Furin Inhibition by Compounds of Copper and Zinc · 2004-05-12 · Furin Inhibition by Compounds of...
Transcript of Furin Inhibition by Compounds of Copper and Zinc · 2004-05-12 · Furin Inhibition by Compounds of...
Furin Inhibition by Compounds of Copper and Zinc
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Furin Inhibition by Compounds of Copper and Zinc
Paul Podsiadlo,† § Tomoko Komiyama,‡ § Robert S. Fuller,‡ Ofer Blum†¶*
†Departments of Chemical Engineering and ‡Biological Chemistry, The University of
Michigan, Ann Arbor, MI 48109, USA and ¶ The Department of Chemical Engineering,
The Technion, Israel Institute of Technology, Haifa 32000, Israel
§ Contributed equally
Corresponding author – Ofer Blum
Phone: +972-(4)-824-6190
Fax: +972-(4)-829-5672
E-mail: [email protected]
JBC Papers in Press. Published on May 12, 2004 as Manuscript M400338200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Summary
Furin, a human subtilisin-related proprotein convertase (SPC), is emerging as an
important pharmaceutical target, because it processes vital proteins of many aggressive
pathogens. It is even being considered as a wide spectrum counter-bioterror drug. Furin
inhibitors reported so far are peptide derivatives and proteins, with the exception of
andrographolides (natural materials). Here we report that the small and highly stable
compounds M(chelate)Cl2 (M = Cu, Zn) inhibit furin and Kex2, with Cu(TTP)Cl2 and
Zn(TTP)Cl2 being most efficient (TTP = 4’-[p-tolyl]-2,2’:6’,2”-terpyridine). Inhibition is
irreversible, competitive with substrate, and affected by substituents on the chelate. The
free chelates are not inhibitors. Solvated Zn2+ is less potent than its complexes. This is
true also for copper and Kex2. However, solvated Cu2+ (kon of 25000±2500 s-1) is more
potent than Cu(TTP)Cl2 (kon = 140±13 s-1), and allows recovery of furin activity prior to a
second inhibition phase. A mechanism that involves coordination to the catalytic
histidine is proposed for all inhibitors. Target specificity is indicated by the fact that
these metal chelate inhibitors are much less potent towards Kex2, the yeast homologue of
furin. For example, kon with Zn(TTP)Cl2 is 120±20 s-1 for furin, but only 1.2±0.1 s-1 for
Kex2. Because recognition of our inhibitors is not dependent on the substrate recognition
system of furin, it can be hoped that their derivatives will enable SPC selectivity.
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Introduction
Furin (also known as PACE and SPC1) is emerging as an important protein target
for therapeutics in the search for antidotes for aggressive biological threats. Furin is a
member of the subtilisin-like pro-protein convertases (SPCs).1 These structurally-related
serine proteases are located within the secretory pathway of the cell, where they cleave
protein precursors at the C-terminal side of single or paired basic residues (1,2,3). The
inactive precursors processed by SPCs are transformed to biologically active hormones,
receptors, growth factors, neuropeptides and enzymes. But SPCs, and especially furin,
also play a major role in pathogenesis (4,5). Human furin processes elements of various
bacterial toxins, including anthrax, diphtheria, shigella and pseudomonas, enabling their
entry into host cells. This is achieved by furin molecules on the outer surface of the host
cell plasma membrane or in the endocytic pathway (4,5). In the trans Golgi network,
furin processes the envelope glycoproteins profusogens of many viruses during their
biosynthesis. In the absence of processing by furin, new viruses released from the host
cell are unable to fuse with new, uninfected cells. Viruses exploiting human furin include
ebola, HIV-1, measles, avian influenza, Newcastle diseases virus, and cytomegalovirus
(4-6). Because furin is expressed in practically all body tissues and cell types (5), and as
it is essential for the effect of so many aggressive pathogens, its inhibitors may be
envisioned as wide-spectrum antidotes against known and even unknown biological
threats (7). Such broad-spectrum intervention may be most useful in case of an
unexpected bioterror attack.
Inhibition of purified furin and the other SPCs has been demonstrated with
proteins and peptides (3,5,6). These include: (a) Natural endogenous inhibitors of SPCs;
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(b) Pro-domains that are part of the inactive pro-SPC. These block pro-SPC proteolytic
activity during synthesis, and are removed by post-translational modification; and (c),
Peptides that have no physiological connection to the activity of SPCs. A few of these
inhibitors are potent, reaching even IC50s values at the low nanomolar range (3,6,8).
The only non-protein, non-peptide inhibitor of furin reported to date is a
neoandrographolide, a diterpene lactone extracted from the medicinally active plant
Andrographis paniculata, and its succinoyl ester derivatives (9). The IC50s reported are
in the high micromolar and low millimolar range. Here we report the inhibition of furin
by copper and zinc complexes of terpyridine derivatives, with IC50s of 5-10 µM (kon of
120-140 s-1). Our compounds are stable at various conditions and are not expected to
pose delivery problems, qualities vital for the development of a wide-scope anti-bioterror
agent, for civil and military uses.
Experimental Procedures
Enzyme, substrate and reagents. Substrate BOC-Arg-Val-Arg-Arg↓MCA
(BOC = t-butoxycarbonyl; MCA = methylcoumarinamide) was purchased from Bachem.
Its concentration was determined according to the released 7-amino-4-methylcoumarin
(AMC) product fluorescence after complete digestion with Kex2. To minimize
degradation, substrate was aliquoted, and kept frozen (-20°C) in dimethylsulfoxide
(DMSO) until use. Furin (10,11,12) and Kex2 (13) were prepared as described
previously. The C217S mutant of Kex2 (Komiyama and Fuller, unpublished) was
prepared according to the same procedure. Salts for buffers and other standard reagents
were from Sigma or Fisher, ACS grade or higher. Solvents for organic syntheses were
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from Fisher or Acros, ACS grade or higher. Chelates and ligands were purchased from
Aldrich (except for 4’-[4-methoxyphenyl]-2,2’:6’,2”-terpyridine (MPT) that was from
Alfa-Aesar) at the highest purity level available, and were used as received.
Zn(chelate)Cl2 compounds were prepared according to published procedures (14,15) that
were slightly modified according to the preparation of Cu(TERPY)Cl2 (TERPY =
2,2':6',2"-terpyridine)(16). Other Cu(chelate)Cl2 compounds were prepared according to
the same modified procedure (16). Compound composition was verified by elemental
analysis (C, H, and N). Stock solutions of free chelates and metal compounds were in
pure DMSO. Buffer pH was adjusted with HCl and NaOH solutions.
