Nghien
CLPU- Ky thuat
Nghien ciru kha nang gan ket cua dan chat benzo[c]phenanthridin va phirc hop topoisomerase I-DNA bang mo hinh
mo ta phan tir docking
Do Thj Ngoc Mai, Huynh Thj Ngoc Phirang, Thai Khac Minh
Bo mon Hoa Dirac - Khoa Duac - Dai Hoc Y Duac TP. Ho Chi MinhOat van de
Trong so edc tde nhan khdng ung thu the hien hoat tinh tren topoisomerase, enzym can thilt cho hoat dpng sao chep, phien ma vd tdi to hgp DNA, cdc alcaloid hp benzo[c]phenanthridin (BCP) dugc trinh bay d Hinh 1 duge biit d i n nhieu (nitidin, fagaronin.
MeO
MeO'
chelerythrin, sangunarin)'''.Ode c h i t tuang ddng BCP da dugc tong hgp va danh gid ddc tinh t l bao cung nhu Id hoat tinh tren topoisomerase loai I (TOP-I). Trong dd, ethoxidin, NK-109 va topoval (ARC 111) Id nhirng chat ed t i l m nang sir dung trong hda trj lieu ung thu'^"^'.
Nitidin R, + R2 = -CH2- Fagaronin R, = H, R2 = Me
R j O O R ,
Chelerythrin Ri = H, R2 = Me Sanguinarin Ri + R2 = CH2
NK109 ARC 111 Hinh 1 : Ciu triic hda hoc ciia cac
\ld\ muc dich nghien eiru kha nang gdn kit cua cdc din chit benzo[c]phenanthridin vdi phirc hgp DNA:TOP-l, md hinh md ta phdn tir docking duge tiln hdnh thuc hien bdng phan mim DOCK 6.2. Kit qua eiia nghien eiru ndy ed t h i irng dung d l thilt k l ra cau tmc cd hoat tinh sinh hpe eao han.
Phuvng phap nghien ciru Casddu>lieu
Tong so 53 din chat BCP dugc thu thdp vdi cde gid trj hoat tinh tren kha nang dc ehl TOP-I vd dpc tinh t l bdo (IC50) ddi vdi ddng t l bdo ung thu mdu RPMI-8402 (lymphoblastic leukemia cell) ' ^ '
ciu triic khong gian 3 chilu
MeCHjSOs
(Topoval) Ethoxidin phan tir BCP c6 khi nang tic ddng lin TOP-I
Cau triic phuc hap DNA:TOP-l
Cau tnjc tinh the tia X eua DNA:TOP- Llndenoisoquinolein (pdb 1SC7) cd dp phdn giai Id 3A dugc tai v l may tinh tir ngdn hang ca sd dir lieu protein (protein data bank http:/Aftww,pdb org)'^'. Ciu tnJc DNA d vj tri ciia guanin thir 11 bj dirt gdy vd thay doi v l cdu dang. Do dd, trude khi tiln hdnh docking, cau triic ciia DNA dugc chinh sira. Oiu tien, nhdm 5'-SH cua G11 dugc chuyin thdnh 5'-0H bdng edeh thay t h i nguyen tir luu huynh (S) ciia G i l bing oxy (0), eau ndi phosphoester bi dirt ciia G12 trong phirc hgp dugc noi lai Todn bp cdu triic ciia DNA dugc toi thilu hda ndng lugng cue bp (chi
Nghien CLPU - Ky thuat
tdi thieu hda cau tnje DNA md khdng Idm thay doi eiu tnJc eiia TOP-I). Cau tmc phirc hgp DNA:TOP-l diing tiln hdnh nghien ciru duge trinh bdy d Hinh 2.
