Strategies in the Design of Small-Molecule Fluorescent Probes for Peptidases Laizhong Chen, Jing Li, Lupei Du, and Minyong Li Key Laboratory of Chemical Biology (MOE), Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Jinan, China Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/med.21316

䉲 Abstract: Peptidases, which can cleave specific peptide bonds in innumerable categories of substrates, usually present pivotal positions in protein activation, cell signaling and regulation as well as in the origination of amino acids for protein generation or application in other metabolic pathways. They are also involved in many pathological conditions, such as cancer, atherosclerosis, arthritis, and neurodegenerative disorders. This review article aims to conduct a wide-ranging survey on the development of small-molecule fluorescent probes for peptidases, as well as to realize the state of the art in the tailor-made probes for  C 2014 Wiley Periodicals, Inc. Med. Res. Rev., 00, No. 0, 1–25, 2014 diverse types of peptidases. Key words: peptidases; small-molecule fluorescent probes; design strategies

1. INTRODUCTION Peptidases, also known as proteolytic enzymes, are important in many aspects of human health and life sciences, considering that their typical biological significance is well illustrated by the fact that 2% genes in each category of organism encode peptidases and/or their homologues.1 The principal function of peptidases is to hydrolyze specific peptide bonds of substrates, which is of importance in protein activation, cell signaling and regulation, as well as in the production of amino acids for protein biosynthesis or for use in other metabolic pathways.2 They are also involved in various pathological conditions, such as cancer, atherosclerosis, arthritis, and neurodegenerative disorders including Huntington’s and Alzheimer’s diseases.3 Consequently, it is tremendously critical to perceive the activities and distributions of peptidases. Peptidases can be divided into six discrete classes (metallo-peptidase, aspartate, glutamate, cysteine, serine, and threonine peptidase), according to the mechanism of catalysis (Scheme 1).4 In general, threonine, serine, and cysteine proteases catalyze the hydrolysis of peptides by using a side chain as the nucleophile. Metallo-peptidase, aspartate, and glutamate proteases maneuver their active-site residues to activate a water molecule for substrate attack.

Correspondence to: Minyong Li, Key Laboratory of Chemical Biology (MOE), Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Jinan, Shandong 250012, China, E-mail: [email protected] Medicinal Research Reviews, 00, No. 0, 1–25, 2014  C 2014 Wiley Periodicals, Inc.

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r CHEN ET AL. Aspartate or glutamate proteases

Zinc metalloproteases

HN

N

N

NH

N

HO NH

O

HO

O

H O H

Zn2+ O H O O H O

R2 N H

R1

H O H O H N H H

O H

NH S H

N

NH

O

Threonine proteases Scheme 1.

N

Serine proteases

Cysteine proteases

Nucleophilic attack modes of the primary families of proteases catalysis.

Up to now, three classes of probes for peptidases, including monoclonal antibodies, isotopelabeled probes, and fluorescent probes, have been designed well. Monoclonal antibodies, obtained in general with high specificity, can be delivered in high concentration to their targets; however, they have the undesirable feature of prolonged clearance of unbound molecules, thus resulting in a high background signal. Besides, most monoclonal antibodies have poor permeability. Although isotope-labeled probes are widely implemented in clinic, their potential radiotoxicity and probable spill are foreseeable issues. Fluorescent nanoparticles are widely studied in these years, but they also have the disadvantage of prolonged clearance of unbound molecules. In contrast, small-molecule fluorescent probes are not only minimally toxic but also precipitously cleared, so as to improve the safety and target-to-background ratio. To this point, several approaches have been employed to generate probes for particular types of peptidases, including switch- and binding-based approaches.

2. BINDING-BASED PROBES A typical binding-based probe has three main components: (i) a mechanism-based structural scaffold to interact with the active site of the peptidase in a covalent or noncovalent manner; (ii) a linker that prevents steric hindrance of fluorophore around the active site; and (iii) a reporter for the visualization and characterization of labeling events. Medicinal Research Reviews DOI 10.1002/med

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

r 3

OH

(a)

H N

OH O O H2N

H N NH

HN

S S

O O

NH H N O

(b)

H N S

O HOOC

H HN N

O O

NH2 O

O

H2N

H N NH

O O

NH H N

fluorescein

Figure 1.

N H

NH2

N O

H HN N

O

O

CNGRC

HN

S S

Cl

H N

OH O

NH2 O

O

CNGRC

N

Cy 5.5

The structures of probe CNP1 (a) and probe CNGRC-Cy5.5 (b).

