Binding Mode Studies of Indenoisoquinoline Analogues into Human Topoisomerase I-DNA Complex Using Flexible Docking

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Human Topoisomerase I-DNA 절개가능 복합체에 대한

Indenoisoquinolin e 유도체들의 결합양상 연구

박인선^†·김보연^†·김춘미·최선^# 이화여자대학교 약학대학 대학원 생명·약학부

(Received June 30, 2009; Revised August 3, 2009; Accepted August 4, 2009)

Binding Mode Studies of Indenoisoquinoline Analogues into Human Topoisomerase I-DNA Complex Using Flexible Docking

In Seon Park

^†

, Boyeon Kim

^†

, Choonmi Kim and Sun Choi

^#

College of Pharmacy and Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea

Abstract— Topoisomerase I (Topo I) participates in the DNA replication, transcription, and repair. Binding of Topo I inhibitor to the Topo I-DNA cleavage complex forms stabilized ternary complex which blocks DNA religation and ultimately causes cell death. Camptothecin (CPT) and its derivatives have been among the most effective anticancer drugs by inhibition of topo I. However, efforts to synthesize non-CPT drugs have been actively going on because the CPT derivatives have several limitations such as poor solubility, short half-life, and side effects. As an indenoisoquinoline, NSC314622 is not as potent as CPT, but its chemical stability and slower reversibility of the cleavage complex made it a good lead compound.

Recently, a series of indenoisoquinoline analogues were synthesized with substituted dimethoxy or methylenedioxy on the aromatic ring and alkylamino on the lactam nitrogen.¹⁾ Some of them showed quite good Topo I inhibitory activity. Using the computer docking program, Surflex-Dock, indenoisoquinoline analogues were docked into the human Topo I-DNA cleavable complex. The docking results showed that the compounds with activity better than NSC314622 intercalated between the -1 and +1 base pairs at the cleavage site, but those with little or no activities did not appear to intercalate. These results could be useful to design new Topo I inhibitors improved than CPT.

Keywords □topoisomerase I-DNA complex, indenoisoquinoline, docking, binding modes

Topoisomerase I(Topo I)^은^세포의^핵에^존재하며 DNA^의^위

상상태를조절하고세포내에서일어나는전사, ^복제, ^그리고^복

구과정에관여하는효소이다.^2-5) Topo I의촉매작용을하는

Tyr723의 hydroxyl group이 DNA 끊어진 가닥의 -1 deoxynucleoside^의 3' ^말단과^{공유결합을}^형성하여^한쪽^가닥의^끊어

짐을유도한다. 그결과 3'-phosphotyprosine(Ptr723) 결합이형 성되면 Topo I^과 DNA^는^절개가능^복합체(cleavable complex)^라

고불리는가역적인복합체를형성하고일시적인 DNA^의^절단

으로끊어진가닥이회전을하여 positively supercoiled DNA는 풀어주고 negatively supercoiled DNA^는^더^꼬아줘서^본래의 DNA ^{이중나선을} ^{유지하도록} ^한다. DNA^를 ^풀어준 ^후 +1

deoxynucleoside^의 5'-OH^가 Ptr723 ^결합을^다시^공격하여^재연

결이일어나 DNA ^{이중나선이}^복구된다.⁶⁾ Topo I ^저해제는 Topo

I-DNA 절개가능복합체를안정화시켜서재연결단계를특이적

으로저해하여세포독성을일으키는것으로알려져있다. DNA

복제도중에 Topo I ^저해제가 Topo I-DNA ^절개가능^복합체와

결합하여안정한삼성분복합체(ternary structure)를이루면복 제포크와충돌하게되어세포의 apoptosis^가^유도된다.⁶⁾

현재알려져있는대표적인 Topo I ^저해제는 Camptothecin (CPT) 유도체이다. 그러나 CPT 유도체는한계점들을가지고있 다. ^첫째, ^{화학적으로}^{불안정하다}. E ^환의 hydroxylactone^이^생

