- Email: [email protected]
Inhibition of protein kinases by bis-ATP mimics
925
Eur J Med Chem (1992) 27,925-930 0 Elsevier, Paris
Inhibition of protein kinases by bis-Al P mimics M Houdin, C Sergheraert* Faculte’ de Pharnzacie,
Institut Pasteur de Lille, URA 1309 CNRS, 1, rue Calmette, BP 245,59019 (Received
22 November
Lille, France
1991; accepted 3 July 1992)
Summary - The primary structure of the different PKC subspecies contains an ATP-binding sequence in the catalytic domain. The a, p, and y isoforms contain an additional ATP-binding consensus sequence whose significance remains unknown. To explain this function and also to prepare new specific PKC inhibitors, bis-ATP molecules have been synthesized. The variation of the mutual position of the ATP mimics is conditioned by the choice of the included spacer. The products generally have an increased inhibitor potency towards PKC, PKA and HL60 tyrosin kinase. The inhibition is always competitive towards ATP but does not allow the conclusion that a simultaneous interaction occurs at both ATP-binding sites. PKC inhibition
/ catalytic
site / ATP-binding
sites / bis-ATP
Introduction The physiological importance of protein kinase (PKC) activation is widely appreciated and well documented; a number of recent reviews have described its role as a signal transducing protein in a plethora of physiological responses [l-5]. Its molecular heterogeneity is proved; to date at least seven subspecies of PKC have been described. The complete structures of four isotypes having a, PI, pz, and y sequences (fig 1) have been clarified by analysis of the cDNA clones obtained from the brain of several mammalian species [6]. These isotypes have also been identified at the protein level in preparations purified from brain tissue. More recently a second group of PKC cDNA clones designated 6, E and < has been isolated from brain libraries using as probes a mixture of cDNAs from the initial clones [7]. Among these new members, only PKC-E has been identified at the protein level in brain while the expression of the remainder has been confirmed only at the mRNA level [8]. The 6, E and c types have a common structure closely related to but clearly distinct from the first group; all lack the conserved C, region which seems to be implicated in the regulation of PKC by calcium [7]. In all isoforms, the C, region contains a Gly-X-Gly-X-X-Gly-X,,-Lys sequence, a consensus
mimics
sequence for ATP-binding sites shared by many other protein kinases and many nucleotide-binding proteins [9, lo]. These residues can form an elbow around the ATP molecule with the first glycine in contact with the sugar moiety and the second glycine lying near the terminal phosphate [ 111. However, the careful examination of the primary structure shows that in the a, p, and y isoforms the C, region contains a second consensus ATP-binding sequence (in the y isoform, an arginine residue is found instead of lysine). Since all PKCs have the same K,,, for ATP, the significance of this second putative ATP-binding site remains unknown [ 121. In order to test whether this second consensus sequence was endowed with functionality towards ATP and possibly to prepare new isotype Group A I.. fJ. and yl Regulatory domain
NH*>cooH
Catalvbc domann
% ,
Ii ATP-binding rite
PS Cvr Group B Id, r. and 5 1 "1
Cl
V2lV3
c3 v4
c4
coon A+P-binding sixe I N”z
*Correspondence
and reprints.
v5
Fig 1. Structural
features
of PKC species.
COOH I
926 consisting of a long hydrophobic chain, The flexibility of this structure allowed this construct to be more tolerant with respect to both the distance and the orientation of the two putative ATP-binding sites. However, the number of methylenes was limited to 12 in order to avoid a negative effect on the solubility of the construct.
specific constructs we have synthesized and tested different PKC inhibitors designed to interact simultaneously with the two ATP-binding sites and potentially present a strongly increased binding energy towards a, p and y isoforms. As a monomer we have selected the compound 1 (scheme 1) obtained by substitution of the primary amine H-9: (N-[2-aminoethyl]-5 isoquinolinesulfonamide) [ 131. 1 was chosen because it is sufficiently active (IC,, = 20 p.M towards PKC) and does not contain a secondary amine like H-7: (1-[5-isoquinolinesulfonyll-2-methylpiperazine), therefore it can be easily covalently linked to a spacer group without loss of activity [ 141. In the absence of information concerning the spatial distance in the folded kinase between the two glycine rich sites, we have used two types of spacer (fig 2). i) In the first hypothesis (compounds 5,6,8,9) we speculated that the two ATP-binding sites might be located in close vicinity. In this case a constrained disubstituted benzene structure was used as the core of the linker thus providing a small spacing between the two mimics while both metu and par-u isomers were used in order to allow different respective orientations. ii) In the second hypothesis (compounds 7 and lo), we have used a longer and more flexible linker
SO2 - NH - (CHZ)Z - NH2
Chemistry The compounds 5, 6, 7 were synthesized as follows: the previously described ester 1 [14] was saponified with methanolic NaOH and after acidification yielded the carboxylic derivative 2 (hydrochloride form) (scheme 1). After protection of the secondary amine by a &butyloxycarbonyl group (Boc): 3, the N-hydroxysuccinimide ester 4 was prepared by reaction with dicyclohexylcarbodiimide (DCC) and hydroxysuccinimide (HOSu). It was then reacted with 1,3-xylylenediamine or 1,4-xylylenediamine or 1,12diaminododecane to yield respectively 5, 6 or 7. Trifluoroacetic acid deprotection of the Boc group and separation by chromatography on silica gel preparative layers gave the final products 8,9 and 10.
SO2 - NH - (CH& - NH - (CH& - COOCI$ 1) NaOH
2)HCl
B-3
b
SO2 - NH - (CH& - N - (CH& -COOH
SO2 - NH - (CH& - NH - (CH& - COOH
Lo A- c(a3)3
Di-e
S02-NH-(CH2)2-N
1,3 - xylylenediamine
-(CH2),-CO-N I
;‘&3)3
<
)
1,4 I,12 -xylylenediamine diaminododecaneb
5 6 ,
Scheme 1. Protection
and activation
of the monomer
1.
