Inhibition of thymidylate synthase by pergularinine ... Journal of Biochemistry & Biophysics...

Ind ian Journal of Biochemistry & Biophysics Vol. 36, December 1999, pp. 442-448

Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine

K Narasimha Rao*, R K Bhattacharya and S R Veankatachalam+

Radiation Biology and Biochemi stry Di vision and +Bio-Organi c Di vision, Bhabha Atomic Research Centre. MUll1h;li 400 OX). India

Receil 'ed 4 December 1998; revised 2 1 lilli e 1999

The acti vity of thymidylate synthase (TS) purified in our laboratory from Lactobacilllls Icic/tlllllllllii was inhihi ted hy pergularinine (PGL) and tylophorinidine (TPD) and deoxytubul osine (DTB) iso lated frolll thc Indian med ici n;ll pl ant, Pergularia pallida and Alallgilllll I{l/llarckii respectively. Cytotoxicity studies showed that cell growth of L. l(' ie/ll/li l/ll/ii \\ ';IS

inhibited (lC5() = 40-45 pM) by all the three alkaloids, the concentrati ons> 80-90 pM resulling in complete loss 01 ' the enzyme ac ti vit y. Ki val ues of the enzyme ca lcul ated from Lineweaver-Burk and Di xon pl ots for PGL. TPD and DTH were lOx I 0-6M, 9x I O-('M and 7x I O--f'M respecti vely. These arc typed as ' non-competiti ve' inhibitors o f TS. All the three ;i1k iiloid, inhibited (IC5o = 50 pM) the elevated TS activity of leukocytes in cancer pat ient s with clinically di ;lgnosed chronic myelocytic leukemia (n= I 0), acute lymphocyti c leukemia (n=8) and metastati c sol id tumours (n=.I).

Thymidylate synthase (TS) (CH2THF: dUMP C-methyltranferase, EC 2.1. 1.45) is ubiquitous and is of prime importance as it provides precursors for DNA synthesis through de novo pathway, thymidylate production being the critical rate- limiting step fo r DNA replication l

. . 1

. TS levels arc highly elevated in chemically or virally transformed cells and mal ignant cells4

.6

. These two important factors therefore make the enzyme a key target for cancer chemotherap/M. Part icularly rich sources of thi s enzy me are the methotrexate (MTX) and dichl oroMTX-resistant bacterial strains LactobacilLus casei, Streptococcus fa eciull1 and vitamin B l2-supplemented Lactobacillus Leichmannit l 5

• TS has long been studied as a target for antiproliferative agents due to its role in DNA synthesis and its rich mechanistic features l 6

.

Compounds that mimic its substrate (dUMP) and

*Author to whom correspondence Illay be add rcssed. Tel: +91-22-550 5050. eXlil 2335: Fax : +91 -22-5505151 ; E-mail: rbbd @magnum.bnrctl .ernet. in Abbreviations IIsed: LlTS and HITS , Lactohacilll/s leic/tll/all ll ii & hu man leukocyte thymidylate synthases: dUMP. 2'-deoxyuridine 5'-monophosphate; (dl )- 1.-THF, (dl )-L-5 ,6,7 ,8-tetrahydrofolate; CH2THF, 5,10-methylenetetrahydro folate: DHF. 7.8-dihydrolo late; dTMP, 2'-deoxythymidine 5'-monophosphale (thymidylate); PGL, pergularininc; TPD, tylophorinidine: DTB. deoxYlubu losine; MTX. met hotrexate; 5-Pd UM P, 5- lluoro-2'-deoxyuridylate; 5-FU, 5-lluorou racil : Ara-C, arabinofuranosyleytos ine; 6-TG, (i-thioguanine; CPA. cyclophosphamide, VNC, vincristine, CML chronic myelocytic leukemia; ALL, acute lymphocYlic leukcmi a: MST, metastatic solidlumours.