리적인 pH^에서^빠르게 carboxylate^으로^전환하여^인간^혈청^알

부민과강하게결합하기때문에, CPT의 99.5% 이상이약효가 없는 carboxylate ^형태로 ^존재하고^약효가^있는 lactone ^형은 0.5% ^미만이^존재한다.⁷⁾^둘째, Topo I-DNA ^절개가능^복합체로

부터빠르게확산되어나오므로삼성분복합체를유지하기위해 지속적인약물주입이필요하다.⁸⁾^셋째, ^백혈구^감소와^같은^부작

#본논문에관한문의는저자에게로

(^전화) 02-3277-4503 (^팩스) 02-3277-2851 (E-mail) [email protected]

†공동제1^저자임.

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용을유발하므로안전성을위해투약용량의제한이필요하다.⁹⁾

이와같은한계점을극복하고자새로운 Topo I 저해제연구가 시작되어, CPT^를 seed^로 ^사용한 National Cancer Institute (NCI) drug screen database^의 COMPARE analysis^에 ^의해 indenoisoquinoline(NSC314622)가 Topo I 저해제로확인되었다.¹⁰⁾ NSC314622^는 indenoisoquinoline ^계열^화합물의^선도^화합물로

서새로운 Topo I ^저해제^개발에^{사용되었다}. NSC314622^은 CPT

와다른생물학적활성을가지므로그한계점을극복하고다른 항암효과프로필을보일수있다. ^첫째, indenoisoquinoline ^계열

화합물인 NSC314622^는^생리적인 pH^에서 CPT^와^달리^화학적으

로안정하다.¹¹⁾둘째, 이물질은 CPT가 Topo I-DNA 절개가능복 합체의가역성으로인해지속적인주입이필요한것에비해, ^절

개가능복합체를더안정화시켜서오래유지되도록한다.¹²⁾^셋째,

이 indenoisoquinoline 화합물은 DNA 절개부위의특이성에서

CPT^와^다르므로, ^다른^{항암스펙트럼을}^제공할^수^있다¹⁰⁾(Fig. 1).

2005^년에 Staker ^등은 CPT, indenoisoquinoline(MJ238), indolocarbazole(SA315F) 등을각각리간드로포함하는결정구 조를발표하였다. ^세^계열^모두^같은^삽입^결합^양상을^가지고

절개부위의 DNA ^염기^쌍에^유사하게^{작용한다는}^것을^확인하

였다. Indenoisoquinoline 계열인 MJ238은 NSC314622의 A 환 에치환된 dimethoxy^기와 D ^환에^치환된 methylenedioxy^기가

수소로치환되어있고 N6의 methyl기가 butylcarboxylic acid로 치환되어있는형태이다. MJ238은절개부위의 +1/-1 염기쌍 사이에삽입되어있고 C ^환과 D ^환은^끊어지지^않은 DNA ^가

닥의염기위에, A 환과 B 환은끊어진 DNA 가닥의염기위에

stacking 상호작용을 하고 있다. C 환의 carbonyl기가 minor groove ^방향으로^향하여 Arg364 guanidino group^의^질소^두^개

와각각수소결합을형성하고있다. B 환의 butyl-carboxylic acid

는 Ala351과 Asn352가 있는 major groove로 향하고 있다.⁶⁾ Marchand ^등은 nor-indenoisoquinoline^을^리간드로^포함하는^결

정구조를발표하고그것을발표된다른구조와비교하여, 수소 결합망을이루는잔기들에일부차이가있으나 CPT, indenoisoquinoline, indolocarbazole, nor-indenoisoquinoline ^계열의^약물

이같은결합양상을보이고있으며같은작용기전을가질것이 라고주장하였다.¹³⁾

본연구에서는 MJ238^의^삼성분^복합체를^통하여^밝혀진 MJ238

의결합양상을근거로 Topo I ^{저해작용을}^나타낼^것으로^예상되

는 indenoisoquinoline 유도체들을인간 Topo I-DNA 절개가능

복합체에 docking^시켜^단백질과^{저해제들의}^{결합양상을}^분석하

고새로운 non-CPT계열약물의개발연구에중요한정보를제공

하고자하였다.

