BBA - Ulasan tentang Cancer 1874 (2020) 188410

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Review

Human MYC G-quadruplex: Dari penemuan hingga target terapi kanker T

a,⁎
Wenmeng Wanga, Shuangli Hua, Yaru Gua, Yunxiao Yana, Daniel B.Stovallb, Dangdang Li ,
a,
Guangchao Sui ⁎

a b
Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Science, Northeast Forestry University, Harbin 150040, China
College of Arts and Sciences, Winthrop University, Rock Hill, SC 29733, Amerika Serikat

INFO ARTIKEL
Kata kunci:
MYC
Ekspresi gen
G-quadruplex (G4)
Agen pengikat G4
peptida
terapi kanker
1. Pendahuluan
ABSTRAK
Ekspresi berlebihan dari onkogen MYC adalah ciri molekuler dari inisiasi dan
perkembangan kanker. Target MYC adalah strategi terapi kanker yang logis dan
efektif. Struktur sekunder DNA khusus, G quadruplex (G4), terbentuk di dalam
wilayah nuclease hipersensitivitas elemen III 1 (NHE III1), terletak di hulu promotor
P1 gen MYC yang menggerakkan sebagian besar transkripsi. Menargetkan struktur
G4 seperti itu telah menjadi fokus terapi antikanker dalam beberapa dekade
terakhir. Dengan demikian, tinjauan komprehensif terhadap struktur MYC G4 dan
perannya sebagai target terapi potensial tepat waktu. Dalam ulasan ini, pertama-
tama kami menguraikan penemuan struktur MYC G4 dan bukti pembentukannya
secara in vitro dan dalam sel. Kemudian, kami menjelaskan peran fungsional G4
dalam mengatur ekspresi gen MYC. Kami juga merangkum tiga jenis protein yang
berinteraksi dengan MYC G4 yang dapat mempromosikan, menstabilkan, dan
melepaskan struktur G4. Akhirnya, kami membahas molekul pengikat G4 dan
aktivitas antikanker dari ligan penstabil G4, termasuk senyawa molekuler kecil dan
peptida, dan menilai potensinya sebagai terapi antikanker baru.
dan metastasis, yang menyebabkan hasil klinis yang buruk [8]. Karena
perannya dalam
mengatur transkripsi global [11], downregulasi MYC adalah mantan
Kanker adalah penyakit utama yang menimbulkan ancaman serius bagi
kesehatan manusia di seluruh dunia, terutama di negara berkembang [1,2].
Pada tingkat molekuler, kanker adalah kelainan genetik dan kejadiannya
terkait erat dengan ekspresi menyimpang dari penekan tumor dan onkogen
[3,4]. Di antara onkogen ini, MYC diekspresikan secara berlebihan hingga
70% dari semua kanker pada manusia, dan terkait dengan sekitar 70.000
kematian per tahun di Amerika Serikat [5–7]. Produk gen MYC adalah
faktor transkripsi utama yang mengikat E-box tertentu di promotor target
saat membentuk heterodimer dengan MAX dan mengontrol ekspresi
banyak gen target, penting untuk banyak kemajuan fisiologis, seperti
proliferasi sel, diferensiasi, adhesi , apoptosis, angio genesis dan
metastasis [6,8-10]. Dengan demikian, peningkatan level MYC secara
langsung berkontribusi pada ekspresi gen yang berubah yang mendorong
perkembangan kanker yang
diharapkan dapat mengurangi proliferasi dan kelangsungan hidup semua
jenis sel. Namun, penghambatan transkripsi MYC menyebabkan hilangnya
fenotipe ganas secara dramatis di sebagian besar sel tumor dengan sedikit
atau tanpa toksisitas pada sel normal [12-15]. Dengan demikian,
penargetan MYC menjanjikan sebagai strategi yang efisien dan aman
dalam pengobatan kanker pada manusia.
Ekspresi MYC yang menyimpang pada kanker terutama diatur pada
tingkat transkripsi [16]. Unsur hipersensitivitas nuklease III 1 (NHE III1),
terletak di hulu promotor P1 MYC, berkontribusi pada 80-90% transkripsi
MYC [16-18]. Khususnya, elemen NHE III1 mengandung daerah kaya G
yang dapat dilipat menjadi struktur sekunder DNA tertentu yang dikenal
sebagai G-quadruplex (G4), yang secara negatif mengatur transkripsi
[19,20]. Sebagai salah satu struktur DNA non-kanonik, G4 umumnya terdiri
dari tiga atau lebih G-quartet (juga dikenal sebagai G

