Subcellular distribution of a nitroxide spin-labeled netropsin-acridine hybrid in living KB cells: Electron Spin Resonance study

Vol. 167, No. 2, 1990 March 16, 1990

Subcellular

distribution

of

netropsin-acridine

Christian INSERM December

a

hybrid

Electron

Received

AN5 BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 798-806

BIOCHEMICAL

Spin

Bailly

and

spin-labeled

living

Resonance

KB

cells:

study

Jean-Pierre

U16, Place de Verdun,

22,

nitroxide

in

Htnichart

59045

Lille

cedex, France

1989

NETGA is an hybrid derivative which possesses an intercalating heterocyclic nucleus related to amsacrine and a miuor groove binding squeletton related to netropsin. Cellular uptake of this drug has been studied by Electron Spin Resonance (ESR) spectroscopy using a spin-label derivative of NETGA (SL-NETGA). ESR determination of the kinetics of the drug repartition between the cytoplasm and nucleus showed that NETGA accumulated very rapidly and predominantly in the nucleus. Analysis of the anisotropic ESR spectra recorded in the nuclear compartment are in agreement with a strong binding of the drug to the DNA besides confirmed by a maximum ATm of 12°C between the spin-label compound-DNA complex and the DNA alone. 01990 Academic PLOSS, Inc. The interest

design

(l-3).

of

By

determinants

basis

the

potency

(4).

known

natural

compounds acridine

design Taking

netropsin

(and

binding

is to be taken sequences of

such

Our

study

appear

as one

in our

laboratory

0006-291X/90 Copyright All rights

compounds

in

will

addition

has addressed of the most

has revealed,

the

that

this

of longer

peptide

of by

intercalation

and

in

particular

Therefore

one

of

numerous and

minor

novel

design

ligands

DNA-binding

problem. fields

With

with unit

this

$1.50 798

longer

a cellufar

capable

in

of investigation.

ligands,

transport

strategy mind,

of

acridine that

the

program

currently

Indeed

by the use of ESR spectroscopy,

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

on

of the

interest.

to the

this

(9).

rings

result

wellhybrids

for the study

characteristics

the

enhanced

anilino-amino-9-

examined

with

emerge.

promising

an

DNA-binding

for the development

biological

transport

intercalating

the

of many

m-AMSA

interest

effectively

and

synthetized

and

drug

current

a rational of

particularly

that (lo),

account

direct

The been

revealed

implicated

into

hybrids

cellular

agents.

of

have,

agents

recently

distamycin)

of

to develop

site specificity

have

antileukemic

linkage

a topic may

possible

is of particular

have

and

both of

has to be considered (3).

binding

techniques are

the

we

analog

to the

Fig.1)

groove

With

related of

groove

features

problem

its

antibiotics

of the

(5-8),

is

chemotherapeutic

the knowledge drugs

(NETGA.

spectroscopic

targeting

account

agents

is now of

of such new compounds

of minor drugs

it

generation

synthetic

DNA-binding

reading

specificity

specificity,

a new

structurally

design on

portion

into

specific

what

that

of

and

moiety

influence these

of

between

The

sequence

understanding

molecular for

DNA

in

use

derivatives

a previous

study

amino-9-acridine

Vol.

167,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

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\\ /” / Iii\ /

R --HN

HN

CH3 NHaNH

CH3

NE’KXR=H

0

SL-NECGA

H3C

R =

CH3 Ly-t

H3C

CH3

N 0

&J

penetrates rather

very

in

rapidly

the

workers

to

cytoplasm

one

to their

not only

is

exhibits

an ESR

as

the

by

kinetics

their

binding

preference

the

constraint

nature

of

which the

concentration of

penetration moiety AND

ESR

of of as

Helene

nucleus and

of

chromophore

and ATAT

compounds

co-

DNA-binding to

of DNA-binding have

spectrum

of

that

When

local

of the compound drug

(15))

a

due but

been

netropsin,

in

also

shown

to

acridine

a nuclear-binding

into

and

NETGA,

moiety

(SL-

by the

reflects the

cells,

We A

have

SL-NETGA as well

shown

comparison

implicates

its

immediate

of the nitroxide

nucleus.