cofactor (CH2THF) have been stud ied as potential inhibitors of TS . Amongst these, 5-f1uorour;lc il (5-FU) and MTX are the well known competi tive inhibitors of TS . However, there are other inhibi tors that clo not mimic either the substrate or co factor. Anti-TS drugs ' di ssimilar' to the substrate ancl co factor are less li kely to have the side effects that are produced by thc nucl eotide and folate mimics l 7

. We report here the inhibitory action of the phcnanth ro indoli zidi ne alkaloids pergularinine (PGL) and tylophorinidinc (TPD) and the B-carbo line-benzoquinoli zidinc alka loid deoxytubulosine (DTB ) (Fig. I ) i:.;o lated from the Indian medicinal plants Pergll/orio I}(tl/it/{/ (N.O. Asclepiadaeceae) and A/angilllli /{/lIwrckii (N.O. Alangiaceae) respectivel/ x.21 on TS purifi cd from B1 rsupplemented L. /eichllw/lnii .

Materials and Methods All the chemicals used were of AR grade. Vitamin

B12, ethylenediaminetetraacetic acid (EDTA) (disodium salt), (dl )-L-tetrahyd rofolate J(dl)-L-THF] . forma ldehyde, 2-mercaptoethanol, RNase A, DNase I. DEAE-cellulose and dextran (av. MW, 100,000-200,000) were from Sigma Chemical Company, St. Louis, Missouri , USA hydroxylapatite from Bio-Rad Laboratories, Ri chllond . Californi a, USA ; 5-eH]dUMP (ammoni um sa lt , 10.6 Ci/lllillole) from Radiochemical Centre. Amcrshalll , UK: Sephadex G-100 from Pharmac ia Fine Chcmi cal s. Uppsala, Sweden ; activated charcoa l and naphthaiene

RAO el al.: INHIBITION OF THYMIDYLATE SYNTHASE

Pergularinine (pG L) Tylophorinidine (TPD)

Obf{" 2 H

H 1-~ #

Deoxytubulosine (DTB)

Fig. I-Molecu lar structu res or thc phcnanthro indo lizidinc and l3-carboline·benzoquinolizidinc alkaloids.

(scintillant grade) from E. Merck, Dermstadt, Germany; vitamin BI2 assay medium from HiMedia Laboratories Pvt Ltd, Bombay, India were used. The chemotherapeutic drugs used in leukemia treatment were daunomycin , arabinofuranosy lcytosine (Ara-C), 6-thioguanine (6-TG), cyc lophosphamide (CPA), vincristine (VNC), adri amycin , L-asparginase, MTX and predni solone which were all standard products marketed by reputed pharmaceutical firms.

Isolation and characterization of PGL, TPD alld DTB PGL and TPD were isolated from the air-dried roots

of P. pallida and characteri zed lR• DTB was iso lated l

,!

from the crude extracts of the flowers of A. lall1arckii and identi fied22

.

Purification and assay ofTS TS was purified fro m the crude extrac ts of

B Irsupplemented L. leichll1all ll ii (A TCC 7830) as described earlier l4

. TS was assayed2.1 by ["'H] release

method using the assay mi x (buffer B)14 in a total volume of 265 /11 reac ti on mi xture. The enzyme protein was measured by the method of Lowry et al.24 after removal of 2-mercaptoethanol by evaporation 14.

Patients with chronic myelocytic leukemia (CML) and acute lymphocytic leukemia (ALL) and metastatic solid tumours (MST) (breast, uterus and colon cancers) selected for the study were all from the Tata Memorial Hospital (Chemotherapy Department) and BARC

Hospital , Mumbai. Normal healthy donors selected were employees of BARe.

Isolation and preparatiol/. or /WIIWII /r! lI /.:o('v l es alld their lysates

Blood ( 10 ml ) from normal healthy donors and cancer patients collected in anticoagulant mediu m ( I

ml) of 1.5% EDTA containing 0.7% NaCL were treated with 3% dextran (S igma) containing O. IS M NaCI ( I :2, v/v). These were processed within 30 min for the isol ati on of leukocytes and their lysate preparation25. All procedures were carried out at 4°e.

