Regulation of human carbonic anhydrase I (hCAI) activity by using a photochromic inhibitor

Article abstract of DOI:10.1002/anie.200802242

Photoswitchable: The activity of a dithienylethene-based human carbonic anhydrase inhibitor (hCAI) can be regulated using light. Converting the flexible, ring-open isomer of the photoswitch into the rigid, ring-closed isomer using UV light reduces the inhibition and increases the activity of hCAI by 50-fold. The inhibitor can be turned back on by using visible light, which has many advantages in biological applications.

Full text of DOI:10.1002/anie.200802242

Communications
DOI: 10.1002/anie.200802242
Light-Regulated Enzyme Activity
Regulation of Human Carbonic Anhydrase I (hCAI) Activity by Using
a Photochromic Inhibitor**
Daniel Vomasta, Christina Högner, Neil R. Branda,* and Burkhard König*
The regulation of enzyme activity is crucial for the metabo-
lism of every organism. In biology, enzymatic control is
typically achieved through the use of allostery[1] or by
covalently modifying the enzyme (by phosphorylation or
dephosphorylation, for example).[2] Some of the original
attempts to artificially influence the activity of enzymes rely
on chemical modifications of the enzyme structure,[3–6] an
approach that is limited by the fact that the regulation is not
reversible. The use of light as a stimulus offers a heightened
level of control, and photoresponsive compounds would
provide the reversibility needed for practical use. Existing
examples of systems that take advantage of the beneficial
properties of light include those that use azobenzene-based
enzyme inhibitors[7] or use thiophenfulgide derivatives cova-
lently linked to the enzyme.[8] Photoinducing changes in the
environment around the enzyme has also been used as a
regulation mechanism by influencing the permeability of a
photoisomerizable polymer containing the enzyme for the
substrate,[8] by controlling the conformation of a specific
domain of the enzyme with surfactants,[9] and by changing the
conditions of the medium (pH or viscosity, for example).[10]
Controlling the activity of carbonic anhydrase is of special
interest as it is an enzyme central to both cellular transport
and metabolic processes. It can be found in virtually every
tissue and cell type, in many subcellular organelles, and in
organisms ranging from unicellular cyanbacteria to mam-
mals.[11] Recently, an azobenzene-based biolabel was used to
photomodulate the activity of carbonic anhydrase (by about
twofold).[12] Although controlling enzyme activity with azo-
benzene derivatives is an elegant concept, the thermal
reversibility that plagues these particular photoresponsive
compounds significantly limits their use in practical applica-
tions. On the other hand, compounds constructed from the
1,2-dithienylethene (DTE)scaffold represent a significant
improvement over most other photoresponsive structures,
primarily because they undergo thermally irreversible photo-
chemical ring-closing and ring-opening reactions (see, for
example, the substructures 1aQ1b in Scheme 1; a: open, b:
closed).[13]
[*] Prof. N. R. Branda
4D LABS at Simon Fraser University
8888 University Drive, Burnaby, BC V5A 1S6 (Canada)
E-mail: nbranda@sfu.ca
Scheme 1. Synthesis and the reversible photochemical ring-closing
reaction ofDTE inhibitor 1a.
D. Vomasta, C. Högner, Prof. B. König
Institute ofOrganic Chemistry, University ofRegensburg
93040 Regensburg (Germany)
Fax: (+49)941-943-1717
E-mail: burkhard.koenig@chemie.uni-regensburg.de
Herein, we describe how this versatile photoresponsive
structure can be used to reversibly control the activity of
carbonic anhydrase by decorating the DTE architecture with
sulfonamide and copper(II)iminodiacetate {Cu(ida)} moiet-
ies. These two moieties were chosen in light of a recent report
by Mallik et al. on a significant increase in the activity of the
weak enzyme inhibitor sulfanilamide (4-aminobenzene-
sulfonamide, 4)upon covalently linking it to a {Cu(ida})
complex.[14]
[**] This work was supported by the Deutsche Forschungsgemeinschaft,
the Fonds der chemischen Industrie, the University ofRegensburg,
the Natural Sciences and Engineering Research Council ofCanada,
the Canada Research Chair Program, and Simon Fraser University.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200802242.
