Species- or Isozyme-Specific Enzyme Inhibitors
Journal of Medicinal Chemistry, 1979, Vol. 22, No. 12
1529
Design of Species- or Isozyme-Specific Enzyme Inhibitors. 3.' Species and Isozymic Differences between Mammalian and Bacterial Adenylate Kinases in Substituent Tolerance in an Enzyme-Substrate Complex Alexander Hampton* and Donald Picker The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111. Received March 30, 1979 N6- and 8-substituted adenosine 5'-triphosphate (ATP) derivatives have been synthesized and studied as potential species- or isozyme-selective inhibitors with Escherichia coli adenylate kinase (AK), the rat liver AK isozymes I1 and 111, and the rat muscle AK isozyme. Substituent tolerance in the enzyme-ATP complexes was assessed from substrate properties, apparent enzyme-inhibitor dissociation constants (Ki values; for inhibitions competitive with respect to ATP), and ZW values (for noncompetitive inhibitions). 8-SCH3-ATP and 8S-n-C3H7-ATPwere not substrates of muscle AK and gave Zw = 6.0 and 6.2 mM, respectively; 8-SC2H5-ATPand 8-S-n-C3H7-ATPgave I s = 5.9 and 4.7 mM, respectively, with E. coli AK. In contrast, 8-SR-ATP (R = CH3, C2H5, and n-C,H,) were substrates (V= 5-16% that of ATP; K M = 0.04-0.18 mM, ATP = 0.09 mM) and gave Ki = 0.05-0.36 mM with AK I1 and 111. 8-SR-ATP [R = n-C4H9,n-C5HII,CH2CH20H,(CH2)30H,and CBH5]gave Ki = 0.06-0.32 mM with AK I1 and 111. N6-(CH2)5NHCOCH21-ATP was a substrate of AK I1 and I11 (Vmu = 21 and 9%, respectively, that of ATP) but not of the muscle AK, and with E . coli AK it gave Is = 4.75 mM. N6-(CH2),NHCOCH3-ATPgave Ki = 4.75 mM with AK I11 and Is = 12.9 mM with muscle AK. These results, and previous findings with thymidine kinase variants, demonstrate the ability of simple substrate substituents to influence binding or lack of binding t o a substrate site in a markedly species- or isozyme-selective manner.
Derivatives bearing a single substituent at any of six different positions of a substrate were recently analyzed kinetically as inhibitors of a bacterial and a mammalian form of the enzyme thymidine kinase.' It was found that certain substituents attached to these positions influenced binding or lack of binding to the substrate site in a manner which was markedly species selective. In the present work, the tendency of single substituents attached to a substrate to produce species-selective effects at the substrate site has been further explored, using bacterial and mammalian forms of a second enzyme, adenylate kinase (AK). This enzyme catalyzes transfer of phosphate from ATP to AMP to form ADP. We report the synthesis of ATP derivatives monosubstituted at either the N6 or C-8 positions and describe their inhibitor and substrate properties with the rat muscle isozyme of AK, the rat liver isozymes AK I1 and AK 111,2*3 and E. coli AK. A second function of the present studies was to determine if the substrate substituents could influence binding to the substrate site in an isozyme-selective manner: such effects were of interest to us in view of the possibility, discussed previously,4 that fetal isozyme-selective inhibitors might constitute useful starting points in the design of antineoplastic agents. The evidence presented here indicates that the substituents attached a t either N6 or C-8 of ATP influence the extent of the enzyme inhibitions and the affinity for the ATP sites of the various adenylate kinases in a markedly species-selective and also isozymeselective manner. Syntheses. The N6-substituted ATP derivatives li and 1j were prepared as previously r e p ~ r t e d . ~ For ? ~synthesis of the 8-substituted ATP derivatives la-h, 8-Br-ATP was prepared by