Inhibition assays. Furin (3 nM) and varying concentrations of either a 1:1
mixture of MCl2 (M = Zn, Cu, Hg) and chelate, or prepared M(chelate)Cl2 complex, or
MCl2 alone were incubated in 96 well plates at 22°C for 2 hours in assay buffer (20 mM
Sodium 2-(N-morpholino)ethanesulfonate (NaMES), pH = 7.0, 0.1% Triton x-100, 3%
DMSO, [NaCl] = 20 mM and [CaCl2] = 1 mM). Substrate BOC-Arg-Val-Arg-
Arg↓MCA (9 µM = Km/2 (11) (Km = Michaelis constant)) was added, and the initial rate
of AMC product release was obtained from the linear change of fluorescence (excitation
at 360 nm, measurement at 460 nm, on an fmax fluorescence microtiter plate reader
(Molecular Devices)) during 10 minutes at 30°C. IC50 values were obtained with
KaleidaGraph 3.0 from a fit of the normalized activity vs. inhibitor concentration data to
∂ ∂ νP t k to obs= ⋅ − ⋅exp[ ] where kobs (the apparent inactivation rate constant) is dependent
on the initial inhibitor concentration ([I]0) according to k k Kobs on m= + [ ]( )( ) ⋅10 0S I[ ] (17)
in the model E I Ei+ →kon , or to k k K Kobs i m= + + [ ]( ){ }( ) ⋅2 0 0 01[ ] [ ]I S I in the model
E I [EI] Ei+ ← → →K ki 2 (P = [product], t = time, vo = rate of uninhibited reaction, [S]0 =
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initial substrate concentration, E = enzyme, I = inhibitor, EI = enzyme-inhibitor
encounter complex, Ei = inhibited enzyme).
Each dose-response experiment was run at least twice in each set, and in at least
two separate sets (often many more). IC50 values obtained for repeat tests within the
same experiment were very close. The same is true for different experiments run within
the same day. Large deviations from the mean IC50 value were observed when a new
batch of substrate was used, or when the substrate batch got older. For this reason,
experiments that were repeated every single time (such as those with solvated Zn2+ and
solvated Cu2+) bear a large error. However, in spite of the errors in the absolute IC50
values, the ratios between the results with the various inhibitors were kept intact in all
experiments. Error values given are of a single standard deviation from the mean.
Inhibition assays with Kex2 were similar to those with furin, but conditions were
slightly different. Kex2 (1.2 nM) and inhibitor were incubated at 22°C for 2 hours in
assay buffer (168 mM 2,2-bis[hydroxymethyl]-2,2’,2”-nitrilotriethanol (Bis-Tris), pH =
7.4, 0.1% Triton x-100, 3% DMSO, [NaCl] = 20 mM and [CaCl2] = 1 mM). Substrate
BOC-Arg-Val-Arg-Arg↓MCA was added ([S]0 = 19 µM = Km (18)), and the product
formation rate was monitored as described for furin. Because the zinc compounds
formed with aryl derivatives of TERPY during the inhibition (or prepared before it) were
fluorescent (15), the activities obtained at high inhibitor concentrations (> 250 µM) were
too low (deviating from the exponential fit). In these cases, IC50 values (all lower than
250 µM) were obtained using only low inhibitor concentration data. This was a problem
only with Kex2, as it was less sensitive to our inhibitors than furin.
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Determination of irreversibility. Furin (15 nM) was incubated at 22°C for 2
hours in assay buffer and either Zn(TTP)Cl2 (TTP = 4’-[p-tolyl]-2,2’:6’,2”-terpyridine)
(at 9, 18 or 34 µM), or Cu(TTP)Cl2 (at 0.6, 2.4 or 9.6 µM) or ZnCl2 (at 12.5, 50 or 250
µM) or CuCl2 (at 0.6, 2.4 or 7.2 µM) or no inhibitor at all. With Cu2+, the experiment
was repeated also with 8 minutes of incubation, leading to identical results as with the
longer incubation. Each inhibitor concentration was run in two independent tubes.
Reactions were then diluted 100-fold by buffer. Substrate (100 µM) was added, and
AMC product release was monitored for two hours as described above for the inhibition
assays (longer monitoring was not useful due to loss of furin activity at 30°C). Rates of
AMC product release after dilution were obtained from a linear fit to data points between
2000 and 7200 s. Activities prior to dilution were determined on an aliquot set aside
from the same reaction mixture, using the procedure described above for the inhibition
assays.
The same procedure was employed with Kex2, with a few differences. Kex2 (6
nM) and either a 1:1 mixture of TTP and ZnCl2 (250 or 1200 µM) or ZnCl2 only (2.5 or 5
mM) or no inhibitor at all were incubated at 22°C for either 30 or 90 minutes in assay
buffer. Incubation periods were short to avoid complete inhibition. Dilution and assay
procedures were the same as with furin. Rates of AMC product release after dilution
were obtained from a linear fit to data points between 1000 and 4000 s.
Determination of kinetic constants. Furin (at 1.5 nM to avoid substrate
depletion) was added to mixtures in 96 well plates that included a constant amount of
inhibitor (30 µM Zn(TTP)Cl2 or 200 µM ZnCl2 or 12 µM Cu(TTP)Cl2 or 100 µM CuCl2),
and a varied amount of substrate (between 4.5 (= Km/4) and 36 µM (= 2Km)) in assay
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buffer. Each substrate concentration was checked in at least two independent wells.
After mixing, the release of AMC product was monitored at 30 °C for at least two hours
as described above for the inhibition assays. The fluorescence emitted by Zn(MPT)Cl2 at
460 nm (the wavelength used to monitor product concentration) prevented us from
reliably monitoring the action of this complex. This was not a problem with Zn(TTP)Cl2.
I n d i v i d u a l t r a c e s w e r e f i t a c c o r d i n g t o
P a v v t v k t kh s o s obs obs= + + + − ⋅ − − ⋅( ) ( ) ( exp[ ]) /ν ν 1 (P = [product], a = vertical
correction, kobs = apparent inactivation rate constant, vo = rate of uninhibited reaction, vs =
product formation rate at equilibrium with inhibitor, vhν = rate of photochemical product
degradation, t = time) (17,19) with vs = 0 whenever the inhibition was irreversible. This
equation simulates slow binding inhibitors. We added the vhν·t term to account for slow
photochemical degradation of the AMC product (20), which is first order in substrate,
and zero order in metal and chelate (19).
We did not examine the kinetics of a 1:1 mixture of M ions (M = Zn2+, Cu2+) and
chelate here, because [M(chelate)]2+ formation is heavily favored. Thus, the changes in
active inhibitor concentration could not be treated under the steady state approximation.
This was not a concern with the pre-incubated experiments, where active inhibitor
formation was complete well before substrate addition.
Kinetic follow up with Kex2 was similar to that with furin. 0.03 nM Kex2 was
added to mixtures that included 19 µM substrate and either Zn(TTP)Cl2 (at 0 – 140 µM,
concentration limited by compound fluorescence), or ZnCl2 (at 0 – 3.5 mM) in assay
buffer. Monitoring the product release rate was as described for furin.