Results and Discussion

Cytotoxicity of PGL , TPD alld DTB The cytotox ic effects of PGL, TPO and OTB on the

growth of L. leichmanllii are shown in Fig. 2. Inset shows the growth inhibition curves in presence of SO

100 >-

.~ 200 c

80 ~ 150

~ 100 L: ..... .... a. ~ 60 0 0 "-en

CIl 40 u ~ .

20

O~~~--~-L~L-~I--~-L--L-~~ 20 40 60 80 100

Alkal oid,}JM

Fig. 2- Inhibition or growth or u icw/i{lcillll.l' it' icllll/llllllii in the presence or increasing concentrations or pCI"glilarin ine (PGLJ. (- e -): tylophorini dinc (TPO). (- X-): ;lIld dc()xytubuloslilc (OTB), (-0-). Contro l growth. (-~-) : in thc ;lbscnl'l: ()r alkaloid is shown in insct. IL. leic/II//{/I/I/ii cclls were gnllvn ;ll 37°C in vitamin B 12 assay growth mcdium supplclllcnted wi th :2 ng B 12 I ml medium l.J . Increasc in turbidit} wi th timc dU ring tile I X hI" growth period prior to thc stationary phase \Vas Illc;lSul"cd (00 ) ;ll

640-700 nlll . 00 (253) in the abscncc or inhibitor ;ll bloids (l nset! was taken as 100% growth acti vity. Inset: ex ponent ial growth ClInc, in the absence, (-~-): and III thc prcscnce or 50 pM PC I.. (-e -); TPO, (-X- ) and OTB. (- 0 - )1

444 INDI AN J BIOCHEM. BIOPH YS., VOL. 36, DECEMBER 1<)<)<)

~ of all the three alkaloids. The three alkaloids showed approximately the same IC50 value of 40-45 ~ and increasing concentrations lip to I 00 ~ resulted in the growth inhibition of the cell s up to 75-85%. Thymidylate synthase provides precursors for DNA biosynthesis through de novo pathway, production of thymidylate being the rate- limiting step for DNA synthesis, and hence a key target fo r cancer chemotherap/. Therefore, inhibition of TS by PGL, TPD and DTB was examined.

Inhibition ofTS by peL. TPD and DTB Time course of inhibition of thymidy late synthase by

PGL, TPD and DTB is presented in Fig. 3. The inhibition was found to be in the order DTB > TPD > PGL. The three alkaloids tightl y bind to TS during the ternary complex formation with the substrate dUMP and cofactor CH2THF20.21 and inhibit the enzyme acti vi ty. ICso values for PGL, TPD and DTB were 45 ~, 42 11M and 40 11M respecti vely. Alkaloid concentrations of 80-90 ~tM resulted in total loss of the enzyme acti vity, thus suggesting that both classes of the alkaloids were potential antitumoLir compound

Inhibitioll COllstants Lineweaver-Burk plots of initi al velocity versus the

specified variable substrate concentrati ons at a series of

React i on time ( min)

Fig. 3-Ti me course or inhi hition or thymidyla te synthase rrol1l L. ieicill//[// /Il i i ror PGL. TPD and DTB . ITS assa) ~ r 4 werc conducted in a total volume or 265 pi reaction mixturcs in Ihc pre~cnce of: (-0-), no alkaloid ; (-X-). 5 pM pGlJrpD/DTI3 and (-0-), 10 pM; (-1'.-). 20 pM: (-e - ). 30 pM or alkaloid . Values presemed arc the averagc or two indcpendcllI cx perilllcnhi.