A. Noncovalent Binding Based Probes Certain varieties of peptidases can serve as receptors and have their natural ligands. Labeling peptidase-specific natural ligands with appropriate fluorophores can generate natural ligandbased probes, which can tag corresponding peptidases and then determine their distribution. For example, aminopeptidase N (APN) isoform has been identified as a critical tumor biomarker that is overexpressed on the surface of various tumors involved in cancer angiogenesis, invasion, and metastasis.5, 6 The cyclic peptide CNGRC, a specific natural ligand of tumor high-expressed APN, cannot be recognized by other types of APN isoforms expressing in normal myeloid and epithelia cells.7 By using the ligand-based probes strategy, Nishimoto et al. used a fluoresceinlabeled CNGRC (CNP1, Fig. 1a) to image tumor cells that express high levels of APN.8 To further demonstrate the importance of CNGRC-based probes, Wallbrunn et al. designed and synthesized a CNGRC-Cy5.5 (Fig. 1b) conjugate,9 which could be used for in vivo imaging of the expression of APN in tumor xenografts in view of the deep tissue penetration capability of near-infrared (NIR) light emitted by Cy5.5 (an NIR fluorophore). Natural ligand-based probes have thoroughly demonstrated significant advantages in imaging proteins.10, 11 However, only a small portion of peptidases have specific natural peptide ligands, which limits the exploitation of natural ligand-based probes. A potent inhibitor normally has high intrinsic affinity and specificity to its target. Therefore, when an appropriate inhibitor is connected to a fluorophore that does not interact with the target, an efficient and specific probe can be produced. For example, compound 23 is an effective APN inhibitor, and an imaging probe Cy5.5–23 (Fig. 2) was synthesized in the Carsten lab by connecting compound 23 and Cy5.5 (an NIR fluorophore) with a short polyethylene glycol (PEG) spacer. This probe Cy5.5–23 could selectively bind to APN-positive BT-549 cells but not to APN-negative BT-20 cells.12, 13 Another example is the development of neprilysin probe. Thiorphan is a mercapto neprilysin inhibitor, whose fluorescent derivative, N-[fluoresceinyl]N -[1-(6-(3-mercapto-2-benzyl-1-oxopropyl)amino-1-hexyl)]thiocarbamide (FTI, Fig. 3), was synthesized by Giocondi and co-workers, which could be also used to assay neprilysin by flow cytometry and photomicroscopy.14, 15 The Brown lab recently presented a fluorescent probe for histone deacetylases (HDAC). Such a probe is based on a potent HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), which contains three parts: a hydroxamate for zinc chelating, a linker, and an anilide cap region. The substitution of the anilide with a fluorescent dansyl group generated a fluorescent HDAC inhibitor (Scheme 2), which provided a useful toolkit for studying the mechanism of the action of HDAC inhibitors.16 Because there are no additional fluorophores that may disturb the behaviors of inhibitors, this type of fluorescent probes are usually suitable for investigating the distribution and other actions for a certain type of inhibitors. Medicinal Research Reviews DOI 10.1002/med

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r CHEN ET AL.

fluorophore SO3H O

HO3S

H3CO

O N

N H

O

N O

O

O

CONHOH

O

HN

OCH3

O O

inhibitor HO3S

N

SO3H

Cy 5.5-23 Figure 2.

The structure of probe Cy5.5–23.

OH

SH

O N H

H N

H N S

O O

OH O

neprilysin inhibitor Figure 3.

fluorescein

The structure of a fluorescent neprilysin inhibitor.

B. Covalent Binding Based Probes Unlike metallo-peptidase, aspartate, and glutamate proteases, threonine, serine, and cysteine proteases catalyze the hydrolysis of peptides by using an amino acid side chain as the nucleophile. These nucleophilic groups (hydroxyl or sulfydryl) can easily react with some electrophiles (such as epoxide, fluorophosphonate, and acyloxymethyl ketone (AOMK)) of inhibitors and form covalent bonds to produce stable complexes between inhibitors and targets. Such an inhibitor that can form a covalent bond with its target is named irreversible inhibitor. Therefore, fluorescence-labeled irreversible inhibitors could be considered as satisfactory probes in view of the stability of the reactions. For example, MeO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH, a highly convincing and selective irreversible cathepsin B inhibitor, was coupled with rhodamine B to generate a highly sensitive and selective probe (Fig. 4). By labeling cathepsin B with an epoxide electrophile, this probe enabled detection of cathepsin B activity in inflammation, tumor invasion, and metastasis.17 It should be noted that this type of inhibitors by using epoxides as the electrophiles have been widely applied in designing binding-based probes.18, 19 In many serine hydrolases, fluorophosphonate is an electrophilic group that can selectively react with serine.20–22 Cravatt and co-workers prepared a binding-based probe (Fig. 5a), Medicinal Research Reviews DOI 10.1002/med

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

Cap region

Linker

r 5

Hydroxamate O

H N

N H

O

OH

inhibitor

O S N O H

O

Scheme 2.

Design of fluorescent HDAC inhibitor.

N H

fluorescent inhibitor

OH

N

N

O

HOOC N

O

H N O

H N

O

O

O N H

HN O

H N

N H

H N S

O HOOC N

linker

inhibitor

Figure 4.

rhodamine B

The structure of a cathepsin B probe.

