,r', ,i$ìffi&bìls r8t-'tt öÚ.'t

(x)21 l()4?/¡r?/01() I -(X¡'ì8/$02. 75/{t

s I rY J o ur ntl rtJ N e urt'( ht tttt RÍìvetr ll'ess, Now Yolk ,';,^* - il';!'l iei ìrtz ln,"tno,ional Society lirr Ncurrrhcmistry f CcËç y drofo late In Vitn¡ Studies of 5, iO- fvf . t h y l e ne tetrah v v\) FolateL Reductase: Il nhibitionnhtbttton byDy Folater olatç Derivatives,L)çr I v cLLr ' Antagonists, and Monoamine Derivatives

Hontmcs"l'h' J' J' M' vAr-t de Wiel' 'r'J. [-' Hollinger' O' R' -f i. C. N. Kok' ancl M' 'l' ' Jansen

N i'i ttt t g c tt t t N t' Í h<' rl u ntl't l )t' ptt rl tttt t t l tll N t' rt rtt lt t gt" "l'l

an inhibitory Abstract: F.r¿rte rn.nogrutarnates and foratc antag()nists .have activitvaclrvrtv .,i"''ìiil'"i"ìiti-ìì"t..t"r'v¡','l-l'l.l,t:.:1:':iì:,'Î,iÎl':'i'il; ¿rctio,r:rctiorr on thetnt: "' ;l'i,i"'iirìi;;.J'f..;; üilrv'trofolic itcitl using the Ihe tYPe of inhibititr alkaloids' the in t,"n'ü"'ì*'iit"t' iu*e of !h9 ni..rnoamine [-ineweavcr.--gr.k reductase, have either a Iif|o pr.oducts of 5,,,,ìì"iiìvi"ìì"ì.,ti,,hy,trnf.rlare Key Words:5'10- or inhibitort on thc en/yrne activity' stinrulittoly "ft¡tt tler-iv¿rtives-F'olate antago- Mcthyle'etetrahytlr..liríirt"."i".1,r.,,,r"-pol¿rtc tt,rttirrg"r J. t. et al. rtt t'itro studies of 5,10- rrists_Monoan'in" ¡",iu,ìi¡u".. ives, fo- l nhibition bv tblate de rivat merhy leneter rur,v¡r.rti, iuìï' i"¡u.ìr.r"," j 642 tlerivatives' ' Ñ''u"'t'htnr' 38' 638- l4tc antagonists, antì "tt'ãtlt*¡"" il9ti2).

lc' with irrclolamines of 1,2,3, -Ietrahydro-p-carbo- 'l'he enzyme 5, l0-methylenetetrahydl-otìrlatc catalyzes the rc¿rçtron: line" s. cluctase lgC. t' 1.68) ;ii,. physiological and pathologica.l significance of H' * monoamine alkaloids is' -5,1O-methylene-TFlIì + NADPH I tn. ïo,lnlr,t¿ehyãe-derived F + NAI)l'' at pl't; sct'tt, being studied' -5-methYl-'l'H --- (1979) clemonstrated the ol' Rommelspachlr' et al' irr tlre pt'csence t he This reaction is revcl-siblc t rahvd ro-13.-c' arbt-rline' (Ktltz-birch irncl -; ti; ;;; o'r o-n vo.o vte nonspecific elcctrtln acccptol's " ^ antl formaldehy.de' both in 'i;;Ñ;¡, acid)' 'l'he alkaloitl fì-on.r serotoniñ l97l) (THþ- - tetrahvtl'ol'olic The alkaloid from tryptarnine ancl tìrr- le(ltlct.irsL' is cirpit' ,rìr,,.t rat. i,ió-î*ìitvLnetctrlhytllo[ol¿tlc ntrìiìüv¿""".r ( l'2,3'4-tetrahvclro-p-carboline) has I e nc tc t Iil h tl ro f'o I il tc' lri; ;ï-i;; i ng 5' I 0- nrc t h v v yivo in rat brain by Balkcr cl al' t'caction yie lds i-,""n l.fé",ifiecl irr *ñi.n because- oi an ec¡uilibl'itrm Folate compounds atlministet'ccl cxoge- rrcitl ( [)onalclson iiçr91. io.*.f¿"tty¿e and tetr¿,rhytlrolirlic to Lause convulsive activity (()b- plcscrìr'c trl' lrigh con- nously are known u"ni'ri"t=titesy, 196 I )'nln thc 1973;Hommes et al'' l97c)).'fhe a cycliz-irtitlrt tll thc tr."t Hommes, ."ntrntionti ott monoami¡rcs' ìr"ìil.iitiri""""¡ tlerivatives 4-rnethoxy