Fig. 4-lnhibit ion or LlTS by (A), pGL; (H). TpD and (e), DTH . I Lincweaver-BLII'k double reciprocal plOis and the replot (in set) 01 UTS inhibition by the three inhibilOrs are shown. Reaction rates were roll owed by measuring the r' H] released or 5-1 ·

111 Id UM p. L1nless specilicd

otherwise, reac tions were ini ti ated by the addit ion or I 0- 15 ~t g enzyme to 250 pi assay mix 14.25 For K; deterlllil:ati on . the reactio n componclll~ were essentiall y same as descri bed in text l4 in a total volume of 265 J.l 1 with the specilied variable substrate dUMP concentrations al di rlCrenl fixed concentrations of the inhibi tors. (- 0 -). no inhibi tor: (·-X-), 5 pM inhi bitor: (-0-). 10 ~tM: (-tl-l. 20 ~IM: (-e -). ~ Il pM. The inhibi tor was preincubated at 30°C for 10 min prior to the substrate addi tion. Slope of each inhibi ted reac tion is plolled a!:,;linsl II1hlbilor (I) in the inset. Values presented in the plots were the average of two independent experiments].

RAO el ai. : INHIBITION OF THYMIDYLATE SYNTHASE 445

-10 o 10 20 Alknlold, "oM

Fig. 5-Dixon plots showing non-competiti ve inhibition of L1TS by (A), PGL; (B), TPD and (C) DTI3 . I All the threc inhi hi t nr~ PGL. TPD and DTB were preincubated at 30°C lor 10 min wi th the TS assay mix (265 ~I ) prior to the su bstrate addition . The dUMP ~lIh~trate

concentrations were 0.24 ~M. (-e -); 0.54 ~M. (-0 -). Values presented in the plots were the average of two inderenuent exrerill1ent~ I·

Table 1- Inhibition of cell growth and th ymidylate synthase activity of LaclObacilllls le ichll1al1llii .

[ICsll values of growth of L. ieichlllClll llii were calcul ated as described in the growth inhihition experiments in the text (see Fig. 2). The Kj values of L1TS lor different inhibitor alkaloids were calculated from the initial veloci ti y experiments as described in the text. These values were deri ved from Lineweaver-Burk and Dixon plots as described.]

Inhibitor

PGL

TPD

DTB

FdUMP

DTMP

MTX

FGlu .1

Growth inhibition IC)II(~M)

45

43

40

K j(M) Reference

lO x 10'(' This study

9x 10'(' Thi s stud y

7 x 10'(' This study

6.7 x 10'(, (39)

2.2 x 10'(' (39)

5 x 10 5 (39)

3.3 x 10') (40)

FGlu.1. folyltriglutamate (pteroyltetraglutamate, unreduced form)

different fixed inhibitor concentrations are shown III

Fig. 4 for PGL (A), TPD (B) and DTB (C) . The Ki values calcu lated26

.27 from the direct linear replots of

the slope vs (I) presented in the insets of these figures were IOx 10-6 M, 9x 10-6 M and 7x 10-6 M for PGL, TPD and DTB respec ti vely. These va lues were reconfirmed by employing the Di xon plots shown in Fig. 5 (A, Band C) using two different substrate concentrati ons of dUMP and the desired graded concentrations of the inhibitor alkaloids (5-30 !JM). The results indicate that the type of TS inhibition by PGL, TPD and DTB with respect to dUMP ( VIl"" 0.55) could be non-competiti ve. DTB, with the lowest Ki value was the most potent inh ibitor. Tab le I presents

1C'iO values of cell growth inhibition or L. leichllwllllii

and the Ki values of L. leichl1wllllii TS (LlTS) for the three alkaloids in comparison with the c lassica l competitive inhibitors 5-FdUMP and MTX. Evidence obtained from di alysis experiments together with the Sephadex G- I 00 chromatographi c data c learl y revealed that the alkaloids are tightl y bound to thymi dy late synthase and that the binding was irreversibIe2

X,2') .