927
/
0 /Q \
CH2
CH2
NH-CC&(CH2)r~-(CH2)~NH-S02
CH2-NH-CO-(CH2)ry-(CH2)+IH-S02
S0,NH-(CH2)2-~-(CH2)&MH-CH2
R
R
I
R : O=C-0-C-(CH& R: H
Fig 2. Chemical
structure
6 9
I
R:
o=c-O-C-W,),
R:
H
of the synthesized
’ 10 bis-ATP
mimics.
Enzymology The inhibitory capacities of the compounds 1, 8,9, 10 towards PKC and CAMP dependent protein kinase (PKA) were tested using histones III, and II* respectively as substrates. Tyrosine kinase activity was studied using HL60 tyrosine protein kinase with angiotensin II as a substrate [ 151.
Table I. IC,, values of the bis-ATP IC,, @‘KC), W
mimics.
IC,, (PKA), ~A.M
IC,, (HL60 tyrosine protein kinase), @I
8
5.5
3
Not active
9
8
7.3
80
Results and discussion
10
25
10.5
Not active
IC,,s determined for the three protein kinases (PKC (p isoform chiefly), PKA and HL60 tyrosine kinase) are given in table I comparatively with the commercial non-specific inhibitor H-7 and the monomer 1.
1
22
12
1700
H-7
6.2
2.3
1500
928 Among the three kinases, only PKC displays a second ATP binding consensus sequence and was thus likely to be more efficiently inhibited by the bis-ATP molecules. An inhibiting potency was maintained for 8, 9, and 10 towards PKC and PKA indicating that the chemical modifications have preserved the capacity of the heterocyclic part to interact with the ATP binding site of these Ser/Thr kinases. Except for 10 which is almost equipotent with monomeric compound 1, both 8 and 9 show an increased activity towards PKC; the inhibition was competitive towards ATP as shown in scheme 2. However, this increase is rather small and is also observed in the case of PKA which is devoid of a second ATP binding consensus sequence. It is thus likely that this slight increase could be attributed to a more efficient interaction with a single ATP-binding domain or its surrounding than to a double-site interaction. The results also show that only 9 has significant inhibitory activity towards the tyrosine protein kinase; although we have no clear explanation for this difference, this result is interesting as 1, the monomeric ATP inhibitor, has no effect on this kinase. While this work was in progress the structure of the catalytic domain of PKA, determined by X-ray crystallography became available [ 161. Due to the fact that the catalytic domains of the PKC and PKA share a very important homology we have been able to localize the second potential ATP binding site of PKC in a region corresponding to the larger lobe of the catalytic domain in the loop located between helix H and helix I. Bir-ATP mimic /ATP Competition
(DIXON melhod)
This model clearly shows that even if the second ATP binding domain is functional, which in view of the spatial structure in this region does not seem likely, the two domains would be too far apart to allow a simultaneous interaction of any of our bis-ATP mimics. However, as PKC enzyme activation involves its translocation to the membrane, the appearance of a high local concentration of the activated enzyme can be expected. In these conditions, interaction of our compounds with a single ATP binding site on two different enzyme molecules could be envisaged at this level, allowing discrimination between activated and inactive cytosolic forms (but no longer dependent on the nature of the isoform). Cellular studies allowing a study of these compounds in more physiological conditions will be necessary to address this hypothesis. Experimental
protocols
Chemistry Melting points were taken on a Gallenkamp apparatus in open capillary tubes. Elemental analyses indicated by the symbols of the elements were within + 0.4% of the theoretical value. FAB ionization spectra were performed at the Universite des Sciences (Lille, France). All found (MH)+ values agreed with the calculated values. ‘H-NMR spectra were recorded on a Bruker 400 MHz spectrometer using TMS as internal standard. Thin layer chromatography was carried out with Merck F254 silica gel. 1,3-Xylylenediamine, 1,4-xylylenediamine and 1,12-diaminodecane were purchased from Aldrich. H-7 was synthesized as previously described [ 133.
Methyl N-(2-isoquinoline-5-suEfonamidoethyl)-3-amino-propionate Z To a solution of 0.71 g (2.83 mmol) of N-(2-aminoethyl)5isoquinolinesulfonamide [14] and 0.787 ml (5.65 mmol) of triethylamine in CH,OH (30 ml) was added 0.308 ml (2.83 mmol) of methyl 3-bromopropionate. The reaction mixture was refluxed for 4 h and evaporated to dryness; water (30 ml) and CH,CI, (30 ml) were added. The organic layer was dried and evaporated to an oily residue which crystallized from light petroleum (yield = 95%; mp = 105’C). R,: 0.30 [EtOAc-MeOH (2:1)] Anal C15HL9N304S (C, H, N, S). NMR (CD,COCD,, 6, ppm): 2.3 (2H, t, CHJOOCH,), 2.4 (4H, m, CH,-NH), 3 (2H, m, CH,-NH-SO,), 3.62 (3H, s, COOCH,), 7.7 (lH, t, isoquinoline), 8.2-8.7 (4H, m, isoquinoline), 9.3 (lH, s, isoquinoline) FAB-MS: (MH)+ = 338.
N-(2-Isoquinoline-S-sulfonamidoethyl)-3-amino-propionic acid 2
Scheme method)
2. Bis-ATP mimic giving Ki = 1.2 pM.
9/ATP
competition
(Dixon
To a solution of 2.57 g (7.6 mmol) of 1 in CH,OH (40 ml) was added NaOH (1.5 g) and the reaction mixture refluxed for 30 min. Upon removal of the solvent, water addition and HCl acidification (pH 3) the aqueous solution was evaporated to dryness. NaCl was discarded by 95% ethanol addition and after evaporation, the oily residue crystallized from acetone. The crystals were used directly in the next step. (Yield = 92%; mp = 182’C) Anal C,,H,,N,O,SCl (C, H, N, S).
