A Universal Assay for Aminopeptidase Activity and Its Application for

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A UNIVERSAL ASSAY FOR AMINOPEPTIDASE ACTIVITY AND ITS APPLICATION FOR DIPEPTIDYL PEPTIDASE-4 DRUG DISCOVERY Arkadiy A. Bazhin, Marc Chambon, Jonathan Vesin, Julien Bortoli, James W. Collins, Gerardo Turcatti, Chieh Jason Chou, and Elena A. Goun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04672 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Analytical Chemistry

A UNIVERSAL ASSAY FOR AMINOPEPTIDASE ACTIVITY AND ITS APPLICATION FOR DIPEPTIDYL PEPTIDASE-4 DRUG DISCOVERY Arkadiy A. Bazhin1, Marc Chambon2, Jonathan Vesin2, Julien Bortoli2, James W. Collins1, Gerardo Turcatti2, Chieh Jason Chou3, Elena A. Goun1* 1 Laboratory

of Bioorganic Chemistry and Molecular Imaging, Institute of Chemical Sciences and Engineering (ISIC), Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland. 2 Biomolecular

Screening Facility (BSF-ACCESS), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland. 3 Microbiome

and Metabolism, Nestlé Institute of Health Sciences SA, 1015 Lausanne, Switzerland

* e-mail: [email protected] ABSTRACT: Aminopeptidases, such as dipeptidyl peptidase-4 (DPP-4, CD26), are potent therapeutic targets for pharmacological interventions because they play key roles in many important pathological pathways. To analyze aminopeptidase activity in vitro (including high throughput screening [HTS]), in vivo, and ex vivo, we developed a highly sensitive and quantitative bioluminescence-based readout method. We successfully applied this method to screening drugs with potential DPP-4 inhibitory activity. Using this method, we found that cancer drug mitoxantrone possesses significant DPP-4 inhibitory activity both in vitro and in vivo. The pharmacophore of mitoxantrone was further investigated by testing a variety of its structural analogues.

Keywords: dipeptidyl peptidase-4, bioluminescent imaging, drug discovery

INTRODUCTION

fluorescent (e.g. aminomethylcoumarin)1,2,6 or absorbing (-

Aminopeptidases are an important class of biomolecules

naphthylamine)7,8 dyes from artificial substrate and are

that cleave peptide bonds at the N-terminal end of a protein

limited to ex vivo applications. In addition, they are time

or peptide. They are involved in many physiological and

consuming and tedious. We developed a quantitative optical-

pathological processes, like

fibrosis2,

based readout method that in addition to overcoming these

and immune

disadvantages, is also sensitive in a variety of applications,

Therefore, several aminopeptidases are potent

including ex vivo, in vitro and in vivo. Using DPP-4 as an

therapeutic targets used for screening of drugs. One such

example, we conducted HTS of 3930 compounds from a

example is dipeptidyl peptidase-4 (DPP-4, CD26), a major

library of known widely used drugs and natural products.

target for the treatment of Type 2 diabetes mellitus (T2DM).

Several hits were identified, and only one compound, a cancer

Despite its vital role in drug discovery, currently there is no

drug called mitoxantrone, possessed significant DPP-4

universal method that can be easily adapted for measuring

inhibitory activity in vivo. This result was re-confirmed by

aminopeptidase activity in formats ranging from in vitro and

classical approach of measuring GLP-1 levels in plasma.

ex vivo analysis to assessment in living animals and high

Investigation of structure activity relationships of multiple

throughput screening (HTS). Most assays used for measuring

analogues of mitoxantrone helped us to identify it’s

aminopeptidase activity are based on the release of

pharmacophore. Even though we demonstrate the application

cancer

development3,

response5.

angiogenesis1,

diabetes

mellitus4,

liver

1

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of this method only in the context of discovering novel DPP-4

synthesis of a short peptide chain ending with D-Cysteine

inhibitors, it can be universally adapted for other important

residue at N-terminus. Such peptides can be produced using

aminopeptidases too.

automated classical solid phase peptide synthesis that is easily accessible, robust, scalable, and cheap.

Bioluminescence (BL) imaging is a widely used optical imaging technique based on luciferase-catalyzed oxidation of

To demonstrate this approach, we choose to work with

a small molecule called D-luciferin resulting in production of

dipeptidyl peptidase-4 (DPP-4, CD26), that is a major target

visible

light9,10.

It has been successfully used for in vivo

for the treatment of T2DM3-6. It also plays important role in

imaging and quantification of many biological processes,

many other biological processes such as T-cell activation,

including uptake of fatty acids and peptides11, measurements

apoptosis and cancer metastasis. Therefore, currently many

of reactive oxygen species12 and various enzymatic activities13-

pharmaceutical companies and research institutes are

16,

gene expression17,18, and tumor cell proliferation19. For many

focusing on developing inhibitors of DPP-425,26. The most

biological applications bioluminescence (BL) imaging is

widely used method for DPP-4 quantification is based on in

superior

and

vitro fluorescent enzymatic assays7-9. However, this assay is

biocompatibility of reagents, deep tissue penetration,

limited to ex vivo applications, as well as time consuming and

to

fluorescence

due

to

low

toxicity

ease to use, and

tedious making the drug discovery process complex and

ability to image and quantify kinetic changes in real-time13-19.

inefficient. In this new approach, D-cysteine, one of the "split

Currently, BL imaging remains the most sensitive imaging

luciferin" parts, is "caged" with DPP-4 specific sequence of

modality in vivo that can be elegantly translated from in vitro

amino acids (Gly-Pro)27 resulting in a tripeptide Gly-Pro-D-

quantification directly to living animals, making it an ideal

Cys (GPc). Proteolytic activity of DPP-4 on this peptide

universal method for evaluating various biological processes.

enables cleavage of D-Cys and subsequent in situ formation of

extremely low signal background

levels11,12,20,

One of the recent application of BL imaging in probing

D-luciferin via reaction with second component of "split

molecular signatures of targeted tissues is caging of D-

luciferin" (CBT) (Fig. 1a). The resulting molecule of D-luciferin

luciferin scaffold with various caging groups that upon

is then oxidized by firefly luciferase resulting in production of

cleavage result in production of bioluminescent light21.

one photon of light, which can be quantified by sensitive CCD

Inspired by multiple recent reports of novel bioluminescent-

camera or plate reader. Since free D-cysteine is required for

based protease probes13,14,22, we decided to develop reagents

the reaction to proceed, no light is observed in the absence of

for quantifying aminopeptidase activity that can be adapted

DPP-4. Also, CBT reacts with endogenous L-cysteine resulting

for a wide range of assays in various formats (e.g. in vitro and

in the formation of L-Luciferin which only produces

ex vivo analysis, HTS, experiments on living animals). Since

background level of light in the presence of luciferase28,29.

aminopeptidases cleave peptide bonds at the N-terminal end

Therefore, the probe named DPP-4 Activated Luciferin (DAL),

of a protein or peptide, we used specific amino acid sequences

enables selective real-time imaging and quantification of DPP-

as a chemical cage for luciferin scaffold or its components.

4 activity by measuring the photon flux from the luciferase-

This is based on "split luciferin" reaction (Fig 1a) where D-

catalyzed oxidation of D-luciferin (Fig. 1a).

cysteine reacts with 6-Hydroxy-2-cyanobenzothiazole (CBT)

EXPERIMENTAL SECTION

to form firefly D-luciferin. It is an example of biocompatible

High throughput screening (HTS)

transformation wherein molecules can selectively react with

HTS was done at the Biomolecular Screening Facility of

each other even inside the living cells or animals.23 The "split

EPFL. In total 3930 compounds were used for screening from

luciferin" approach along with caging D-Cysteine component

Prestwick library and a natural products collection (selection

with aminopeptidase-specific peptide sequence has several

of compounds from Analyticon and InterBioScreen).

advantages over caging the full luciferin scaffold. Unlike the

100 nL stock 10 mM solution compounds in DMSO,

latter approach, it is more practical, versatile, avoids complex

sitagliptin control in DMSO, or DMSO control were dispensed

low-yielding synthetic procedures24 , and only requires 2


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Analytical Chemistry by an Echo 550 acoustic dispenser (Labcyte Inc.) into a 96-well

0.91 mM mitoxantrone (3 mg/kg) and 27.3 mM GPc (55 mg/kg)

plate according to a general scheme (Scheme S1). The

in PBS. Subsequently, mice were anesthetized with isoflurane

following solutions were added to each well: 90 µL 11% (v/v)

and injected i.p. with 100 µL of 10 mM (5.9 mg/kg) CBT in 30%

human plasma solution in PBS, 10 µL of a mixture of 400 µM

v/v PEG400:70% water.

GPc and 1 mM CBT in 5% DMSO:95% PBS. After 30 min, 10 µL

DAL administration

80 µg/mL firefly luciferase solution was added to each well.

Mice were injected i.v. with 200 µL 30 mM GPc peptide in

Resulting mixture was incubated at room temperature for 30

PBS (55 mg/kg). In 15 min mice were anesthetized with

min followed by the measurement of the resulting BL signal

isoflurane and injected i.p. with 100 µL 10 mM (5.9 mg/kg) CBT

using a Tecan Infinite 500.

in 30% v/v PEG400:70% water.

The procedure for the "control" experiment was the same as

Bioluminescence imaging

the high throughput screening experiment described above

Immediately after CBT injection, the mice were placed in an

with one exception: 10 µL 200 µM D-luciferin was used instead

IVIS Spectrum (PerkinElmer). BL signal from whole body was

of 10 µL solution containing GPc and CBT.

measured every 18 sec over the first 5 min, and then every

Measurement of DPP-4 activity in vivo using DAL probe

minute over the following 35 min. Regions of interest were

Mice

identified and quantified, photons s−1 (total photon flux),

Pathogen-free FVBTg(CAG-luc,-GFP)L2G85Chco/J (FVB-

using the Living Image software (PerkinElmer).

Luc) mice were purchased from Jackson laboratory and bred

GLP-1 measurement in plasma.

in the Center of PhenoGenomics (CPG), EPFL. Male and

Mice, fasting for 8 h prior to the experiment, were injected

female mice weighing between 20-40 g were housed

i.v. with 200 µL 1 mM mitoxantrone in PBS, 3 mM saxagliptin

separately in HEPA-filtered cages with sterilized beddings,

in PBS, or PBS. After 15 min, mice received an oral gavage of

food, and water. Experiments were repeated on 2 separate

300 µL of 40% glucose solution and yoghurt. 10 min post

occasions with 3-8 mice per group. 10 weeks old female

gavage mice were anesthetized with isoflurane and 700-1000

FVB/NJ mice were purchased from Jackson laboratories and

µL blood was taken, by cardiac puncture, into a syringe

used for GLP-1 measurement in plasma. They were housed

containing 100 µL 1 mM saxagliptin (to stop GLP-1 truncation),

separately in HEPA-filtered cages with sterilized beddings,

7 µL protease inhibitors cocktail (Sigma, P8340), and 28 mM

food, and water. Experiments with these mice were repeated

K3EDTA in PBS. Blood was then transferred into vacutainer

on 2 separate occasions with 8 mice per group.

tubes (BD, 368861) and centrifuged at 0°C. Subsequently,

All animal experiments were approved by the Veterinary

plasma was harvested, aliquoted, and stored at -70°C prior to

authority of the Canton Vaud, Switzerland (license #2849c,

analysis. ELISA kits (Millipore, EZGLP1T-36K and EGLP-35K)

3147).

were used to measure the concentration of active and total

Treatment of mice with DPP-4 inhibitors

GLP-1 according to manufacturer instructions.

Mice were treated with either sitagliptin, saxagliptin, or PBS

Statistical analysis

by oral gavage, at doses ranging from 0.01 mg/kg to 2 mg/kg.

Data presented graphically are presented as mean ± SD. A

All inhibitors were administered 45 min prior to the

Mann-Whitney test was used to analyze all data, using the

administration of CBT.

commercially available software GraphPad Prism (version 6.07); P value of less than 0.05 was considered as significant.

Mitoxantrone treatment and DAL co-administration Mice were injected i.v. with 220 µL of a solution containing

3



Figure 1. (a) Design strategy for DPP-4 mediated formation of firefly luciferin from DAL probe. Uncaging of D-cysteine by DPP-4 via cleavage of corresponding tripetide (Gly-Pro-(D-Cys)) and subsequent reaction with CBT results in formation of firefly luciferin, followed production of light. (b) Ex vivo measurement of DPP-4 inhibition by sitagliptin (known inhibitor) using two different reagents – novel DAL probe and conventional GP-AMC fluorogenic reagent (Bachem).

Supplementary

experimental

procedures,

synthetic

photon flux all the way to the background level (Fig. S1).

procedures and characterization of GPc peptide can be found

We then decided to compare in vitro performance of DAL

in Supplementary Information.

with the commercial fluorogenic probe widely used to quantify DPP-4 activity in vitro and based on caged

RESULTS AND DISCUSSION

aminocoumarin scaffold (GP-AMC, Bachem, USA)25. To

Validation of DAL-based assay in vitro and in vivo.

mimic physiological conditions, pooled human plasma was

In the first step, we established that DAL is indeed a

used as the source of DPP-4 and IC50 values of sitagliptin were

substrate for DPP-4 protease using an in vitro assay with

determined using both probes in side-by-side experiment

human recombinant DPP-4. In the presence of DPP-4, BL

These values were found to be 9.3 ± 1.9 nM and 9.0 ± 0.6 nM

signal from DAL increased 20-fold above the DPP-4 free

(95% CI) for DAL and GP-AMC, respectively (Fig. 1b). This

control (Fig. S1). Further addition of 0.1 µM and 1 µM

result suggest that the DAL can be used as a valuable

sitagliptin, a known DPP-4 inhibitor approved for T2DM

substitute for the GP-AMC probe and therefore suitable for

treatment30, attenuated the photon flux in dose dependent

screening of compounds with DPP-4 inhibitory activity in

manner. Also, addition of 10 µM of sitagliptin reduced the 4


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Analytical Chemistry vitro. Also, coumarin-based fluorescent probes are known to

inhibitors was observed in a dose-dependent manner (Fig. 2b).

produce high autofluorescence. As a result, they require

Importantly, the median effective dose (ED50) of saxagliptin

laborious processing in live tissues and whole blood leading to

was ~9-fold less than that of sitagliptin (0.13 mg/kg and 1.14

big experimental variations. Since DAL is based on BLI

mg/kg respectively), which is consistent with previously

readout bereft of these drawbacks, it can potentially measure

published data using two other independent methods32-34.

DPP4 activity directly in whole blood and live tissues,

This is the first report to successfully measure DPP-4 activity

significantly

in living animals with high sensitivity and accuracy using non-

minimizing

sample

processing

time

and

experimental errors.

invasive

imaging.

Another significant advantage of bioluminescent-based DAL over a fluorescence coumarin-based probe is its potential application in real-time imaging and quantification of DPP-4 activity directly in living animals. To study its utility in living animals, we first determined whether the route of administration of the two DAL components (GPc and CBT) affects the resulting BL signal in mice. For this experiment we used transgenic FVB-luc+ mice that ubiquitously express luciferase under β-actin promoter31 (FVB-Tg(CAG-luc,GFP)L2G85Chco/J mice, commercially available from Jackson laboratory (USA)). When both CBT and GPc were injected i.p., the resulting BL signal from the whole mouse body was almost the same as background signal resulting from CBT injection along. Alternatively, when GPc was administered i.v., the whole-body

BL

signal

increased

17-fold

above

CBT

background (Fig. S2). Therefore, i.v. injection of GPc and i.p. injection of CBT was the preferred route for administering DAL. Besides, fast clearance of residual BL signal after DAL administration allows longitudinal imaging of the same mouse (Fig. S3). To assess DPP4-specificity of the signal, mice received oral gavage of sitagliptin at a dose of 10 mg/kg (19 µmole/kg) or vehicle alone (PBS) before injecting the probe.

Figure 2 Evaluation of DAL probe in FVB-luc+ mice. (a) Oral gavage of sitagliptin (10 mg/kg) resulted in 6 fold reduction in total photon flux from DAL (GPc i.v.+CBT i.p.) treated mice. (b) In vivo measurements of median effective dose (ED50) in FVB-luc+ mice of two known DPP-4 inhibitors (saxagliptin and sitagliptin). Relative luminescence is expressed as a percentage of total photon flux obtained from mice treated with a DPP-4 inhibitor, compared to naïve mice. X axis— negative logarithm of an inhibitor concentration in 100 μL of gavaged solution. Each data point is mean ± SD, n=3. ED50 of saxagliptin and sitagliptin are 0.13 mg/kg and 1.14 mg/kg, respectively.

The resulting BL signal from mice treated with inhibitor was comparable to the background level (Fig. 2a), indicating a strong link between the signal intensity in vivo and DPP-4 activity. In the next step, we studied dose-dependent signal suppression mediated by sitagliptin and saxagliptin, another potent inhibitor of DPP-4. Mice received oral gavage of varying concentrations of sitagliptin or saxagliptin (0.03-10 mg/kg) for 30 min before injecting DAL. Significant reduction of signal produced from mice treated with both DPP-4

High-throughput screening and validation of hits

aid in discovery of potent inhibitors of DPP-4 activity, a

Adapting DAL-based assay for high throughput screening

current focus of many pharmaceutical companies. We

(HTS) followed by in vivo validation of the resulting hits would

selected a total of 3930 compounds from two compound 5



Page 6 of 9

libraries readily available at our University (EPFL) – the

human recombinant DPP-4 was measured for each of these

Prestwick chemical library of known drugs (1280 compounds)

compounds in the concentration range of 1-500 µM. Although

and a natural products collection library (2650 compounds).

all three compounds clearly inhibited DPP-4 activity in a dose-

A schematic representation of the screen is depicted in Figure

dependent manner with IC50 of 54, 103, and 146 µM for

3a and b. Sitagliptin, a known inhibitor of DPP-4, was used as

mitoxantrone, nisoldipine, and disulfiram, respectively (Fig.

