and, more pronounced, 7diethylamino substitution of 4-methylcoumarin led to a reduced reactivity in alkaline hydrolysis [19] , whereas introduction of an electron-withdrawing phenyl group at position 3 accelerated this reaction [20] .

Besides their bioactivities, the importance of coumarins rests upon their fluorescent properties as short-wavelength reactive dyes. They have been widely applied for the detection of proteins or other biological targets. The parent compound 1 (Fig. 3) is a weak fluorophore with low quantum yield. However, a combination of an electron-withdrawing substituent at 3-position and an electron-donating group at 7-position produces high fluorescence quantum 7-aminocoumarin is released from the 7-peptidylcoumarin through the protease-catalyzed hydrolysis of the amide bond. Compounds 4 and 5 (AMC) exhibit maxima for absorption and emission of 404 nm and 445 nm (in ethanol) and of 354 nm and 435 nm (in ethanol), respectively [23] . J o u r n a l P r e -p r o o f

Regarding the photophysical behavior of 7-aminocoumarins, a planar, highly emissive intramolecular charge-transfer (ICT) excited state and a non-fluorescent twisted intramolecular charge-transfer (TICT) state have been rationalized (Fig. 4) . Preventing this twisting process increases the quantum yield and restores fluorescence in aqueous media, as can be achieved through rigidization of the amino group in one or two rings [11, 26] . In coumarin 343 (8) , longer wavelengths of excitation (440 nm) and emission (480 nm, in aqueous solution) appear [27] . The extension of the π-electron system due to coupling in position 3 with a heteroaryl substituent and sulfonation at position 6 resulted in a bathochromic shift, high quantum yield and good water solubility of the resulting dye 9 with an absorption maximum of 426 nm and an emission maximum of 481 nm (in aqueous solution) [10] . The incorporation of coumarin moieties into the side chain of amino acids has frequently been carried out and utilized for the assembly of fluorescent probes. Two prototypical structures are exemplary depicted in Figure 3 . In compounds 10 (X = N, O, OR; n = 1-4), the linkage is realized through an amide bond [28] [29] [30] [31] , while in 11, an alkylidene chain connects the coumarin C-4 with the -carbon of the amino acid [32] [33] [34] [35] [36] [37] .

The pharmacology of coumarins is closely related to hemostatic processes, mainly conducted by serine proteases, which are, however not directly affected by most of the 7 such as Aspergillus fumigatus led to the formation of dicumarol (12, Fig. 5 ) [39] . This coumarin dimer turned out to be a VKOR inhibitor, hence decreasing antithrombotic proteins.

In 1941, dicumarol was established as anticoagulant and was the first approved coumarincontaining drug [38] . The coumarin derivatives phenprocoumon and warfarin are still on the market as VKOR inhibitors.

There are reports on coumarin-based compounds acting as serine protease inhibitors [40] , which will be discussed in the following. [43] [44] [45] . Factor Xa acts in the clotting cascade by converting prothrombin into thrombin in the presence of phospholipids and calcium. Factor Xa is a connecting link between internal and external ways of coagulation. Factor Xa inhibition has become a particular successful strategy in the development of new anticoagulants. Thrombin represents the terminal serine protease of the blood clotting cascade. It acts as a stimulator of platelet activation and as the direct converter of the glycoprotein fibrinogen to fibrin. Thrombin amplifies its own generation by feedback activation of factors V, VII and IX. Moreover, thrombin also activates factor XIII by cleaving an activation peptide segment. The resultant transglutaminase introduces the covalent crosslinks into the fibrin clot [46] . Compound 15 show a pronounced inhibition of chymotrypsin [47] , rather than of thrombin and factor Xa. Coumarin 16 exhibited a secondorder rate constant of thrombin inactivation of 37,007 M -1 s -1 . Activity against chymotrypsin was reduced in the order 15 to 17, and the latter compound was more potent against thrombin (k i /K i = 3,457 M -1 s -1 ) than against chymotrypsin [45] . The introduction of a para-guanidino group into the phenoxy part of such coumarins led to water soluble derivatives [48] .