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While the sulfonamide group in 1a acts as the inhibitor,
the role of the {Cu(ida)} component is to reversibly coor-
dinate to the imidazole side chains of the histidine residues
exposed on the protein surface close to the ZnII active site of
the enzyme and help dock the sulfonamide inhibitor group
into the catalytic center. Given the fact that the activity of any
two-pronged enzyme inhibitor is directly dependent on the
distance and relative orientation of the two groups (in this
case, {Cu(ida)} and the sulfonamide) and the fact that the
as it is kept in the dark, and the colored state did not revert to
its colorless form even after six months. Irradiation of the
colored solution with visible light (l > 420 nm)converts the
ring-closed isomer back into 1a and regenerates the original
absorption spectrum. This ring-closing/ring-opening cycle can
be repeated at least seven times without any sign of
degradation.[16]
The human carbonic anhydrase I (hCAI)-catalyzed
hydration of carbon dioxide [Eq. (1)] is a convenient probe
DTE architecture can be toggled between a flexible, ring-
CO þ H O hCAl HCO ꢁ þ Hþ
ƒƒ!
ð1Þ
[15]
2
2
3
open (1a)and rigid, ring-closed ( 1b)isomer,
we designed
compound 1 to reversibly photoregulate enzyme activity
without having to resort to chemical modifications or changes
in the natural environment of the enzyme. The synthesis of
compound 1a[16] started with the stepwise coupling of the acid
chloride of cyclopentene 2[17] with diester 3 and sulfanilamide
(4; Scheme 1). After removal of the two tert-butyl groups with
acid, treatment with CuCl2 under basic conditions afforded 1a
in good yield.
that can be used to investigate the inhibitory effect of the
photoresponsive DTE compound in its ring-open (1a)and
ring-closed (1b)states. [16] The known inhibitor, sulfanilamide
(4),[19] and photoresponsive compounds 6–8 provide excellent
controls for comparison. All results are presented in Figure 2
and Table 1.
Irradiating an aqueous solution of 1a (5% DMSO,
tris(hydroxymethylamino)methane (Tris) sulfate buffer,
[18]
20 mm, pH 8.3 at 258C)with 312 nm light
resulted in the
immediate changes in the UV/Vis absorption spectra that are
typical for photoresponsive DTE derivatives (Figure 1). The
Figure 2. Change in the activity (%) ofhCAI when the concentrations
ofcompounds 1, 4, 6, and 7 in their ring-open and ring-closed forms
are varied.[16] The data were obtained in an enzymatic assay that
monitored the reaction ofcarbon dioxide and water to generate
hydrogen carbonate [Eq. (1)].[22]
Figure 1. Changes in the UV/Vis absorption spectra ofan aqueous
solution of 1a (1.08”10ꢁ5 m) in DMSO (5% v/v) and Tris sulfate
buffer (20 mm, pH 8.3) when irradiated with 312 nm light. Irradiation
periods are 0, 3, 5, 8, 10, 14, 17, 20, and 34 s. The inset illustrates the
change in color ofthe solution rfom colorless to purple as the ring-
closed isomer is generated.