transporter

KB

spin-label

produced

environment. cells

in living

a nitroxide

by the position the

NETGA

a nitroxide

motion

subcellular probe

containing

incorporated

is determined

the

GC (14)

of this upon

discernible.

spectrum

an acridine

the

(11,16).

derivative

the

by the

on grounds

Both

of the uptake

Because readily

transport.

into

used

amplify

of

(respectively

cellular

an examination

amino-9-acridine MATERIALS

complementarity of

been

to

justified

NETGA.

concentrate

also

and

well

motion,

preferential

have

penetration

is particularly

a spin-labeled

environment

Acridines

linking

Fig.1).

rotational

preferentially

covalent

We report NETGA,

(11).

and

The

because

using

cells

and spin-labeled

(12-13).

a nuclear

cells,

the

cellular

base-specific

crucially have

in

facilitate

oligoribonucleotides netropsin

formulae of NETGA

: Structural

the

the of

the

anilino-

system.

METHODS

Chemistrv : IR spectra were recorded on a Perkin-Elmer 177 spectrometer and only the sharply defined peaks are given. FAB mass spectra were determined on a Kratos layer MS-50 RF mass spectrometer arranged in an EBE geometry. Thin 799

Vol.

167,

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2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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chromatography (TLC) was carried out using silica gel 60F-254 Merck precoated UVsensitive plates. Svnthesis of the snin-labeled derivative : 4-[[[(4-amino-1-methyl-pyrrole-2yl]carbonyl]amino]-1-methyl-pyrrole-2-carboxylic acid (9) (0.5g, 1.28mmol) was coupled to 3-carboxy-2,2,5,.5-tetramethyl-1-pyrrolidinyloxy (3-carboxy-proxyl, Aldrich) (0.24g, 1.28mmol) by a classical procedure using dicyclohexylcarbodiimide (DCC) and lH-hydroxy-1,2,3-benzotriazole (HOBt) at 0°C for 3h then at room temperature overnight in a mixture of dimethyl formamide/dichloromethane (DMF/CH2C12) (1/5,v:v). After complete evapora tion of DMF, the CH2C12 solution was washed in turn with 1N HCl, H20 then 1M NaHC03 to eliminate the unreacted and side products. Dicyclohexylurea was discarded by careful precipitation with cold acetone. After evaporation of the solvent, the residual solid was dissolved in a minimum amount of CH2C12 (3ml) then precipitated by addition of diethylether (IOml). The desired compound was obtained as a pure white powder (380mg,67% yield) as judged by TLC analysis. Rf(CHC13/MeOH,8/2,v:v in a saturated NH3 atmosphere):OXl; IR vmax (KBr) 1350,1580,1650, 1670,1690,1720, 2940-2990,334O cm-l; MS-FAB,m/z, 445(M++l). Conversion of this methyl ester (300mg,0.675mmol) into the corresponding acid was conducted in a hydro-methanolic solution containing NaOH (lOOmg,2.7mmol) of the solvent gave a white powder which was for 18h at 20°C. Evaporation partitioned between H20 and CH2C12 to remove the remaining ester. Acidification of the aqueous layer to pH 3.5 with dilute HCl and extraction with ethyl acetate (3 x 25ml) afforded 220mg of the chromatographically pure acid. 76% yield; Rf(CHC13/MeOH,8/2,v:v in a saturated NH3 atmosphere):O-0.15, Rf(CHC13/MeOH,2/8, v:v):O.75; IR vmax (KBr) 1460,1640,1650,1740, 2950-3000 cm-l; FAB-MS,m/z, 431(M++l). 