실험방법

모든 실험은 SYBYL molecular modeling program package (version 7.3.3)로 RHEL 4.1 Intel Xeon processor 5050 Linux workstation에서수행하였다.

Docking을위한Topo I-DNA X-선결정구조의선택과처리 인간 Topo I-DNA 절개가능 복합체에저해제인 indenoisoquinoline(MJ238)^이^결합된^삼성분^복합체^결정^구조의 1SC7.pdb

를선택하였다. 1SC7.pdb는 3.0 Å의해상도를가지며 Tyr723이

DNA^의 -1 site^인 T10^과 phosphotyrosine ^결합으로^연결된^구조

이다.⁶⁾

1SC7.pdb를 docking에적합한상태로만들기위해 X-선결정 구조를수정하였다. ^활성부위^잔기에서^벗어나^위치한 glycol^은

제거하고리간드의원자형을확인, ^{수정하였다}. DNA^의^안정한

결정구조를만들기위해 G11의 5'-OH기가 5'-SH기로치환^2,4)된

5'-thio-2'-deoxy-guanosine phosphonic acid(TGP) ^구조로^되어

있어 황을산소로 수정하였다. ^그리고 SYBYL^의 Biopolymer Structure Preparation Tool을사용하여 atom types, backbone, N-, C-termini, hydrogen bonding, bump ^등을^고려하여 docking

실험에적합하도록 구조를처리하였다. ^그리고, ^{단백질에는} Kollman-all-atom charge를, DNA와 리간드에는 Gasteiger- Hückel charge^를^주었다. ^원래 1SC7.pdb^의 DNA ^서열과^최종

수정된 DNA ^서열은^다음과^같다.

수정전 5'-AAAAAGACTT TGPGAAAAATTTTT-3' ^잘리는^쪽

수정전3'-TTTTTCTGAA-C CTTTTTAAAAA-5' ^잘리지^않는^쪽

수정후 5’'-AAAAAGACTT GGAAAAATTTTT-3' ^잘리는^쪽

수정전3'-TTTTTCTGAA-CCTTTTTAAAAA-5' ^잘리지^않는^쪽

Docking에사용할화합물의처리

실험에사용된화합물들은 Cushman ^등에^의해 NSC314622

를선도화합물로하여합성된것으로 A 환, D 환, 및 N6에치 환기를달리하였고 Topo I 저해활성에대한자료가반정량적으 로(semi-quantitatively) ^나타나^있다. ^추가로^현재^{임상개발을}^위

한 NCI Developmental Therapeutics Program의 '0'상 단계에 있는 NSC725776과 NSC724998도 docking에사용하였다.

Fig. 1 −Structures of camptothecin (A) and indenoisoquinoline derivative, NSC 314622 (B).

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Docking ^실험에^사용된 12^개의 indenoisoquinoline ^유도체^구

조는결정구조의 MJ238^을^추출하여 MJ238^의 4-^환^모핵을^바

탕으로 sketch하고 CONCORD로정리하였다. 생리적조건에서

protonation ^또는 deprotonation ^될^수^있는^것들은 protonation

상태에맞게조절하고그에상응하는 formal charge를주었다.

그리고 Tripos force field, Gasteiger-Hückel charge를사용하여 에너지를 최소화하였다(Method, Powell; gradient, 0.001 kcal/

(mol*A); maximum iterations, 100000). 재현성실험을위해결 정구조의리간드를수정한후위방법과마찬가지로처리하였다.

Topo I-DNA결정구조에대한리간드 docking 실험

Surflex-Dock^14,15)에서는단백질구조로부터가상으로리간드들 이배열하는수용체와의결합공간을나타내는 protomol^을^만든

다. Protomol 생성후, Surflex-Dock의기본 setting을사용하여

(단, additional starting conformations per molecule은 5로설정)

각리간드당 30^개의 pose^가^{도출되도록} indenoisoquinoline ^유

도체의 docking 실험을수행하였다. Scoring fuction은 Surflex score^외에 ChemScore, G_Score, D_Score, PMF_Score^가^포함되

어있는 CScore(consensus score)^를^추가로^{계산하였다}.