Illhibitioll of TS inleukocYles Levels of human leukocyte TS (HITS ) in cancer

patients with CML, ALL and metastat i(: solid tumours that include breast, uterine and colon cancers (B. C and o respecti vely) compared to normal hea lth y controls (A) are shown in Fi g. 6. TS levels were elevated 65-fold in CML patients (n= I 0) and 3S-fold in ALL patients (n=8) when compared with normal healthy control subjects (n= I 2) whose baseline act ivity was negligibly low. TS levels of the leukocytes of patients with the metastat ic so lid tumours (n=3) were 8-rold elevated. HITS activ ity of untreated pati ents selected for the study was hi gher compared to that of the patients respond ing to chemotherapy. and it good correlation between the leukemic leukocyte count. blast cell value of myelob lasts/lymphoblasts and the enzyme activity has been recorded1o

. .1 1 thus clearl y revealing the biochemical strategy of the tumour marker enzyme acti vity that accurately monitors status of the disease and the clinical diagnos is of the hematologic malignancy. About 50% of the HITS ac ti vity was inhibited by PGL. TPD and DTB at 50 11M concentrations in the three types of cancers under study (Fig. 6). These results show that PGL TPD and DTB

446 INDIAN J BIOCHEM . BIO PHYS. , VOL. 36, DECEMBER 1999

25 0 No inhibitor atkatoid

0+ PGL

~ +TPO

~ +OTB

~15 > -u

"' Vl 10 ~

5

0 A B C D

Fig. 6--lnhibition of human leu kocyte thymidylate synthase (HITS ) by PGl, TPD and DTB in (A), control: (B), chroni c mye locyti c leukemi a (CML); (C), acute lymphocytic leukemi a (ALL) and (D), mctastati c so lid tu mours (MST) (of breast, uterus and ·o lon ). Bars. SD. I Activi ty in leukemic patients was determined from the % of bl ast cell s (myelocytes and lymphocytes) recorded from the hone marrow (ohtai ned hy sternal puncture and aspiration) whereas the blast values of the solid tumour patients and the norma l hea lt hy donor:; wen: those of the peripheral blood. Percentage bl ast cells was determined by the standard ligh t microscopic examinati on of Gicmsa stained smeal·s. O ne hal f o f the leukemic patients (CML and ALL. n=5 and 4 respectively) and all the three metastatic solid tumour patient s under stud y were freshly admitted and have not been given treatment , whereas the remaining half (CML and ALL. n=5 and 4 respecti ve ly) were Linder therapellt ic treatment. Therapeutic regimens: daunomycin . 20 mg/m"/d: arabinofurn osylcytosine (Ara-C), 100 mg/m' /d : 6- thi oguani ne ((, -TGl. 100 mg/m2/d ; cyclophosphamide (CPA ). 100 mg/m2/d : vi ncri st ine (VNC), 1.4 mg/m2

; adriamycin , 20 mg/m' : L-asparg inase. 6.0()() IUlm' /d: intrathecal MTX , 10 mg/m2

; predni so lone, 40 mg/m2/d . Human leukocytes and their Iysates were prepared" as descrihed in Methods. l ilTS

was assayed by e H] release assay l ~ and expressed as r' H] re leased, cpm I 0·1/mg protein/hr at 30°C. Reacti on mi xtures with added inh ihitors

consisted of 50 11M PGL, TPD and DTB separatel y].

inhibit the activity of thymidylate synthase and could serve as potential anticancer agents.