4-(9-acridinylamino)-N-[4-[[[4-[[3-(2,2,5,5-tetramethyl-l-pyrrolidinyloxycarbonyl] amino]-1-methyl-pyrrol-2-yl]carbonyl]amino]-1-methyl-pyrrol-2-carbonyl] glYcY1 aniline (SL-NETGA) was synthetised by the reaction of 0.35mmol. (150mg) of the acid described above and 0.3mmol. (102mg) of 4-(9-acridinylamino)-N-glycylaniline (17) in the presence of DCC/HOBt as coupling agent and in DMF/CH2C12 (l/l,v:v) for 4h at 0°C then 18h at 2O’C. Evaporation of the solvent gave a red powder which was triturated with CH2C12 then dissolved in MeOH (3ml) and precipitated by addition of cold acetone (50ml). This operation was repeated twice to obtain 95mg (42% yield) of SL-NETGA. Rf(CHC13/MeOH,2/8,v:v):0.83; no distinct m.p. (decompose upon heating); JR vmax (KBr) 1520,1630,1650,1740,2910,3300-3400 cm-l; FAB-MS,m/z, 756(M++2). At each step of the synthesis, the presence of the nitroxide was confirmed by ESR. 3-carboxy-proxy1 (Aldrich) was used as reference for the penetration and localization of the nitroxide moiety. Cell Culture : KB cells were grown as suspension cultures in Jodlik modified Eagle Medium (Seromed, Munich, FRG) supplemented with 5% heat-inactivated Colt serum at 4 x lo5 cells/ml concentration. Suin labeling and cell fractionation : SL-NETGA (18.9mg) was added to cell culture (500ml) at 50pM (final concentration) for various incubation times. Cell fractionation was performed as previously described (11,18). The purity of the fractions was checked by electron microscopy and reveals that the fraction refered as the cytoplasmic one was absolutely free of nuclei but contains intracellular membranes (endothelial membranes, lysosomes and mitochondria). The nuclear fraction contained almost exclusively undegraded nuclei. ESR Snectra : Prior to ESR examination, the cellular fraction were defrosted and sonicated (two 5 set burst with microtip probe of a Branson sonifier (Danburry, Connecticut), maximum power), then treated with H202 in the presence of sodium phosphotungstate to point out a biological reduction in situ (19,20). ESR measurements were recorded on a Varian E 109 X-band spectrometer with a E 238 cavity operating in the TM110 mode. A 100 KHz high frequency modulation was used with a 20 mW microwave power. The samples were examined in a flat quartz cell.The degree of immobilization of the spin-labels as a result of binding was evaluated by the correlation time (Tc), the constant 2A,, and the ratio K. The correlation time (Tc) was calculated by the empirical expression : Tc = C . AH0 [(m+ m ) -21 set, where I+, IO and I- are the amplitudes of the low-field, central and high-field resonance lines respectively, and A HO is the width of the central line in Gauss (G). The constant C for the nitroxyl radical is C = 6.6x10-10 G/S (21). The constant 2A,z 800

Vol.

167,

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2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

represents the distance in Gauss between the two extrema of the ESR spectrum. K is a ratio between the intensities of the low- and high-field line (K=I+/I-). DNA thermal denaturation determinations were recorded with a Uvikon-Kontron 810/820 spectrophotometer and realized in 0.1 M SSC buffer (0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) as previously described (22). RESULTS Time

course

of untake

- The

time

was measured (5 0p.M). 3

with

of

the

drug

SL-NETGA

was

with

This

the

increased level

sites

2).

detected

both

after

preponderance

in the nucleus 2. The

fold

in

NETWcyt,,,

the

nucleus

than

rapid in

5h

(nasopharengal be

very

cytoplasmic intracellular

both of

to

the

since

and the

transport.