평가기준

Surflex-Dock ^실험 ^결과로 ^각 ^{리간드마다} ^도출된 30^개의 conformation 중에서함께계산된 Surflex_Score, CScore를분 석하여각리간드의 conformation^을^{선택하였다}. CScore(Chem_

Score, G_Score, D_Score, PMF_Score)^는 docking ^결과로^나온 30개 conformation에서개별 score들의평균값을각각계산하여 개별 score^에서^평균값^이상을^가지면 1^을^주고^그^이하의^값을

가지면 0^을^준^후^개별 score^에서^얻은^값의^합이다. ^본^실험에

서는 CScore가 4~5로높은 conformation에서 G_Score¹⁶⁾값이

Topo I ^저해^활성 ^정도와^비교적^좋은 ^상관성을^{보였으므로}

CScore^가^높으면서 G_Score^가^가장^좋은 conformation^을^선택

하였다. G_Score는 genetic algorithm을 이용한 docking program^인 GOLD(Genetic Optimization for Ligand Docking)^의 scoring function을통해도출된 score이다. 위와같이선택된

conformation을 Topo I-DNA 절개가능 복합체와 indenoiso-

quinoline ^유도체의^{상호작용을}^리간드의^모핵^환이^절개^부위의

염기사이에서 staking 상호작용을형성하며삽입형태로결합하

고있는지, 리간드가활성부위잔기와근접하여결합하고있는 지의두가지를중심으로분석하였다.

실험결과 및 고찰

X-선결정구조리간드의재현성실험

재현성 실험은 docking을 수행하기 전에 사용한 docking

protocol^에^의해^본래의^결정^구조가^얼마나^정확하게^재현되는

지를검증하기위한실험으로, X-^선^결정^구조의^리간드와^그^리

간드를 docking하였을때를비교하는것이다. Topo I-DNA 절개 가능복합체의 X-^선^결정^구조 1SC7.pdb ^에서^리간드를^제거하

고그구조에제거한리간드를 docking하였다. 원래결정구조 에 docking한결과모두리간드가 DNA 염기와 stacking 상호작 용을했고, intercalative mode^로^{결합하였다}. RMSD^는 1.48 Å^으

로 Surflex-Dock의좋은재현성을확인하였다(Fig. 2).

Topo I-DNA절개가능복합체와indenoisoquinoline유도체의 docking결과분석

Docking에사용한 indenoisoquinoline 유도체의구조와그 Topo I ^저해^활성값을 Table I^에^{나타내었다}. NSC314622^를^기준으로

하여 ++는 NSC314622와유사한활성을보이는것이고, +++, ++++는 NSC314622보다높은활성을나타내는것이며, +, ±

는 NSC314622^보다^약한^{활성이거나}^활성이^없는^것이다.

NSC314622의docking결과 −Docking 결과 NSC314622는

DNA^의^끊어진^가닥의 +1/-1 ^염기(G11/T10) ^사이에 intercalative mode^로^{위치하였으며} methylenedioxy^기가^있는 D ^환은^끊어진

가닥에향해있고 dimethoxy기가있는 A 환은끊어지지않은가 닥으로향하였다.

NSC314622^와 Arg364, Asn722^사이의^{수소결합은} Topo I^을^저

해하는중요한상호작용으로생각된다(Fig. 3). Arg364는 CPT

내성에관여하는잔기로 CPT^와^비슷한^{작용기전을}^가지는 Topo I ^{저해제와도}^중요한^결합을^할^것으로^예상되며, Asn722^를 Ser

으로 mutation하여약물내성을시험해보았을때 CPT와마찬 가지로 NSC314622^도^또한^교차내성(cross-resistance) ^현상을

나타내었다.¹⁰⁾

NSC314622보다높은활성을갖는화합물의docking결과 −

NSC314622^보다^활성이^좋은^화합물은^{공통적으로} DNA^의 +1/

-1 ^염기(G11-T10) ^사이에서^염기^쌍과^평행하게 intercalative mode

결합을하였으며인접한 Topo I 잔기들과수소결합을이루었다.

