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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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).


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.


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.


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).

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36741–36749


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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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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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36

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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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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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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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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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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42

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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furin inhibition by compounds of copper and zinc · 2004-05-12 · furin inhibition by compounds of...

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