The

nucleus,

(i.e.

nucleus

matrix

to

a

meanwhile as judged

is approximately

([SL-NETGA],,,l.,

a

after

corresponding

of SL-NETGA

cytoplasm

fast

ug/m I

fractions

compartments

incubation

cells)

of 37.5

and less in the cellular

concentration in

and

in the cytoplasm

shown

higher

a

slightly

by the curve

in Fig

proved

nuclear

indicates

cells

at a concentration

cells

in both

a plateau (Fig.

by KB3

the drug

into

observed

spin-label

of the receptor

a larger

the

was

reaching

of SL-NETGA

the cells

incubation.

of

cytoplasm) -

of

signal

min

saturation

of uptake

by incubating

ESR

concentration and

course

Penetration

significant only

and localization

18h/[

three S L-

18h =W.

I.‘..I.“.,....I.*“I..‘.I”‘,. I; ,

Time

(h)

u : Kinetics of penetration of SL-NETGA into the nucleus (0) and the cytoplasm (0) of living KB human tumor nasopharengal cells. Drug concentrations have been obtained by integration of the total surface of the ESR spectra and comparison with an ESR spectrum of a 50uM drug stock solution.Values are the mean of 3 independent experiments. 801

Vol.

167,

No.

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

a’

RESEARCH

COMMUNICATIONS

bi

tot Fip. 3 : Time dependence of the ESR spectra of SL-NETGA at room temperature in different environments : a) in the cytoplasm, b) in the nucleus. Instrumental gain conditions : *5x104, **4x104, ***3.2x104, ****1.6x104.

Environment

of

the

probe

broadened triplet ESR signal characterized by : a somewhat - In the cytoplasm a correlation time T, of 0.55-0.8 nsec and a K ratio of 0.8, was observed (Fig. 3a, Table I).

These

even

signals

after

are characteristic

: the ESR signal,

became more and more time T, and the decrease the

constant

3b)

reflects

bound nuclear

an unrestricted,

freely

rotating

nitroxide

label

24 h exposure.

_ In the nucleus

typical

of

2Azz the

of

the

remain

to a molecule DNA.

It

free (from

3 min to 45 min

incubation)

anisotropic as reflected by the increase of the correlation in the K ratio (Table I) while, as for the cytoplasmic spectra, constant

appearance presence

first

to 32+1

of a tightly of

at least

which

might

is noteworthy

that

Gauss. The bound

two

species,

be attributed in

specie, one

shape of the ESR spectra The

last

freely

to the binding

the same

conditions,

observed

rotating

and

of SL-NETGA the nitroxide

(Fig.

spectra the

other to the

alone

Table I : ESR parameters of the crude cellular extracts, the nuclear and cytoplasmic compartments spin-labeled with SL-NETGA at various incubations times .~ Cellular extracts Nuclear Fractions Cytoplasmic Fractions -___ -Time K K K Tc Tc Tc (nsec) (nsec) (ns& control 3 min. 15 min. 30 min. 45 min. 1 h

2h 3h 6h 12 18 24 30

h h h h

0.80 0.92 0.92 0.90 0.91 0.87 0.80 0.82

0.20 0.43 0.40 0.37 0.42 0.48 0.72 0.91

0.80

1.03

0.78 0.75 0.75 0.74

1.15 1.38 1.42 1.51 -

0.80 0.92 0.91 0.88 0.86 0.80 0.77 0.65 0.67 0.60 0.59 0.57 0.57

0.20 0.31 0.34 0.47 0.88 1.00 1.23 1.48 1.39 2.03 2.17 2.44 2.40

0.80 0.83 0.84 0.86 0.84 0.80 0.82 0.81 0.72 0.78 0.76 0.76 0.76

0.20 0.36 0.58 0.55 0.55 0.57 0.62 0.64 0.64 0.75 0.58 0.75 0.80

The ratio K and the correlation time Tc were determined as described in Materials and Methods and are the mean of 3 independant experiments. Control is refered to the free drug in solution. 802

are

(3-

Vol.