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Results
Enzyme inhibition experiments included incubation of chelate and metal salt (or
of the pre-assembled metal-chelate complex) together with the enzyme at 22°C, followed
by addition of the substrate BOC-Arg-Val-Arg-Arg↓MCA. Inactivation was quantified
from the initial rate of fluorescent AMC product release. The results with zinc and its
complexes are presented in table 1 (see also scheme 1), and those with copper in table 2.
Table 1 here please
Scheme 1 here please
Inhibition by zinc and its compounds.
The trends among the various chelates were similar for furin and Kex2, but furin
was more susceptible to the inhibition by about two orders of magnitude (table 1, and see
also kon values below). Zinc complexes of the aromatic derivatives of TERPY were the
only inhibitors more efficient than solvated Zn2+. Yet, all zinc-chelate combinations
afforded some degree of inhibition, except for Zn(t-Bu3-TERPY)Cl2 (t-Bu3-TERPY =
4,4’,4”-tri-tert-butyl-2,2’:6’,2”-terpyridine), which is probably too bulky. The free
chelates by themselves had no effect on enzyme activity. As found also with copper and
Kex2 (19), inhibition enhancement was maximized at a 1:1 ratio of metal to tridentate,
suggesting that the active inhibitor has only one tridentate bound to zinc. In supporting
of this conclusion is the observation that inhibition by pre-assembled complexes was
practically the same as, or slightly better than inhibition by the corresponding mixtures of
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solvated Zn2+ and chelate (which formed the complexes in-situ). These findings justify
the use of preassembled complexes to study the inhibitory effects. We did so whenever a
free tridentate chelate was insufficiently soluble, or when kinetic constants were
obtained.
Inhibition is irreversible. Dilution experiments reveal that furin and Kex2
inhibitions by Zn(TTP)Cl2 were irreversible. To examine reversibility, the enzymes were
pre-incubated with the inhibitor, reactions were diluted 100-fold by buffer, and substrate
was added. Reaction monitoring was initiated immediately afterwards (figure 4 shows a
similarly looking experiment with solvated Cu2+). When product formation was resumed
after the dilution, product accumulation rates were linear with time. However, the slopes
were not parallel - the higher the initial inhibitor concentration, the slower the product
formation rate. This indicates that even after dilution, furin retained a substantial fraction
of the inhibitor bound. The linearity of the kinetic traces indicates that inhibitor
dissociation (resulting in higher active enzyme concentration and increasing reactivity)
did not take place after enzyme activity was resumed. The residual enzyme activity after
dilution (calculated from the slopes of the kinetic traces) and before dilution (calculated
from dose-response correlation using a set aside portion of the non-diluted solutions)
shows that there was no increase in active enzyme concentration after the dilution
(product formation rates differ according to the dilution factor). This holds true for both
furin and Kex2 at all the concentrations examined.
Furin and Kex2 inhibition by Cu(TTP)Cl2 was similar to that by Zn(TTP)Cl2, and
likewise irreversible. But this was not the case with the solvated metal ions. With furin,
the residual activity after dilution was higher than before dilution by a factor of 7 for Zn2+
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(at 250 µM, the highest inhibitor concentration examined, 65% of the initial uninhibited
activity was observed instead of 8%). With Cu2+, the activity after dilution was higher by
a factor of 3 (22% at 7.2 µM Cu2+ instead of 7%). This indicates that some of the bound
metal ions (but not the complexes) dissociate from the protein during the dilution process.
Metal dissociation is complete within the time needed for the protein to resume product
formation. This suggests two modes of inhibition by Zn2+ - one irreversible (linear
kinetic traces), the other reversible (rapid but partial metal dissociation upon dilution).
Partially reversible inhibition by the solvated metal ions was found also with Kex2. With
2.5 mM Zn2+ the activity was 46% prior to the dilution, but 53% after. With 5 mM Zn2+
the values were 7.5% and 22%. The metal-chelate complexes were fully irreversible with
Kex2.
Competition with substrate. Enzyme inhibition by Zn(TTP)Cl2 and by solvated
Zn2+ was competitive with substrate – it was attenuated by higher substrate
concentrations (figures 1,2).
Figures 1 and 2 here please
Kinetic data. Data obtained by kinetic follow-up of furin inhibition at seven
substrate concentrations (between 4.5 (= Km/4) and 36 µM (= 2Km)) was linearly fit in a
plot of [I]0/kobs vs. [S]0 (S = substrate, I = inhibitor) (17). Using the simplified model
E I Ei+ →kon (E = enzyme, Ei = inhibited enzyme) we got kon values of 120±20 s-1 for
Zn(TTP)Cl2 and 5.4±0.6 s-1 for Zn2+. The same [I]0/kobs vs. [S]0 plot can be used also to
extract Ki = k-1/k1 and k2 for the simplified model E I [EI] Ei+ ← → →K ki 2 (EI = enzyme
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+ inhibitor encounter complex). However, in spite of the good linear fit, the values
obtained had no physical meaning (one of the resulting values – Ki or k2 is negative), and
the latter model was rejected. Also with Kex2, only the model E I Ei+ →kon was
consistent with all our data, yielding kon values of 1.2±0.1 s-1 for Zn(TTP)Cl2 and
0.28±0.03 s-1 for Zn2+. To validate our kinetic model, Kex2 was inhibited also with
varied [I]0, keeping [S]0 constant. For this experiment, each kinetic model leads to a
different plot. The standard plot for the E I [EI] Ei+ ← → →K ki 2 model ([I]0/kobs vs. [I]0)
resulted in poor correlation. A plot of kobs vs. [I]0 (resulting from the E I Ei+ →kon
model) yielded kon values very similar to those of the first experiment.
Table 2 here please
Unlike our findings with Kex2 and copper (19), the effect of chloride on Kex2
inhibition by Zn2+ and its compounds and on furin was negligible.
Mutational analysis. Cys217 of Kex2 is adjacent to the catalytic His213. It is not
known to take part in the catalysis (21), but it is a potential site for metal binding within
the active site. A C217S mutant of Kex2 was constructed, expressed and purified (data
not shown). The mutant enzyme was enzymatically active, although activity was reduced
(roughly five-fold; characterization of the effects of this mutation on catalysis will be
published elsewhere). Using normalized inhibition tests, (table 3) we found that the
mutation had some effect on the inhibition by solvated Zn2+ and Cu2+, but none on the
inhibition by the complexes [Cu(TERPY)Cl]+ and Zn(TTP)Cl2 (table 3).
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Table 3 here please
Inhibition by copper and its compounds.
Furin inhibition with solvated copper ions and copper complexes was similar to
that with zinc and to our previous observations with Kex2 (19) in being competitive with
substrate (figures 1, 2, 3, and 5), in being irreversible with complexes, and in having two
metal ions populations – one reversibly bound, and one irreversibly bound (dilution
experiments, figure 4). As we have seen with zinc, all copper-chelate compounds
exhibited some degree of furin inhibition (table 2), except for [Cu(TERPY)2]2+, which has
no coordination sites available for target binding. Copper compounds of TERPY chelates
substituted at the 4’ position by an aryl group were consistently the best here too, but this
time the advantage in modifying TERPY was very small (table 2).