929 N-(2-Isoquinoline-S-sulfonamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionic acid 3 Di-tert-butyl dicarbonate (7.62 g, 35 mmol) was added dropwise over 1 h to a well-stirred solution of 2 (4.6 g, 11.6 mmol) in KHCO, 1 N (12.5 ml) and tert-butyl-alcohol (26 ml) while pH was kept between 8 and 9 by adding 1 N Na,,CO,. After stirring overnight, the aqueous phase was acidified to pH 2.5 (1 N HCl) and extracted with ethyl acetate (2 x 40 ml). The combined extracts were dried (Na,SO,) and evaporated to dryness. The oily residue was chromatographed on silica gel preparative layers eluting with CH,Cl,/MeOH (9:l) to yield white crystals of 3 (yield = 61%; mp = 52°C). R,: 0.1 (ethyl acetate). Anal C,,H,O,N, S Cl (C, H, N, S). NMR (CDJOCD,, 6, ppm): 1.35 (9H, s, t-butyl), 2.4-3.2 (8H, m, CH,-N), 7.8 (lH, t, isoquinoline), 8.3-8.6 (4H, m, isoquinoline), 9.4 (lH, s, isoquinoline). FAB-MS (MH)+ = 424. N-(2-Isoquinoline-5-su~onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionic acid N-hydroxy-succinimide ester 4 A solution of 3 (1.3 g, 3 mmol) and N-hydroxysuccinimide (0.345 g, 3 mmol) in CH,Cl, (50 ml) was cooled in an ice-water bath and dicyclohexylcarbodiimide (0.618 g, 3 mmol) was added with stirring. The mixture was kept at O’C overnight. The separated N,N’-dicyclohexylurea was removed by filtration and the solvent evaporated in vacuum. The crude product was recrystallized from isopropanol (yield = 81%; ;np ; 8$‘C$ R,: 0.4 [CH,Cl,/MeOH (9:1)]. Anal C,,H,,N,O, S , , , , bis [(2-Isoquinoline-5-sul$onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionate] I ,3-xylylenediamine 5 To a solution of 4 (0.795 g, 1.5 mmol) in CH,Cl, (10 ml), 100 pl (0.75 mmol) of 1,3-xylylenediamine in CH,Cl, (200 ml) was added dropwise. After stirring for 24 h, at room temperature the solvent was evaporated, CH,OH was added and the precipitate (N-hydroxysuccinimide) collected. The concentrated filtrate was chromatographed on silica gel preparative layers eluting with CH,ClJMeOH (9:l) to yield white crystals of 5 (yield = 16%; mp = 104°C dec). R,: 0.35 [CH,Cl,/MeOH (9:1)]. Anal C,,H,,N,O,,S, (C, H, N, S). NMR (CDJOCD,, 6, ppm): 1.35 (18H, s, t-butyl), 2.45 (4H, t, CH,-C(=O)NH), 3.01 (4H, t, CH,-NRC(=O)O), 3.25 (4H, t, CH,-NR-C(=O)O), 3.3 (4H, t, CH,-NHSO,), 4.3 (4H, d, benzyl), 7.2 (4H, m, benzene), 7.8 (2H, t, isoquinoline), 8.3-8.6 (8H, m, isoquinoline), 9.4 (2H, s, isoquinoline). FAB-MS: (MH)+= 947. In the same conditions 6 and 7 by condensation of 4 respectively with 1 ,Cxylylenediamine and 1,12-diaminododecane were obtained. bis [(2-Isoquinoline-5-sul$onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionate] I,4-xylylenediamine 6 White crystals (yield = 20%; mp = 110°C dec). R,: 0.55 [CH,Cl,/MeOH (9:1)]. Anal C,,H,,N,O,,S, (C, H, N, S). NMR: (CD,COCD,, 8, ppm): 1.35 (18H, s, t-butyl), 2.45 (4H, t, CH,-C(=O)NH), 3.1 (4H, t, CH,-NR-C(=O)O), 3.25 (4H, t, CH,-NRC(=O)O), 3.3 (4H, t, CH,-NH-SO,), 4.3 (4H, d, benzyl), 7.2 (4H, s, benzene), 7.8 (2H, t, isoqumoline), 8.3-8.6 (8H. m. isoauinoline). 9.4 (2H. s. isoauinoline) FAB-MS: (MI-i)+ = 947.’ ’ bis [(2-Isoquinoline-5-su~onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionate] 1,12-diaminododecane 7 White crystals (yield = 25%; mp = 58°C). R,: 0.59 [CH,Cl, MeOH (9.5:0.5)]. Anal C,&,N,O,,S, (C, H, N, S). NMR: (CDJOCD,, 8, ppm): 1.2 (16H, -(CH,),), 1.35 (18H, s,