ó.1¡i

TNO 1 6422 drd;rÊ{, ,i ¡#

lN Vl'fRO S'lL/Dtl''S Ot" 5,t0 Ilt',1'ttl'1,þ)NI:"l l:'l'llAlly'l)ROl''Ol,A'l'l'; llLl)ll("1'ASI:- ó39

asslrnlption tlrat the corìvrllsive bch¿rvitlul. irttlt¡cccl 'f'AlJl-h, l. K, t'ultrt.s tt.l li¡lult dtrirtttitas ttttl t I tt I tt t t I tt tt i,¡ 1.¡ t r t I t t 5, I 0- t t tt t I t t lt tt t' I t' I rtt- try tblate is dcpentle nl upotì the fìllnt¿rtiort of'¿rlklt- lì t' 11o li It¡'lnt litIttte t ( I u( l tt.\( Ioitls as ¿ì r'esult of rcductase activity. 50% inhibition MATERIAl-S AN D M[ì'I'TIOI)S t,lo.lll." c()nccntfation 5 l"('Inrethyltetlirhydrofìrlic ¿rcitl brrt ittnt sirll, spe cilic 1lll1"Ì,,',"', r' activity -57 ¡nCi/nrn"rol. was pulchasctl fi'oltt I{atlioche nti' l)ilrydnrlìrlic acitl 7.0 ¡ l(l | c¿rl (-'cntre, Anret'sharlr, Iirtglancl. fiolic acicl, tlihyrllofìrlic I.'olic acitl l.-5 x l0 'Ictlahydrofitlic acitl, tetrirhytllofi¡lic acitl, ttrcthott cxirte, pyt-intct hartlirrc, acitl ' 1.0 x l0'l 5 t -gltrtamic Mclhot|cxato 1.7 x l0 sotliunl glutarnate, ancl N-¡rat'lr at.tti nobcrrztlyl- l purchasctl (lhenrical Pyrrmethamine 3.7 x l0 acid were from Signra Co.4- l.[l x l0¡ tlerivative, .! ldcnosyl-t methioninc Methoxydopamine HCll, 4-rnethoxydoparninc N-¡r-anrinobenzoyl'r.-glutanric acitl Ncl inhibition epirrinc, epinine dcrivative, and 1.2,3,4-tctr-ahydlo-2,9- SotliLlrn glutamate Ntl inhibition dinrcthyl-p-carboline wcle gilis fì'onr P. L¿tdur-on, Jansse tl [)harrnaceutica, Bcerse, Belgiunt. [jach valt¡c rcpÌesenls the incubation concentrutitrn (rnolt:s/liter) ot inhibitor nccessary to procluce a 50%, Preparation of rat brain homogenates inhibition of e nzynte activity with 5-[!rC]rnethyltett'a- Wistal r'¿rts ol 160- 200 g botìy weight wele killetl by hydrolìrlic acitl (1.74 nnrol/litcr) üs methyl donor antl decapitation. 'lheir brains were rapidly removcd ¿tnd 4-nrcthoxydopamine as the fìrlmalclehytle-bincling sub- washetl in 0.25 tr¿-ccllcl sucrose. 'lhe conrpletc bt'ain was stancc in rat blain homogcnates. homogenized lbr I nrin in l0 vol. o1'0.2.5 tvt-cold sucr()sc with a Potter-Elvejhern hornogcnizer. 'l'he suspensittn was ccrrtriluged for 20 min at 6009 to Icmove ccll cleblis. bintling subst¿rncc.'l'he incubation mixture cc¡ntainecl ptrosphate pH 6.4, 150 'Ihe supernatanl was storetl at 20"C until t-cquircd fìrr' 100 ¡rl t).5 tr¡-sotliurlr buttet', ¡rl (jnzyrne assays. glass-distilled walel or I00 ¡rl of'various concentrations of firlatc delivatives ol rnonoarnine derivatives, and 50 ¡rl Enzyme assays in rat brain hornogenatcs glass-distillecl watcr'. 100 ¡rl 4-methoxytlopamine 0. I tr¡, 50 'l'he -5, lO-methylenetetlahydr-ofìrl¿rte reduclasc w¿rs cs- ¡rl -5-lr'('lnrcthyltetlahyth-ofìrlic acid ( 10.0 ¡¿Ci), antl 100 tirnated in the revelse dircction. We clid nr)t r-rsc ¿ur clec- ¡rl cnz-ynre pr-eparatitln. The blank was prepared without tron acceptor in the incubation lnixttl-e becattse wc usctl enzynrr or with boilctl cnzynrc. Aficr incubation for 2 h in clude enzyrne pl'e parâtions. Cl'utle homogcnates con- a w¿rter bath at 37"(', the reaction was stopped by adding tainccl oxidizing substances, such as IìAD,'strflìcient that 1.0 rul ol'ir 0.-5 n¿.sodium borate buf'ler pH 10.0.'l'he leac- addition of such elect¡on ¿ìcceptors did not firlher s¡imu- tion plotlucls wcrc extt'actcd in I0.0 ml of a tol- late the enzyme activily. Only purilìed enz-yme dctnon- ucne isoanryl alcohol 2:3 rnixtur-e tì'tlm the aqt¡eous strated a requilemcnt fbr oxidizing substances in thc en- phrrsc. ¡rlcviously satur-atecl with lg sodiunr chloritle. zyme ¿rss¿ry (stcbbins et al., 1976). Alìcl nrixing t¡n ¿r Vor-tex mixer for I min, the tubes were 'Ihe method we used was based on lhat of l-atlttnrn e I ccntlifugctl fìr' l-5 nrin at l-545 g. F-oul nrilliliters of the al. ( 1975) with 4-methoxydopamine as the tìrlm¿rldchyrle- olganic phasc wcr.c adtled to a vial with 10.0 ml trnstagel