Figures 3 and 4 here please
Unlike our previous examples, all metal-tridentate complexes tested with furin
were much inferior in potency to the solvated Cu2+ ions. As a result, the mixture of
solvated Cu2+ and free chelate was superior as a furin inhibitor to the pre-prepared
complex (by at least one order of magnitude) due to remaining uncomplexed copper ions
in the mixture. This became obvious when pre-incubation of copper and tridentate
chelate in the absence of enzyme led to reduced inhibition under any of the following
conditions: longer pre-incubation times, higher TERPY/copper ratios, and higher TERPY
+ Cu2+ concentrations (at constant TERPY/copper ratios).
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Furin inhibition by solvated Cu2+ was unique also in revealing two phases during
kinetic follow-ups (figure 5). The first phase seemed like all the other inhibitions we
observed, although it was completed much faster (within ~ 1000 s). However, furin
resumed product formation after a lag. This recovery was more pronounced at higher
substrate concentrations. A second, slower inhibition phase was observed within the
duration of our experiment (figure 5). At substrate concentrations higher than those
shown, only one inhibition phase is apparent. Observation of the recovery of furin
activity after inhibition is limited to a narrow window of solvated Cu2+ concentrations.
At 2.5 µM Cu2+ (and 9 µM substrate) there is no recovery of activity after inhibition,
whereas at 20 nM (1.33 : 1 inhibitor to furin ratio) we see very little inhibition (figure 6).
It should be noted that our dose response results with solvated Cu2+ (table 2) were
completed within 10 minutes after substrate addition, well before furin activity recovery
was noticeable.
Figures 5 and 6 here please
Treating the kinetic follow up data for the first phase only (t < 1000 s) by the
same model we used for zinc and its compounds (E I Ei+ →kon ) yielded kon of
25000±2500 s-1 (figure 7). Using the same model for Cu(TTP)Cl2 we got kon = 140±13 s-1
(figure 8). As we found earlier with zinc, the simplified model E I [EI] Ei+ ← → →K ki 2
yielded values with no physical meaning, and the model was rejected. Applying the
E I Ei+ →kon and the E I [EI] Ei+ ← → →K ki 2 models for the second phase of the
inhibition by Cu2+ (t > 3000 s) did not yield reasonable results (kon too big or Ki negative,
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respectively). This and the shapes of the kinetic traces (figure 5) suggest the latter phase
to be reversible. Due to the slow photochemical decomposition of the product (19,20,
and figures 1-3) and because vs ≠ 0, we do not have sufficient data to extract the kinetic
parameters using a fully reversible kinetic model.
Figures 7 and 8 here please
Inhibition by solvated mercury ions Hg2+
We wanted another demonstration of the peculiar sequence seen for the inhibition
of furin by solvated Cu2+ (figures 5 and 6). Because mercury ions inhibit Proteinase K (a
homologue of Kex2 and furin) (22) and because mercury has higher cysteine affinity than
either copper or zinc, it seemed logical to test furin inhibition by solvated Hg2+.
Inhibition (IC50 = 200 nM) was somewhat less efficient than with solvated Cu2+, but
better than with solvated Zn2+. Yet, we were unable to find conditions under which an
inhibition-recovery-inhibition sequence (in similarity to figures 5 and 6) could be
observed with mercury.
Discussion
Mechanism of inhibition
Inhibition by [Zn(tridentate)]2+. We found close similarity between the
inhibitions of furin and Kex2 by [Zn(tridentate)]2+ (table 1). For both proteins, the
inhibition: (a) was irreversible; (b) was competitive with substrate; (c) exhibited
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kinetics following the simplified model E I Ei+ →kon ; (d) was as efficient or slightly
more efficient with pre-assembled metal-chelate complexes than when complexes were
formed in situ from a mixture of Zn2+ and the tridentate chelate; and (e), exhibited the
same trends with the various inhibitors. These similarities imply that both enzymes are
inhibited by similar mechanisms. The major difference between furin and Kex2
inhibition was in the two orders of magnitude higher potency of all zinc-based inhibitors
towards furin. According to the suggested mechanism, this is due (at least in part) to the
higher solvent accessibility of the catalytic His194 in furin. In Kex2, Tyr212 is situated
between catalytic His213 and the solvent, but there is no residue with similar positioning in
furin (21,23).
Inhibitor binding involves metal coordination. None of the free chelates tested
inhibited Kex2 (at 800 µM or below) and furin (at 400 µM). Yet, all combinations
involving zinc afforded some inactivation (table 1), indicating metal coordination to
protein in all cases. Comparison to our copper data (table 2 and reference 19) suggests a
lower protease affinity for zinc. Binding was sufficiently strong to overcome all ligand
interferences (except with too bulky t-Bu3-TERPY). The strength of the zinc-protein
binding is manifested also in the irreversibility of the inhibition.
Inhibitors bind to protease active site. Inhibition is competitive with substrate,
indicating inhibitor binding at the enzyme active site. The active site has residues that
can bind divalent zinc and copper well - Cys217, His381 and catalytic His213 in Kex2 (21,24)
and the analogous Cys198, His364 and catalytic His194 in furin (23,24). His381 in Kex2 (and
the analogous His364 in furin) is suggested to help the catalytic histidine in polarizing the
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hydrolyzing water molecule through another intervening water molecule (21). So far, the
role of the cysteine residue was not elucidated.
Zn(tridentate)Cl2 complexes and solvated Zn2+ differ in binding to Kex2. The
similarity between the IC50 values obtained with the 1:1 Zn2+ to tridentate mixtures and
the Zn(tridentate)Cl2 complexes suggests a [Zn(tridentate)]2+ moiety as part of the active
inhibitor. Inhibition of Kex2 by Zn(TTP)Cl2 and Cu(TERPY)Cl2 was insensitive to the
C217S mutation (table 3), indicating that Cys217 is not involved in inhibition by Zn-metal
chelates. This was not the case with the solvated ions of Cu2+ and Zn2+. C217S Kex2
was somewhat less susceptible than the wild-type (WT) enzyme to inhibition by solvated
copper, whereas inhibition by solvated zinc was facilitated (table 3). This implies a
difference between the Kex2 binding modes of the complexes and the solvated ions
during inhibition.
Binding of Zn(tridentate)Cl2. An obvious difference between the complexes
and the solvated metal ions is in the availability of more sites to coordinate to the enzyme
on the ions. The tridentate chelate occupies three coordination sites in each complex.
Because zinc is usually present in tetrahedral environment when coordinated to proteins
(25), the complexes are most likely to form only a single coordination bond to Kex2.
