Hala Issa

Supervised by: Prof. Rudolf K. Allemann

Dr. David Miller

Inhibition of μ-calpain; towards

treatment of rheumatoid arthritis

Calpains: Domains and Activation

Calpain Family

Cytosolic cysteine proteases

Calcium dependant

15 enzymes all share in common the active site (Cys, His, Asn)

However, 2 major enzymes have been under much investigation

due to their ubiquitous prevalence in the human body.

μ- and m- calpains are heterodimeric enzymes with 60% similarities

Their small subunit is identical, it is composed of two domains, the glycine rich

domain (DV) and PEF(S) domain D(VI)

Calpains: Domains and Activation

Moldoveanu, T.; Hosfield, C. M.; Lim, D.; Elce, J. S.; Jia, Z.; Davies, P.

L., A Ca(2+) switch aligns the active site of calpain. Cell 2002, 108 (5),

649-60.

The large subunit

contains the catalytic

domains DI and DII,

along with C2L

domain DIII, and

another PEF(L)

domain DIV, as well

as an N-terminal

anchor helix.

Calpains: Domains and Activation

Hosfield, C. M.; Elce, J. S.; Davies, P. L.; Jia, Z., Crystal structure of calpain reveals the structural basis for Ca(2+)-

dependent protease activity and a novel mode of enzyme activation. EMBO J 1999, 18 (24), 6880-9.

Calcium binding to PEF(L) and PEF(S) cause slight conformational changes.

Calpains: Domains and Activation

http://calpain.net/3dstructure/index.html

Calpastatin

•70 kDa with no sequence similarity.

•Binds activated calpains.

•4 domains, 3 subdomains.

Calpain exerts modulatory effect on several substrates due to its

ubiquitous presence in human body.

Its overactivation is implicated in inflammatory diseases such as

rheumatoid arthritis (RA) and neurodegenerative diseases such as

Parkinson’s and Alzheimer’s disease, due to calcium ions imbalance

Substrates:

fodrin,

53,

spectrin,

talin,

fibronectin

Role of Calpains

Calpain and neutrophil spreading andmigration

Miller, D. J.; Adams, S. E.; Hallett, M. B.; Allemann, R. K., Calpain-1 inhibitors for selective treatment

of rheumatoid arthritis: what is the future? Future Medicinal Chemistry 2013, 5 (17), 2057-2074.

Neutrophils

predominantl

y express μ-

calpain

Rheumatoid Arthritis

•chronic inflammatory

autoimmune disease.

•synovium is infiltrated by

inflammatory cells.

•Release of cytokines and

increased calcium influx.

•Neutrophils present in synovial

fluid.

Normal vs. Arthritic joint

http://www.intechopen.com/source/html/43387/media/image1.png

Calpain Inhibitors

Mono-halogenated α-mercaptoacrylates derivatives are

among the most potent inhibitors of calpains. They react

against PEF(L) and PEF(S) domains and bind in their

hydrophobic pocket.

PD150606 and PD 151746 represent the precursors for

modified α-mercaptoacrylates synthesis.

2 derivatives were studied against the PEF(S) homodimer revealed the following:

Bind in the same pocket as PD150606.

The volume of the pocket depends on the size of the ring and the halogen.

Majority of interactions take place inside the pocket

Compound B showed 2 different conformations in the hydrophobic pocket.

NH specific interactions are not important for tight binding

Calpain Inhibitors

Aim of the study

Study the interactions of one of the newly synthesized

inhibitors against PEF(L) domain.

Materials and Methods

Transformation and expression of PEF(S) and PEF(L)

pET21d vector containing PEF(L) and PEF(S) was transformed

with BL21-CodonPlus(DE3)-RP ®

66.2

45

35

25.5

18.4

SDS polyacrylamide gel visualization indicating the presence of

PEF(S) (left) with a MW of 20,000 and PEF(L) (right) with a MW

23,000.

Purification of PEF(S)

Anion Exchange Chromatography and Size Exclusion

Chromatography

M FT CE 23 22 21 20

Size exclusion chromatography Anion exchange chromatography

CE PEF(S) FT PEF(L,S) BSA

Purification of PEF(L):PEF(S) complex

Ni-column for the PEF(L):PEF(S) complex and dialysis buffer

200 200 200 50 10 W M CE

SDS-PAGE analysis of the fraction products from Ni-NTA

column with the small box showing the desired protein of

the proper band size for both PEF(L) protein MW 23,000

and PEF(S) protein MW 20,000 in the first elution with

200 mM imidazole buffer

66.2

45

35

25.5

18.4

SDS-PAGE analysis of the pure product obtained after

dialysing PEF(L):PEF(S) complex in dialysis buffer

overnight. The two bands correspond to PEF(L) (MW

23,000) and PEF(S) (MW 20,000)

Circular Dichroism Spectroscopy

PEF(L):PEF(S) complex and PEF(S) had a concentration of 86.3 µM (3.71

mg/ml) and 840 µM (16.795 mg/ml) respectively.