x D¡hydrofolic ac¡d o Tetrahydrolol¡c acid + fo¡ir acid in vitro . À'letlìotrexale o Pyrioìetlì¿nìine 5, I0 - methylenetetrahydrofolate adenosyl ÍÌethron¡ne reduclase æt¡vity against c0nc. z€ro + 5 l- r2q

I I

\a-- . -__\ -. -\_o ---+---o--ì\

\--

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concentrat¡on in mol / I FlG. 1. lnhibition of the 5,10-methylenetetrahydrofolate reductase activtty by various concentrations of folate derivatives and folate antagonists in rat bra¡n homogenates in vitro. Reductase activity was measured with 4-methoxydopamine as the formaldehyde-binding substance.

J. Nt'ttrochttn., Vol. 18, N,t. 3, 1932

, tÌ.i ,' .¡t. .., .1 . _i:.,, "i: l : ¡ ':nll¡,, ()40 J. 1.. II()l.l.lNGI.ll l''1' Al antl thc r-atlioactivity wils lllc¿lstll c(l with ¿r lit¡rritl se intillir tiort ct¡utttcl. 'Ilre cnz-yrnc irclivity wirs (\lll'u'sÙLl ¡lì rtantlntolesrrf'ptotlttcts pct ttottt pel !irillìl ti\stle . Michaelis- Menten kinetics Whenevcl kinetic plopcltics sinlil¿rl to Michire lis Mertten kinctics were tlbtaine d' thc pirllrntctct.s h,,, ittttl V,,,,,. werc detctnlinetl by means of thc I-inewcrrvct Burk transltlt-nl¿tt¡on (l.ine we ¡vet irntl []trlk. l9l4).

RESUI-'fS Kinetic properties rlf 5, l0-methylenetetrahydroftrlate reductase of rat tissue The v¿rlues filt'K,,' ( 1.4-5 ::: 0.25 ¡rrrl) ¿tttd V"',,' (7.43 t 0.33 nmol/h/g tisstte, ûìean t s.l).) were deter- mined by measuring the reaction velocities trt clif- fèrent concentrations of 5-[rrC]rnethyltetr-ahyclro- f'olic acid with crucle enzyrnc pleparations ll'onr rat brain. We have been unable to obtain ft-orn the lite¡- ature K,n and y,,ìa* values fbr the 5,10-methyl- enetetrahydrofolate reductase activity of crucle rat brain homogenates.