t-butyl), 1.4 (4H, t, CH,-CH,-NR-C(=O)), 2.4 (4H, t, CH,-C(=O)NH), 3.05 (4H, t, CH,-NH-C(=O)), 3.1 (4H, t, CH,-NR-C(=O)O), 3.25 (4H, t, CH,-NR-C(=O)O), 3.3 (4H, t, CH,-NH-SO,), 7.8 (2H, t, isoqumoline), 8.3-8.6 (8H, m, isoquinoline), 9.4 (2H, s, isoquinoline). FAB-MS: (MH)+ = 1011. Synthesis of the deprotected compounds 8,9 and 10 (trijuoroacetate form) from 5,6 and 7 The product 5 (0.140 g, 0.15 mmol) was stirred in a 50% mixture TFA/CH,Cl, (20 ml) for 30 min. The solution was evaporated to dryness, anhydrous ether was added (three times) and removed. The residue was dissolved in water and lyophilized to give white powder of 8 (trifluoroacetate form; yield = 53%; mp = 200°C). Anal C,H,,NsO,&F,, (C, H, N, S, F). NMR: (D,O, 6, ppm): 2.8 (4H, t, CH,-C(=O)N), 3.3 (8H, s, CH,-N-), 3.4 (4H, t, CH,NHSO,), 4.4 (4H, s, benzyl), 7.3 (4H, m, benzene), 8.2 (2H, t, isoquinoline), 8.8-9 (8H, m, isoquinoline), 9.8 (2H, s, isoquinoline). FAB-MS: (MH)+ = 747. 9 and 10 (trifluoroacetate form) were obtained by the same procedure. 9 white powder (yield = 55%; mp = 195°C). Anal C,H,N,0,,S,F,2 (C, H, N, S, F). NMR: (D,O, 6, ppm): 2.8 (4H, t, CH,-C(=O)NH), 3.3 (8H, s, CH,-N-), 3.4 (4H, t, CH,NHSO,), 4.4 (4H, s, benzyl), 7.3 (4H, s, benzene), 8.2 (2H, t, isoquinoline), 8.8-9 (8H, m, isoquinoline), 9.8 (2H, s, isoquinolink). FAB-MS: (MH)+ = 747. 10 white crvstals (vield = 37%: mu = 135°C). Anal C,,H,,N,O,,S,F;, (C, g, N, S, F). NkR:‘(D,O, 6, ppm): 1.2 (16H, s, (CH,),), 1.6 (4H, s, CH,-CH,-NHC(=O)), 2.8 (4H, t, CH,-C(=O)NH), 3.2 (4H, t, CH,-NHC(=O)), 3.3 (8H, s, CH,-N-), 3.4 (4H, t, CH,-NH-SO,), 8.2 (2H, t, isoquinoline), 8.8-9 (8H, m, isoquinoline), 9.8 (2H, s, isoquinoline). FAB-MS: (MH)+ = 8 11. Enzyme assay Histones II, and III,, phosphatidylserine and diolein were purchased from Sigma, [r3*P]-ATP was from Amersham and Aqualyte reagent was from Baker. PKA was purchased from Sigma; it was the catalytic subunit isolated from bovine heart. PKC was kindly provided by H Coste (Glaxo Laboratories, Les Ullis, France). It was purified from bovine brain on DEAE cellulose then on phenyl-Sepharose and consisted chiefly of the B subspecies; activity was 98% strictly Ca*+ and phospholipiddependent. PKC inhibition was assayed in a reaction mixture (80 pl) containing 50 mM Tris/HCl buffer (pH 7.5), 5 mM MgCl,, 0.5 mM CaCl,, 50 yg/ml phosphatidylserine, 5 pg/ml diacylnlvcerol, 10 UM Tr *Pl-ATP (2000-4000 cpm/pmol), 0.5 I.& PKC, histones III; as substrate and inhibitors at ‘differem concentrations. PKA inhibition was assayed in a reaction mixture (80 pl) containin 50 mM Tris/I-ICl buffer (pH 7.0) 5 mM MgCl,, 10 pM [y f *PI-ATP (radioactive specific activity: 565 Ci mol-I), 1 pg catalytic subunit of PKA, histones II, as a substrate and inhibitors at different concentrations. For each kinase, reactions were run at 30°C for 7 min and stopped by trichloroacetic acid (TCA) (12% w/v) in presence of bovine serum albumin (0.9 mg) as a carrier. After centrifugation (10 min at 3000 rpm) sunematant containing l$*Pl-ATP and unurecinitable inhibitois were discarded and &e pellet was dissolved-in 1 M NaOH and precipitated a second time by TCA. Radioactivity incorporated into histones was counted by scintillation spectrometry with Aqualyte reagent. All experiments were carried out in triplicate.
930 I-IL60 tyrosine kinase inhibition was tested as previously described [15]. Briefly, 20 pl of partially purified enzyme was incubated 30 min at 30°C in a buffer containing Hepes 50 mM, pH 7.3, MgCl, 5 mM, MnCl, 5 mM with 10 p.1 of a saturated solution of NaCl, 10 pl of [~32P]-ATP and 10 p.1 of the inhibitor solution in DMSO (1% maximal final concentration). The reaction was started by 10 pl of angiotensin II (Neosystem) (2 mg/ml final). It was stopped by 500 pl of 10% (v/v) trichloroacetic acid containing 10 mM sodium pyrophosphate. 30 pl of the solution was injected in a Waters system HPLC equipped with a Microbondapak column.
3 4
Thanks are due to J Boutin (Institut Servier, Suresnes, France) for inhibition studies on HL60 tyrosine kinase, P Lemiere for skillful technical assistance, and to C Desruelle for efficient secretarial assistance.