Moreover, Zn-N bonds are stronger in tetrahedral as compared to octahedral environment
(26,27). A well known example - when bound only to histidines in zinc fingers, zinc ions
bind to four such residues. This effect is more prominent in the presence of multiple
metal-nitrogen bonds. Our complexes have three Zn-N bonds, and a fourth forms upon
binding to a Kex2 histidine. It is true that our compounds are penta-coordinate in the
solid state (as Zn(tridentate)Cl2), but the very similar rates by which the complexes and
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the 1:1 mixture of Zn2+ and chelate react suggest that in buffered solutions, chloride
dissociation is rapid, and a tetrahedral environment is formed. Reasons to deviate from
tetrahedral geometry, such as a gain of chelate effect related energy (as in
[Zn(tridentate)2]2+) or of solvation energy (stabilizing cationic [Zn(tridentate)Cl]+ as
neutral Zn(tridentate)Cl2 in apolar media) do not apply to the Kex2-inhibitor complex.
His213 is the most likely site of [Zn(tridentate)]2+ binding to Kex2 upon inhibition.
Binding to Cys217 is ruled out by the identical IC50s of C217S and WT Kex2. His381 is too
far away from the catalytic or substrate docking residues to inhibit Kex2 activity.
We found that Zn(TTP)Cl2 binds irreversibly to Kex2. Irreversibility in this case
is probably due to a combination of poor lability of the zinc to aromatic nitrogen bond,
and to blocking the pathway for ligand exchange on zinc after binding to the protein.
Inhibition by [Cu(tridentate)]2+. The results with Cu(TTP)Cl2 and the other
copper complexes (table 2, figure 3) were similar in every regard to those with the zinc
complexes. Hence, a similar mechanism is indicated.
Inhibition by solvated Zn2+. Furin inhibition by solvated Zn2+ ions showed close
similarity to the inhibition of Kex2, just as we found with the zinc-chelate complexes
(table 1). For both enzymes the inhibition was: (a) irreversible with most of the solvated
Zn2+ population, but reversible with the rest; (b) competitive with substrate; and (c),
with kinetics that followed the simplified model E I Ei+ →kon . The similar
observations suggest that furin and Kex2 are inhibited by solvated Zn2+ by a similar
mechanism. However, because the mutational analysis was done only with Kex2, we had
to elucidate the mechanism of inhibition by solvated Zn2+ for this enzyme first.
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Unlike [Zn(chelate)]2+, solvated Zn2+ can replace more than a single water ligand
by stronger metal-protein coordination bonds. Proteinase K (a homologue of Kex2 and
furin) forms two strong bonds (and some weaker ones) to Hg2+ by stepwise binding to the
catalytic histidine and to a subsequently exposed, nearby conserved cysteine (22,28). If
the Zn2+/Kex2 analogy to Hg2+/proteinase K system holds, a His213-Zn-Cys217 bonding is
expected in zinc inhibited Kex2.
In the Hg2+/proteinase K system a second population of bound mercury is
observed (22,28). The second binding site involves the proteinase K analogues of Kex2
Cys217 and His381, and is only partially occupied. Also in Kex2 (and furin) we find
evidence for a second population of inactivating Zn2+. A complete analogy to the
proteinase K system suggests a His213-Zn-Cys217 bonding in Kex2, concomitant with zinc
binding to His318. But this model does not explain why dissociation of the reversibly
bound zinc allows recovery of Kex2 activity in the dilution experiment (His381 is too
distant from the catalytic triad and the substrate-binding site). The model also does not
predict the that C217S Kex2 could be inhibited by solvated metal ions.
An explanation in keeping with all our observations could be developed with the
aid of the recently published structure of Kex2 (21). The better zinc and copper binding
residues at the vicinity of the Kex2 active site - His213, Cys217 and His381 form an almost
equilateral triangle. The distances between the zinc coordinating atoms (N and S) are
given in table 4. These should be treated as guidelines only, because the imidazole rings
can turn around the histidine C(α)-C(β) axis. The distances given allow zinc binding
between any two of the three amino acids mentioned. Differences between the three
options would largely originate from the different exposure of Kex2 His213, Cys217 and
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His381 to solvent (and inhibitor). His381 is fully accessible to solvent. The imidazole ring
of His213 is largely masked from solvent by Tyr212. Cys217 is fully buried, and can bind to
Zn2+ only after the ion is bound to another residue, in analogy to the binding of mercury
to the cysteine in proteinase K (22).
Table 4 here please
To elucidate which of the three binding options is operative in the Zinc/Kex2
system, it is possible to analyze how each will affect our observations:
(a) His213-Zn-His381: This is the only option available for inhibition of the C217S mutant
of Kex2. IC50s for C217S and WT Kex2 due to inhibition by this mode are expected
to be very similar.
(b) Cys217-Zn-His381: This binding option is not expected to inhibit Kex2. However, it is
the only option that could explain the somewhat higher IC50 of WT Kex2 as compared
to the C217S mutant. Removal of this binding option by the mutation increases the
fraction of enzyme available for inhibition. Concomitant binding of additional zinc to
His213 provides the most reasonable source for a reversibly bound zinc population,
which is detected in the dilution experiment. Kex2 can regain its activity upon zinc
dissociation from His213.
(c) His213-Zn-Cys217: This option should increase the IC50 for C217S Kex2, as compared
to the native enzyme, in contrary to observation. It is also the least likely from a
kinetic point of view, because Cys217 is buried, and His213 is partially masked from the
solvent (21).
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A combination of the first two inhibition options is necessary to explain our
observations for Kex2 inhibition by Zn2+. There is no reason to invoke a role for binding
option c. However, the somewhat higher (20%) IC50 for the inhibition of C217S Kex2 by
Cu2+ indicates that option c is of importance for the inhibition of Kex2 by solvated Cu2+.
This parallels the higher affinity of Cu2+ to thiolates (and cysteine) as compared to zinc
(29).
The best Zn2+ binding amino acid residues in furin are arranged like an equilateral
triangle, just like in Kex2 (table 4). Since other observations regarding furin inhibition
by solvated zinc are similar to those with Kex2, a mechanism in the same lines to those
suggested for Kex2 will be in keeping with our findings with furin. As suggested for the
metal-chelate complexes, we associate the easier inhibition of furin as compared to Kex2
to the higher solvent accessibility of the catalytic histidine in furin.
Inhibition by solvated Cu2+. Our data suggests that solvated Cu2+ inhibits Kex2,
in a similar manner to Zn2+, with slight differences regarding the different binding options
operating (see above). Yet, solvated Cu2+ seems unique in its high speed of furin
inhibition, and in the recovery of enzyme activity, which follows the inhibition prior to a
second inactivation (figure 5). Nevertheless, the mechanism suggested for solvated Zn2+
is strongly corroborated by allowing an explanation of the otherwise difficult to
rationalize peculiarities of copper.