The postulated interaction of mechanism-based inhibitors of type 15-17 is initiated by the attack of the active-site serine hydroxyl nucleophile at the lactone carbon, which leads to a ring-opened structure with a covalently bound serine (Fig. 6 ). The formation of such an acyl enzyme is not irreversible. Slow hydrolysis can occur to relieve the enzyme and produce the 'destroyed', ring-opened compound. Indispensable for the suicide mechanism is a good leaving group in position 6 linked via a methylene group. Its elimination can create a paraquinone methide derivative, able to subsequently react in an irreversible way with an activesite nucleophile. Such a covalent mechanism was already confirmed by the crystal structure of the complex of 7-hydroxycoumarin bound to γ-chymotrypsin where 2,4-dihydroxycinnamic acid is acylating the active site serine [49] , PDB entry 1K2I. the specificity of a certain protease. On the other hand, based on a given substrate specificity, so far unknown substrates of a protease can be assumed. Moreover, the preferred amino acid sequence around the scissile peptide bond can be employed to create specific and highly active peptidomimetic inhibitors for further drug development. Besides the application as drugs, pharmacological and biochemical tool compounds, in particular activity-based probes can be designed and employed to study the protease of interest in detail.

A commonly used principle for the identification of favored substrate sequences and the generation of such substrates is the implementation of a positional scanning synthetic combinatorial library (PS-SCL) [54, 55] . In a peptide sequence, for example with four amino acids P1-P4, one position is fixed with a known amino acid, while the others contain an equimolar or isokinetic mixture of natural amino acids. The expansion by unnatural amino acids led to hybrid combinatorial substrate libraries (HyCoSuLs) [56, 57] . In a fluorogenic PS-SCL, the C-terminal P1 amino acid is coupled to a fluorophore, which is buried in the S1´ pocket. Protease-catalyzed cleavage of the amide bond between the P1 amino acid and the dye results in time-dependent increase in fluorescence, amenable to kinetic measurements. By means of this technique, it is possible to study the impact of the known amino acid at the desired position independent of the other ones. Thus, PS-SCLs provide information on the particularly preferred amino acid residues at the corresponding positions and allow for the deconvolution to obtain an optimal peptide sequence. The resulting substrates can be characterized by the specificity constant k cat /K m with high values corresponding to suitable substrates. As a matter of course, such an active site mapping is limited to the non-primed positions [56, 58] .

Coumarin derivatives are prevailingly applied fluorophores for such attempts. Owing to small size, they are well acceptable in the S1´ pocket. 7-Amino-4-methylcoumarin (AMC; structure 5, see This practice allows the incorporation of the unmodified P1 amino acids and thus the positional profiling of this position [59, 60] . The involvement of the P1 amino acid to reconstruct the specificity profile is also possible by applying 7-amino-4carbamoylmethylcoumarin (ACC; structure 7, see Fig. 3 ). ACC is bifunctional and can be coupled to the C-terminus of the peptide via the scissile amide bond. For the connection to a Rink amide resin, for example, the 7-amino-Fmoc protected free acid of ACC can be used.

Cleavage from the resin can be achieved under acidic conditions with triisopropyl silane as reducing scavenger of carbenium ions to generate of the final coumarinyl peptide. ACC exhibits a 3-times higher quantum yield compared to AMC, making ACC-based assays more sensitive [54, 55] . AMC and ACC-based PS-SCLs have been utilized for the substrate mapping of several proteases, also including the type II metalloprotease aminopeptidase N [61] . In the following coumarin substrates for serine and cysteine proteases are reviewed [9, 56, 58] , and some recent and significant examples are discussed, including human, bacterial and viral proteases.