Singkatan: ADAR1, Adenosine deaminase yang bekerja pada RNA 1; AML, Acute myelocytic leukemia; BCL2, B cell leukemia / lymphoma 2 ; BLM, protein sindrom
Bloom; CD, Circular dichroism; ChIP, Chromatin immunoprecipitation; CLSM, Mikroskopi pemindaian laser confocal; CNBP, protein pengikat asam nukleat jari seng tipe
CCHC; DDX5, Asp-Glu-Ala-Asp (DEAD) -460 kotak polipeptida 5; DMS, Dimetilsulfat; EMSA, Uji pergeseran mobilitas elektroforetik; FRET, transfer energi resonansi
fluoresensi; FUSE, elemen urutan hulu jauh; G4, G-quadruplex; HIF-1α, Faktor yang diinduksi hipoksia 1-alfa; hnRNP K, Ribonukleoprotein nuklir heterogen K; hPARP-
1, poli (ADP-ribosa) polimerase-1; ITC, kalorimetri titrasi isotermal; NAD, Nicotinamide adenine dinucleotide; NCL, Nucleolin; NF Y, Nuclear factor Y; NHE III 1, Nuclease
hipersensitivitas elemen III1; NMR, Resonansi magnetik nuklir; NPM1, Nukleofos min; PDGF-A, faktor pertumbuhan A yang diturunkan dari trombosit; RBD, domain
pengikat RNA; RECQ5, RecQ seperti helicase 5; RGG, Arginin-glisin-glisin; SP1, Kekhususan protein 1; SPR, Resonansi permukaan plasmon; TNBC, kanker payudara
triple-negatif; TSS, Situs inisiasi Transkripsi; WRN, protein sindrom Werner; XRCC1, perbaikan silang sinar-X melengkapi 1; ε-PLL, ε-poly-l-lysine; α, ε-PLL, α, ε-poly-l-
lysine; 2-Ap, 2-aminopurine
⁎
Sesuai penulis.
Alamat email: lidd@nefu.edu.cn (D. Li), gcsui@nefu.edu.cn (G. Sui).

https://doi.org/10.1016/j.bbcan.2020.188410
Diterima 29 Juni 2020; Diterima dalam bentuk revisi 21 Juli 2020; Diterima 21 Juli 2020
Tersedia online 19 Agustus 2020
0304-419X / © 2020 Elsevier BV Semua hak dilindungi undang-undang.
W. Wang, dkk. BBA - Review tentang Cancer 1874 (2020) 188410
Gbr. 1. Struktur G-quadruplexes. A.Struktur kuartet G yang dibentuk oleh susunan koplanar empat guanin yang dipegang oleh pasangan basa Hoogsteen antara N1
dan O6 mereka (disorot dengan warna merah), dan distabilkan oleh kation logam univalen pusat (M +), dengan preferensi untuk monovalen kation dengan urutan K + >
Na+ > Li+. B dan C. Varietas topologi dari G-quadruplexes intramolekul dan antarmolekul. D. Daerah loop dari struktur G4. Kepala panah menunjukkan arah untaian
DNA; guanine (G) terlihat pada gambar dengan lingkaran biru.