a positive control in the screening assay. Z’ factor, an indicator

3c), with mitoxantrone being the most active. To further

of assay reliability, was calculated for each plate separately.

validate the hits, the same experiment was performed using

The mean Z’ factor was 0.89 ± 0.05 (mean ± SD, n=104),

fluorogenic substrate GP-AMC that is a commonly used test

demonstrating a very high degree of reliability (Fig. S4).

for assessment of DPP-4 activity. The results were in

Reproducibility

independent

agreement with DAL assay and confirmed that mitoxantrone

experiments where IC50 of sitagliptin was measured using

is indeed the most active compound (Fig. S5). While DPP-4

human plasma as the source of DPP-4. As expected, sitagliptin

inhibitory activity of mitoxantrone was lower than commonly

inhibited DPP-4 activity in a dose dependent manner (Fig. S4)

used DPP-4 inhibitory drugs (example, sitagliptin and

and the mean IC50 was 7.29 ± 0.57 nM (mean ± SD, n=6) with

saxagliptin), the compound represents new class of molecules

very low CV of 7.8% which demonstrates excellent assay

that could be further investigated and optimized for this new

reproducibility.

application. To evaluate whether mitoxantrone would inhibit

was

evaluated

in

three

Sixty-seven hits were identified from the HTS screen (Tab

DPP-4 activity in living animals, we used the DAL-based assay

S1). In the next step, we wanted to ensure that the signal

previously validated for in vivo use. Mice were divided in

attenuation was indeed specific to DPP-4-activity. DAL

several groups and treated with corresponding compounds

readout is based on enzymatic luciferin-luciferase reaction

followed by administration of DAL. Since mitoxantrone has

and might get affected by the presence of a certain

low DPP-4 inhibitory activity, we used varying concentration

drug/natural product tested in the screen. To correct this

ranging from higher therapeutic dosage to its LD50.

anomaly, a common practice in the field is to use free luciferin

Mitoxantrone

as a control experiment and plot normalized values12,35,36. In

suppression (32%) of BL signal when compared to PBS control

line with this practice, another HTS was performed using free

(Fig. 3d). We observed no interference between mitoxantrone

luciferin instead of DAL (Tab. S1). Significant DPP-4-

and the assay itself in vivo (Fig. S6). The inhibitory activity of

independent signal suppression from luciferin-luciferase

mitoxantrone was further validated by measurements of GLP-1

reaction was observed for flavone and iso-flavone derivatives

concentration in plasma of drug-treated mice following oral

and therefore these hits were excluded from further studies.

nutrient stimulation. In this experiment, mitoxantrone-

These results re-iterate the importance of the control

treated mice showed a 5-fold increase in the ratio of active

experiment and normalization of the two signals. The data

GLP-1 to total GLP-1 compared to PBS-treated mice (Fig. 3e).

shown in Table S1 represent normalized values where the

This result clearly demonstrates the reduction of BL signal

"DAL light output" for each of the compound was normalized

observed using the DAL-based assay is due to mitoxantrone-

to "free luciferin" control. The resulting values reported in

mediated inhibition of DPP-4 activity in live animals.

at

3.1

mg/kg

demonstrated

significant

Table S1 reflect the DPP-4-specific activity of each compound

Structure-activity relationship of mitoxantrone

in the screen.

In order to find out which elements of the mitoxantrone

Interestingly, the top three most active compounds were

structure are essential for it’s DPP-4 inhibitory activity we

commonly used drugs against hypertension, cancer, and

performed structure-activity relationship (SAR) study (Fig. S7,

alcoholism (nisoldipine, mitoxantrone, and disulfiram,

S8, Table S2). Based on the results, we concluded that the

respectively). In the next step, dose dependent inhibition of

pharmacophore lies within two aminoethyl side arms at 5th and 8th position of mitoxantrone. Moreover, the cytotoxic 6


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Analytical Chemistry properties of mitoxantrone are independent from DPP-4 inhibition.

Figure 3. (a) Flow chart of the study. (b) Assay design strategy and 96-well plate layout for HTS. (c) Dose response curves of the three top hits. Mitoxantrone, disulfiram and nisoldipine were tested using DAL assay and human recombinant DPP-4 enzyme in the concentration range from 1 nM to 500 µM. Each point of a curve is mean ±SEM, n=2. (d) Measurement of mitoxantrone DPP4 activity in vivo (FVB-luc+ mice). Total photon flux was measured over 40 min post administration of mitoxantrone (3.1 mg/kg, iv.) followed by i.p. injection of DAL reagent. Values are mean ± s.e.m (n=5). ** - p

A Universal Assay for Aminopeptidase Activity and Its Application for

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