Inhibition of the 5, 1O-methylenetetrahydrofolate reductase by folate derivatives and fmethotrexate. S-adenosyl-l-nrethiolline dihydrofolic acid 2 x t¡: K-3.11 pM; y-u,7.30 nmol/h g shown to inhibit the enzyme activity' For sotlit¡m- ti ssue. glutamate, which is epileptogenic t.rnly in high closes, and N-para-arn i no be nz-oy l-r,-glu tamic ac icl, a constituent part of the folate nlolectlle' no inhibititln to be better substrates (lower K,',) thttrt was shown. Using the f -incweavet' tstrrk tr¿rtrsl'ot- bccn showlt mation, dihydrofblic acitl was tìluncl to show conl- rnonoglutanlate s fbr fblate-dependent enzymes, and petitive inhibitory activity on the enzyme (Fig. 2). lnay fìtnction as natural substrates and as regulattlrs ol' fol¿rte metabolising enzymes (Coward et al.. Influence of monoarfrine derivatives on brain 1974: Cìheng et al., 1975). We have performecl our 5, I O-methylenetetrahydrt¡f'olate red uct ase stuclies on the inhibition of 5,10-methylenetetrahy- activity in vitro dro{olate lecluctase. however, with tblate mono- Difïerent efTects on the -5,10-methyletre tctrtthy- glutan.rates, because polyglutamates were not avail- tlrofolate reductase activity wel'e observecl with the able. 'I'hc inhibition by díhydrofolic acid was found cyclizecl monoamine clcrivatives ('lable 2). 'l'hc ilo- to be competitive with respect to 5-methyltetrahy- pamine clerivative was shtlwn to inct.e¿tse the drofìllic acid, in agreement with the observations of enzyme activity whereas the cpinine deriv¿rtive antl Matthews antl Haywood (1979), who used purified the B-carboline derivative had inhibitory ef'tects. lecluctase tiom pig liver. The more pronouncetl in- Epinine had no efïect. hibition by dihydrofblic acid than that caused by fblic acid or tetrahydrofblic acid is probably due to the fonnation of an intermediate product, an DISCUSSION N"-methyldihydrofblate derivative, in the enzyme Cellular folates are present mostly as their reaction (M¿ttthews and Haywood 1979). polyglutamate derivatives. ln this form they have The assumption that folate compounds exert their

.1. Nturo< han.. Vol- 38, No. J, I9ll2 rI '{+Èir.

tN Vl'l'lìO ,\'t'(ll)ttts ()l:5,t0'l\1t'.tltl'l,l\Nl','ll;'llltlllYl)ll()LOl,A'l'1, ll.tl)t.i("lASI" 64t

t'Alll-H 2. Í-l.lttt.t o.l tttttrttttrntittt tltritttli¡'t't tttt tltL' .i,10-tnctltvlr'tt(l(lt-ttlt\'(lt().lrtlttlt rtlrtclt!.\r (t(l¡t'it.\'

Enzyntc ¿rcllvrty irs Pcl cclllitge St¡bstirrtec ('()rtccrìl¡irlir)rl ol lhc colt!ttll

4-Mcthoxydop¿tntrte tlc¡ivirtive" I ¡. l() 1u l(X) Irl0:rrr 186 rtvt I ^ l0 116 l)oparnine tlcrivalivci' l.7l l0't ¡r 204 ". I l.7l ' l0 nr 120 1.72 r l() I Nr l0-5 lìpinine' 1.{r l x l0 'r ti,t 100 lÌpinine clel.ivative'i 1.5.ì x l0 'r vr I I ¡ 1..5J x l0 rø 47 1.5.ì x l0 r'Nr 92 p-Calboline tlcrivltivc' 1.94 x l0 'j rt l-5 1.9,1 ¡ l0 r¡vr 2'/ Lt)4 x l0 i i\,r 16