tetrad), masing-masing terdiri dari empat guanin planar yang dihubungkan 2

oleh ikatan hidrogen Hoogsteen dan distabilkan oleh kation monovalen
(terutama K+) di pusatnya (Gbr. 1A) [21-24]. Baru-baru ini, sebuah studi G4
ChIP-seq menggunakan BG4, sebuah antibodi yang mengenali struktur G4,
mengungkapkan sekitar 10.000 struktur G4 di promotor dan 5 'daerah yang
tidak diterjemahkan dari gen yang sangat ditranskripsikan dalam genom
manusia [25,26]. Selain itu, banyak penelitian menunjukkan bahwa struktur
DNA khusus ini dapat langsung divisualisasikan dalam sel manusia oleh
antibodi spesifik G4 (BG4 dan 1H6) dan probe khusus. Secara fungsional,
struktur G4 terlibat dalam berbagai proses biologis, termasuk pemeliharaan
telomer, transkripsi, replikasi DNA, terjemahan, stabilitas mRNA, dan
banyak lagi [21,27-31]. Para promotor banyak gen mengandung struktur
G4. Di antara mereka, yang paling banyak dipelajari dan paling
berkarakteristik adalah MYC G4 [32]. Yang penting, beberapa kelompok
baru-baru ini menemukan sejumlah besar ligan pengikat MYC G4,
sebagian besar senyawa molekul kecil dan gelombang pasang, dengan
aplikasi potensial dalam terapi kanker [12,33-39]. Saat ini, penekanan
transkripsi MYC melalui stabilisasi struktur G4 yang dimediasi ligan
merupakan rute yang menarik dalam pengobatan kanker
W. Wang, dkk. BBA - Review tentang Cancer 1874 (2020) 188410
[12,33,40]. Dalam ulasan ini, kami merangkum penemuan, karakterisasi
struktural, fitur fungsional dan protein pengikat MYC G4, dan
mendiskusikan ligan spesifik yang menargetkan struktur MYC G4 sebagai
terapi kanker yang menguntungkan.
2. Penemuan dan karakteristik struktural utama MYC G4
Struktur asam nukleat memainkan peran penting dalam mengatur
berbagai fungsi sel dan peristiwa biologis. Sekarang sudah mapan bahwa
DNA dapat membentuk berbagai struktur sekunder selain struktur heliks
ganda tangan kanan dari DNA bentuk-B (B-DNA) [31]. Khususnya, urutan
DNA kaya G spesifik dalam genom dapat dilipat menjadi struktur G4 yang
sangat stabil [21,22,41]. Dalam beberapa tahun terakhir, struktur G4 telah
dipelajari secara lebih menyeluruh dalam kimia dan biologi, terutama
karena struktur molekulnya yang khusus dan beragam (Gambar 1B dan C).
Berdasarkan jumlah molekul DNA yang terlibat dalam pembentukan
struktur, struktur G4 dapat dikelompokkan menjadi dua kategori utama:
intramolekul (disebut juga unimolekuler) G4 yang dibentuk oleh satu
molekul DNA, dan antarmolekul
G4 dibentuk oleh dua atau lebih molekul DNA (Gambar 1B dan C)
[21,42,43]. Untuk kasus G4 intramolekul, motif yang dilestarikan adalah
G≥3N1-7G≥3N1- 7G≥3N1-7G≥3 (N mewakili nukleotida apapun). Jadi, promotor
yang mengandung empat saluran G kontinyu, masing-masing terdiri dari
setidaknya tiga guanin, dapat dengan mudah dilipat menjadi G4
intramolekuler [21,41]. Namun, Shklover et al. mengusulkan bahwa struktur
G4 antarmolekul mungkin hadir di daerah promotor gen [44]. Dailey dkk.
juga menunjukkan bahwa promotor yang mengandung lebih dari empat
saluran G dapat membentuk beberapa kuartet diskrit yang tetap dalam
kesetimbangan dinamis satu sama lain [45]. Dalam studi in vitro baru-baru
ini menggunakan ukuran eksklusi chromato graphy dan uji NuPAGE,
Rauser dan Weinhold mengungkapkan bahwa struktur multi-timer G4
benar-benar diubah menjadi G4 monomer dengan perlakuan 150 mM
NaOH diikuti dengan netralisasi menggunakanK + buffer[46]. Dengan
demikian, struktur G4 antarmolekul yang dibentuk oleh promotor dalam
dua penelitian ini dapat dihasilkan dari kondisi mental percobaan yang
tidak optimal dari uji in vitro, meskipun kondisi eksperimental ini lazim
digunakan dalam penelitian G4.
Selanjutnya, menurut orientasi relatif untai DNA selama proses
pelipatan, struktur G4 dapat mengasumsikan konformasi paralel,
antiparalel, hibrid, dan tingkat tinggi (Gbr. 1B) [21,47,48]. Selain itu,
keragaman topologi struktur ini juga muncul dari jumlah G-kuartet, sifat
loop, stoi chiometry dan polaritas untai, sudut torsi ikatan glikosidik, sifat