bromination of ATP6and treated in aqueous methanol with the appropriate alkylthio anion; formation of la-h could be accelerated by catalytic amounts of dibenzo-18-crown-6. The products were isolated as their tetrasodium salts, which were homogeneous as indicated by ultraviolet extinction coefficient, paper electrophoresis, paper chromatography, high-pressure LC, anion-exchange column chromatography, and elemental analysis. Adenylate Kinase Studies. The present series of 8and N6-substituted ATP derivatives were studied as inhibitors of the adenylate kinase catalyzed conversion of AMP to ADP. The inhibition constants obtained are presented in Table 11. In those cases in which the inhib-
NHR'
I
I
I
OH O H
la, R' b. R' e,. R' d, R' e, R' f; R'
= SCH,;R2 = H = SC,H.;RZ = H = S(dH;),CH,; R Z = H = S(CH,),CH,; R 2 = H = S(CH,),CH,; R2 = H = SCH,CHzOH;R2 = H g, R' = S(CH,),CH,OH; R Z = H h , R' = SC,H,; R2 = H i, R' = H; R 2 = (CH,),NHCOCH,I j, R' = H; R 2 = (CH,),NHCOCH,
itions are competitive with respect to ATP, these constants are given as Ki values, and for noncompetitive inhibitions they are given as IW values. The ATP derivatives were studied as their monomagnesium complexes in order to preclude possible contributions to inhibition caused by depletion of magnesium ions required for the catalyzed reaction. Some of the present compounds were also tested for their ability to substitute for ATP as a phosphate donor with the adenylate kinases, and these findings are given in Table 111. The 8-(methy1thio)-ATP derivative la was a substrate of AK I1 and AK I11 and was a competitive inhibitor with respect to ATP for both isozymes. The KMvalues were only ca. twice the KIMof ATP and ca. half the respective enzyme-substrate dissociation constants (Ki values), suggesting that the methylthio group has little effect on affinity for the ATP sites of AK I1 and 111. Compound la inhibited the rat muscle AK isozyme 20-fold more weakly and in noncompetitive fashion, indicating that the methylthio group hinders or possibly prevents binding to the ATP site and that la adsorbs to a form of the enzyme which is other than the free enzyme form to which ATP binds. This view is supported by the lack of detectable substrate activity of la with the muscle isozyme (Table 111).
0022-262317911822-1529$01.00/0 0 1979 American Chemical Society
Hampton. Picker
1530 Journal of Medicinal Chemistry, 1979, Vol. 22, X ( I , 12
Table I. Physical Properties of 8-Substituted Derivatives of Adenosine 5'-Triphosphate _______ ________~.____ _.___ ~ . ~ ~.~ ~ . HPLC u v 'ma, electrophoresis' X fsystem retn yield, ( p H 7),n m ____ time, no. '70 (C Y lo3) pH 7.5 pH 3.5 lh 2b minC I'ormula anal ..._______________ ~-..~ ~-.~~ .~ - . _ la jL4 281 (18.8) 0.80 1.0 0.33 C , , H I,h ,OliP,SNa, 61 281 ( 1 8 . 5 ) 0.83 0.90 0.59 C , 2 H l , , N . 0 1 , 1 P 3 S N a , ~ 3 1 - l ~C, 0 H,N. S lb IC 60 281 (18.6) 0.80 0.90 0.29 0.44 16.0 C , , H l , 5 N , 0 , , P , S N a ,BH:O , C. 11, N , S Id 56 281 (18.9) 0.76 0.32 0.%5il C,,H,,,N,O,,~P,SNa;H:O C, H. N le 55 281 ( 1 8 . 4 ) 0.76 0.S5 0.14 0.61 19.0 C,.H,IN,O,,P,SNa, c, N,N If 39 281 ( 1 6 . 7 ) 0.82 0.90 0.12 0.26 C,,I-I,,~N,O,,P,SNa;1H,O (1, H, N , S lg 43 282 ( 1 7 . 3 ) 0.80 0.85 0.34 15.5 C,,H:,N\',O,,P,SNa;ZH,O c'. €1. N lh 57 284 ( 1 6 . 0 ) 0.80 0.90 0.34 18.25 C,,,H,,N.O,,P,SNa;I-I:O C. H.N ~
~
~~
~
~~
~
~~
_
~
Mobilities relative t o ATP ( = 1.0). Compositions given under the Experimental Section. mental Section; HPLC high-pressure liquid chromatography.
('
._
~
~
~
~
________
~.
rat AK IIb
K,,d mhl
no.
rat AK IIIb
co mpd
compd concns, niM
Kj, niXI
concns, mM
~
la lb
0.32 0.08 0.055 0.075 0.08 0.25 0.115 0.32
compd concns, mM
I