NSC314622, compound 3, 6, 8^은 N6 ^치환기가 minor groove^의

빈공간으로향하고있는데, 빈공간이 N6 치환기들이들어갈정 도로충분하고주위의 Topo I이나 DNA 잔기들과상호작용이가 능하기때문에모핵의염기쌍사이에서의 stacking ^{상호작용을}

방해하지않는다는전제하에, 유의한결합양상으로판단되었다.

모두 Arg364와 수소결합을 형성하고 있었고 A, D 환에

dimethoxy^기, methylenedioxy^기가^치환된^{화합물들은}^공통적으

로 methylenedioxy기가결합되어있는 D 환이끊어진가닥방 향으로향하여 Asn722와수소결합을형성하였다. 이는 methylenedioxy^기가 dimethoxy^기에^비해^{입체장애를}^덜^일으키기^때

문인것으로보여졌다. A, D 환에치환기가없는구조들은모핵

환의 stacking 상호작용을통한 intercalative mode에의존하고

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Fig. 2 −Docking result of MJ238 into Topo I-DNA cleavable complex. A, binding interactions of the crystal ligand MJ238 and its docked conformation at the active site; B, side view of the active site in the presence of the crystal ligand MJ238 and its docked conformation.

Active site generated by Connolly surface and colored by electrostatic potential. The crystal ligand MJ238 is illustrated in capped stick, and its docked conformation is in ball and stick. Carbon atoms in the crystal ligand MJ238 are in green, and those in the docked conformation are in magenta. All hydrogen atoms are in cyan. Hydrogen bonds are displayed as green dashed lines in the crystal ligand MJ238 and as black dashed lines in the docked conformation, and the participating residues are marked.

Table I −Structures of indenoisoquinoline analogues and their inhibitory activity on human Topo I-DNA cleavable complex

Compound R1 R2 R3 Topo I inhibition^a

1(NSC314622) OCH3 OCH2O CH3 ++¹¹⁾

2(NSC706744) OCH3 OCH2O (CH2)3NH(CH2)2OH ++++¹²⁾

3 OCH3 OCH2O (CH2)3NH(CH3)2 ++++¹⁷⁾

4 H H ++++¹⁾

5 H H +++¹⁾

6 OCH3 OCH2O (CH2)3NH2 +++¹¹⁾

7 H H (CH2)3NH(CH3)2 +++¹⁾

8 H H (CH2)4NH2 ++¹⁾

9 H OCH2O (CH2)3Br +¹⁾

10 H OCH2O (CH2)3NH2 +¹⁾

11 H H (CH2)2Cl ±¹²⁾

12 OCH3 OCH2O CH2CH3 ±¹¹⁾

MJ238 H H (CH2)2COOH N/A⁶⁾

aThe compounds were tested at concentration ranging up to 10^µM. The activity of the compounds to produce Topo I-mediated DNA cleavage was expressed semiquantitatively as follows: +: weak activity; ++: similar activity as NSC314622; +++ and ++++:

greater activity than NSC314622.

(5)

있고 N6 alkylamino group 치환기의추가적인상호작용으로삼 성분복합체의안정화를유지한다고추정되었다(Fig. 4).

NSC314622보다낮은활성을갖는화합물의docking결과 −

NSC314622보다활성이좋지않은화합물은공통적으로 DNA

염기사이에 intercalative mode^로^결합을^못하거나^{불안정하게}

한것으로나타났다.^모핵의^{일부분만이} DNA ^염기^위에^있거나 DNA 염기와평행을이루지못해서 stacking 상호작용을형성하 지못하였다.

Compound 9, 10은경우 D 환에만 methylenedioxy기가치환 되어있는형태이고, compound 11은 A, D 환양쪽에모두치 환기가없다. ^이로^인해 stacking ^{상호작용이}^약해지고^활성^부

위잔기들과의수소결합이줄어듦과함께 R3의치환기가수소 결합을 이루지 않거나, ^적절하지 ^않은 ^{수소결합을} ^이루어 intercalating mode^를^이루지^못했다. Compound 12^는 A, D ^환

에각각 dimethoxy기와 methylenedioxy기가치환되어있으나

R3^의 ethyl^기가^모핵의 stacking^을^방해하여^역시 intercalating mode^를^{만족시키지}^못하였다(Fig. 5).

결 론

본 연구에서는 non-camptothecin 계열의 Topo-I 저해제인

indenoisoquinoline ^{유도체들을}^인간 Topo I-DNA ^절개가능^복합

Fig. 3 −Docking result of compound 1 into human Topo I-DNA cleavable complex. A, binding interactions of compound 1 at the active site.

Compound 1 is illustrated in ball and stick, protein and DNA are in capped stick. Carbon atoms in compound 1 are in magenta.

Hydrogen bonds are displayed as black dashed lines, and the participating residues are marked; B, side view of the active site in the presence of the docked compound 1. Multi-channel MOLCAD surface (Connolly) was generated for the protein and DNA and colored by cavity depth, where the deepest cavity of the hydrophobic pocket is in orange. MOLCAD surface (van der Waals) was generated for compound 1, where the hydrogen donor is in red and the hydrogen accepter is in blue.

Hydrogen bonds are displayed as black dashed lines, and the participating residues are marked; B, side view of the active site in the presence of the docked compound 3. Multi-channel MOLCAD surface (Connolly) was generated for the protein and DNA and colored by cavity depth where the deepest cavity of the hydrophobic pocket is in orange. MOLCAD surface (van der Waals) was generated for compound 3, where the hydrogen donor is in red and the hydrogen accepter is in blue.

(6)

체인 1SC7.pdb^에 docking^하여^{결합양상을}^{분석하였다}. Indeno- isoquinoline 유도체들을선도화합물인 NSC314622보다활성이 더좋은화합물과좋지않은화합물로분류하여분석한결과, NSC314622^보다^활성이^좋은^{화합물들은}^대부분 DNA ^염기^사

이에 intercalative mode로결합하는것으로나타났다. 몇몇화 합물들은모핵의 N6^의^위치가 X-^선^결정^구조와^달리 minor

groove로 향해 있었으나 모핵 환이 DNA 염기 사이에서

intercalative mode로결합하고주위의아미노산들및 DNA와수 소결합을형성하고있어타당한 결합양상으로판단되었다. NSC314622보다 활성이 좋지 않은 화합물들은 intercalative mode로결합하지못하거나수소결합형성에실패하였다. R1, R2

모두에각각 dimethoxy^기와 methylenedioxy^기가^치환된^경우 stacking과수소결합을통해 intercalative mode를잘형성하고 R1,

R2에치환기가없을경우에도 R3의치환기가적절할경우모핵

의 stacking^과 R3^의^{수소결합만으로} intercalating mode^를^안정시

켜주고, R1, R2에치환기가없고 R3의치환기도적절하지못할 경우 intercalating mode^를^유지하지^못하는^것으로^나타났다.

이상의결과로부터결합양상과 Topo I ^활성^저해^효과^사이에

밀접한상관관계가있음을발견하였고 docking 연구에서제시된 결합양상은새로운 non-CPT 약물을개발하는데에구조적정보

를제공하여 Topo I^을^표적으로^하는^항암제^개발에^기여할^수

있을것으로판단된다.

감사의 말씀

본연구는 2^단계^두뇌한국 21 ^사업, ^{이화여자대학교}^세포신호

전달계바이오의약연구센터를통한교육과학기술부/^{한국과학재}

단국가핵심연구센터, 한국학술진흥재단(No. KRF-2005-005- J01501), ^{이화여자대학교} Research Grant of 2006^의^지원으로

수행되었습니다.

참고문헌

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