empat alur, dan lokasi linker dan loop. (termasuk loop di agonal, lateral dan
baling-baling) (Gbr. 1D) [21,49,50]. Keragaman struktural ini membedakan
struktur G4 satu sama lain dalam genom manusia, yang memungkinkan
kita merancang ligan spesifik yang dapat mengenali dan mengikat struktur
ini secara berbeda untuk memodulasi ekspresi gen yang sesuai.
Selanjutnya akan diringkas penemuan dan karakterisasi MYC G4 dalam
mengatur ekspresi gen MYC.
2.1. Bukti in vitro struktur MYC G4
Regulasi transkripsional dari gen MYC sangat kompleks dan
melibatkan empat promotor, termasuk P0, P1, P2 dan P3, dan dua lokasi
awal skrip trans (Gbr. 2A) [16,51-53] . Di antara promotor ini, sebagian
besar transkrip diproduksi oleh promotor P1 dan P2, dengan 75-90%
transkrip MYC berasal dari promotor P2 [51-53]. Selain itu, dua elemen cis
penting terletak di hulu promotor P1 dan P2, yang ditunjuk sebagai elemen
urutan hulu jauh (FUSE) dan NHE III 1, dan mengatur transkripsi MYC
dengan cara yang memikat (Gbr. 2A) [16,51]. FUSE adalah elemen cruise
control yang berfungsi sebagai sensor fisik dari transkripsi gen MYC yang
sedang berlangsung, sedangkan elemen NHE III 1 terlibat dalam
mengaktifkan dan membungkam transkripsi [16,51]. Banyak penelitian
telah berfokus pada elemen NHE III 1 , karena mengatur 80-90% dari
keseluruhan transkripsi MYC [16-18]. NHE III1 berisi urutan kaya purin 27-
bp (Pu27) yang terletak -142 hingga -115 bp di hulu promotor P1.
Khususnya, Pu27 terdiri dari lima traktat G con sekutif; tiga dari saluran ini
terdiri dari empat guanin dan dua saluran terdiri dari tiga guanin (Gambar
2A) [40,54], dalam menentukan potensi tingginya untuk membentuk
struktur G4.
Sejak tahun 1998, Simonsson et al. pertama kali mengusulkan bahwa
Pu27 yang kaya G dapat membentuk struktur G4 intramolekul dengan
kuartet G antiparalel dalamK + larutan[55]. Struktur G4 terdiri dari tiga
kuartet G yang terbentuk dari saluran-G 1, 2, 4 dan 5 yang dihubungkan
dengan loop diagonal pusat dan dua loop lateral. Namun, Siddiqui-Jain et
al. melaporkan bahwa wilayah ini dilipat menjadi setidaknya dua struktur
G4 yang berbeda [19]. Dimetilsulfat (DMS) dapat memetilasi N7 guanin
dalam ss dan dsDNA, tetapi N7 dalam struktur G4 ini tidak dapat diakses
oleh DMS karena pembentukan ikatan hidrogen Hoogsteen [56,57].
Dengan demikian, uji footprinting DMS dapat mengidentifikasi guanosin
mana yang dapat diintegrasikan ke dalam struktur G4. Dengan
menggunakan uji ini, struktur MYC G4 mayor melibatkan saluran-G 2, 3, 4
dan 5, dan pembentukan G4 minor melibatkan saluran-G 1, 2, 4 dan 5
(Gbr. 2A, B dan D) [19]. Kedua struktur G4 ini juga ditunjukkan untuk
membentuk struktur paralel tipe baling-baling intramolekul oleh kelompok
lain (Gbr. 2A, B dan D) [58]. Demikian pula, menggunakan footprinting
DMS,
W. Wang, et al. BBA - Review tentang Cancer 1874 (2020) 188410
3
dichroism melingkar (CD), dan pendekatan resonansi magnetik nuklir,
Yang et al. menunjukkan bahwa Pu27 dapat membentuk struktur G4
beruntai paralel "propeller-type" yang dominan di tramolekul oleh G-tracts 2
sampai 5 pada ujung 3 'dari urutan [59]. Menariknya, studi pencetakan kaki
DMS dari Sun dan Hurley menunjukkan struktur G4 beruntai paralel yang
melibatkan saluran-G 1 sampai 4 dalam sistem plasmid superkoil dalam
larutan air (Gbr. 2A dan C) [60,61]. Meskipun urutan Pu27 dari promotor
MYC dapat membentuk tiga jenis struktur G4 secara in vitro, struktur G4
paralel tipe baling-baling intramolekul utama melibatkan saluran G 2-5 (G7-
G9, G11-G13, G16-G18, dan G20-G22 ) dan berfungsi in vivo (Gbr. 2D)
[19]. Memang, Ambrus et al. mensintesis oligonukleotida 22-nukleo
pasang, dinamai Pu22, menggunakan urutan saluran-G 2-5 dengan G4,
G14 dan G23 diganti oleh T. Ketika diperiksa dengan resonansi magnetik
inti (NMR), Pu22 sebagian besar dapat membentuk konformasi G4 paralel
intramolekul (Gbr. 2A dan E) [20]. Struktur ini terdiri dari tiga kuartet guanin
dan tiga loop samping, dengan dua loop nukleotida tunggal dan satu loop
cleotida nu ganda, untuk menghubungkan empat untai guanin dan
menjaga struktur tetap stabil (Gbr. 2E). Urutan 3′-flanking membentuk
konformasi fold back stacking yang stabil yang membatasi ujung atas
struktur G4, sedangkan urutan 5′-mengapit menutup ujung bawah G4,
dengan adenin yang ditumpuk sempurna ke dalam kuartet bawah [20].
Yang penting, struktur G4 ini stabil secara termodinamika, dengan suhu
leleh yang serupa (> 85 ° C) dengan MYC Pu27 [20]. Konsisten dengan
struktur air yang dideteksi oleh NMR, Stump et al. baru-baru ini
menyelesaikan struktur utama Pu22 MYC G4 dengan difraksi sinar-X pada
larutan 2,35 Å [62]. Antara struktur berair dan kristal, mereka menunjukkan
perbedaan besar dalam konformasi nukleotida mengapit 5 'dan 3', tetapi
tidak pada struktur pusat G-kuartet. Baru-baru ini, hasil penelitian ini
didukung lebih lanjut oleh kelompok kami menggunakan CD, uji
pergeseran mobilitas elektroforetik (EMSA) dan penelitian pewarnaan DNA
perak [63-65]. Oleh karena itu, data ini sangat menyarankan bahwa NHE
III1 dalam promotor MYC membentuk struktur G4 paralel intramolekul stabil
utama yang melibatkan saluran-G 2-5 secara in vitro.
2.2. Bukti berbasis sel struktur MYC G4
Pembentukan struktur MYC G4 secara in vitro dalam kondisi fisiologis
telah dibuktikan dengan jelas. Selain itu, bukti dari berbagai kelompok
penelitian menunjukkan keberadaannya dalam sel hidup.
Pertama, BG4 adalah antibodi yang direkayasa dengan spesifitas tinggi
dan afinitas pengikatan ke struktur G4, tetapi pengikatan tidak terdeteksi ke
jepit rambut RNA, ssDNA atau dsDNA [27]. Hänsel-Hertsch dkk.
mengembangkan protokol G4 ChIP-seq menggunakan BG4 untuk
memetakan lokasi seluruh genom dari struktur G4 dalam kromatin sel
HaCaT, kera tinosit manusia diabadikan [25]. Data mereka memverifikasi
pengayaan struktur G4 dalam promotor MYC dalam sel HaCaT
dibandingkan dengan keratinosit epi dermal manusia normal. Kedua,
nukleolin (NCL) adalah fosfoprotein nukleol multi fungsi 100-kDa dengan
ikatan selektif pada struktur G4 beruntai paralel, terutama MYC G4 [66,67].
Yang penting, NCL hanya mengikat pada struktur G4 dari promotor MYC
daripada bentuk ssDNA atau dsDNA [66,67]. Studi ini memberikan bukti
yang kuat tentang keberadaan struktur MYC G4 dalam konteks seluler
menggunakan antibodi G4 dan protein pengikat G4. Dalam beberapa
tahun terakhir, probe fluorescent molekul kecil tertentu telah muncul
sebagai pendekatan alternatif untuk memvisualisasikan dan melacak
struktur G4 [21,30]. Misalnya, sensor "nyala" fluoresen yang peka terhadap
lingkungan mikro, disebut sebagai BTC f, dilaporkan oleh Panda et al.
karena kemampuannya untuk secara spesifik mendeteksi MYC Pu22 G4
versus bentuk dsDNA Pu22 dengan peningkatan intensitas fluoresensi dan
pergeseran biru [68]. Yang penting, mikroskop pemindaian laser confocal
(CLSM) dapat mendeteksi pewarnaan selektif oleh BTC f dalam inti sel
HepG2 [68]. Kemudian, Chauhan et al. mensintesis dua fluorescent
binaphthyl amines (ligan 8 dan 12) dengan dua cincin triazole. Ini memiliki
afinitas yang lebih tinggi ke MYC G4 dan berinteraksi dengan kuartet G
ujung 3, mungkin melalui restrukturisasi pembatasan TAA-3 [69]. Selain itu,
dua probe ini
Gbr. 2. Struktur G4 promotor gen MYC. A. Struktur promotor dari gen MYC dan elemen terkait yang berbeda. Unsur-unsur ini termasuk urutan 27-mer (Pu27) dari
wilayah NHE III 1 di hulu promotor P1 dan urutan yang dimodifikasi: (1) MYC-1245 berisi G-traktat nomor 1, 2, 4, dan 5, dan Nomor saluran-G 3 diganti dengan “TTTT”;
(2) MYC-1234 berisi G-tracts nomor 1, 2, 3, dan 4; (3) MYC-2345 berisi G-tracts nomor 2, 3, 4, dan 5, dan mutasi G4 menjadi T; (4) Pu22 (4/14 / 23T) berisi G-tracts
nomor 2, 3, 4, dan 5, dan mutasi G-ke-T pada G4, G14 dan G23. MENJADI. Gambar skema struktur MYC-1245, MYC-1234, MYC-2345, dan Pu22 (4/14 / 23T) G4.
Kepala panah menunjukkan arah untai DNA 5'-ke-3 '; Guanin, adenin, dan timin (G, A, dan T) masing-masing ditunjukkan oleh lingkaran biru, kotak tertutup, dan kotak
terbuka.