Factor VII activating protease (FSAP) is a human serine protease implicated in thrombosis, atherosclerosis, stroke and cancer. FSAP plays a dual role in hemostasis, being involved in both procoagulant and fibrinolytic pathways. The liver enzyme is secreted as inactive 70 kDa zymogen (pro-FSAP) into the blood plasma. The circulating enzyme seems to conduct an auto-activation, which is triggered through ingredients of damaged cells and on positively and negatively charged macromolecules [62, 63] . Once activated, FSAP can be inhibited by several endogenous inhibitors such as 1-proteinase inhibitor, 2-plasmin inhibitor, antithrombin, C1 inhibitor, plasminogen activator inhibitor-1 [64] . The activated FSAP was reported to cleave single-chain urokinase-type plasminogen activator (scuPA) and

several other substrates, such as platelet-derived growth factor, basic fibroblast growth factor/epidermal growth factor, histones, high-molecular-weight kininogen and protease The k cat /K m value of this probe for DPP4 (850,000 M -1 s -1 ) is similar to that of H-Gly-Pro-AMC (760,000 M -1 s -1 ) [77] . Likewise, a ratiometric two-photon fluorescent probe to monitor DPP4 was developed, based upon the cleavage of H-Gly-Pro-OH from 6-amino-2-butyl-1H- Fig. 9 . Activity-based probes for flaviviral serine proteases with biotin as detectable tag and diphenyl phosphonate as warhead [84] .

The Moreover, the effects of cathepsin S in nociception and atherogenesis might be mediated due the cleavage of a precursor of fractalkine, a large, membrane-bound cytokine protein [85, 86] .

The 329-amino acid pre-proenzyme of cathepsin K is activated through auto-or heterocatalysis to the mature 215-amino acid protease. It is mainly expressed in osteoclasts, while its expression rate is triggered by receptor activator of NF-κB ligand (RANKL) and reduced by estrogen and calcitonin. Collagen type I represents 90% of the organic extracellular bone matrix. Cathepsin K, which is stored in cytosolic vesicles, is released in the resorption lacunae, and efficiently cleaves triple helix and telopeptide type I collagen as well as osteonectin. Also proteins, occurring outside of the bone matrix, are substrates of cathepsin K such as aggrecan, type II collagen, and elastin. Its involvement in bone resorption and disease states like osteoporosis and osteoarthritis turned cathepsin K into an outstanding therapeutic target. However, the development of the most promising drug candidate, odanacatib, has been discontinued because of the increased risk of cerebrovascular accidents [87, 93] .

Out of several reports on mapping of the non-primed substrate specificity, three will be discussed here. They all used combinatorial peptide libraries with a terminal ACC (7, see 

The cleavage of plasminogen into plasmin is mediated by two types of activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA).

Besides their fibrinolytic profile, they are implicated in tissue proliferation and cellular Roubinet et al. reported on a uPA substrate [10] , which was assembled with coumarin 9 (see Fig. 3 ) as solubility-mediating fluorescence donor and a nitro-azobenzene derivative as acceptor with a quenching range between 394 and 506 nm. The enzymatic reaction was followed with excitation and emission wavelengths of 400 nm and 480 nm, respectively. The principle of such quenched substrates is shown for this uPA substrate in Figure 10 . (Fig. 11) . Moreover, almost no intrinsic 19 F NMR signal is detectable in living organisms. The probe consists of a godalinum-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (Gd 3+ -DOTA) complex, the caspase-3 substrate sequence Asp-Glu-Val-Asp and the coumarin derivative 6 (see Fig. 3 ). Substrate cleavage at the C-terminus results in the release of 6 and an increase in fluorescence. The Gd 3+ -DOTA complex in the intact probe quenches the 19 F NMR signal through a paramagnetic relaxation enhancement (PRE) effect, which disappears upon cleavage. The dual probe exhibited a V max /K m value of 7.61 × 10 -3 s -1 compared to 9.91 × 10 -4 s -1 for the Ac-DEVD-AMC substrate [106] . inflammation, infections, cardiovascular diseases, cancer and autoimmune vasculitis [109, 110] .