How can the catalytic activity of furin be recovered after inhibition? Of the three
amino acids in the vicinity of the active site of furin with predicted high Cu2+ affinity
(His194, Cys198, His364), catalytic His194 is the only one to which binding is likely to result
in furin inhibition. Hence, a recovery of furin activity should involve a free His194. The
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reactivated enzyme is different from an enzyme that was not inhibited at all, because it
takes much longer to inhibit in the second phase, and the second inhibition seems
reversible (figures 5, 6). A mechanism in keeping with these demands involves rapid
initial inhibition by solvated Cu2+ binding to His194, which is more exposed to solvent
than partially buried His364, and fully buried Cys198 (23). A stepwise mechanism in which
the bond to His194 is replaced by His364-Cu-Cys198 configuration regenerates the catalytic
activity. Now free, the catalytic His194 can bind another copper ion and bring upon the
second phase of furin inactivation. However, there is now no free nearby amino acid that
can anchor the copper irreversibly (His364 and Cys198 are bound to the first copper), thus
inhibition is reversible.
The proposed formation of His364-Cu-Cys198 can take place through either one of
two intermediates: His194-Cu-His364 or His194-Cu-Cys198. The distances between the
residues involved allow both (table 4). In analogy to our proposed mechanism, the
transfer of copper between the yeast copper chaperone ATX1 and the copper transporter
CCC2 is suggested to take place by thiol exchange, following the associative sequence
(CysATX1)2-Cu(I) � (CysATX1)2-Cu(I)-CysCCC2 � CysATX1-Cu(I)-CysCCC2 � CysATX1-
Cu(I)-(CysCCC2)2 � Cu(I)-(CysCCC2)2 (30). In our case, reduction to Cu(I) is likely to take
place upon binding of the solvated ion to Cys198 of furin (31,32). Bonds to Cu(I) are
much more labile than bonds to Cu(II) (24).
Cu2 + vs. Zn2+. It is possible that inhibition-recovery-inhibition sequence
described for solvated Cu2+ applies also to solvated Zn2+, but is not distinguishable
graphically due to a much smaller overall effect, as is the case with lower Cu2 +
concentrations (figure 6). The smaller effect with zinc may be due to a combination of
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the following reasons: (a) the weaker affinity of Zn(II) to cysteine (29); (b) the lower
lability of Zn(II) as compared to Cu(I) (Zn(II) cannot be reduced by cysteine); and (c), a
less favorable metal binding geometry (copper forms a linear His-Cu-His or His-Cu-
Cys geometry, whereas zinc forms non-linear connections within the set distance of the
amino acids, resulting in longer zinc-amino acid bonds). Unlike Zn(II), Hg(II) has a
higher affinity to cysteine than Cu(II), and it also give rise to linear His-Hg-His or His-
Hg-Cys geometry. However, mercury bonding to histidines and cysteines is not labile.
We were unable to achieve an inhibition-recovery-inhibition sequence with solvated
Hg2+, adding evidence that copper reduction by cysteine takes place prior to the recovery
of furin catalytic activity.
The lower affinity of Zn(II) to ligands in general (as compared to Cu(II)) (33)
probably accounts also for the higher degree of dissociation of the second bound zinc ion
as observed in the dilution experiment.
His381 and Cys198 in furin catalysis.
His381 in Kex2, and possibly also its furin analogue His364, is suggested to have a
role in polarizing water in support of the catalytic action (21). We have suggested above
that furin regains its catalytic activity with Cu2+ bound to His364. These two statements
are not necessarily contradicting. A metal ion bound to the polarizing histidine will
probably be more efficient in polarizing the water chain than the water molecule it had
displaced.
Regaining the catalytic activity after the initial phase of inhibition by solvated
Cu2+, and the retention of 20% of the catalytic activity by the C217S mutant of Kex2
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mutant, both indicate that Cys198 has no active role in furin catalysis, in spite of being in
proximity to the active residues, and being conserved throughout the SPC family. So
why have a conserved cysteine that does not participate in S-S bonding so close to the
catalytically active center? An intriguing possibility is that the recovery of furin action
after inhibition by copper could actually be of use for protein function in-vivo. The
relatively high affinity of furin to copper should not pose a threat to catalysis inside cells,
where metal concentration and movement are under tight control (34,35). However, this
does not seem to be the case outside the cell, and possibly also inside the secretory
pathway and the endosomes, the locations of furin operation.
Protein selectivity of the inhibitors.
The reported inhibitors of the subtilisin-like pro-protein convertases (SPCs) (with
the exception of neoandrographolide (9)) are all peptide or protein-based. Rendering
them selective to a single SPC has proven difficult (3,6), because they all utilize the
inherent recognition system of the proteins (the Pn and Sn pockets) that possibly was not
designed to give complete differentiation between the SPCs (5, with reference 8 being an
exception). The 100 fold faster inhibition of furin as compared to Kex2 places our
M(chelate)Cl2 metal-compounds (M = Cu, Zn; chelate = MPT, TTP), within the small
group of inhibitors that show some degree of SPC selectivity. Other examples include
polyarginines (36) and Elgin c derivatives (8,10). Unlike the peptidyl inhibitors, our
compounds are not restricted to the inherent recognition elements of the target to achieve
selectivity. Hence, there is reasonable hope that our compounds can provide an
appropriate scaffold for the development of fully-selective and higher-affinity SPC
inhibitors. This said, it should be noted that our motivation for the development of furin
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inhibitors is to stop aggressive viruses after infection, to prevent the damage of toxins,
and possibly to find an effective wide range anti-bioterror agent. For these purposes,
SPC selectivity is not required.
Acknowledgments. We thank Laura M. Rozan (University of Michigan) for preparing
Kex2, the NIH for partial support through GM39697 (RSF) and the University of
Michigan for support (OB).
References
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Footnotes
* To whom correspondence should be addressed: [email protected]
1 The abbreviations used are: SPC = pro-protein convertase; BOC = t-
butoxycarbonyl; MCA = methylcoumarinamide; AMC = 7-amino-4-
methylcoumarin; DMSO = dimethylsulfoxide; MPT = 4’-[4-
methoxyphenyl]-2,2’:6’,2”-terpyridine; TERPY = 2,2':6',2"-terpyridine;
NaMES = Sodium 2-(N-morpholino)ethanesulfonate; Bis-Tris = 2,2-
bis[hydroxymethyl]-2,2’,2”-nitrilotriethanol; TTP = 4’-[p-tolyl]-2,2’:6’,2”-
terpyridine; Me2-4’-TTP = 4,4”-dimethyl-4’-[p-tolyl]-2,2’:6’,2”-terpyridine;
t-Bu3-TERPY = 4,4’,4”-tri-tert-butyl-2,2’:6’,2”-terpyridine; Cl-TERPY = 4’-
chloro-2,2’:6’,2”-terpyridine; PyCH2PP = 1-[2-pyridinylmethyl]-piperazine;
DPA = di-[2-picolyl]amine; Me3-[9]aneN3 = 1,4,7-trimethyl-1,4,7-
triaazacyclononane; Im = imidazole; OH-TERPY = 4’-hydroxo-2,2’:6’,2”-
terpyridine; WT = wild-type.