consisting of a fluorophosphonate and biotin linked together via an alkyl chain, which had the ability to label serine hydrolases in biological samples.23 The substitution of biotin with a fluorescent group could directly acquire a visible probe for proteasome complex (Fig. 5b).24, 25 Notably, the sensitivities of fluorescent probes are at least 100-fold higher than that of biotinconjugated probes. AOMK was reported to have low reactivity toward weak nucleophiles, but high reactivity towards cysteine proteases due to the fact that sulfur atom in the active site of a cysteine protease is much more nucleophilic than the oxygen atom in a serine protease.26 By linking AOMKlabeled substrates to biotin, Bogyo and his colleagues prepared a series of binding-based probes that can selectively bind to cysteine protease by formation of covalent bonds with the thiol of the active site (Fig. 6a), which allowed direct biochemical profiling of protease activity.27 Using the same strategy, van der Hoorn et al. designed a specific fluorescent binding-based probe (Fig. 6b) that could display various AvrPphB (an avirulence (Avr) protein from the plant pathogen Pseudomonas syringae) isoforms in bacterial extracts.28 Like AOMK, chloromethyl ketone (CMK) and fluoromethyl ketone (FMK) can also form covalent bonds with cysteine proteases.29, 30 Z-Val-Ala-Asp(OMe)-FMK is a widely used caspase inhibitor that displays good cellular permeability. Z-Val-Ala-Glu(OMe)-FMK, an analog of Z-Val-Ala-Asp(OMe)-FMK, Medicinal Research Reviews DOI 10.1002/med

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r CHEN ET AL. (a) H N

S

H N

NH HN

O

O F P O O fluorophosphonate

O

biotin

OH

(b) H N O F P O O fluorophosphonate Figure 5.

H N

H N S

O O

OH O fluorescein

The structures of two serine protease probes.

was synthesized by Forrest et al. and further labeled with an IR780 fluorophore to yield an NIR caspase probe (shown in Fig. 7). This probe can be used to monitor live cells undergoing apoptosis31 and even detect cells apoptosis in living nude mice by photoacoustic imaging (PAI).32 Covalent binding based probes for some categories of peptidase can be prepared from their substrates by adding electrophilic warheads, such as epoxide, fluorophosphonate, and AOMK. However, which type of warhead should be selected and where it should be placed still remain critical. In the case of dipeptidyl peptidase I (DPP-I, also named cathepsin C) that is a cysteine protease and solely responsible for NH2 -prodipeptide removal, AOMK and epoxides are widely used as warheads in designing its probes. For instance, DCG-04 (Fig. 8a) can unselectively label a number of papain family members without selectivity.19 Yuan et al. considered norvalinehomophenylalanine dipeptide free amine as the selective substrate to retrieve a highly selective binding-based probe to DPP-I. To minimize disturbance of the dipeptide recognition module, they preferred a vinyl sulfone moiety as the warhead for three reasons: (i) the free amine of the dipeptide was necessary for the selectivity; (ii) the epoxysuccinate used in DCG-004 had to be attached to the N-terminus of the peptide; and (iii) the prime site element of AOMK acted as a leaving group upon covalent binding to the target protein. As a result, they found a probe FY01 that could selectively identify DPP-I in rat liver homogenates and intact cells (Fig. 8a, b).33 Other types of electrophiles, such as isocoumarins,34 β-lactams,35 and sulfonyl fluoride,36 can also be applied as the warheads for labeling peptidases with low selectivity. By taking advantages of stable interactions of covalent binding based probes and their targets, Michael et al. converted a noncovalent binding based probe to a covalently labeling probe by adding a UV crosslinker (Fig. 9). The probe and its target peptidase could be linked covalently and irreversibly after photolysis of the UV crosslinker. As a result, this probe could identify an aminopeptidase in a complex proteome.37 This strategy was also successfully applied to γ -secretase probes.38 Other photolabile groups, such as diazirine, can also be used.39 Medicinal Research Reviews DOI 10.1002/med

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

r 7

O

(a) HN

O

biotin (reporter)

NH

HN S O N H

O

H N

O

H N

O

O

O

substrate-AOMK (inhibitor)

OH

(b)

O

R

N

O

O

rhodamine

COO

N

NH COOH O

H N

N H

O

Figure 6.

H N

O O

O O substrate-AOMK (inhibitor)

The structures of a series of AOMK-based cysteine protease probe (a) and an AvrPphB probe (b).

IR780 fluorophore N

O NH

H N

NH O

O N H

O

H N O

O

O F

Z-Val-Ala-Glu(OMe)-FMK N

Figure 7.

Structure of IR780–linker–Val-Ala-Glu(OMe)-FMK.

Medicinal Research Reviews DOI 10.1002/med

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r CHEN ET AL. (a)

O

O

H N

H2N

N H

O

O

H N

OEt

N H

O

O

O

HO

HN

(b)

DCG-04

HN

O H2N

O N H

S O O

N H FY01

O

H N

NH2

O

OH

NB N F F

=

O

O

HN

MeO

Figure 8. (a) The structures of DCG-04 and FY01. (b) FY01 is a selective marker of cathepsin C (DPP-I). DCG-04 could label multiple cathepsin in rat liver homogenates (left panel), whereas FY01 is selective for DPPI (right panel). All of the binding-based labeling can be blocked by pretreatment with PM-Oet, a papain family protease inhibitor. Source: Reference 33 copyright (2006), American Chemical Society.

UV crosslinker

H N

HN H2N

O

O O

O

H N

N H

O

O

O O

NH2

NH

O S

N H

O

HN

OH O non-covalent inhibitor

NH

O

NH

OOC fluorophore N

Figure 9.