In a combinatorial approach to convolute the P1´ and P2´ specificities of cathepsin G, as part of the so-called CBA complex, regulates gene expression by the activation of NF-κB transcription factors and controls transcript stability [113] . For full-length MALT1, an IQF substrate was developed with structure ACC-AHX-Ala-Leu-Val-Ser-Arg-Gly-Thr-Lys(DNP)-Gly-OH (with AHX = 6-aminohexanoic acid, the P1 amino acid is underlined) exhibiting a k cat /K m value of 6,300 M -1 s -1 [114] .

In the following probe, a coumarin was again positioned at the N-terminus of the peptide. Yuan et al. employed 7-diethylamino-coumarin-3-carboxylic acid as energy donor and a tetraphenylethenethiophene moiety as energy quencher, connected through a caspase-3 specific peptide (Fig. 12) . The probe is non-fluorescent by itself due to the energy transfer and the dissipation caused by the molecular motion of the quencher. Substrate cleavage renders not only the coumarin to a non-quenched fluorophore, but also the tetraphenylethene derivative to a fluorescent compound. In the absence of the energy transfer, its intramolecular rotation is restricted, leading to fluorescence after excitation at the same wavelength. In the cellular environment, caspases 3 and 7 were able to turn-on the fluorescence of this probe [115] . Fig. 12 . A probe to detect caspase activity with a dual signal output [115] . 

The M -1 s -1 ) was designed with lysine acetylated with 7-hydroxy-4-methyl-coumarin-3-yl-acetyl acid as donor and with ornithine acylated with coumarin 343 (8, see Fig. 3 ) as quencher [122] .

An overview on the protease substrates discussed in the review is given in the Supplementary material which can be found online at ...

Fluorescent activity-based probes have become a powerful tool in enzyme research, e.g. for the localization and imaging of enzymatic activities in biological systems by virtue of the high sensitivity, nondestructive analysis, and real-time detection abilities of the probes.

ABPs enable the analysis of only the active form of the target enzyme, which is beneficial, because the enzymatic activity does not necessarily correlate with the expression level of the protein [13] . Coumarins do not represent the most frequently applied fluorophores to generate ABPs for serine and cysteine proteases, but relevant recent examples will be discussed in the following.

In order to design an ABP for the serine protease matriptase-2, substrate mapping approaches and the consideration of the endogenous substrate were used as point of origin.

Because the S1 and the S3/S4 subunits prefer basic amino acids, in particular arginine, two guanidinophenyl groups were introduced as arginine mimetics. A coumarin label was inserted into corresponding phosphonate inhibitors leading to probe 20 (Fig. 14) [123] . The Matriptase, one of the best characterized TTSPs and a close relative of matriptase-2, is mainly expressed in epidermis, salivary gland, thyroid, stomach, kidney, prostate and ovaries as an inactive zymogen and has to be converted in an autocatalytic manner into its active form. Matriptase processes several proteins, such as hepatocyte growth factor/scatter factor, uPA and protease-activated receptor 2 which play critical roles in tumorigenesis and trigger other signaling pathways related to cancer proliferation and metastasis [124] . An ABP for matriptase (21, Fig. 15 ) also included the diphenyl phosphonate warhead and two arginine mimetics expected to occupy the S1 and S2 subunits. The coumarin fluorophore was assumed to be orientated towards the S3/S4 region. The second-order rate constant for inactivation of matriptase (k inac /K i = 576 M -1 s -1 ) was one order of magnitude higher than that for matriptase-2. Probe 21 was successfully employed for direct fluorescence readout after SDS-PAGE and HPLC size-exclusion chromatography coupled to fluorescence detection [125] . Fig. 15 . A matriptase-directed ABP for in-gel and HPLC fluorescence detection [125] .

Sulfonyloxyphtalimides are mechanism-based inhibitors of serine proteases whose interaction involves a nucleophilic attack of the active-site serine, ring opening, Lossen rearrangement of the O-sulfonyl hydroxamic acid intermediate, and trapping of the resulting isocyanate by water or a second adjacent active-site nucleophile [126] . This principle was adapted for the design of the ABP 22 targeting human neutrophil elastase (Fig. 16 ) [127] . The coumarin tag of the probe allowed for the in-gel fluorescence detection of HNE with a lownanomolar probe concentration. The reaction of porcine pancreatic elastase with 22 was followed by analyzing the fluorescence kinetics. A FRET was applied by exciting the anthranilic acid fluorophore at 320 nm and detecting the emission of coumarin 343 at 490 nm.

