inhibition of μ-calpain; towards treatment of rheumatoid arthritis
TRANSCRIPT
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
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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
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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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