N

The structure of a crosslinker-based aminopeptidase probe.

3. ACTIVABLE PROBES The above-mentioned probes for peptidases are mainly dependent on high-affinity or firm covalent binding to their targets, which can be delivered at high concentrations to the target. Nevertheless, there are still some undesirable unbound molecules remaining that lead to a high background signal. By contrast, activable probes, which are nonfluorescent themselves and Medicinal Research Reviews DOI 10.1002/med

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

(a)

r 9

(b)

(c)

fluorescent

Scheme 3. Schematic depiction of three types of activable probes. (a) Substrate–fluorophore probes, (b) substrate–linker–fluorophore probes, (c) FRET probes.

become fluorescent when degraded by their targeting peptidases, can minimize the background signal. The regular activable probes characteristically comprise two parts, a fluorophore and its corresponding quencher. So far, there are three types of activable probes, including substrate– fluorophore probes, substrate–linker–fluorophore probes, and fluorescence resonance energy transfer (FRET) probes as depicted in Scheme 3. A. Substrate–Fluorophore Probes for In Vitro Activity Assay Fluorescent detection of peptidase activity primarily relies on a substrate containing an amide functionality formed with peptidase specific residue as the acid and a fluorophore as the amine. For instance, Leu-AMC (leucine-(4-methyl-7-coumarinylamide)) is extensively used in APN and LAP (leucine aminopeptidase) activity assays, while tripeptidyl-peptidase II inhibitors are based on Ala-Ala-Phe-AMC, and so on.40–42 Because there are numerous classifications of enzymes in tissues, in the event that the binding-based probe is able to identify the activities and distributions of peptidases in vivo, many approaches should be considered to improve the selectivity of probes, and one feasible approach is to find specific substrates. For example, to prescribe a selective probe that could monitor DPP-I activity in living cells, a series of (dipeptidyl)2 -rhodamine substrates were well designed and synthesized by Harris et al. One of them, (NH2 -aminobutyric-homophenylalanine)2 – rhodamine, exhibited functional reactivity and selectivity in the fluorescence-activated cell sorting (FACS) analysis (Scheme 4).43 Fibroblast activation protein (FAP; EC: 3.4.21.B28) and dipeptidyl peptidase IV (DPP-IV; EC: 3.4.14.5) are both type II transmembrane serine proteases. They can hydrolyze N-terminal Medicinal Research Reviews DOI 10.1002/med

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r CHEN ET AL.

DPP-I O H2N

H N

N H

H N

O

O

O

H2N

NH2

O

O N H

NH2

O O

O O non fluorescent

fluorescent

Mechanism of detecting DPPI activity.

Scheme 4.

N

N HN R

O

FAP or DPP-IV

N

H2N

O

N

SO3

SO3

SO3 non-fluorescent

SO3

fluorescent

R = GPGP APAP TSGP Ac-GP Ac-GPGP

2SBPO

Scheme 5.

Mechanism of detecting DPP-IV and FAP activity.

dipeptides after a proline residue.44 In view of a high sequence similarity between FAP and DPP-IV, it is difficult to examine the activity of one in a mixture containing both of them. Recent studies have demonstrated that glycylprolyl dipeptide (GP) is an excellent substrate for DPPIV; however, it could in addition be recognized and hydolyzed by FAP.45 To obtain a probe to differentiate between these two proteases, five peptide-disulfonated benzo[a]phenoxazine (2SBPO) substrates were synthesized by Tung et al. Fortunately, two molecules, GPGP-2SBPO and Ac-GPGP-2SBPO, could discriminate the activities between FAP and DPP-IV in a cellular assay (Scheme 5).46 Chymotrypsin substrates have been employed to evaluate prostate-specific antigen (PSA) activity. To track down a PSA-specific probe, a series of peptides were conjugated to AMC by Isaacs et al. in 1997.47 A selectivity study with these probes revealed that sequence HSSKLQ had powerful specificity for PSA (i.e., cannot be recognized by chymotrypsin and some other tested proteases), which was also confirmed by Jones et al. in 2006.48 With this selective sequence in hand, Isaacs et al. synthesized Mu-HSSKLQ-Leu-Doxorubicin, a PSA-based prodrug, which showed very low cytotoxicity to PSA-nonproducing TSU human prostate cancer cells in vitro.49 Medicinal Research Reviews DOI 10.1002/med

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

r 11

B. Substrate–Self-Immolative Linker–Fluorophore Probes Substrate–fluorophore probes in general are dependent on the direct linkage between a fluorescent reporter and a protease recognition part. Nevertheless, the absence of a spacer between the two components may result in two key issues: (i) substrate–fluorophore probes for enzymatic detection sometimes cannot be recognized due to steric hindrance of the bulky fluorophore near the cleavage site that prevents binding to the active pocket of the protease; (ii) the fluorophores are limited to aromatic amines, such as rhodamine and hydroxymethyl rhodamine green,50 which do not cover a wide range of detection wavelengths in the visible and NIR spectra.51, 52 By contrast, substrate–self-immolative linker–fluorophore probes can overcome the above-mentioned disadvantages. It should be noted that the current type of probes employ a self-immolative linker to connect the substrates and fluorophores for preventing the steric hindrance around the cleavage site. Furthermore, as illustrated in Scheme 3b, the selection of fluorophores is augmented since the chemical bonds between linkers and fluorophores are no longer restricted to amide bonds. A self-immolative linker, also named a traceless linker, was first introduced to the design of prodrug by Katzenellenbogen and co-workers in 1981.53 In 2006, Jones et al. had successfully reported a self-immolative linker that could be utilized in the design of selective image contrast agents for serine protease PSA.48 As depicted in Scheme 3b, after hydrolysis of the amide bond between peptide and linker, the bond between linker and fluorophore can spontaneously split under physiological conditions to release the fluorophore. Since then, a large number of articles reported the importance of self-immolative linkers in the design of probes as listed in Table I. To drive the phenol-based fluorophores feasible in the design of peptidase probes, Renard et al. employed the Waldmann self-immolative linker to secure the coumarin fluorophore and peptide sequence to synthesize a probe for determining penicillin G acylase (PGA).62 Unlike its application in combinatorial chemistry,63, 64 the peptide sequence–Waldmann self-immolative linker–coumarin probe was unstable under the simulated physiological conditions due to the leaving-group ability of the 7-hydroxycoumarin fluorophore.62 The same phenomenon also occurred in the design of self-cleavable chemiluminescent probes for PGA.58 To enhance the stability of probes, ether and carbamate bonds were used to conjugate the phenol-based fluorophores and linkers. For example, Renard et al. replaced the Waldmann linker with a para-aminobenzyl alcohol (PABA) linker to generate a phenylacetic acid–PABA–7-hydroxycoumarin probe. After adding immobilized PGA to the phenylacetic acid–PABA–7-hydroxycoumarin solution, a strongly fluorescent signal was generated at 460 nm, indicating the hydrolysis of the amide bond and the liberation of free 7-hydroxycoumarin dye (Scheme 6). Apart from PABA, some analogs, such as ortho-aminobenzyl alcohol (OABA),54–56 (5-aminopyridin-2-yl)methanol,57 and (3-aminopyridin-2-yl)methanol,57 are also acceptable self-immolative linkers. Because of the lightweight structural variation among these linkers, the spontaneous decomposition rate are varied: (5-aminopyridin-2-yl)methanol > PABA, (3-aminopyridin-2-yl)methanol > OABA, and PABA > OABA.54, 57 There are other linkers that combine the function of PABA and OABA, the self-immolative dendrimers.65, 66 This type of probe can usually connect one substrate with two or three reporters. Shabat and Danieli developed a dual output probe for peptidases by using this self-immolative dendrimer, which could examine the activity of PGA under both UV and fluorescence conditions (Scheme 7).67 All of the current substrate–fluorophore detection of peptidase activities is largely based on substrates containing an aromatic amide bond. This is explained by the fact that only the amino group that directly connects to a conjugated system can play a “quench–dequench” role in a fluorophore. In fact, most of the natural peptides rely on an aliphatic amide bond. A self-immolative linker enables aliphatic amide bonds to be used in detecting peptidases activi¨ ties. Enzyme-labile linker alkyloxy(phenyl)methanamine was originally invented by Bohm et al. Medicinal Research Reviews DOI 10.1002/med

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Table I. Disassembly Mechanisms of Self-Immolative Linkers Self-immolative linker

Release mechanism

O Trigger

X

Z

48, 54–58

X

H2 N

X = C or N

X = C or N NH2

Z

Trigger

OR

Fluorophore

Ref.

OR

Fluorophore

O

54–57

X

X

X = C or N

X = C or N

O

O

Fluorophore

O

OR

Trigger

58

OMe

OMe H 2N

OMe

OMe

COOH

COOH

51 S

Trigger

Fluorophore

H2N

S

R

59 N

N

NH 2

Trigger

H2N

Trigger

N O

O Fluorophore

R O

60

N O O

H2N

O Trigger

O

O

Fluorophore

O

OR

61

for the reversible coupling of alcohols to solid supports.68 Based on this mechanism, Romieu and co-workers designed a new self-immolative linker involving a thioalkyl moiety and a selfcyclization linker as shown in Scheme 8.51 In vitro fluorescent test and liquid chromatography– mass spectrometry (LC-MS) study showed that the aliphatic amide bond based probe with the thioalkyl N-methyl-N-[2-(methylamino)ethyl] carbamate linker was not liable to nonspecific hydrolysis and was suitable to be used in PGA detection, although the enzyme-triggered domino reaction became much slower. A kinetic study of the PABA-based or hemithioaminal counterparts based self-immolative linkers was carried out by Yves et al.69 As depicted in Table II and Figure 10, the decomposition of PABA-based self-immolative linkers was faster than those with the hemithioaminal counterparts. This result indicated that the 1,6-benzyl elimination occurs Medicinal Research Reviews DOI 10.1002/med

r 13

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

O

HOOC O

O O

low stability O

O

NH OMe

H N

O

O

OMe

O

PGA

O O O

OH

O

NH2

spontaneously

O

Design of a PABA-based PGA probe.

Scheme 6.

O NH

O O2 N

O N O O

N

O

N

N

N N

N

O O H N N

O

O

N

O

N

N

OH O O O NH

5000 GEP HN

O

N

O

NH PGA

N

O

N

O

O

5000 GEP HN

O

H N

O

H N N

CO2

O

N

O2N

O

HO

HO spontaneously

H N

O CO2

N

N N

N

N

OH O

+

O2N

O

+

N NH2

HO 5000 GEP HN O

Scheme 7.

A dual output probe for PGA.

more rapidly than the thioalkyl carbamate intermediate involved fragmentation–cyclisation process. And because the carbamate decarboxylation occurring during the domino reaction provides an additional thermodynamic driving force, probe 5 was in a position to release 7-hydroxycoumarin even faster than the parent PABA-based fluorogenic probe 4. Other aliphatic amide bond based self-immolative linkers are summarized in Table I.

3.3. Fluorescence Resonance Energy Transfer Probes Some types of endopeptidases can only hydrolyze amide bonds of nonterminal amino acids (i.e., within the peptide molecule); therefore, their activities cannot be examined by substrate– fluorophore and/or substrate–linker–fluorophore probes. To overcome this limitation, a Medicinal Research Reviews DOI 10.1002/med

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r CHEN ET AL. COOH

COOH

O N H

S

O

O

O

O

PGA H 2N

O

O

S

O

O

O

O

COOH

HN

O S

O

+

HO

O

O

S

O

O

O

O

O

Scheme 8. Mechanism of detecting the activity of PGA by using a thioalkyl-contained self-immolative linker based probe.

Figure 10. Fluorescence emission time-course of pro-fluorescent probes 2–6 (concentration: 3.0 mM) with recombinant PGA (0.12 U, 37°C) in PBS buffer at 460 nm (Ex = 360 nm). Source: Reference 69 copyright (2010), Royal Society of Chemistry.

Medicinal Research Reviews DOI 10.1002/med

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

r 15

Table II. Structures and Disassembly Mechanisms of Some Self-Immolative Linkers O

self-immolative spacer

O

O

O

R PGA cleavage site

Compounds

Self-immolative linker

Release mechanism COOH

COOH

2

O

O O

S

N

H2N

N

O

S

COOH

COOH

O O

S

N

N

OF

O

O

3

N

H2N

N

O O

S

N

N

OF

N

OF

O

O

4

OF O

H2N

O O

5 O

N

O

N O

N

O

H2N O

6

O

HN

N

OF

N

R = H for compounds 2–5 and Me for compound 6; OF = 7-hydroxycoumarin.

fluorescent donor and a fluorescent or a nonfluorescent acceptor molecule were tethered through a protease substrate peptide linker to generate a so-called FRET probe (Scheme 3c), which has been extensively used in the study of protein interactions. In such an occasion, the acceptor can absorb the light emitted by the excited state donor due to the FRET effect; consequently, the probe is nonfluorescent at the donor emission wavelength. After the degradation of the peptide by peptidase, the FRET influence will disappear, and the donor will retrieve fluorescence.70 So far, the FRET approach has been well accommodating to examining various peptidases, and one case in point is the development of fluorescent probes of the matrix metalloproteinases (MMPs). MMPs are well recognized to be critical in normal tissue remodeling as well as in diverse pathological states, such as lung diseases, atherosclerosis, and carcinoma.61, 71, 72 Among the MMP family, MMP-7 has been a persuasive target due to its overexpression in pancreatic, colon, and breast cancers.73 Bearing in mind the endopeptidase activity of MMP-7, Tung and co-workers developed a FRET probe consisting of a enzyme-selective peptide VPLSLTMG, an NIR fluorescence donor (Cy5.5), and an NIR fluorescence absorber (NIRQ820).74 Considering that NIRQ820, Medicinal Research Reviews DOI 10.1002/med

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KO3S SO3K

O3S

HO3S

SO3K N

N

HOOC

O3S O

Cl NIRQ820

N (CH2)5

N Cy5.5

HN Gly

O

Val

O

NH

N

S

Pro Leu Ser Leu Thr Met Gly Cys O CONH2 MMP-7

KO3S SO3K

O3S

HO3S

SO3K N HOOC

N Cl

O3S N (CH2)5

N

O HN Gly

O S

Val Pro Leu Ser

Scheme 9.

O

NH

N

Leu Thr Met Gly Cys O CONH2

Schematic representation of a FRET probe for MMP-7.

a cyclohepta polymethine fluorochrome (λex /λem = 790/820 nm), could absorb the light released by excited state Cy5.5 (λex /λem = 675/694 nm), the probe displayed no fluorescence at 690 nm (excitation at 667 nm). Upon adding MMP-7 to the solution of the probe, a noteworthy fluorescence occurred at 690 nm based on the split-up of two fluorophores, which reflected the decomposition of the VPLSLTMG sequence and the hydrolytic activity of MMP-7 (Scheme 9). A shortcoming in the function of a fluorophore as an acceptor is that the emission light produced by the acceptor fluorophore may sometimes act as visual “noise” to interfere with the measurement.75 Compared to a fluorophore quencher (acceptor), the dark-quencher (nonfluorescent acceptor) has no native fluorescence and therefore can capture the light emitted by fluorescence donor. Eventually, this quenched fluorogenic substrates may result in a feasible high-throughput screening that allows for a rapid and continuous measurement of enzyme activity. Another example in the MMP family is MMP-13 that overexpresses in degenerating cartilage.76 To image osteoarthritis development in vivo, BHQ-3 (a dark-quencher, the acceptor) and Cy5.5 were linked to the MMP-13 substrate GPLGMRGLGK to make a FRET probe, which enables the visualization of MMP-13 in vitro and in the osteoarthritis-induced rat models (Scheme 10).77, 78 There are many commercial available dark-quenchers, such as dabcyl (490 nm), BHQ-0 (493 nm), EclipseTM(522 nm), BHQ-1 (534 nm), BHQ-2 (579 nm), BHQ-3 (672 nm), and so on, which can quench a certain wavelength fluorophore separately. In 2009, Peng et al.79 reported an extremely useful dark-quencher, IRDye QC-1, which efficiently quenches fluorescence from a wide range of fluorophores spanning the visible to NIR spectrum (500–800 nm). However, the dark-quencher-based FRET probe cannot reflect the distribution of the uncleaved probe in cell imaging. To address this issue, Geoffray et al. considered a FRET probe with a chemically deactivatable quencher (acceptor). As indicated in Scheme 11, these probe Medicinal Research Reviews DOI 10.1002/med

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SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES FRET

N N Gly Pro Leu Gly

Met Arg Gly Leu Gly

N (CH2)3CO

N

BHQ-3

Lys NH2

KO3S SO3K MMP-13

SO3K O3S

Gly NH2

Leu OH Gly Pro

Met Arg Gly Leu Gly

N (CH2)5

N

Cy5.5

Lys NH2

O

Scheme 10.

A dark-quenched FRET probe for MMP-13.

HOOC

O N

N

O O

N H

COOH O H N O

OH

FRET

N H

H N O

O

N

O

N

H N

O OH

+

HN

O

O

COOH NH2

COOH

e-3

pas

cas HOOC N

N

O O

OH

N H

COOH O H N O

N H

H N O

O N H O COOH

O H N

O

N

dit

H N

hio

nit

e

O

COOH

NH2

O O

acceptor

substrate

donor OH

Scheme 11.

N H

COOH O H N O

N H

H N O

O N H O COOH

O H N

H N O

COOH

Principle of FRET-based probe with a chemically deactivatable quencher.

molecules can be activated by caspase-3 hydrolysis. After detecting the activity of caspase-3, dithionite, which can chemically deactivate the quencher, was added to the imaging system. All of the unreacted probes were then turned on to display their distribution.80 Other commonused FRET pairs are Cy3 and Cy5, Alexa488 and Cy3, Alexa488 and Alexa555, and FITC and rhodamine. More information about FRET pairs and their application can be found in recent reviews.75, 81

4. SWITCHABLE BINDING BASED PROBES A. Activable Binding-Based Probes Activable probes can minimize the background signal by using switch-off and switch-on strategies, nonetheless they cannot precisely locate their targets because of diffusion of the fluorophores. To address this issue, Bogyo and co-workers synthesized a panel of activable binding-based probes by using the FRET strategy as depicted in Scheme 12a.82, 83 After covalent modification of the targets, the quenching groups were cleaved and fluorescently labeled enzymes were acquired. The same strategy was successfully used again by Bogyo and coworkers to design an activable binding-based probe for imaging legumain (a lysosomal cysteine protease).84 These probes can exactly present the distribution of their targets with remarkable low noise; however, they can only be applied to cysteine, serine, and threonine peptidases. Medicinal Research Reviews DOI 10.1002/med

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r CHEN ET AL. (a)

FRET Fluorophore

Fluorophore

O O O

peptide

S

peptide

quencher O

SH

O

quencher O

O R

(b)

X

X

HN Peptidase

Nu

O quencher

Nu O

O

O

O Fluoro phore

Fluoro phore

Fluoro phore

FRET

Scheme 12.

Mechanism of activable binding-based probes.

Another brand of activable binding-based probes was intended by the Yao group using quinone (or quinolimine) methide as the electrophilic group (Scheme 12b),85 which could be used for, but not limited to, cysteine, serine, and threonine peptidases. Upon enzymatic turning on the switch, an 1,6-elimination reaction resulted in the liberation of the quencher group, thus releasing a fluorescent labeled quinone/quinolimine methide intermediate. The intermediate could generate the covalent bonds with nucleophilic groups of the targets, as well as provide fluorescence-labeled enzymes.

4.2. Environment-Switchable Binding Based Probes It needs to be noted that all activable probes are mainly based on enzymatic reaction, whose switchable property significantly reduces the background signal. To expand the scope of switchable peptidase probes, Tan et al. recently described a type of environment-switchable fluorescent probes.86, 87 The heart of this approach is a highly environment-sensitive fluorophore, 4-sulfamonyl-7-aminobenzoxadiazole (SBD), which presents weak fluorescence in polar and protic environments but turns out to be strongly fluorescent in hydrophobic surroundings. By connecting an SBD derivative with a reversible competitive inhibitor of trypsin, benzamidine, an environment-sensitive trypsin fluorescent probe could be generated (Scheme 13). Because the binding site of trypsin is hydrophobic, this probe demonstrated a dramatic 17-fold fluorescence enhancement after incubation with trypsin. Medicinal Research Reviews DOI 10.1002/med

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low fluorescent High fluorescent

HN H2N hydrophobic site HN binding

NH2

dissociation

receptor

receptor HN N O N SBD

O S O HN

O

H2N

Scheme 13.