However, at an excitation wavelength of 258 nm for the enzyme's tryptophan, a double-FRET system was realized. By monitoring the later stages of the reaction, the half-life of the anthranoyl enzyme 23 ( 

Cysteine cathepsins represent a promising target for activity-based probing. For the purpose of establishing an ABP with cathepsin S preference, an ,-unsaturated Michael acceptor was used as a soft-electrophile warhead (Fig. 17) . The formation of a covalent sulfur-carbon bond led to an irreversible inhibition. In the predicted binding mode, the vinyl sulfone moiety extended towards the S1´ pocket and the hydrophobic S2 subsite was occupied by phenylalanine. Probe 24 was selective for cathepsins S and L (k inac /K i = 49,000 M -1 s -1 and 16,900 M -1 s -1 ) over cathepsins K and B (k inac /K i < 900 M -1 s -1 ). The feasibility of compound 24 for direct in-gel fluorescence detection and for labeling of native cathepsin S in the protein extract from human placenta was demonstrated [128] . Fig. 17 . A tripeptidomimetic ABP for cysteine cathepsins [128] .

Dipeptide nitriles are well-established covalent inhibitors of cysteine proteases forming thioimidates with the active-site cysteine [87] . The inhibition is not irreversible, and the adduct can dissociate and release the enzyme. This is the reason that peptide nitriles are not suitable as ABPs for in-gel detection. However, the labelling with a coumarin can be performed to study the cellular uptake by fluorescence measurements. A prototypical cathepsin inhibitor structure was equipped with coumarin 343 leading to 25 (Fig. 18 ) [129] .

The integration of an isobutylsulfonylcysteine at P2 provides particular affinity for cathepsin S, while the tetracyclic coumarin fits in the deep S3 pocket of cathepsin K. Accordingly, a dual-active, sub-micromolar inhibitor was obtained. HEK cells were treated with 25 for different incubation times and cell lysates were analyzed with an excitation wavelength of 450 nm and an emission wavelength of 492 nm. The protein concentration was used as a relative benchmark for the cellular uptake of 25 [129] . 

None

Synthesis and antihyperglycemic activity of novel N-acyl-2-arylethylamines and Nacyl-3-coumarylamines

Recent progress in the development of coumarin derivatives as potent anti-HIV agents

Synthesis and biological evaluation of novel coumarin derivatives with a 7-azomethine linkage

Design, synthesis, and biological evaluation of imidazolyl derivatives of 4,7-disubstituted coumarins as aromatase inhibitors selective over 17-α-20 lyase

Synthesis and biological evaluation of hydroxylated 3-phenylcoumarins as antioxidants and antiproliferative agents

Structure-based optimization of coumarin hA 3 adenosine receptor antagonists

Alkynyl-coumarinyl ethers as MAO-B inhibitors

Exploring basic tail modifications of coumarin-based dual acetylcholinesterasemonoamine oxidase B inhibitors: Identification of water-soluble, brain-permeant neuroprotective multitarget agents

Current strategies for probing substrate specificity of proteases

A synthetic route to 3-(heteroaryl)-7-hydroxycoumarins designed for biosensing applications

Coumarin-based small-molecule fluorescent chemosensors

On the size of the active site in proteases. I. Papain

Activity-based probes for the study of proteases: recent advances and developments

Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging

Topological resonance energies of thiocoumarins

Energetics of coumarin and chromone

1-Benzothiazin-4-ones and 3,1-benzoxazin-4-ones: Highly different activities in chymotrypsin inactivation

Kinetics and mechanisms of lactonization of coumarinic acids and hydrolysis of coumarins

Alkaline hydrolytic lability of some hydroxy-and methoxycoumarins and related anticoagulants

