This document summarizes research on calpain inhibition for the treatment of rheumatoid arthritis. Calpains are calcium-dependent cysteine proteases implicated in inflammatory diseases. The large subunit of μ- and m-calpains contains catalytic domains and calcium binding domains. Calpastatin inhibits activated calpains. Overactivation of calpains is associated with rheumatoid arthritis due to calcium imbalance. Newly synthesized mono-halogenated α-mercaptoacrylate derivatives potently inhibit calpains by binding calcium binding domains. This study examines the interactions of these inhibitors with the PEF(L) and PEF(S) calcium binding domains.

1. Hala Issa
Supervised by: Prof. Rudolf K.Allemann
Dr. David Miller
Inhibition of μ-calpain; towards
treatment of rheumatoid arthritis

2. 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.

3.  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.

4.  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.

5.  Calcium
binding to
PEF(L) and
PEF(S) cause
slight
conformational
changes.
Calpains: Domains and Activation
http://calpain.net/3dstructure/index.html

6. Calpastatin
•70 kDa with no sequence similarity.
•Binds activated calpains.
•4 domains, 3 subdomains.

7.  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 andAlzheimer’s disease, due to calcium ions imbalance
 Substrates:
 fodrin,
 53,
 spectrin,
 talin,
 fibronectin
Role of Calpains

8. 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

10. 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

11. 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.

12.  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

13. Aim of the study
 Study the interactions of one of the newly synthesized
inhibitors against PEF(L) domain.

14. 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.

15. Purification of PEF(S)
 Anion Exchange Chromatography and Size Exclusion
Chromatography
M FT CE 23 22 21 20
Size exclusion chromatographyAnion exchange chromatography
CE PEF(S) FT PEF(L,S) BSA

16. 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)

17. 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
θMRE/degcm2dmol-1
wavelength/ nm
-8000
-6000
-4000
-2000
0
2000
4000
6000
8000
190 210 230 250 270 290 310 330 350
θMRE/degcm2dmol-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.

18. 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
Absorbance(280nm)/mAU
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
Absorbance(280nm)/mAU
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
Absorbance(280)nm/mAU
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
Absorbance(280nm)/mAU
Volume/ ml
PEF(L):PEF(S) MW (43,000)
PEF(L):PEF(S) MW (43,000)
D

19. 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 μMANS and mM of CaCl2 were
added to 20 mMTris-HCl to make a total volume of 3 ml, inhibitor was added
with increasing gradient

20. 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
Fluorescence/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

21. 0
100
200
300
400
500
600
380 430 480 530 580
Fluorescence/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 )

22. 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

23. Acknowledgments
 Professor Rudolf K.Allemann
 Dr. David Miller
 Dr. Sarah E.Adams
 Friends