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2, 1990

-0.0

AND

BIOPHYSICAL

04

0.2

RESEARCH

0.6

0.8

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10

IDrugl/[DNAj ratio E&& : Comparative effects of NETGA (0) and SL-NETGA Q on the ATm of the helix coil transition of calf thymus DNA (open symbols, 100 uM in base pairs) and poly[d(AT):d(A-T)] (filled symbols, 25 uM in base pairs), in O.lM SSC buffer. Each point _^- _^^^ -. .L^ -^^__^I__^ ..c -3 -I^t^-:^ar:---

carboxy-proxyl) nucleus

was

(slow

nucleic

than

nitroxide

the

those

reported

substituent

potentially

ionic

of SL-NETGA

and

bond

thus

observed

site)

with

an isotropic

with

was found the

are certainly

induced

a high

compound.

loose

of

the cause

and

in the

signal.

a large positive of

12°C

strength

to be lower

the unlabeled

particularly

ESR

a maximum

representating

ATm

in the cytoplasm

as NETGA

temperature

complex,

However

NETGA

melting

both

exhibiting

in Fig. 4, SL-NETGA

acids

NETGA-poly(dAT:dAT) binding.

concentrations

penetration),

: As shown

in

in equal

progressive

DNA-binding increase

found

for

and

ATm

the

presence

a positive of the lower

of

extent

in the presence

The

SL-

of SL-

of the bulky

charge site (i.e. a intensity of binding

as opposed to NETGA

DISCUSSION The accounts

radical of the

of the way

in which

During of

16 Gauss

between

moiety rotational

the

the first

of

the

spin-labeled

freedom

the binding hour

of incubation,

adjacent

lines)

unpaired

electron

and

increasing

incubation characteristic

times, of

acts

as a reporter

environment

in

group

which

it

giving

resides

and

occurs.

between

asymetric,

dye

of the local

the

the nuclear a high

the isotropic

result

degree

from nuclear

spectra of 803

the

ESR

spectra

anisotropic

spin become

immobilization

of

the

broader

(with

a splitting

hyperfine

interaction

nitrogen and

atom.

more

of the nitroxide.

With

and

more

Such

a

Vol.

167,

No.

spectrum and

(Fig.

f)

BIOCHEMICAL

2, 1990

5) is the sum of two

represent

the

signal

intranuclear

medium

low

of the partially

motion

by an outer

The

the immobilized

to

(bound

of the drug as being binding

DNA.

The

the

membranes

which

might be one the cytoplasmic nsec)

of

between and

the drug. be taken

reflect Thus into

(d)

l/3

free)

to

G (2Tl)

(the

can be calculated

possible

and 0.80

sites

of action

nsec,

restricted of the

after

the

in

total

cannot

drug

surface 82(rt2) % expected

after

suggests

24h

these

experiments, The

derivatives

cellular (24,25)

the spectra corresponding to Their correlation times T,, that

and/or

of the control i.e. nuclear

a weak

(T,=0.20 binding

membranes

account.

*T, Typical ESR spectra after 24 h of incubation of SL-NETGA prelabeled KB cells. This nuclear fraction has been obtained as 5 :

with

nucleus of

described under Materials and Methods. The spectra represent a composite absorption consisting of bounds labels designated by a,c,e and unbounds labels b,f. Since the low-field peak, f, of the unbound spin resonance is distinctly separated from the bound resonance, e, its surface (shaded area) was taken to estimate the relative amount of the free label. Instrumental gain : 1.6 104.

804

of

should

. Fig.

of

area in

rationally

eliminated.

9-amino-acridine

than

to the

the total

of the

species

of the nitroxide,

to endothelial

in

splitting

24h exposure,

However,

be absolutely

are greater

(b

characterized

while

to a macromolecule

of

bands

of the signals

integration

parameters of this possibility.

rotation

drug

by

nuclear-bound

cannot

rotate

peak f (shaded

concentration

degradation.

targets

to

hyperfine

narrow

suggests that,

of a significant

narrow

of SL-NETGA,

the superimposition

spin-label

is bound

two

free

inner

area of the high

intracellular

to others

a fewly binding

from

The

COMMUNICATIONS

(a and e) can be attributed moiety

these targets. The ESR fractions strongly suggest 0.36

bands

nitroxide

of the free

survival

are

remaining

results

The

in the nucleus

drug

spin-label

Such an evaluation

resistance

of

included

(23).

recovered

a substantial

band

plus

of the ESR spectra

tracings.

of 25.3

species.