high-energy band (lmax = 290 nm)decreases in intensity and
an absorption band in the visible spectral region (lmax
=
545 nm)appears as the solution changes from colorless to
purple because of the formation of the ring-closed isomer 1b
(a smaller band at lmax ꢀ 360 nm also appears). These spectral
changes are complete after irradiation for 34 s (at a concen-
tration of 1.08 ” 10ꢁ5 m), and a photostationary state contain-
ing at least 99% of the ring-closed isomer is generated
according to HPLC analysis of the reaction mixture. This
effective photoconversion attests to the versatility of the
dithienylethene backbone as a photoresponsive architecture
on which to build practical devices. The large amount of 1b in
the photo-generated mixture is highly beneficial, and a lower
amount would make the differences in enzyme inhibition
significantly less pronounced. The solution containing the
ring-closed isomer is very stable at room temperature as long
Sulfanilamide (4)has an IC 50 value of 0.46 mm, which is in
good agreement with that reported in the literature.[20] The
inhibition effect of the ring-closed DTE isomer 1b is
comparable (IC50 = 0.4 mm). The similarity of the inhibitor
strength of 4 and 1b suggests that only the sulfonamide
component interacts with the active site of the enzyme in the
latter compound. This is likely because the planar and rigid
backbone in 1b prevent the simultaneous binding of the
{Cu(ida)} and sulfonamide components, and will be elabo-
rated on later in this communication.
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Table 1: IC50 values and Ki binding affinities of compounds 1, 4, and 6–8
in their ring-open and ring-closed forms.
Inhibitor
IC50 [mm]
ring-closed ring-open
Ki [mm][a]
ring-closed
ring-open
0.46ꢂ0.01
4
1
6
7
8
0.29ꢂ0.007
0.008ꢂ0.0003 0.40ꢂ0.005 0.005ꢂ0.0002 0.30ꢂ0.003
0.53ꢂ0.007
1.55ꢂ0.8
–
0.57ꢂ0.01
1.46ꢂ0.15
–
0.34ꢂ0.005
1.16ꢂ0.05
–
0.35ꢂ0.008
1.00ꢂ0.01
–
[a] The values of Ki were obtained using the Cheng–Prusoff equation.[16]
On the other hand, the ring-open counterpart (1a)inhibits
the enzyme much more significantly, and its inhibition activity
is two orders of magnitude higher (IC50 = 8 nm)than that of 4
and 1b. This increase can be attributed to the structural
flexibility of 1a, which allows both recognition components to
bind to the enzyme and leads to a higher overall binding
affinity. The photoresponsive bis(sulfonamide) 6 shows simi-
lar inhibition as sulfanilamide 4, and no difference between
the activity of the ring-open (IC50 = 0.53 mm)and ring-closed
isomers (IC50 = 0.57 mm)can be observed. [21] In the case of the
photoresponsive bis(iminodiacetate) 7, the IC50 value is lower
than that of sulfanilamide 4 and but once again, no significant
difference between the ring-open (IC50 = 1.55 mm)and the
ring-closed isomers (IC50 = 1.46 mm)is measured. The photo-
responsive bis(ethyleneglycol) 8 was synthesized to inves-
tigate whether the dithienylethene unit itself has an influence
on the enzyme activity. This compound shows no inhibition in
the hCAI-catalyzed hydration of carbon dioxide. All
observed changes in the enzymeꢀs activity can, therefore, be
ascribed to the synergistic roles the sulfonamide and the
{Cu(ida)} groups play as well as to their relative spatial
orientation to each other. The binding affinities (Ki)of all
compounds show similar trends (Table 1). The exception is
ring-open isomer 1a, which more effectively binds to the
enzyme (Ki = 0.005 mm for 1a as compared to ꢀ 0.29–1.16 mm
for 1b, 4, 6a, 6b, 7a, and 7b). The reversible DTE ring-closing
and ring-opening cycle, converting 1a into 1b and back, is also
possible in the presence of the enzyme.[16]
Figure 3. Illustration ofthe catalytic center ofhCAI, containing a
sulfanilamide, an IDA, and one of the surface-exposed imidazole
groups. The distance between and relative positioning ofthe sulaf nila-
mide and IDA groups can only be satisfied by the ring-open form of
compound 1, which can adopt the productive parallel conformation.
The structure ofthe enzyme with sulaf nilamide in the active site was
derived from crystal structure data, generated and rendered with the
program PYMOL from Graph Pad.[23] From Mallik’s results[14] the
length of the inhibitor to guarantee a high binding affinity is known.