Protein concentration diluted down to 20 µM

-2000

-1000

0

1000

2000

3000

190 210 230 250 270 290 310 330 350

θ M

RE

/ d

eg

cm

2 d

mo

l-1

wavelength/ nm

-8000

-6000

-4000

-2000

0

2000

4000

6000

8000

190 210 230 250 270 290 310 330 350 θ

MR

E/

deg

cm

2 d

mo

l-1

Wavelength/ nm

The CD spectrum of the PEF(S) (left) and the heterodimer PEF(L):PEF(S) complex

(right) with peak minima at 222 nm and 208 nm is consistent with α-helical secondary

structure.

Analytical size exclusion chromatography

0.00

50.00

100.00

150.00

200.00

250.00

300.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Ab

sorb

anc

e (

280

nm

)/ m

AU

Volume / ml

Trypsin (MW 23,000)

Trypsin (MW 23,000)

A

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

0.00 5.00 10.00 15.00 20.00 25.00

Ab

sorb

anc

e (

280

nm

)/m

AU

Volume/ml

PEF(S) (MW 20,125)

PEF(S) (MW 20,125)

B

0.00

50.00

100.00

150.00

200.00

250.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00

Ab

sorb

anc

e (

280)

nm

/m

AU

Volume / ml

BSA (MW 66,400)

BSA (MW 66,400)

C

0.00

50.00

100.00

150.00

200.00

250.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Ab

sorb

anc

e (

280

nm

)/ m

AU

Volume/ ml

PEF(L):PEF(S) MW (43,000)

PEF(L):PEF(S) MW (43,000)

D

Interaction of (Z)-3-(4-bromophenyl)-2-mercaptoacryilic

acid with PEF(S) and PEF(L):PEF(S) complex

Fluorescent compound ANS has absorption maximum at 370 and emission

maximum at 470

Emission spectra were obtained between 380 nm and 610 nm.

A final concentration of 2.5 μM of protein, 2.5 μM ANS and mM of CaCl2 were

added to 20 mM Tris-HCl to make a total volume of 3 ml, inhibitor was added

with increasing gradient

Interaction of (Z)-3-(4-bromophenyl)-2-mercaptoacryilic acid with

PEF(S) and PEF(L):PEF(S) complex

0

50

100

150

200

250

300

350

400

380 430 480 530 580

Flu

ore

sce

nc

e/

FU

Wavelength/nm

PEF(S)-ANS

PEF(S)-ANS-Ca2+

0.1 μM inhibitor

0.5 μM inhibitor

1.0 μM inhibitor

1.5 μM inhibitor

2.0 μM inhibitor

3.0 μM inhibitor

4.0 μM inhibitor

5.0 μM inhibitor

6.0 μM inhibitor

7.0 μM inhibitor

9.0 μM inhibitor

10 μM inhibitor

A

0

100

200

300

400

500

600

380 430 480 530 580

Flu

ore

sce

nc

e/

FU

Wavelength/nm

PEF(L):PEF(S) complex- ANS

PEF(L):PEF(S) complex- ANS-Ca2+

0.1 μM inhibitor

0.5 μM inhibitor

1.0 μM inhibitor

1.5 μM inhibitor

2.0 μM inhibitor

3.0 μM inhibitor

4.0 μM inhibitor

5.0 μM inhibitor

6.0 μM inhibitor

7.0 μM inhibitor

9.0 μM inhibitor

10 μM inhibitor

B

Interaction of (Z)-3-(4-bromophenyl)-2-mercaptoacryilic acid with PEF(S)

and PEF(L):PEF(S) complex

Fluorescence spectra of the fluorescent probe ANS bound toPEF(S) (A) and

PEF(L):PEF(S)complex (B )

Conclusion and Future Work

It is the hydrophobic functional groups in the α-mercaptoacrylates that are responsible for the inhibition reaction.

Sulfhydryls and carboxylic acids don’t seem to have much of importance. This can be a place for manipulation of this unit.

Flexible pocket gives room for larger aromatic rings and halides to be introduced.

After establishing the mechanism of action of the newly synthesized monohalogenated α-mercaptoacrylates on PEF(S), a deeper look into their interaction with PEF(L) needs to take place.

Rerun the experiment for CysPc cloning and establish if these compounds do react with the active site

Acknowledgments

Professor Rudolf K. Allemann

Dr. David Miller

Dr. Sarah E. Adams

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