lnhibitoly ancl stinrulatory ellects ()l rìror)oarìrinc delivatives on the ,5,l(ln-rethylcnetetratryclrolòlate rcduclilsù irctivity fìrlrn ir singlc onzy¡ne source prcpared tìom pooled lat brairts. lìeductasc activity was measured with 4-lncthoxytlopanrirrc ¿rs the lìrrnraltlehydc-binding sub slance. " 1,2,J.4-'l'etralrytlro-7-rrtcthoxy-6-isoquinolinol- t' 1,2,3,4-'l-etrahytlr-o-6,7-isoquinolinediol. '' 4-l-2-( Methylanrino)ethyl lpyt ocatechol. '1 1,2,3,4-'l'elrahydro-2 nrethyl-6,7-isoquinolirrediol. '' 1,2,3,4-'f etrahyclro-2.9-rlirnet hyl-/3 carboli ne.

convulsant effect by means tlf slimulatiott of' rnecliatc proclrrct, the N;'-methykìihydrofolate, is mclnoamine alkaloid fot'¡nation is not conlìr'med by fornretl (Matthcws and Haywood, 1979). We oru rcsulls. suggest that flonr this product the enzyme coulcl 'fhe various monoamine derivatives h¿rvc tlilÏ'er- tlansfèr- a orre-carbon unil (in the fbl'rn of formal- e nt effècts on the 5,10-methylenetetrahycllofolate dehyde) to rnt¡ltt¡anrines. rccluctase activity. The doparnine clerivative, which is anticonvulsive, stin'rttlates the l'ecluct¿tsc :rctivity. 't'he epinine clerivative and the p-carboline dcriva- REFERENCES tive. which werr: convulsive, inhibit the enzyrnc trc- tivity. lJllker S.;\.. ll¡r¡isort li. H., tsr'own Cì. ts., antl Christi¿ul S. I. 'l'hese results al'e the contt'ary of what we hittl ( I 97t)) ( i¿ts c hr rrnratogrilplìic nìass/spect¡'t¡nretric evitlencc lìr thu idcntilic¿rtion of I .2,-j.4-tetrahytlro-p-carbolinc as an expected. We hacl expectecl that the doptrnrirte tle- in vivo corì:ititucnt t¡l' ¡ ¡t brain. I]ilx h¿ttt. Biophv:. Ra.s. rivative, which is anticonvulsive, shoulcl inhibit the ( ()tttttttttt. tì7, 146 l-54. reductase activity. 'l'he epinine derivative antl the ('heng Ir. W., Shane B., atrcl Stokstatl E. t.. R. (197-5) Perìta- p-carboline derivative, being convulsant, should ghrtlrììate tlcrivativcs of folate as substrate for rat liver let- stimulate the t'etiuctasc activity. rahytlroptc royl glutamate rnethyltransferase and -5, I 0- mcthylcnctctr¿rhytlrofblate reductase. C¿ttt. .l . Bit¡the¡n. 53, It remains uncertain if the rnonoamine alkaloitls 1020 t027. clemonstrated in brain tissue (tsarker et al., 1979; ('oward J. K., Palanrcswalan K. N., Cashmore A. R., and Ber- Rommelspacher et al., 1979), and in human plasmit tino J. R. ( 1974) 7,8-Dihydropteroyl oligo-y-r.-glutamates: and platelets (Kari et al., 1980) are proclucecl by Synthesis antl kinetic stutlies with purifierl díhytlrofolate re- tluctase llom m¿tnrmalian sources. Biot ltetnislr.¡, 13, reductase, which would require the means of the Itì99 1903. reductase to act in the reverse of its nornl¿rl physitl- I)o¡raltlson K.0. antl Kcresztesy J. C. (1961) Further evirlence logical direction. ()n thc n¿rtule ol prefolic A. ßiochetn. Biopht,s. Rat. Kutzbach and Stokstad ( 197 I) calculntccl that the ( t'tttttttttÌ. 5. lll9 :92. llonrrrcs O. R.. I-lollinger J. [.., Jansen M. J. T., Schoofs M., enzymatic reduction of -5, l0-methylenetetlahydrt-r- V¿¡n tle Wiel 'l-h., and Kok J. C. N. (1979) Convulsant folate to 5-methyltetrahyclrofblate has a K,.,, - l0 ploperties of fìrlats compounds: Some consitleratic¡ns and at pH 7-thus, under normal physi