Hinh 2: Ciu triic tinh thi phirc hgp DNA.TOP-I (pdb 1SC7) sau khi chinh sira DNA
Xay dimg cau true khong gian 3 chieu dan chat BCP
Ciu triie 3 chilu cua 53 dan chit benzo[c]phenanthridin xdy dung bing each sip dung chirc nang ve eiu tnJc cua phin mim ChemBioOffice2008 (2D va 3D) dudi djnh dang mol2'®'. Nguyen tir trong phan tir duge xae djnh dien tich dudi dang dien tich Gasteiger-Huckel, Ciu triie phdn tir duge tdi thilu hda ndng lugng din mire su thay doi ndng lugng eao nhit Id 0,001 keal.mor'A"'.
Chuang trinh tdi thieu hda ndng lugng (minimize energy-ME) vd ddng lue hpe phdn tir (molecular dynamic-MD) trong phin mim ChemBioOffiee2008 dugc sir dung d l thu duac ciu dang cd mire ndng lugng toi thilu todn phin. Kit qua qud trinh ndy dugc phdn tieh vd eiu dang ed mire ndng lugng thip nhit duge luu vdo dLr lieu diing cho qud trinh docking.
Docking
Docking Id phuang phap thilt k l thuoc dua vdo eiu triie mue tieu tde ddng, nghien ciru kha ndng gin kit eua mdt hay nhilu phdn tii' thude (hay eau tip, ligand) Idn tren mue tieu tde ddng (thu the, enzym hay edc protein, DNA...) trong khdng gian 3 chilu.
Hien nay ed nhilu phin mim docking nhu FlexX, Autodock, GOLD... Chuang trinh DOCK 6.2, phin mim docking miln phi cho nghien ciru, duge sir dung d l tiln hdnh thi nghiem''''. Qua trinh docking dugc thue hien theo sa dd 1.
Diem tac dgng
Diem tde ddng tren phirc hgp DNA:TOP-l (pdb 1SC7) duge xde ^nh Id tit ea eae acid amin vd acid nucleic trong pham vi khdng gian 6A tinh tir trung tdm phdn tip indenoisoquinolein.
Cau true inuf tieu t;<c dona
Tiio be in.U c;ui true inuc tieu t-.ic d^iia
Tijo klioi cau bao quanh (liem tac dona Tao hop \ vl luiri
Kiem tra lai voi ket quS cfpt bien protein Kilm tra lai dilm tac dpng
Cau true lisand 2D
Xay dung can tnic ligand .^D Toi thieu lion ii.iug hioiii; (EM) DiSna luc hoc phnii tu (MI))
Phan tich kit qua Mo hinh gan ket 6' muc do phan tii'
Diem so docking chuc nang
Sc dd 1: Tiin trinh docking
50 TAP CHi DUQC HQC - 12/2009 (SO 404 NAM 49)
Nghien CLFU - Ky thuat
Danh gia docking
Cau triie indenoisoquinolein duge xdy dung vdi phuang phdp tuang tu nhu d p h i n xdy dung e i u triie khdng gian 3 chieu cua cdc d i n chit benzo[c]phenanthridin. Mat khae, c i u triic indenoisoquinolein cung dugc trieh ra tir e i u triic 1SC7, sau dd ehmh sira lai cho dimg k i l u dinh dang nguyen tip (atom type), sau dd dugc luu trtr vdo dir lieu vd sir dung nhu la c h i t ddi chilu cho qud trinh docking.
Kit qua docking dugc ddnh gia dua tren edc diem so chipc ndng. D i u tien, docking indenoisoquinolein xdy dung vd indenoisoquinolein tinh the (su dung ddi chilu,
ehuan ndi) dugc phdn tich. K i t qua docking eiia e i u dang phdn tCr indenoisoquinolein xdy dung vd indenoisoquinolein doi chieu cd d i l m sd ke d khde biet nhd d mire 0,28 kcal/mol (- 52,73 so vdi -53,01 kcal/mol) ehirng td phuang phdp xdy dung e i u triie khdng gian 3 chieu ciia cae phdn tip, phuang phdp docking ed t h i dp dung dugc d l phdn tich k i t qua docking ciia ede c h i t khac dugc t i l n hdnh ddng thdi. C i u dang eua cdc cdu triic duge gidng hang vd trinh bdy d Hinh 3.
(A) '^ (B)
Hinh 3: (A) Ciu dang sau 'dock' ciia indenoisoquinolein xiy dirng (carbon xanh) va ciu dang indenoisoquinolein (carbon xim) trong phirc hgp kit tinh tinh thi DNA.TOP-l.indenoisoquinolein gidng hing vdi nhau trong diim tic ddng. (B) Ciu dang sau 'dock' cua indenoisoquinolein ddi chiiu (carbon tim hdng) vi ciu dang indenoisoquinolein (carbon xim) trong phirc hgp kit tinh tinh thi DNA.TOP- Lindenoisoquinolein gidng hang vdi nhau trong diim tic dong. Miu ciia cac nguyen tir: oxy (do), nita (xanh duong).
Khi nang gan ket a mirc dp phan ttr Cde chit (rc e h l hoat ddng eua enzym TOP- I hidn nay duge ehia Idm 2 nhdm. Thir nhit Id nhdm chit kim ham (suppressor) hoat tinh enzym TOP-I. Nhdm ndy Id nhirng c h i t dc e h l hoat ddng enzym TOP-I nhung khdng Idm b i n trang thdi trung gian cua phirc hgp DNA:TOP-l.
Cdc chat nhdm kim hdm hoat ddng TOP-I thudng g i n k i t vdi DNA Idm cho TOP-I khdng the nhdn dien ra d i l m gdn k i t chuyen biet tren DNA vd k i t qua Id ire e h l s u thdnh lap phirc hgp giua TOP-I vd DNA. Ca e h l kim ham ndy hogt ddng theo ea e h l tuang tranh (thuoc vd TOP-I canh tranh g i n k i t tren ea c h i t Id DNA) vd enzym v i n ed hoat tinh nhung khdng cd ca chit d l hoat ddng. K i t qua Id Idm ngirng chu trinh t l bdo. Thir hai Id nhdm gdy ddc TOP-I (poison). Nhdm ndy bao gom nhirng chat ed tde
ddng chong s u tdch ra eua phirc DNA:TOP-l sau khi TOP-I den g i n k i t vd cdt doan DNA, Cdc c h i t ddc tinh vdi TOP-I ed the hoat ddng theo 3 ea e h l bao gdm (i) g i n k i t vdi TOP-I bdng phirc hgp thuoc:DNA, (ii) nhdn dien vd gdn k i t vdi phirc DNA:TOP-l, (lii) g i n k i t vdi DNA trong bdng phirc hgp thudc:TOP-l. Cde c h i t gdy ddc TOP-I hoat ddng theo nguyen t i e Id g i n k i t vdi ea hai, enzym TOP-I lan DNA, trong phirc hgp DNA:TOP-l bdng lien k i t ddng hda tri, b i n hda phirc va Idm vd sgi kdp DNA Su hien dien cua sgi DNA dan dugc nhdn dien vd tin hieu ndy lam ngirng chu trinh te bdo. K i t qua cudi ciing Id su huy bao'^', Trong nghien CiPU ndy, sau khi t i l n hanh docking phipc hgp DNA:TOP-l:BCP dugc phan tich tren kha ndng g i n k i t vdi phiPC hgp hay DNA rieng le
Diem s6 chirc nang
Nghien
CLFU- Ky thuat
Docking bdng chuang trinh DOCK 6.2 truy x u i t ra k i t qua dua vdo 2 loai diem sd docking chirc nang. Dd Id (i) D i l m he thdng ke d (grid score) Id d i l m sd dugc tinh dua tren cae tuang tac khdng lien k i t b i n g tinh toan trudng luc ca hpc phdn t i i ' ; (ii) Diem sd tuang tac (contact score) Id tong sd tuang tac giira cae nguyen tir trong ligand vd diem tac ddng. Trong nghien eiru ndy, ca hai gid trj d i l m sd docking chirc nang dugc phdn tieh. Gia tri he thong ke d dugc SLP dung cho danh gia x i p hang cac c h i t BCP va phdn tieh mdi lien quan vdi hoat tinh sinh hpe.
Ket qua va ban luan
c i u true khdng gian 3 chilu cua d i n c h i t BCP
B i n g cdch SLP dung chuang trinh ve c i u triic, tdi thilu hda nang lugng vd ddng luc hgc phdn tip vdi phdn mem ChemBioOffiee 2008, e i u dang b i n n h i t vdi mirc ndng lugng tdi t h i l u todn p h i n duge tao ra vd luu vdo ca s d dir lieu de chuan bj cho docking.
c i u triic tinh t h i phiPc hap TOP-I: DNA:
ligand
Hien nay tren ngdn hdng ea s d dir lieu c i u triic protein (PDB) cd 4 c i u triic phirc hgp TOP-I: DNA: ligand' dugc cdng bd dd Id phirc hgp eiia DNA:TOP-l vdi Indenoisoquinolin (pdb 1SC7"^'. Indolocarbazol (pdb ISEU)'^', Vanadat ion (VO/") (pdb 2B9S)'^', Topotecan (pdb 1K4T)''°'. Trong t i t ca c i u true phirc hgp, t i t ca d i u cd s u thay doi c i u dang d vj tri cua guanin vj tri 11. D i l u nay chirng td vj tri ciia guanin nay quan trong cho qud trinh ire e h l TOP-I vi ly do tac q u y l n nen nhdm nghien ciPU dd giir lai.
Trong cde cdu true nay, nghien ciru t i l n hdnh docking tren e i u triic 1S07 vdi do phdn giai Id 3A, Cau triic ciia DNA dugc tdi xdy dung mpt edeh hgp ly vd k i t qua docking cung phii hgp vdi nhdn dinh ndy.
Kha nang g i n k i t cua BCP a mi>c do phan tip b i n g docking
Tong so 53 d i n chat BCP docking thdnh cdng vdo phirc hgp. Diem sd thay doi tir -57,57 kcal/mol d i n -36,27 kcal/mol. T i t ca cac c i u dang trong c i u triic phire hgp d u g c phdn tich
va so sdnh nhdm giai thich moi lien quan giira cdu triie hda hpc vd tac dung sinh hpc. Phdn tieh k i t qua docking eho thay t i t ca cdc d i n chdt BCP d i u cd kha nang tao lien k i t TT-TT vdi Guanin G11. He thong vdng cua ddn chat BCP cd xu hudng ndm d vj tri song song va xen vdo giua cde cap base G l 1/0112 va A113/T10 cua c i u triic DNA vd han c h i kha nang g i n k i t cua DNA vdi TOP-I (vdi ATGO Id ten eua cdc nucleotid). K i t qua docking ciing cho thay nhdm BCP cd xu hudng can trd khdng gian do tao lien k i t b l mat vdi DNA n h i l u han kha nang gdn k i t vdi TOP-I. D i l u ndy cd the giai thich do c i u triic 'eirng' cua he thong vdng vd kha nang chen vao cau true DNA. Ben canh dd, mdt sd BCP cung cd kha nang tao lien k i t vdi TOP-I d cdc acid amin Asn352, Arg364, Asn722, Glu356, Lys425. Tuy nhien, k i t qua docking cho t h i y xu h u d n g g i n k i t rd ret ciia nhdm BOP len DNA n h i l u han la TOP-I. K i t qua ndy cho thdy da sd cdc chat trong 53 d i n c h i t BCP d u g e tien hanh docking hoat ddng theo ea c h i kim ham hoat tinh TOP-I (suppressor).
Trong cdc d i n chdt da thue hien docking, nhdm d i n c h i t ciia ARC-111 the hien kha nang g i n k i t tot nhit. D i l u nay phii hgp vdi cde gid trj sinh hoc v l IC50 cung n h u kha nang ire e h l topoisomerase 1 la cao n h i t trong nhdm. ARC- 111 cd kha nang tao 2 lien k i t n-n song song vdi Guanin 11. Base nita cua ARC-11 tao lidn k i t hydro vdi Guanin. Cdc c h i t cd the tao lidn k i t hydro vdi Guanin d u g c cho la cd hoat tinh iPC e h l t l bdo tdt vi s u gdn k i t ciia cdc chat vao DNA phu thupc vdo s u hien dien cua guanin vd nhdm 2-amino ciia guanin Id quan trpng trong viec hinh thdnh phirc hgp ben cua thudc-DNA'"'. Kha nang g i n k i t d mire dd phdn t u cua ARC-111 giai thich cho kha ndng ire e h l hoat ddng eiia TOP-I bdng cdch canh tranh vdi TOP-I trong viec hinh thdnh phirc hgp vdi DNA, can trd hoat ddng ciia TOP-I trong vide t i l p can vd g i n k i t vdi DNA va lam ngirng qud trinh sao chep, phien ma vd tdi to hgp DNA. Md hinh g i n k i t d mirc dp phdn tCr trong khdng gian 3 chieu eiia ARC-111 vdi phirc hgp DNA:TOP-l d u g c trinh bay d Hinh 4A (hinh tao bdi ehirang trinh Chimera'"') vd md hinh tuang irng the hien kha ndng gdn k i t d khdng gian 2 chieu d u g c trinh bdy d Hinh 4 8 (hinh tao bdi chuang trinh M O E " ^ ' ) .
52
TAP CHi DUQC HQC - 12/2009 (SO 404 NAM 49)
Nghien CLPU - Ky thuat
(A) ( B )
Hinh 4: (A) Md hinh gin kit d mire do phin tir trong khdng gian 3 chiiu eua ARC-111 vdi phirc hgp DNA:TOP-l.
(B) Md hinh biiu diin khi nang gin kit 2D ciia ARC-111 vdi phire hgp DNA:TOP-l.
Mdi lien he giiPa diem s d docking va hoat tinh sinh hoc
Mdi lien he giua d i l m so docking vd hoat tinh tren TOP-I ciia 53 d i n c h i t tuang ddi t h i p (r^=0,41, Hinh 5A). Mdt d i l m can luu y d ddy la gid trj hoat tinh sinh hoc tren TOP-1 dugc xdc djnh bdng cdch do mire dp eiia edc manh DNA bj vd sau khi cho ligand vdo hdn hgp gdm DNA vd TOP-1 (Topoisomerase-mediated DNA cleavage assay)'^"*', D i l u ndy cd nghTa Id gid tri hoat tinh trdn TOP-1 xdc djnh bdng phuang phdp ndy chi t h i hien eho kha ndng gdy ddc TOP-1 md khdng xet d i n co c h i kim hdm. Mat khde, k i t qua docking eho t h i y eae d i n c h i t BCP g i n k i t ehu y l u tren DNA vd cd t h i hoat ddng chinh theo ca e h l kim ham hoat tinh
enzym TOP-I. Do dd, s u tuang quan t h i p giira diem so docking vd gia tri ire e h l TOP I ndy Id hgp ly.
T i l p tue nghien ciru mdi lien he giira d i l m so docking va kha ndng gdy dde tinh trdn ddng t l bdo RPMI8402 (IC50) eua nhdm d i n c h i t BCP thi thu he sd hdi qui Id Id r^=0,70. Ddy Id k i t qua khd b i t ngd va c i n thdi gian d l phdn tieh t i l p theo. Tuy nhien khdng loai trir trudng hgp tac ddng kim ham tren TOP-I bdng g i n k i t tren DNA ciia edc c h i t BCP Id y l u td quylt djnh cho ddc tinh t l bdo. N l u d i l u nay xay ra dudng b i l u d i l n s u lien he giCpa d i l m sd docking vd ddc tinh t l bdo (IC50) cd the giiip tien dodn ddc tinh t l bdo eiia edc c h i t tuang dong BCP mdi.
A
R2 = 0.41
log REC
B
R' = 0.70
/
loglC^
Hinh 5: (A). Mdi liin hi giua diim sd docking vi hoat tinh ire chi TOP-I ciia 53 din chit BCP.
(B). Mdi liin hi giua diim sd docking vi doc tinh ti bio (ICso) cua 53 din chit BCP
Nghien CLFU - Ky thuat
Ban luan luan k i t qua va trien vpng
Cde chit ire e h l hoat ddng eua enzym TOP-I cd t h i tde ddng theo ea e h l gay dpc hoac kim ham. K i t qua docking cho t h i y cdc c h i t tuang ddng BCP nghien ciru ed xu hudng g i n k i t tren DNA va tac ddng ire e h l TOP I theo k i l u kim ham. Tuy nhien, gid trj hoat tinh sinh hpc xae djnh theo ca e h l kim ham thi r i t it tai lieu cdng bd.
Kha nang ire c h i hoat dpng TOP-I cua mpt chit cd the do 3 ca e h l sau: (i) gdn k i t vdi DNA vd can trd TOP-I d i n g i n k i t (canh tranh vdi TOP-I tai DNA, ca c h i thudng t h i y cua cae c h i t kim ham hoat ddng TOP-I); (ii) gin k i t vdi TOP-I, Idm thay doi dilm tac ddng lam cho enzym khdng cdn kha nang nhdn dien va g i n k i t vdi ea c h i t Id DNA (canh tranh vdi DNA tai enzym); (iii) g i n k i t vdi ca hai DNA vd TOP-I trong phiPC hgp TOP- l:DNA vd lam b i n hda phire nay (ea e h l gay ddc TOP-I). Chit tac ddng theo ca e h l eanh tranh vdi DNA tai enzym TOP-I (ca e h l ii) it dugc n h i c d i n trong tdi lieu tham khao. Dua vdo c i u true tinh t h i cua TOP-I cd the irng dung thilt k l thudc dua vao c i u triic muc tieu d l sdng loc vd thilt k l ra chat ire e h l ehuyen biet tren TOP-I. Ben eanh dd, n l u dieu kien cho phep nen sir dung mdt so chuang trinh docking khac nhu FlexX, GOLD, AutoDock.,, tiln hdnh nghien ciru n h i m so sanh k i t qua va thLPC hien nghien ciPU t i l p theo.
K§t luan
Nghien ciPU kha nang lien k i t cua phdn tip tren muc tieu tae ddng b i n g md hinh md ta phdn tip vdi su hd trg cua mdy tinh Id hudng nghien ciPU mdi trong Unh vue hda duge n h i m mue dieh tim ra thuoc mdi. Trong nghien ciru nay, kha nang gdn k i t ciia cde d i n c h i t BCP vdi phiPC hgp DNA:TOP-l dugc phdn tieh bdng chuang trinh DOCK 6.2. Kha nang g i n k i t d mipc dp phdn tip ciia BCP len phipc hgp cho thdy BCP cd xu hudng g i n k i t vdi DNA han Id TOP-I vd c i n thip nghiem t i l p theo d l khdng djnh lai. Ben eanh y nghTa khoa hpc, d l tai cdn xdy dung dugc protocol thuc hien qua trinh docking bdng each k i t noi cae chuang trinh m i l n phi vd ipng dung triln khai d qui md phdng thi nghiem.
Summary
Human topoisomerase I (TOP-I) is the molecular target of a set of diverse anticancer compounds, including camptothecins, indolocarbazoles, indenoisoquinolines and
benzo[c]phenanthridines (BCP). Based on the crystal structure of DNA:TOP-I complex, 53 BCP derivatives were docked into the complex by DOCK 6.2 program. Docked BCP:DNA:TOP-l complexes indicated that BCPs tended to establish stronger interaction with DNA than with TOP-I. Moreover, a relationship between the biological activities and the docking scoring function was analyzed as well. Maybe, the information are useful for designing new BCP compounds of higher affinity.
Keywords: docking, topoisomerase, benzolcjphenanthridine, drug design, cytotoxicity, anticancer
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