Studies of 7-hydroxycoumarins

Improvement of fluorescence characteristics of coumarins: syntheses and fluorescence properties of 6-methoxycoumarin and benzocoumarin derivatives as novel fluorophores emitting in the longer wavelength region and their application to analytical reagents

Synthesis and fluorescence properties of substituted 7-aminocoumarin-3-carboxylate derivatives

Synthesis and fluorescent properties of some heterobifunctional and rigidized 7-aminocoumarins

Recent advances and therapeutic journey of coumarins: current status and perspectives

Comparative pharmacokinetics of vitamin K antagonists: warfarin, phenprocoumon and acenocoumarol

Solvent effects on emission yield and lifetime for coumarin laser dyes. Requirements for a rotatory decay mechanism

Coumarin-modified gold nanoprobes for the sensitive detection of caspase-3

In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids

Fluorescent peptides labeled with environment-sensitive 7-aminocoumarins and their interactions with lipid bilayer membranes and living cells

Fluorescently labeled amino acids as building blocks for bioactive molecules

Clickable coumarins as fluorescent labels for amino acids

Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

A very short route to enantiomerically pure coumarin-bearing fluorescent amino acids

Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore

Minimalist probes for studying protein dynamics: thioamide quenching of selectively excitable fluorescent amino acids

A concise preparation of the fluorescent amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine and extension of its utility in solid phase peptide synthesis

Synthesis of fluorescent amino acids via palladium-catalyzed allylic alkylations

Discovery of anticoagulant drugs: a historical perspective

The biosynthesis of 4-hydroxycoumarin and dicoumarol by Aspergillus fumigatus Fresenius

Coumarin and isocoumarin as serine protease inhibitors

Novel 3-carboxamidecoumarins as potent and selective FXIIa inhibitors

Synthesis, evaluation and structure-activity relationship of new 3-carboxamide coumarins as FXIIa inhibitors

Pochet, 3,6-disubstituted coumarins as mechanism-based inhibitors of thrombin and factor Xa

Investigation of mechanism-based thrombin inhibitors: Implications of a highly conserved water coumarins within the S pocket

Mechanismbased thrombin inhibitors: design, synthesis, and molecular docking of a new selective 2-oxo-2H-1-benzopyran derivative

Design of small-molecule active-site inhibitors of the S1A family proteases as procoagulant and anticoagulant drugs

Esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid as inhibitors of alphachymotrypsin: significance of the "aromatic" nature of the novel ester-type coumarin for strong inhibitory activity

Discovery and assessment of water soluble coumarins as inhibitors of the coagulation contact pathway

Crystal structure of gamma-chymotrypsin in complex with 7-hydroxycoumarin

Toward the first class of suicide inhibitors of kallikreins involved in skin diseases

A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation

Proteases: History, discovery, and roles in health and disease

Positional scanning synthetic combinatorial libraries for substrate profiling

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

Emerging challenges in the design of selective substrates, inhibitors and activity-based probes for indistinguishable proteases

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Active site mapping with peptidic substrates and inhibitors

An alkanesulfonamide "safety-catch" linker for solid-phase synthesis

Synthesis of positionalscanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library

Factor VII-activating protease: Hemostatic protein or immune regulator?

Protease activated receptors (PAR)-1 and -2 mediate cellular effects of factor VII activating protease (FSAP)

Analysis of the substrate specificity of Factor VII activating protease (FSAP) and design of specific and sensitive peptide substrates

Activation of single-chain urokinase-type plasminogen activator by platelet-associated plasminogen: a mechanism for stimulation of fibrinolysis by platelets

Fluorescent activitybased probe for the selective detection of Factor VII activating protease (FSAP) in human plasma

New chromogenic substrates of human neutrophil cathepsin G containing nonnatural aromatic amino acid residues in position P(1) selected by combinatorial chemistry methods

Application of a novel highly sensitive activity-based probe for detection of cathepsin G

Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly

Toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils

Matriptase-2: Monitoring and inhibiting its proteolytic activity

Iron metabolism and iron disorders revisited in the hepcidin era

Probing the substrate specificities of matriptase, matriptase-2, hepsin and DESC1 with internally quenched fluorescent peptides

Critical role of dipeptidyl peptidase IV: A therapeutic target for diabetes and cancer

,4,5-trifluorophenyl)butan-2-amine: A potent, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

A time-resolved fluorescence probe for dipeptidyl peptidase 4 and its application in inhibitor screening

A highly specific ratiometric two-photon fluorescent probe to detect dipeptidyl peptidase IV in plasma and living systems

The mitochondrial Lon protease: Novel functions off the beaten track?