Cl (1985) Biochim Biophys Acta 822,219 U, Kishimoto A, Nishizuka Y (1989)
Annu Rev Biochem 58,3 1 5 6 7 8 9
Acknowledgments
Ashendel Kikikawa
10 11 12 13
Parker PJ, Kour G, Marais RM, Mitchell F, Pears C, Schaap B, Stabel S, Webster C (1989) Mel Cell Endocrinol65, 1 Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y (1988) J Biol Chem 263,6927 Ono Y, Fujii T, Ogita U, Kikkawa U, Igarashi K, Nishizuka Y (1987) FEBS Lett 226, 125 Konno Y, Ohno S, Akita Y, Kawasaki H, Suzuki K (1989) J Biochem 106,673 Hanks SK, Quinn AM, Hunter T (1988) Science 241, 42 Wierenga RK, Ho1 WG (1983) Nature 302,842 Sternberg MJE, Taylor WR (1984) FEBS Lett 175,387 Huang KP (1989) TINS 12,11,425 Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984)
Biochemistry 23,503&5043 14
Ricouart
A, Gesquiere
JC, Tartar A, Sergheraert
C (1991)
J Med Chem 34,73 15
References
16 1 2
Nishizuka Nishizuka
Boutin
JA, Ernould
AP, Genton A, Cudennec
CA (1989)
Biochem Biophys Res Commun 160,1203 Y (1986) Science 233,305
Y (1988) Nature 334,661
Knighton DR, Zheng J, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM (1991) Science 253, 407
Eur J Med Chem (1992) 27,925-930 0 Elsevier, Paris
Inhibition of protein kinases by bis-Al P mimics M Houdin, C Sergheraert* Faculte’ de Pharnzacie,
Institut Pasteur de Lille, URA 1309 CNRS, 1, rue Calmette, BP 245,59019 (Received
22 November
Lille, France
1991; accepted 3 July 1992)
Summary - The primary structure of the different PKC subspecies contains an ATP-binding sequence in the catalytic domain. The a, p, and y isoforms contain an additional ATP-binding consensus sequence whose significance remains unknown. To explain this function and also to prepare new specific PKC inhibitors, bis-ATP molecules have been synthesized. The variation of the mutual position of the ATP mimics is conditioned by the choice of the included spacer. The products generally have an increased inhibitor potency towards PKC, PKA and HL60 tyrosin kinase. The inhibition is always competitive towards ATP but does not allow the conclusion that a simultaneous interaction occurs at both ATP-binding sites. PKC inhibition
/ catalytic
site / ATP-binding
sites / bis-ATP
Introduction The physiological importance of protein kinase (PKC) activation is widely appreciated and well documented; a number of recent reviews have described its role as a signal transducing protein in a plethora of physiological responses [l-5]. Its molecular heterogeneity is proved; to date at least seven subspecies of PKC have been described. The complete structures of four isotypes having a, PI, pz, and y sequences (fig 1) have been clarified by analysis of the cDNA clones obtained from the brain of several mammalian species [6]. These isotypes have also been identified at the protein level in preparations purified from brain tissue. More recently a second group of PKC cDNA clones designated 6, E and < has been isolated from brain libraries using as probes a mixture of cDNAs from the initial clones [7]. Among these new members, only PKC-E has been identified at the protein level in brain while the expression of the remainder has been confirmed only at the mRNA level [8]. The 6, E and c types have a common structure closely related to but clearly distinct from the first group; all lack the conserved C, region which seems to be implicated in the regulation of PKC by calcium [7]. In all isoforms, the C, region contains a Gly-X-Gly-X-X-Gly-X,,-Lys sequence, a consensus
mimics
sequence for ATP-binding sites shared by many other protein kinases and many nucleotide-binding proteins [9, lo]. These residues can form an elbow around the ATP molecule with the first glycine in contact with the sugar moiety and the second glycine lying near the terminal phosphate [ 111. However, the careful examination of the primary structure shows that in the a, p, and y isoforms the C, region contains a second consensus ATP-binding sequence (in the y isoform, an arginine residue is found instead of lysine). Since all PKCs have the same K,,, for ATP, the significance of this second putative ATP-binding site remains unknown [ 121. In order to test whether this second consensus sequence was endowed with functionality towards ATP and possibly to prepare new isotype Group A I.. fJ. and yl Regulatory domain
NH*>cooH
Catalvbc domann
% ,
Ii ATP-binding rite
PS Cvr Group B Id, r. and 5 1 "1
Cl
V2lV3
c3 v4
c4
coon A+P-binding sixe I N”z
*Correspondence
and reprints.
v5
Fig 1. Structural
features
of PKC species.
COOH I
926 consisting of a long hydrophobic chain, The flexibility of this structure allowed this construct to be more tolerant with respect to both the distance and the orientation of the two putative ATP-binding sites. However, the number of methylenes was limited to 12 in order to avoid a negative effect on the solubility of the construct.
specific constructs we have synthesized and tested different PKC inhibitors designed to interact simultaneously with the two ATP-binding sites and potentially present a strongly increased binding energy towards a, p and y isoforms. As a monomer we have selected the compound 1 (scheme 1) obtained by substitution of the primary amine H-9: (N-[2-aminoethyl]-5 isoquinolinesulfonamide) [ 131. 1 was chosen because it is sufficiently active (IC,, = 20 p.M towards PKC) and does not contain a secondary amine like H-7: (1-[5-isoquinolinesulfonyll-2-methylpiperazine), therefore it can be easily covalently linked to a spacer group without loss of activity [ 141. In the absence of information concerning the spatial distance in the folded kinase between the two glycine rich sites, we have used two types of spacer (fig 2). i) In the first hypothesis (compounds 5,6,8,9) we speculated that the two ATP-binding sites might be located in close vicinity. In this case a constrained disubstituted benzene structure was used as the core of the linker thus providing a small spacing between the two mimics while both metu and par-u isomers were used in order to allow different respective orientations. ii) In the second hypothesis (compounds 7 and lo), we have used a longer and more flexible linker
SO2 - NH - (CHZ)Z - NH2
Chemistry The compounds 5, 6, 7 were synthesized as follows: the previously described ester 1 [14] was saponified with methanolic NaOH and after acidification yielded the carboxylic derivative 2 (hydrochloride form) (scheme 1). After protection of the secondary amine by a &butyloxycarbonyl group (Boc): 3, the N-hydroxysuccinimide ester 4 was prepared by reaction with dicyclohexylcarbodiimide (DCC) and hydroxysuccinimide (HOSu). It was then reacted with 1,3-xylylenediamine or 1,4-xylylenediamine or 1,12diaminododecane to yield respectively 5, 6 or 7. Trifluoroacetic acid deprotection of the Boc group and separation by chromatography on silica gel preparative layers gave the final products 8,9 and 10.
SO2 - NH - (CH& - NH - (CH& - COOCI$ 1) NaOH
2)HCl
B-3
b
SO2 - NH - (CH& - N - (CH& -COOH
SO2 - NH - (CH& - NH - (CH& - COOH
Lo A- c(a3)3
Di-e
S02-NH-(CH2)2-N
1,3 - xylylenediamine
-(CH2),-CO-N I
;‘&3)3
<
)
1,4 I,12 -xylylenediamine diaminododecaneb
5 6 ,
Scheme 1. Protection
and activation
of the monomer
1.