In rapidly proliferating cell popUlations, adequate supplies of thymidylate are critical to DNA synthesis, and TS inhibition had been examined for application in

h 781? TS ' h'b" h cancer t erapy ... -. -Ill I Itlllg agents are t e most effective compounds with either potential antimicrobial or antitumour action or both ' ;. TS inhibitor anal ogs have structural similarity to either the substrate dUMP or the cofactor CH2THF and as a result, by virtue of their affinity towards TS binding sites, their accumulation in the cells is favoured . Recently, some compounds non-structurally related to substrate or cofactor but with a fairl y good affinity towards the TS binding sites have been synthesized3H1

. The design of such TS inhibiting anticancer drugs have resulted in a wealth of biochemical and structural data together with

d I · I ' 1(, 14 A 'f I TS some propose cata ytlC mec lafllsms ". ntl 0 ate inhibitors such as naphthalimide, benzoquinazoline and dideazafol ate compounds have been extensively invest igated for their potenti al applicati on as anticancer

t 1.1.37-W agen s

Studies on S-FdUMP showed that this inhibitor in the presence of cofactor CH2THF forms a covalent linkage with TS during the ternary complex formation

and since the dissociation constant of the comp lex is lower than the practica l concentrations of the enzyme, binding is stoichiometric and the inhibitor is. in effect, an active site titrant 3R

. It would therefore be inrerred that L1TS homodimer has two acti ve binding sites . one

h b . ,9 40 d ' " I' d I I per eac su UllIr ' an It IS Il11p Icate t lat two 1110 es of each of the inhibitor alka loid possibly bind per 1110 1e

enzyme. Furthermore, our resu lts showed that L1TS binding to the phenanthroindolizidine inhibitor alkaloids PGL and TPD and the /3-caboline-benzoquinolizidine inhibitor alkal oid DTB

is very tight through a possi ble covalent bond and is irreversible2R29

. 2,6-Diam inobenz[cd]indole type and 2,4-d iami no-S-su bsti tu ted-pyrro I o[2,J-d] py ri 111 i dine type inhibitors of TS and dihydrofolate reduc tase (DHFR) respectively are some such nonclass ica l noncompetitive inhibitors41

-12 Alkaloid binding to

HITS could be quite similar to that of UTS. Human TS has been purified from the ce ll lines or CC RF-CEM human lymphocytic l ellkemi a~' and frol11 the blast ce ll . ..; of patients with acute myelocyt ic leuk 'Illi a (ANI L )~~ and partially characteri zed. Locks hin e,' ({ f. showed lil al human leukemic TS (M,., 66,0000) is a hOl11 ml i Iller and binds 2 moles of FdUMP per mole enzyme ~ ; .

RAO el al. : INHIBITION OF THYMIDYLATE SYNTH ASE 447

Our findings on the highly elevated human leukemic leukocyte TS acti vity as measured by the CH] release assay are in corraboration with those reported by Silber et at 5

. In human leukemias, blood plasma (leukocyte fraction) TS46 and the blood serum thymidine kinase (TK)47 levels serve as markers in the progression of malignancy.

Although some physico-chemical properties of the phenanthroindolizidine alkaloids have been elucidated48

, very little is known about their cytotoxic and anti tumour activity. Of these alkaloids, most important are tylophorine, tylocrebrine, cytopleurine and a congener of tylophorinidine reportedly having anti tumour acti vity in diffe rent spec ies48. Inhibition of DNA synthes is by tylocrebrine and cytopleurine has been demonstrated by two groups of investigators independentl / <),50. Some of the chemical and spectral properties of emetine alkaloids have been extensively reviewed22

. However, information on their biochemical and biological properties is scanty. Considerable interest in the emetine alkaloids such as tubulos ine has been evoked with the di scovery of their antileukemic5

!

and amebicidaI52.5.1 acti viti es that are mainl y associated

with the transfer reaction in protein biosynthesis. Biological properties other than anti tumour acti vity of the Alangilll l1 emetine alkaloids and demethyicephaeline that are of pharmacologic and cl inical signi ficance are their anti platelet aggregation, antihypotensive and sedati ve action2

<J . Common structural determinants of biological acti vity have been pointed out fo r both phenanthro indoli zidine and the emetine alkaloids54.

Acknowledgement The authors express their gratitude to Drs. J.G. Satav

and A.S . Subbaraman fo r their va luable help.

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