Bacterial proteolytic complexes as therapeutic targets

Leveraging peptide substrate libraries to design inhibitors of bacterial Lon protease

Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor

Inhibitors of the Zika virus protease NS2B-NS3

Profiling of flaviviral NS2B-NS3 protease specificity provides a structural basis for the development of selective chemical tools that differentiate Dengue from Zika and West Nile viruses

Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response

Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes

Can cysteine protease cross-class inhibitors achieve selectivity?

Cysteine protease cathepsins in cardiovascular disease: from basic research to clinical trials

Proteases as activators for cytotoxic prodrugs in antitumor therapy

Cathepsin-sensitive nanoscale drug delivery systems for cancer therapy and other diseases

Real-time monitoring of drug release

A review of small molecule inhibitors and functional probes of human cathepsin L

Cathepsin K inhibitors for osteoporosis: biology, potential clinical utility, and lessons learned

Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

Fluorescent probes towards selective cathepsin B detection and visualization in cancer cells and patient samples

Selective imaging of cathepsin L in breast cancer by fluorescent activity-based probes

Serum based fluorescent assay for evaluating dipeptidyl peptidase I activity in collagen induced arthritis rat model

Prodrug-inspired probes selective to cathepsin B over other cysteine cathepsins

Cell penetrable, clickable and tagless activity-based probe of human cathepsin L

Substrate specificity profiling of SARS-CoV-2 Mpro protease provides basis for anti-COVID-19 drug design

Components of the plasminogen-plasmin system as biologic markers for cancer

Thioamidebased fluorescent protease sensors

Allosteric modulation of caspases

caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis

Design and application of a fluorogenic assay for monitoring inflammatory caspase activity

Dual-function probe to detect protease activity for fluorescence measurement and 19 F MRI

A novel three-fluorophore system as a ratiometric sensor for multiple protease detection

Activity based fingerprinting of proteases using FRET peptides

Neutral serine proteases of neutrophils

Processing and regulation mechanisms within antigen presenting cells: a possibility for therapeutic modulation

Development of sensitive cathepsin G fluorogenic substrate using combinatorial chemistry methods

Internally quenched fluorogenic substrates with unnatural amino acids for cathepsin G investigation

Holding all the CARDs: How MALT1 controls CARMA/CARD-dependent signaling

Determination of extended substrate specificity of the MALT1 as a strategy for the design of potent substrates and activity-based probes

A FRET probe with AIEgen as the energy quencher: Dual signal turn-on for self-validated caspase detection

A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases

Characterization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic substrate with increased specificity constants for collagenases and tumor necrosis factor converting enzyme

A coumarin-based fluorescence resonance energy transfer probe targeting matrix metalloproteinase-2 for the detection of cervical cancer

Membrane-bound FRET probe visualizes MMP12 activity in pulmonary inflammation

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters

Three wavelength substrate system of neutrophil serine proteinases

Phosphono bisbenzguanidines as irreversible dipeptidomimetic inhibitors and activity-based probes of matriptase-2

Specifically targeting cancer proliferation and metastasis processes: the development of matriptase inhibitors

A fluorescent-labeled phosphono bisbenzguanidine as an activity-based probe for matriptase

Sulfonyloxy)phthalimides and analogues are potent inactivators of serine proteases

Design of an activity-based probe for human neutrophil elastase: Implementation of the Lossen rearrangement to induce Förster resonance energy transfers

A coumarin-labeled vinyl sulfone as tripeptidomimetic activity-based probe for cysteine cathepsins

Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors