1. Introduction

Enzymatic assays are not used very often in High throughput Screening (HTS) because there are very few assays available in high scale format even though enzymes are implicated in a wide range of diseases or physiological reactions.

Cyclophilin D (CypD) is a Peptidyl Prolyl Isomerase (PPi) expressed in the mitochondrial matrix where is has a regulatory role in the functions of the mitochondrial permeability transition pore. Opening of the mitochondrial permeability

pore is a stepstone event in cell death leading to disruption of the mitochondrial membrane. Cyclosporin A (CsA) is an immunosuppressant drug used in many instances from organ transplant to prosiaris. But its Adverse Drug Reactions

(ADR) are numerous and severe: convulsions, pancreatitis, nephrotoxicity, hepatotoxicity among others. When CsA binds to CypD, it inhibits the mitochondrial permeability transition pore opening and prevents cell death.

Several companies are working today on compounds which could replace CsA in its actions without the severe ADR. The issue is that this enzymatic reaction, like many enzymatic reactions, is very fast and implies a slowing down of

cellular activity by a decrease in temperature. With a classic PMT-based reader, it takes around 30min to read one 96 well plate which is very far from the throughput needed in primary screening. We report here a fluorescent method to

monitor CypD activity in 384 well plates based on a method described by Mori et al. In 2009 on Cyclophilin A* with a reading time of 4min per plate allowing the use of this enzymatic screening in HTS capacity.

4. Cyclosporin A dose response

The second set of validation experiment is a CsA dose response with CypA and CypD in the FDSS7000. These dose responses allowed us to calculate

pIC50 for each enzyme: 7,8 for CypA and 7,35 for CypD which is, once again, in accordance with literature (Fig. 3)

7. Conclusion

The aim of this study was to validate on Cyclophilin D the screening method described by Mori et

al. In 2009 on Cyclophilin A* We used the FDSS7000 which allows for simultaneous injection and

measurement of all the 384 wells in a plate, this feature is critical in fast enzymatic reactions like

the one studied here. Validation steps performed here allowed us to determine CypD characteristic

constants (KM, Kcat, pIC50 …) all in accordance with literature. These data demonstrate that this

protocol is adapted to CypD or A study for Real time Fluorescence Monitoting.

Our study was taken a step further with a small library screening (143 compounds) which yielded

25 positive hits. Taken together, these results demonstrate that the FDSS7000 can be successfully

used to screen for inhibitors of Cyp A or D in HTS.

2. Material and Methods

Assays are performed on the FDSS7000 obtained from

Hamamatsu Photonics. This instrument allows dispensing

and measurement in fluorescence and luminescence of

384 well plates at once.

Screening is performed on Cyclophilin A and D (CypA and

CypD respectively) with substrate Suc-Ala-Ala-Pro-Phe-

AMC where Suc stands for succinyl and AMC for 4-me-

thylcoumaryl-7-amide. The increase in 7-amino-4-methyl-

coumarine (AMC) liberated by proteolysis is monitored

fluorometrically with excitation λ ex=365nm and emission

λ em=460nm during 4 min. For the first 30sec an interval

of 150 msec is used then an interval of 400 msec is used

for the rest of the measurement.

5. Validation with tool compounds

GSK library contains several compounds with known affinity for CypD and 3 of those were used to validate the assay protocol in the FDSS7000.

Fig. 4 shows that the compounds called here GSK A, B and C generate pIC50 of 6,78; 4,16 and 4,78 respectively which are the values expected by GSK according to

previous experiments,

3. Substrate titration

The first set of experiments performed is a substrate titration between 0 and 160µM. The aim of this experiment is to determinate CypA and CypD KM. KM (Michaelis constant)

is a specific value that indicates the enzyme’s affinity for its substrate. It represent the substrate concentration at which the reaction rate is half its maximum (or Vmax). Kcat is

also calculated in this experiment and represents the number of substrate molecules converted into product per enzyme molecule and per time unit. These two values allowed

calculation of the ratio Kcat/KM which is specific for each enzyme. Values for KM, Kcat and Kcat/KM are displayed in Fig.2 and are all in accordance with literature.

6. Screening

After validation of the assay pro-

tocol on the FDSS7000, a small

set of compounds was screened.