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Figure & Table Legends
Table 1. Furin and Kex2 inhibition by solvated Zn2+ alone, in the presence of mixtures
containing chelates and solvated Zn2+ at a 1:1 ratio , and by preprepared dichloro-zinc
complexes of these chelates. Mixtures were incubated for 2 hours at 22°C prior to
substrate addition. Initial product formation rates were obtained from fluorescence
measurements at 30°C. IC50 values were obtained from a fit of the relative activity vs.
inhibitor concentration data to ∂ ∂ νP t k to obs= ⋅ − ⋅exp[ ] (17). Conditions - with furin:
[Furin] = 3 nM, [S]0 = 9 µM = Km/2 (11). Buffer was 20 mM NaMES, pH = 7.0, 0.1%
Triton x-100, 3% DMSO, [NaCl] = 20 mM and [CaCl2] = 1 mM. With Kex2: [Kex2] =
1.2 nM, [S]0 = 19 µM = Km (18). Buffer was 168 mM Bis-Tris, pH = 7.4, 0.1% Triton x-
100, 3% DMSO, [NaCl] = 20 mM and [CaCl2] = 1 mM.
Table 2. Furin and Kex2 inhibition by solvated Cu2+ alone, in the presence of chelates at
a 1:1 ratio to Cu2+, and by dichloro-copper complexes of these chelates. Conditions were
identical to those listed under table 1.
Figure 1. Furin inhibition progress curves at different initial substrate concentrations
([S]0) (4.5 – 36 µM) at fixed initial Zn2+ concentration ([Zn]0) (200 µM). Reactions were
initiated by enzyme addition at 30°, without any prior incubation.. [Furin] = 1.5 nM.
Buffer was as stated under table 1 for furin. The loss of product fluorescence that is
observed at higher inhibitor is due to product photochemical degradation (19,20). This
loss is first order in AMC, but zero order in inhibitor.
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Figure 2. Kex2 inhibition progress curves at different [S]0 (4.25 – 38 µM) at fixed
[Zn2+]0 (2 mM). Reactions were initiated by enzyme addition at 30°, without any prior
incubation. [Kex2] = 0.03 nM. Buffer was as stated under table 1 for Kex2.
Table 3. Inhibition of Kex2 and its C217S mutant by copper and zinc. Conditions are
identical to those listed under table 1 for Kex2. The large errors associated with the IC50s
of solvated Cu2+ and Zn2+ are due to the large number of times these experiments were
conducted (see experimental section). The error associated with the actual comparison
with the mutant is smaller by an order of magnitude.
Figure 3. Furin inhibition progress curves at different [S]0 (4.5 – 36 µM) at fixed initial
Cu(TTP)Cl2 concentration (12 µM). Reactions were initiated by enzyme addition at 30°,
without any prior incubation.. [Furin] = 1.5 nM. Buffer was as stated under table 1 for
furin.
Figure 4. Product formation by furin after 100-fold dilution of incubations with solvated
Cu2+ at 0.6, 2.4 and 7.2 µM, or with no inhibitor at all (control). Enzyme (15 nM) and
inhibitor were incubated for 2 hours at 22°C. Reactions were diluted 100-fold by buffer,
substrate (100 µM) was added, and product formation was monitored. Product formation
rates after dilution were obtained from a linear fit to data points between 2000 and 7200
s. Activities prior to dilution were determined with aliquots set aside from the same
solutions, according to the procedure outlined under table 1.
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Figure 5. Furin inhibition progress curves at different initial substrate concentrations
(4.5 – 36 µM) at fixed [Cu2+]0 (100 nM). Conditions were as stated under figure 1 for
furin. Traces at 6.75, 9, and 18 µM substrate are not shown for clarity.
Figure 6. Furin inhibition progress curves at different initial inhibitor concentrations
(0.02 ≤ [Cu2+] ≤ 2.5 µM) at fixed substrate concentration ([S]0 = 9 µM). The two phases
of furin inhibition can be seen clearly only at 500 nM and 100 nM inhibitor. Conditions
were as stated under figure 1 for furin.
Figure 7. Derivation of kon for the inhibition by solvated Cu2+. Data from figure 5 at
times < 1000 s was used. kon can be derived separately from either the slope (kon(1)) or
the intercept (kon(2)). The kon value reported is the average of the two values. The error
value reported is the larger of the two values obtained.
Figure 8. Derivation of kon for the inhibition by Cu(TTP)Cl2. Data from figure 3 was
used. kon can be derived separately from either the slope (kon(1)) or the intercept (kon(2)).
The kon value reported is the average of the two values. The error value reported is the
larger of the two values obtained.
Table 4. Distances between the possible zinc (and copper) coordinating atoms at the
vicinity of the active sites of furin and Kex2. Distances were obtained from the PDB
files of the crystal structure of furin (23) and Kex2 (21) using RasMol Macintosh version
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no. 2.7.2.1. The distances should be treated as guidelines only, because the imidazole
rings can rotate around the histidine C(α)-C(β) axis. This is well demonstrated in the
structure of furin, which in this case is an octamer. The distance between the same atoms
in different units differs by more than 0.1Å (for this reason only a single digit beyond the
decimal point is given). In solution, upon imidazole rotation around the histidine C(α)-
C(β) axis in combination with ligand binding, the distances will change by much more.
It should be noted that the table refers to 5 atoms (2 nitrogens from each histidine
imidazole, and 1 cysteine sulfur), but for binding the metal ions we need only a single
nitrogen from each histidine, and the sulfur. Hence, the longer distances from Nδ(1) of
His194 does not matter.