927
/
0 /Q \
CH2
CH2
NH-CC&(CH2)r~-(CH2)~NH-S02
CH2-NH-CO-(CH2)ry-(CH2)+IH-S02
S0,NH-(CH2)2-~-(CH2)&MH-CH2
R
R
I
R : O=C-0-C-(CH& R: H
Fig 2. Chemical
structure
6 9
I
R:
o=c-O-C-W,),
R:
H
of the synthesized
’ 10 bis-ATP
mimics.
Enzymology The inhibitory capacities of the compounds 1, 8,9, 10 towards PKC and CAMP dependent protein kinase (PKA) were tested using histones III, and II* respectively as substrates. Tyrosine kinase activity was studied using HL60 tyrosine protein kinase with angiotensin II as a substrate [ 151.
Table I. IC,, values of the bis-ATP IC,, @‘KC), W
mimics.
IC,, (PKA), ~A.M
IC,, (HL60 tyrosine protein kinase), @I
8
5.5
3
Not active
9
8
7.3
80
Results and discussion
10
25
10.5
Not active
IC,,s determined for the three protein kinases (PKC (p isoform chiefly), PKA and HL60 tyrosine kinase) are given in table I comparatively with the commercial non-specific inhibitor H-7 and the monomer 1.
1
22
12
1700
H-7
6.2
2.3
1500
928 Among the three kinases, only PKC displays a second ATP binding consensus sequence and was thus likely to be more efficiently inhibited by the bis-ATP molecules. An inhibiting potency was maintained for 8, 9, and 10 towards PKC and PKA indicating that the chemical modifications have preserved the capacity of the heterocyclic part to interact with the ATP binding site of these Ser/Thr kinases. Except for 10 which is almost equipotent with monomeric compound 1, both 8 and 9 show an increased activity towards PKC; the inhibition was competitive towards ATP as shown in scheme 2. However, this increase is rather small and is also observed in the case of PKA which is devoid of a second ATP binding consensus sequence. It is thus likely that this slight increase could be attributed to a more efficient interaction with a single ATP-binding domain or its surrounding than to a double-site interaction. The results also show that only 9 has significant inhibitory activity towards the tyrosine protein kinase; although we have no clear explanation for this difference, this result is interesting as 1, the monomeric ATP inhibitor, has no effect on this kinase. While this work was in progress the structure of the catalytic domain of PKA, determined by X-ray crystallography became available [ 161. Due to the fact that the catalytic domains of the PKC and PKA share a very important homology we have been able to localize the second potential ATP binding site of PKC in a region corresponding to the larger lobe of the catalytic domain in the loop located between helix H and helix I. Bir-ATP mimic /ATP Competition
(DIXON melhod)
This model clearly shows that even if the second ATP binding domain is functional, which in view of the spatial structure in this region does not seem likely, the two domains would be too far apart to allow a simultaneous interaction of any of our bis-ATP mimics. However, as PKC enzyme activation involves its translocation to the membrane, the appearance of a high local concentration of the activated enzyme can be expected. In these conditions, interaction of our compounds with a single ATP binding site on two different enzyme molecules could be envisaged at this level, allowing discrimination between activated and inactive cytosolic forms (but no longer dependent on the nature of the isoform). Cellular studies allowing a study of these compounds in more physiological conditions will be necessary to address this hypothesis. Experimental
protocols
Chemistry Melting points were taken on a Gallenkamp apparatus in open capillary tubes. Elemental analyses indicated by the symbols of the elements were within + 0.4% of the theoretical value. FAB ionization spectra were performed at the Universite des Sciences (Lille, France). All found (MH)+ values agreed with the calculated values. ‘H-NMR spectra were recorded on a Bruker 400 MHz spectrometer using TMS as internal standard. Thin layer chromatography was carried out with Merck F254 silica gel. 1,3-Xylylenediamine, 1,4-xylylenediamine and 1,12-diaminodecane were purchased from Aldrich. H-7 was synthesized as previously described [ 133.
Methyl N-(2-isoquinoline-5-suEfonamidoethyl)-3-amino-propionate Z To a solution of 0.71 g (2.83 mmol) of N-(2-aminoethyl)5isoquinolinesulfonamide [14] and 0.787 ml (5.65 mmol) of triethylamine in CH,OH (30 ml) was added 0.308 ml (2.83 mmol) of methyl 3-bromopropionate. The reaction mixture was refluxed for 4 h and evaporated to dryness; water (30 ml) and CH,CI, (30 ml) were added. The organic layer was dried and evaporated to an oily residue which crystallized from light petroleum (yield = 95%; mp = 105’C). R,: 0.30 [EtOAc-MeOH (2:1)] Anal C15HL9N304S (C, H, N, S). NMR (CD,COCD,, 6, ppm): 2.3 (2H, t, CHJOOCH,), 2.4 (4H, m, CH,-NH), 3 (2H, m, CH,-NH-SO,), 3.62 (3H, s, COOCH,), 7.7 (lH, t, isoquinoline), 8.2-8.7 (4H, m, isoquinoline), 9.3 (lH, s, isoquinoline) FAB-MS: (MH)+ = 338.
N-(2-Isoquinoline-S-sulfonamidoethyl)-3-amino-propionic acid 2
Scheme method)
2. Bis-ATP mimic giving Ki = 1.2 pM.
9/ATP
competition
(Dixon
To a solution of 2.57 g (7.6 mmol) of 1 in CH,OH (40 ml) was added NaOH (1.5 g) and the reaction mixture refluxed for 30 min. Upon removal of the solvent, water addition and HCl acidification (pH 3) the aqueous solution was evaporated to dryness. NaCl was discarded by 95% ethanol addition and after evaporation, the oily residue crystallized from acetone. The crystals were used directly in the next step. (Yield = 92%; mp = 182’C) Anal C,,H,,N,O,SCl (C, H, N, S).