143 compounds were tested in

single shot at 100 µM and the

25 compounds active in both

replicate were then assayed in

dose response.

Cyclophilin Screening in a Real Time Fluorescence Monitoring System

Beatriz María Rodríguez-Miquel (1), Maria Concepcio Cid-Calzada(1) Jean Marc D’Angelo (2),

Christelle Catone (2), Sunao Hisada (2), Shouming Du

GlaxoSmithKline, Centro de Investigación Básica, Parque Tecnológico de Madrid,

Santiago Grisolía, 4, 28760 Tres Cantos, Madrid, Spain

Hamamatsu Photonics : contact jmdangelo@hamamatsu.fr

* (Use of a Real-Time Fluorescence Monitoring System for High-Thoughput Screening for

Prolyl Isomerase Inhibitors, Mori T. et al., J. Biomolecular Screening, 2009).

% inhibition in SS

Compound Copy 1 Copy 2 DR results

GSK E 38.98 30.16 40% @ 100 µMGSK F 30.59 112.21 20% @ 100 µMGSK G 105.38 93.00 138.20 µMGSK H 60.06 86.92 11% @ 100 µMGSK I 101.90 124.71 8.32 µMGSK J 42.11 34.50 9 µMGSK K 73.09 85.51 32.56 µMGSK L 53.61 88.74 NAGSK M 63.04 34.75 33% @100 µMGSK N 57.57 70.44 129.31 µMGSK O 64.81 79.61 21% @ 100 µMGSK P 43.99 171.85 25% @ 100 µMGSK Q 39.30 76.62 39% @ 100 µMGSKR 38.16 30.98 NAGSK S 69.46 73.48 68.47 µMGSK T 67.13 51.87 23% @100 µMGSK U 92.26 63.77 4 µMGSK V 81.56 55.86 NAGSK W 31.08 42.31 28% @100 µMGSK X 73.46 72.37 28.83 µMGSK Y 32.37 74.80 49.2 µMGSK Z 86.67 72.96 95% @10 µMGSK AA 39.26 49.75 15% @100 µMGSK AB 48.21 65.78 NAGSK AC 234.58 91.21 14.1 µM

KM app (µM) Kcat (s-1) Kcat/KM

CypD 34,7 159 4,6CypA 45,7 210 4,6

Fig. 2: Values for KM, Kcat and Kcat/KM Fig. 1: Left: Chymotrypsin rection with its compound. Right: Schematic procotol of the reaction.

Fig. 3: Csa dose reponse for CypA (left) and CypD (right) Fig. 4: Dose response curves of 3 GSK compounds

Fig. 6 : ScreeningFig. 5: FDSS7000EX Hamamatsu Photonics

Suc-Ala-Ala-Pro(cis)-Phe-MCA

CypA or CypD

Chymotrypsin

Suc-Ala-Ala-Pro(trans)-Phe-MCA

Suc-Ala-Ala-Pro(cis)-Phe-COOH + AMC

Compound

10 µl @ 530 µM in 5,3 % DMSO

+

CypA or CypD

20 µl @ 80 nM in 1 % DMSO

Transfer plates into FDSS7000

Chymotrypsin

20 µl @ 125 µM @ 4° C

Substrate

3 µl @ 706 µM (prepared @ 4° C)

Total volume 53 µl with

30 nM CypA or D / 47 µM Chymotrypsin / 40 µM substrate / 1 % DMSO

Measurement

30 sec @ 150 msec interval

3 min 30 sec @ 400 msec interval

λ excitation 365 nm / λ emission 465 nl

Incubation 15 min @ 4° C

nM10 100 1000

pIC50 6.78GSK A

pIC50 4.16GSK B

nM10 100 1000

% in

h

% in

h

% in

h

1

pIC50 4.78GSK C

nM10 100 10001

µM peptide

mM

AM

C

mM

AM

C

0 20 40 60 80 100 120 140 1600

2

4

6

Cyp A

µM peptide

0 20 40 60 800

1

2

3

Cyp D

µM peptide

mM

AM

C

mM

AM

C

0 20 40 60 80 100 120 140 1600

2

4

6

Cyp A

µM peptide

0 20 40 60 800

1

2

3

Cyp D