Biomedical Engineering Reference
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(or long isoform), mainly expressed in the smooth muscle and non-muscle cells,
respectively, derive from a single gene (
mylk
1) and result from alternative initiation
sites (Hong et al.
2011
). The smooth muscle isoform of MLCK is composed of an
actin-binding domain, a proline-rich region and a fibronectin domain (whose func-
tions are unknown), a kinase domain (the catalytic domain), a calmodulin-binding
domain (the regulatory domain), an auto-inhibitory domain and a C-terminal im-
munoglobulin domain (Hong et al.
2011
). MLCK is inactive when not bound to
Ca
2+
/calmodulin (auto-inhibited state). Upon binding to Ca
2+
/calmodulin, the auto-
inhibitory domain is displaced from the kinase domain, thereby allowing substrate
access. The kinase domain binds to ATP and phosphorylates residue Ser19 (and sub-
sequently Thr18) in the regulatory light chain of myosin II (Hirano et al.
2003
). This
phosphorylation increases the ATPase activity of myosin II and is thought to play
major roles in a number of biological processes, including smooth muscle contrac-
tion, through the interaction of activated myosin II with actin filaments (Takashima
2009
). MLCK (and in particular the non-muscle isoform) is also a key regulator
of tight junction permeability (Turner et al.
1997
; Shen et al.
2010
; Cunningham
and Turner
2012
) and has revealed a role in barrier dysfunction, in response to
inflammatory mediators (Rigor et al.
2013
).
Inhibitors of MLCK have been proposed as therapeutics (1) acting as potential
vasodilators for pathological conditions like vasospasm (Sasaki
1990
; Kerendi et al.
2004
), (2) decreasing the intestinal epithelial permeability (Feighery et al.
2008
), for
disorders such as ulcerative colitis (Liu et al.
2013
), or (3) overcoming infectious
agents such as herpes simplex virus type-1 (Antoine and Shukla
2013
). Two MLCK
inhibitors, the serine/threonine protein kinase inhibitors ML-7 and ML-9 (Fig.
3.4a
),
are used in most of the studies devoted to the physiological role of MLCK (Saitoh
et al.
1987
; Ishikawa et al.
1988
). However, the therapeutic utility of these structural-
ly related compounds is limited, since they also inhibit other kinases such as protein
kinase A and protein kinase C (Saitoh et al.
1987
). Peptidic antagonists have revealed
a better specificity towards MLCK, such as the membrane-permeant inhibitor of
MLCK (PIK, Fig.
3.4a
), identified within a peptide library derived from the auto-
inhibitory sequence of MLCK (IC
50
50 nM; Lukas et al.
1999
; Owens et al.
2005
).
However, the low stability of the peptide in vivo has limited its applications, and ana-
logues have been designed to enhance the resistance to protease while maintaining
activity and selectivity, such as D-PIK and D-reverse PIK (Owens et al.
2005
).
In 1996, in the course of a screening of microorganisms to identify MLCK in-
hibitors as potential vasodilators and bronchodilators, Yano et al. reported that MS-
271 (i.e. siamycin I; Tsunakawa et al.,
1995
; Fig.
3.4b
) inhibited the chicken gizzard
MLCK with an IC
50
of 8 μM (Yano et al.
1996
). Chicken and turkey MLCKs, abun-
dant and easily purified from the gizzard tissue, have been used extensively to study
MLCK, although they lack a portion of the proline-rich region found in mammalian
MLCKs (Olson et al.
1990
; Hong et al.
2011
). Propeptin did not inhibit cyclic AMP-
dependent protein kinase, protein kinase C or calcium-/ calmodulin-dependent
cyclic nucleotide phosphodiesterase at concentrations up to 400 μM (Yano et al.
1996
). Non-peptidic inhibitors, such as dehydroaltenusin (IC
50
0.69 µM), were also
reported by this group (Nakanishi et al.
1995
).
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