929 N-(2-Isoquinoline-S-sulfonamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionic acid 3 Di-tert-butyl dicarbonate (7.62 g, 35 mmol) was added dropwise over 1 h to a well-stirred solution of 2 (4.6 g, 11.6 mmol) in KHCO, 1 N (12.5 ml) and tert-butyl-alcohol (26 ml) while pH was kept between 8 and 9 by adding 1 N Na,,CO,. After stirring overnight, the aqueous phase was acidified to pH 2.5 (1 N HCl) and extracted with ethyl acetate (2 x 40 ml). The combined extracts were dried (Na,SO,) and evaporated to dryness. The oily residue was chromatographed on silica gel preparative layers eluting with CH,Cl,/MeOH (9:l) to yield white crystals of 3 (yield = 61%; mp = 52°C). R,: 0.1 (ethyl acetate). Anal C,,H,O,N, S Cl (C, H, N, S). NMR (CDJOCD,, 6, ppm): 1.35 (9H, s, t-butyl), 2.4-3.2 (8H, m, CH,-N), 7.8 (lH, t, isoquinoline), 8.3-8.6 (4H, m, isoquinoline), 9.4 (lH, s, isoquinoline). FAB-MS (MH)+ = 424. N-(2-Isoquinoline-5-su~onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionic acid N-hydroxy-succinimide ester 4 A solution of 3 (1.3 g, 3 mmol) and N-hydroxysuccinimide (0.345 g, 3 mmol) in CH,Cl, (50 ml) was cooled in an ice-water bath and dicyclohexylcarbodiimide (0.618 g, 3 mmol) was added with stirring. The mixture was kept at O’C overnight. The separated N,N’-dicyclohexylurea was removed by filtration and the solvent evaporated in vacuum. The crude product was recrystallized from isopropanol (yield = 81%; ;np ; 8$‘C$ R,: 0.4 [CH,Cl,/MeOH (9:1)]. Anal C,,H,,N,O, S , , , , bis [(2-Isoquinoline-5-sul$onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionate] I ,3-xylylenediamine 5 To a solution of 4 (0.795 g, 1.5 mmol) in CH,Cl, (10 ml), 100 pl (0.75 mmol) of 1,3-xylylenediamine in CH,Cl, (200 ml) was added dropwise. After stirring for 24 h, at room temperature the solvent was evaporated, CH,OH was added and the precipitate (N-hydroxysuccinimide) collected. The concentrated filtrate was chromatographed on silica gel preparative layers eluting with CH,ClJMeOH (9:l) to yield white crystals of 5 (yield = 16%; mp = 104°C dec). R,: 0.35 [CH,Cl,/MeOH (9:1)]. Anal C,,H,,N,O,,S, (C, H, N, S). NMR (CDJOCD,, 6, ppm): 1.35 (18H, s, t-butyl), 2.45 (4H, t, CH,-C(=O)NH), 3.01 (4H, t, CH,-NRC(=O)O), 3.25 (4H, t, CH,-NR-C(=O)O), 3.3 (4H, t, CH,-NHSO,), 4.3 (4H, d, benzyl), 7.2 (4H, m, benzene), 7.8 (2H, t, isoquinoline), 8.3-8.6 (8H, m, isoquinoline), 9.4 (2H, s, isoquinoline). FAB-MS: (MH)+= 947. In the same conditions 6 and 7 by condensation of 4 respectively with 1 ,Cxylylenediamine and 1,12-diaminododecane were obtained. bis [(2-Isoquinoline-5-sul$onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionate] I,4-xylylenediamine 6 White crystals (yield = 20%; mp = 110°C dec). R,: 0.55 [CH,Cl,/MeOH (9:1)]. Anal C,,H,,N,O,,S, (C, H, N, S). NMR: (CD,COCD,, 8, ppm): 1.35 (18H, s, t-butyl), 2.45 (4H, t, CH,-C(=O)NH), 3.1 (4H, t, CH,-NR-C(=O)O), 3.25 (4H, t, CH,-NRC(=O)O), 3.3 (4H, t, CH,-NH-SO,), 4.3 (4H, d, benzyl), 7.2 (4H, s, benzene), 7.8 (2H, t, isoqumoline), 8.3-8.6 (8H. m. isoauinoline). 9.4 (2H. s. isoauinoline) FAB-MS: (MI-i)+ = 947.’ ’ bis [(2-Isoquinoline-5-su~onamidoethyl), N-(tert-butyloxycarbonyl)-3-amino-propionate] 1,12-diaminododecane 7 White crystals (yield = 25%; mp = 58°C). R,: 0.59 [CH,Cl, MeOH (9.5:0.5)]. Anal C,&,N,O,,S, (C, H, N, S). NMR: (CDJOCD,, 8, ppm): 1.2 (16H, -(CH,),), 1.35 (18H, s,
t-butyl), 1.4 (4H, t, CH,-CH,-NR-C(=O)), 2.4 (4H, t, CH,-C(=O)NH), 3.05 (4H, t, CH,-NH-C(=O)), 3.1 (4H, t, CH,-NR-C(=O)O), 3.25 (4H, t, CH,-NR-C(=O)O), 3.3 (4H, t, CH,-NH-SO,), 7.8 (2H, t, isoqumoline), 8.3-8.6 (8H, m, isoquinoline), 9.4 (2H, s, isoquinoline). FAB-MS: (MH)+ = 1011. Synthesis of the deprotected compounds 8,9 and 10 (trijuoroacetate form) from 5,6 and 7 The product 5 (0.140 g, 0.15 mmol) was stirred in a 50% mixture TFA/CH,Cl, (20 ml) for 30 min. The solution was evaporated to dryness, anhydrous ether was added (three times) and removed. The residue was dissolved in water and lyophilized to give white powder of 8 (trifluoroacetate form; yield = 53%; mp = 200°C). Anal C,H,,NsO,&F,, (C, H, N, S, F). NMR: (D,O, 6, ppm): 2.8 (4H, t, CH,-C(=O)N), 3.3 (8H, s, CH,-N-), 3.4 (4H, t, CH,NHSO,), 4.4 (4H, s, benzyl), 7.3 (4H, m, benzene), 8.2 (2H, t, isoquinoline), 8.8-9 (8H, m, isoquinoline), 9.8 (2H, s, isoquinoline). FAB-MS: (MH)+ = 747. 