N

N N

N H

Mechanism of environment-switchable binding based probes.

5. CONCLUSIONS AND PROSPECTIVE All categories of small-molecule fluorescent probes that can be employed for the detection of peptidases have been mentioned throughout this review article. Fluorescent probes of various types have proven to be useful to detect a variety of peptidase types; for example, substrate– fluorophore probes have been extensively used for the activity assays for various peptidases at the enzymatic level. However, if the activity of a single peptidase needs to be evaluated in an intact cell, a selective substrate–fluorophore probe is urgently required in the presence of many other types of intracellular peptidases. FRET probes are necessary for evaluation of endopeptidase activity because substrate–fluorophore probes and substrate–self-immolative linker–fluorophore probes are only suitable for exopeptidases activity assay. Because of the diffusion of the fluorophores, activable probes cannot precisely locate their targets. Covalent binding based probes have outstanding advantages in the distribution study of threonine, serine, and cysteine proteases. Noncovalent binding based probes can also be utilized to locate metallo-peptidase, aspartate, and glutamate proteases. However, common binding-based probes have intrinsically high background signals. Therefore, more and more activable binding-based probes should be considered for cell and tissue imaging because they can examine both activities and distributions of peptidases with low background signal, in the meanwhile environmentswitchable fluorescent probes in protein imaging should also be developed. There are still many attempts to improve the current peptidases probes, and one active attempt is the development of ratiometric probes. For example, because the activity assay of peptidases proceeds in buffer or culture medium that may significantly affect the spectral properties of fluorophores, two types of ratiometric fluorescent probes for APN were designed and synthesized in our laboratory.88, 89 These APN probes can in vitro and in cellulo detect the activity of APN with minimal environmental influence. Hitherto, many types of ratiometric probes have been considered for the detection of ions since they can supply a built-in correction for environmental effects.90–94 However, only a few ratiometric peptidase probes have been reported up to now. Activable binding-based probes have many advantages in tissue imaging; nevertheless, some types of peptidases cannot be labeled via covalent bonds, as well as covalent labels may result in cytotoxicity. Noncovalent activable binding based probes, which use their conformation changes before and after binding upon their targets to switch on/off the fluorescent reporters, can overcome these difficulties and should be further developed. It should be underlined that such noncovalent activable binding based probes have been employed in carbonic anhydrase, COX-2, and RNA detection, but not in peptidase detection hitherto.95–97 Medicinal Research Reviews DOI 10.1002/med

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Futhermore, in view of many intracellular biomarkers and bioactive small molecules, a multifunctional and/or multimechanism peptidase probe that can react with these specific molecules will be superior in cell and tissue imaging.98, 99

ACKNOWLEDGMENTS The present work was supported by the Program for New Century Excellent Talents in University (NCET-11-0306), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13028), the Shandong Natural Science Foundation (JQ201019), the Independent Innovation Foundation of Shandong University, IIFSDU (2014JC008), and the Graduate Independent Innovation Foundation of Shandong University, GIIFSDU (yzc12096).

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Medicinal Research Reviews DOI 10.1002/med

SMALL-MOLECULE FLUORESCENT PROBES FOR PEPTIDASES

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Laizhong Chen obtained his bachelor’s degree at School of Pharmacy, Shandong University, in 2009. And he stayed in Shandong University to complete his PhD in the Li lab. His main research interests include APN (Aminopeptidase N) probe design and their biological application. Jing Li received her bachelor’s degree at School of Pharmacy, Shandong University, in 2010. And she joined the Li group and pursued her master’s degree at the same school. Her research interests mainly focus on the design and bioactivity study of bioluminescent probes. Lupei Du is currently an associate professor at School of Pharmacy, Shandong University. She obtained her PhD degrees from China Pharmaceutical University, in 2006, and got her postdoctoral training at Department of Chemistry, Georgia State University (2006–2009). She joined Shandong University in 2009. Her main research interests include the rational design and synthesis of medicinal molecules and bioactive probes. Minyong Li is a professor at School of Pharmacy, Shandong University. He received his PhD degree from China Pharmaceutical University in 2005. He began his academic career in 2005 with Dr. Binghe Wang at Department of Chemistry, Georgia State University, as postdoctoral research associate. From 2007 to 2009, he was promoted to be a research assistant professor at Department of Chemistry, Georgia State University. In 2009, he relocated to his current institution as a full professor. His research interests are in the general areas of medicinal chemistry and chemical biology.

Medicinal Research Reviews DOI 10.1002/med

Strategies in the design of small-molecule fluorescent probes for peptidases.

Peptidases, which can cleave specific peptide bonds in innumerable categories of substrates, usually present pivotal positions in protein activation, ...
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