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Tables
Table 1
Tridentate
with 1:1 Zn2+
IC50 (µµµµM)
Furin
IC50 (µµµµM)
Kex2
Zinc complex IC50 (µµµµM)
Furin
IC50 (µM)
Kex2
Zn2+ alone 21±3 890±110 Zn2+ alone 21±3 890±110
MPT 10±2 85±8 Zn(MPT)Cl2 9±1.2 75±1.5
TTP 10±2 100±7 Zn(TTP)Cl2 9±1.1 95±19
Me2-4’-TTP a a Zn(Me2-4’-TTP)Cl2 14±2 115±6
TERPY 29±5 1050±160 Zn(TERPY)Cl2 - 890±60
t-Bu3-TERPY a a Zn(t-Bu3-TERPY)Cl2 No inh.b No inh.b
Cl-TERPY a a Zn(Cl-TERPY)Cl2 70±7 2200±190
PyCH2PP 19±3 870±200 - - -
DPA 100±10 1030±160 - - -
Me3-[9]aneN3 55±10 >2500 - - -
a Free ligand was insufficiently soluble
b Inh. = inhibition
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Furin Inhibition by Compounds of Copper and Zinc
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Table 2
Copper complex IC50 (µµµµM)
Furin
IC50 (µµµµM)
Kex2 (19, this work)
Cu2+ alone 0.14±0.06 95±35
Cu(MPT)Cl2 5.1±0.6 10±0.07
Cu(TTP)Cl2 5.0±0.6 11±0.01
Cu(Me2-4’-TTP)Cl2 14±1.5 16±0.09
Cu(TERPY)Cl2 7.7±0.5 25±4
[Cu(TERPY)Cl](OCl4) 6.9±0.5 25±2
[Cu(TERPY)2](OCl4)2 No inhibition No inhibition
[Cu(TERPY)(Im)(H2O)](BF4)2 Incomplete inhibitionb 120±13
Cu(OH-TERPY)Cl2 7.2±0.7 280±60
Cu(DPA)Cl2 38±3 165±40
a Free ligand was insufficiently soluble
b Inhibition leveled at 63%. IC65 = 4.0, whereas for the other TERPY complexes it was 1.8 – 2.3 µM
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Table 3
Enzyme IC50 µM
Solvated Zn2+
IC50 µM
Zn(TTP)Cl2
IC50 µM
Solvated Cu2+
IC50 µM
[Cu(TERPY)Cl]+
Kex2 (WT) 890±110 95±20 95±35 25±2
C217S Kex2 490±20 95±4 115±4 25±1
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Table 4
Atoms involved
Furin
Distance
(Å)
Atoms involved
Kex2
Distance
(Å)
Nδ(1) His194 – Sγ Cys198 4.4 Nδ(1) His213 – Sγ Cys217 4.4
Nε(2) His194 – Sγ Cys198 5.2 Nε(2) His213 – Sγ Cys217 4.8
Nδ(1) His364 – Sγ Cys198 5.3 Nδ(1) His381 – Sγ Cys217 5.4
Nε(2) His364 – Sγ Cys198 4.7 Nε(2) His381 – Sγ Cys217 3.9
Nδ(1) His194 – Nδ(1) His364 7.4 Nδ(1) His213 – Nδ(1) His381 6.5
Nδ(1) His194 – Nε(2) His364 5.7 Nδ(1) His213 – Nε(2) His381 6.3
Nε(2) His194 – Nδ(1) His364 6.9 Nε(2) His213 – Nδ(1) His381 5.2
Nε(2) His194 – Nε(2) His364 4.9 Nε(2) His213 – Nε(2) His381 5.4
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Furin Inhibition by Compounds of Copper and Zinc
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Figures & Schemes
Scheme 1
NN N
NN N
Cl
NN N
OH
NN
N
MPT
NN N
O
NN N
Me2-4'-TTP
TERPY Cl-TERPY OH-TERPY
PyCH2PP
NN N
NN N
H
DPAt-Bu3-TERPY
TTP
NN N
N
NN
Me3-[9]aneN3
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Furin Inhibition by Compounds of Copper and Zinc
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Figure 1
0
1
2
3
4
5
6
7
8
0 5000 10000 15000 20000
Acc
umul
ated
Pro
duct
Flu
ores
cenc
e(a
rbitr
ary
un
its)
Time (s)
[S]0 = 36 µM; kobs = 3.7·10-4 s-1
[S]0 = 27 µMkobs = 3.9·10-4 s-1
[S]0 = 18 µM; kobs = 4.3·10-4 s-1
[S]0 = 13.5 µMkobs = 4.7·10-4 s-1
[S]0 = 9 µM; kobs = 4.9·10-4 s-1
[S]0 = 4.5 µM; kobs = 5.5·10-4 s-1
[S]0 = 6.75 µMkobs = 5.1·10-4 s-1
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Furin Inhibition by Compounds of Copper and Zinc
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Figure 2
0
1
2
3
4
5
6
7
8
0 1000 2000 3000 4000 5000 6000 7000
Acc
um
ula
ted
Pro
du
ct F
luo
resc
ence
(arb
itra
ry u
nit
s)
Time (s)
[S] = Km/4, kobs = 2.5·10-4 s-1
[S] = Km/2, kobs = 1.7·10-4 s-1
[S] = Km = 19 µM,
kobs = 6.4·10-5 s-1
[S] = 2Km,
kobs = 4.6·10-5 s-1
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Furin Inhibition by Compounds of Copper and Zinc
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Figure 3
0
5
10
15
0 5000 1 104 1.5 104
Acc
umul
ated
Pro
duct
Flu
ores
cenc
e(a
rbitr
ary
un
its)
Time (s)
[S]0 = 36 µMkobs = 1.7·10-5 s-1
[S]0 = 4.5 µM; kobs = 1.6·10-3 s-1
[S]0 = 6.75 µM; kobs = 1.2·10-3 s-1
[S]0 = 9 µM[S]0 = 13.5 µM; kobs = 9.3·10-4 s-1
[S]0 = 18 µM; kobs = 8.3·10-4 s-1
[S]0 = 27 µMkobs = 6.3·10-4 s-1
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Figure 4
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0
10
20
30
40
50
0 2000 4000 6000 8000 10000 12000
[S]0 = 36 µM
[S]0 = 27 µM
[S]0 = 13.5 µM
[S]0 = 4.5 µM
Acc
um
ula
ted
Pro
du
ct F
luo
resc
enc
(arb
itra
ry a
nit
s)
Time (s)
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0
5
10
15
20
25
30
35
0 2000 4000 6000 8000 10000 12000
Acc
umul
ated
Pro
duct
Flu
ores
cenc
e(A
rbitr
ary
Uni
ts)
Time (s)
[Cu2+]0 = 2.5 µM
[Cu2+]0 = 500 nM
[Cu2+]0 = 100 nM
[Cu2+]0 = 20 nM
No inhibitor
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0 100
2 10-5
4 10-5
6 10-5
8 10-5
1 10-4
1.2 10-4
0 1 10-5 2 10-5 3 10-5 4 10-5
[I] 0
/kob
s (
M·s
)
[S]0 M
ko n(1) = 1/(Km·slope) =
1/(18·10- 6·2.0417) = 27210 ± 2491 s- 1
ko n(2) = 1/intercept = 22190 ± 1800 s- 1
ko n(Cu2 +) = (27210 + 22190)/2 = 24700 ± 2491 s- 1
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0
0.005
0.01
0.015
0.02
0.025
0 1 10-5 2 10-5 3 10-5 4 10-5
[I] 0
/kob
s (
M·s
)
[S]0 (M)
ko n(1) = 1/(Km·slope) =
1/(18·10- 6·423.21) = 131.27 ± 9.49 s- 1
ko n(2) = 1/intercept = 148.50 ± 13.20 s- 1
ko n(Cu(TTP)Cl2) =
= (131.27 + 148.50)/2 = 139.83 ± 13.20 s- 1
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Paul Podsiadlo, Tomoko Komiyama, Robert S. Fuller and Ofer BlumFurin inhibition by compounds of copper and zinc
published online May 12, 2004J. Biol. Chem.
10.1074/jbc.M400338200Access the most updated version of this article at doi:
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