As alluded to throughout this communication, we can
explain the differences in inhibition and binding affinity of the
two photoisomers of 1 by comparing the differences in their
conformational flexibility. The flexible ring-open form 1a was
designed to allow the simultaneous docking of the sulfona-
mide and the {Cu(ida)} components onto the enzyme surface.
ꢁ
This is possible because of the free rotation around the C C
likely candidate for bivalent binding to the enzyme. The
planar, rigid backbone found in the ring-closed isomer 1b
forces the two components away from each other in a
nonproductive manner, allowing only one of the components
to bind to the enzyme at a time. This reduces binding and
inhibition.
We have demonstrated that by using a well-designed, two-
pronged inhibitor and appropriate wavelengths of light, the
enzyme activity can be reversibly and significantly enhanced
by toggling the DTE between a high- and a low-affinity
conformation. The thermal stability, nearly quantitative
formation of each photoisomer, and activation with visible
light makes the system a suitable tool for the reversible
single bonds joining the two thiophene heterocycles to the
central cyclopentene ring, which allows the inhibitor to adopt
a geometry appropriate for bivalent binding only when in its
ring-open form. The structure of the enzyme active site
(containing both sulfonamide and {Cu(ida)} components;
Figure 3)clearly reveals the need for geometric adaptation.
The distance between the two binding components (ca. 10 Š)
and the way they project in space can only be satisfied by 1a.
Although the distance between the sulfonamide and
{Cu(ida)} components does not change when the antiparallel
conformation of 1a is converted into its ring-closed counter-
part (Figure 3), it is the parallel conformation of 1a that is the
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Chemie
Wiley-VCH, Weinheim, 2001, pp. 37 – 62; M. Irie in Photo-
regulation of enzyme activity by light. The use of visible light
to activate the inhibitor is particularly important as it will
allow better penetration into tissue and reduce the amount of
damage caused by higher energy UV light.
chromic and Thermochromic Compounds, Vol. 1 (Eds.: J. C.
Crano, R. J. Guglielmetti), Plenum, New York, 1999, pp. 207 –
222; H. Tian, S. Yang, Chem. Soc. Rev. 2004, 33, 85 – 97; H. Tian,
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[14] A. L. Banerjee, D. Eiler, B. C. Roy, X. Jia, M. K. Haldar, S.
Mallik, D. K. Srivastava, Biochemistry 2005, 44, 3211 – 3224. Use
of reversible coordination in molecular recognition: M. Kruppa,
B. Kꢁnig, Chem. Rev. 2006, 106, 3520 – 3560.
Received: May 13, 2008
Published online: September 2, 2008
Keywords: carbonic anhydrase · enzyme catalysis · inhibitors ·
molecular switches · photochromism
[15] Two conformations (parallel and antiparallel)of the ring-open
isomer of the DTE architecture coexist, typically in a 1:1 ratio, in
solution (see the Supporting Information). The photoactive form
is the antiparallel conformation.
[16] See the Supporting Information for details.
[17] T. B. Norsten, N. R. Branda, J. Am. Chem. Soc. 2001, 123, 1784 –
1785.
[18] Standard hand-held lamps used for visualizing TLC plates (from
Herolab, 6 W)were used to carry out the ring-closing reactions
at 312 nm. The ring-opening reactions were carried out using the
light of a 200 W tungsten source that was passed through a
420 nm cutoff filter to eliminate higher energy light.
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[21] The photoreactions of compounds 6a, 7a, and 8a are shown in
the Supporting Information.
[22] During the enzyme-catalyzed reaction, the pH of a buffered
solution (5% DMSO, Tris sulfate buffer, pH 8.3)containing a
pH indicator (phenol red, 5.6 ” 10ꢁ4 m)dropped from 8.3 to 6.3,
which is the end point of the reaction. The time required for this
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