RESEARCH

superimposed

immobilized

middle

is proportional

the

BIOPHYSICAL

G). The wide

splitting

and free

concentration

of

(2Azz=33

hyperfine

be measured). Fig.5)

AND

Vol.

No. 2, 1990

167,

Estimation

BIOCHEMICAL

of

the

total

of the areas of the nucleus a 50 uM This

SL-NETGA

high

Comparing parent

Nt

that

(fast moiety,

i.e.

netropsin

part

transport

of

already

been

(16)

cells to

penetration

of

The

detected (i)

the

heterocycle:

close

to the longitudinal

forced

into

coplanar

the

ligand

This

study

and

design

and

DNA-binding

correlation

the other

hand if NETGA

concerning

the

in vivo

group

only

an acridine the

into

obtained

incubation

of

the

cell

is

to two

is

to

between attached

of this

hydrophobic

to the the amide

bond,

has a restricted

rotation.

sequence

specific

of a DNA

drugs

can

such

a

of

studies

and

hydrophobic very

position

of the

region

and is

moiety.

intermolecular

This linkage

which

because

binding

drugs

the

biological

activity

remain

have shown that NETGA is able to form a II (Saucier d, unpublished results). On

seems to be a weakly antitumor

the

N-methyl-

be associated. However, if the nuclearagent are now well characterized, new

data

DNA-topoisomerase

the

netropsin

namely

design

:

intercalation

the

group,

that

been

at a position

of

the nitroxide

and

had

its

at the N-terminal

relative

the

the

times

factors

by

attached

located

that

a part

by

with

spin-label

which

directly

(exactly

configuration

Preliminary

with

had

molecule.

moiety

physicochemical

complex

the

part

of

nature

properties

to elucidate.

the

to control bleomycin

those

identical

heterocycle

nitroxide

only

targeting

cleaving

of

facilitates

be attributed

whole

of the ligand

demonstrates

difficult

might

acridine

favored

character

between

of the

acridine

linkage

from

motion

acridine

trailing

tighly is

of nuclear

can

acridine

affinity

While

(22,28)

(under

nitroxide

coplanar

and

double-bond

the

end

almost

(16)

of the

the

is wedged an

the

the

5 is different

restricted

the

planar

whereas

configuration

between of its

of

the

layers,

agent

of the

seems

of

26,27),

we

of penetration nuclear

analogs.

(0.1%.

difference

binding

position

pair

backbone),

drug no

of

of

base Nt

rates

in Fig.

This

presence

the

bleomycin

chelating

depicted

signals).

the strength

(ii)

an effect

small

practically

(isotropic

increases

tropism

Such

SL-netropsin

where

(ll),

nuclear

cell.

those of its

drug.

spectrum

binding

conditions)

hybrid

the

cell.

the

nucleus,

affecting

acridine

inside

to those

from

the cells.

the

acridine speed

own with

of with

the

its

inside

drug

comparable

rate and

comparison

obtained

SL-NETGA

the

the

bleomycin-like

the

ESR

a very

hybrid

probe

and

without

previously

in

a

of

through

observed

the

the

influence

the

are more

increasing

because

of

moiety

chromophore,

drug

the

of the studied

parameters

(by

spectrum

of the drug is found

reflects

penetration

shows

to an ESR

43(*5)%

neptrosin

Thus

acridine

the

intercalator

pyrrole

the

the measured the

penetrates

the

concentration

spectra

that

of penetration

penetration).

nuclear

solution)

facilitating

compounds,

drug

concentration

the kinetics

conclude ring

stock

in

intracellular

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