9 and 10 (trifluoroacetate form) were obtained by the same procedure. 9 white powder (yield = 55%; mp = 195°C). Anal C,H,N,0,,S,F,2 (C, H, N, S, F). NMR: (D,O, 6, ppm): 2.8 (4H, t, CH,-C(=O)NH), 3.3 (8H, s, CH,-N-), 3.4 (4H, t, CH,NHSO,), 4.4 (4H, s, benzyl), 7.3 (4H, s, benzene), 8.2 (2H, t, isoquinoline), 8.8-9 (8H, m, isoquinoline), 9.8 (2H, s, isoquinolink). FAB-MS: (MH)+ = 747. 10 white crvstals (vield = 37%: mu = 135°C). Anal C,,H,,N,O,,S,F;, (C, g, N, S, F). NkR:‘(D,O, 6, ppm): 1.2 (16H, s, (CH,),), 1.6 (4H, s, CH,-CH,-NHC(=O)), 2.8 (4H, t, CH,-C(=O)NH), 3.2 (4H, t, CH,-NHC(=O)), 3.3 (8H, s, CH,-N-), 3.4 (4H, t, CH,-NH-SO,), 8.2 (2H, t, isoquinoline), 8.8-9 (8H, m, isoquinoline), 9.8 (2H, s, isoquinoline). FAB-MS: (MH)+ = 8 11. Enzyme assay Histones II, and III,, phosphatidylserine and diolein were purchased from Sigma, [r3*P]-ATP was from Amersham and Aqualyte reagent was from Baker. PKA was purchased from Sigma; it was the catalytic subunit isolated from bovine heart. PKC was kindly provided by H Coste (Glaxo Laboratories, Les Ullis, France). It was purified from bovine brain on DEAE cellulose then on phenyl-Sepharose and consisted chiefly of the B subspecies; activity was 98% strictly Ca*+ and phospholipiddependent. PKC inhibition was assayed in a reaction mixture (80 pl) containing 50 mM Tris/HCl buffer (pH 7.5), 5 mM MgCl,, 0.5 mM CaCl,, 50 yg/ml phosphatidylserine, 5 pg/ml diacylnlvcerol, 10 UM Tr *Pl-ATP (2000-4000 cpm/pmol), 0.5 I.& PKC, histones III; as substrate and inhibitors at ‘differem concentrations. PKA inhibition was assayed in a reaction mixture (80 pl) containin 50 mM Tris/I-ICl buffer (pH 7.0) 5 mM MgCl,, 10 pM [y f *PI-ATP (radioactive specific activity: 565 Ci mol-I), 1 pg catalytic subunit of PKA, histones II, as a substrate and inhibitors at different concentrations. For each kinase, reactions were run at 30°C for 7 min and stopped by trichloroacetic acid (TCA) (12% w/v) in presence of bovine serum albumin (0.9 mg) as a carrier. After centrifugation (10 min at 3000 rpm) sunematant containing l$*Pl-ATP and unurecinitable inhibitois were discarded and &e pellet was dissolved-in 1 M NaOH and precipitated a second time by TCA. Radioactivity incorporated into histones was counted by scintillation spectrometry with Aqualyte reagent. All experiments were carried out in triplicate.
930 I-IL60 tyrosine kinase inhibition was tested as previously described [15]. Briefly, 20 pl of partially purified enzyme was incubated 30 min at 30°C in a buffer containing Hepes 50 mM, pH 7.3, MgCl, 5 mM, MnCl, 5 mM with 10 p.1 of a saturated solution of NaCl, 10 pl of [~32P]-ATP and 10 p.1 of the inhibitor solution in DMSO (1% maximal final concentration). The reaction was started by 10 pl of angiotensin II (Neosystem) (2 mg/ml final). It was stopped by 500 pl of 10% (v/v) trichloroacetic acid containing 10 mM sodium pyrophosphate. 30 pl of the solution was injected in a Waters system HPLC equipped with a Microbondapak column.
3 4
Thanks are due to J Boutin (Institut Servier, Suresnes, France) for inhibition studies on HL60 tyrosine kinase, P Lemiere for skillful technical assistance, and to C Desruelle for efficient secretarial assistance.
Cl (1985) Biochim Biophys Acta 822,219 U, Kishimoto A, Nishizuka Y (1989)
Annu Rev Biochem 58,3 1 5 6 7 8 9
Acknowledgments
Ashendel Kikikawa
10 11 12 13
Parker PJ, Kour G, Marais RM, Mitchell F, Pears C, Schaap B, Stabel S, Webster C (1989) Mel Cell Endocrinol65, 1 Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y (1988) J Biol Chem 263,6927 Ono Y, Fujii T, Ogita U, Kikkawa U, Igarashi K, Nishizuka Y (1987) FEBS Lett 226, 125 Konno Y, Ohno S, Akita Y, Kawasaki H, Suzuki K (1989) J Biochem 106,673 Hanks SK, Quinn AM, Hunter T (1988) Science 241, 42 Wierenga RK, Ho1 WG (1983) Nature 302,842 Sternberg MJE, Taylor WR (1984) FEBS Lett 175,387 Huang KP (1989) TINS 12,11,425 Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984)
Biochemistry 23,503&5043 14
Ricouart
A, Gesquiere
JC, Tartar A, Sergheraert
C (1991)
J Med Chem 34,73 15
References
16 1 2
Nishizuka Nishizuka
Boutin
JA, Ernould
AP, Genton A, Cudennec
CA (1989)
Biochem Biophys Res Commun 160,1203 Y (1986) Science 233,305
Y (1988) Nature 334,661
Knighton DR, Zheng J, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM (1991) Science 253, 407