,1 . N<'untt'htn., Vol.3tl. No.3. l9tl2

.it .¡.t! i{::'(r I .'í lr) ' ' ! 642 J. 1.. IIOI,I.TNCIIR TiI'A1.. I

enetet¡'¿¡hydt.()lìrlirte I,cduetasc. parf ial put.if.icati()n, pt1)l) tlonol fìrr biogcnic anrincs: Þìnzynratrc lìrr.nratiurr elties anrl inhihitior¡ by .!-atlenosyl-,nethi.,ni,,c. IJit¡t.ltrnt. ol. lìlr. nraltlchydc. St it'ttt t' l|l, lj l t-:.3. ßio¡th.ts. At.tu 2SO, 459 4j7. '['. ()bbcns li. A. M. antl Honlmcs ( L.utlulon_P- M., Ver.rvintp M. O. R. 197,]),l'he e pilcprogcnie F., J¿¡nsscn l). Ë,'. Nl.,lntl (junlr¡re cfï'cct lìrlate re n W. R_ (1975) 'l'issue of dcrivatives in thc ¡.ttt..l . N<,utlol.'.tr¡. Ztl, fracrionation in r.irt brilin, kitlncy 223.229. and livcr. I. lnlr-acellular I()c¿rliz¿rrion,,f ii' j-i,,"rny,lt"r,,,_ hyilrol'olic acitl Ronrnrclspirchcr H., Honccker. 'litnte r.equir.ing cnlymc. ßi,,,.itiì,tir). 1.,.rtr,,¡t,1,, H., Iìar.be y M., antl Mcinkc fl. 57,251 260. ( 1979) 6-Hydroxy-lctrahydxrnorh¡rrnranc (ó-hytlr.ory, |.,¿tuwers tclr'írhydro,p-carbolinc), W., l-eysen J., Vcrht¡cven ll., antl l_atlLll.on p. ( a new active ute tabolitc of indole_ ldcntitìcation 197.5) irlkylanrines of ulkaloids; the conrle nslti.,n'pr..,.lr,ct. uf in man and ¡-¿tl. Nuutt.\,tt St.ltttrit,lt,bct gt Art.lt. biogenic Phttttnttt rmines with lìrr.ntaltlchytle. enzymaticirlly f.orrnetl ol . 3 10, l_5 4i. fiom 5-mc'thylter rahyttroflrlic Roscngalte i¡iitl. ll i t t t t t c d. iì u.r.r. S p - n [.1., Meller. Iì., antl l,ì.icdholT A. J. I1975) Synthcsis tntm. 2, l5-22. ", of tetrahydro-B-carbolines .l.he fì.t¡nr intjolcanrincs via cnzyntalic Lineweaver Il. and Brr¡k I). ( 1934) fìr¡'nralit¡n clctcrrnin¿rtion of.cnzynro of for-nraldehyde fì.o¡n _5 mcthyltefrahydiolìrlic rlissociation constants. J. A¡¡t. Chcttt.,l",. ià, O,S¡f OAO. aeid. ßiorhin. plturnutol. 24, lTSg 1i62, Matthews R. C. antt ltayrvood B. J. (1979) tnhibiri,irì-of pig tivcr Ste methylenetett.ahytlr.ofirlare bbin-s R. t)., Meller. Iì., Rosengar.rcn H., l..rietlhofi A. J.. and retluctasc by tlihyttrofolate: Silbcl ( Sonre mechanislic A. 1976) t{entifìcition .l' N",N,,r-'¡ethylcnc_ and r.egulator.y i,npti.aii.rn*.''a ¡()(.lt(, nt¡s- tetlahytlrolìllate ,r.y 18, reductase as the enzyme involvctl in thc 4845 - 485 I . 5-nìethyltetlahydr'olì)late-dependenf Meller,E., Rosengarren lìlrmatit¡n ()f a p I,1., FriedhotïA. J., Stcbbins R. l)., and carboline Silber R. ( derivative of 5-hydroxy-tryptânìine in hr¡nran 197-5¡ .S-ys¡¡rlretr-ahytlrofirli. u.l.j'i, a merhyl platelcts. ",,, An h. Biot hcttt. Biopl¡¡,3. l:.3, 673-6jg.

J. Neuro<:hen., Vol. 38, No. 3, lqg2

ì:,:;,iYJ+.r, rJ.l-