dipamerkan fluoresensi "turn-on" tanggapan dengan MYC G4 dan diwarnai 4

inti dengan warna biru dalam sel [69]. Pada 2019, kelompok lain
merancang probe guanosin fluoresen yang menembus sel, bernama ADG,
yang menunjukkan preferensi pengikatan ke MYC Pu22 G4 dibandingkan
dengan G4 dan dsDNA lainnya. ADG terikat ke dua terminal G-kuartet dari
MYC Pu22 G4 dan berinteraksi dengan saluran kaya G yang berdekatan
[70]. Menggunakan confocal micro scopy, ADG diamati menembus
membran sel dan terlokalisasi di inti sel dengan fluoresensi biru [70]. Selain
itu, Zhai et al. menghasilkan probe fluoresen menyala, 9CI, yang
menampilkan lektivitas tinggi dan fluoresensi yang kuat untuk MYC Pu22
G4 in vitro dan dalam sel [71]. Secara mekanis, 9CI dapat mengikat erat ke
loop T20-A22 dan pada kuartet G ujung 3. Khususnya, analisis spektral CD
mengungkapkan bahwa 9CI tidak mengubah struktur topologi MYC G4
[71]. Yang penting, probe ini memiliki sitotoksisitas yang dapat diabaikan
dalam sel dan fluoresensi yang diaktivasi G4 hampir hilang dalam lisat sel
[71]. Baru-baru ini, Deiana et al. mensintesis probe rotor light-up rakitan
khusus lokasi, 4b, dengan kekuatan pengikatan, sensitivitas, selektivitas,
dan stabilisasi yang menonjol untuk struktur MYC Pu22 G4 secara in vitro
[72]. Probe 4b ini juga terikat ke struktur MYC G4 melalui penumpukan
eksternal. Dalam sel HeLa, 4b bisa terlokalisasi secara istimewa di
kompartemen subnuklir yang kaya G4 [72]. Tiga kelompok tambahan juga
mengembangkan probe DAOTA-M2, IZFL-2 dan DDG1 untuk mendeteksi
struktur MYC G4 secara in vitro [73-75]; bagaimanapun, aplikasinya untuk
memvisualisasikan struktur G4 dalam sel hidup tidak divalidasi. Secara
keseluruhan, penelitian ini sangat menyarankan adanya struktur G4 di
promotor MYC dalam sel hidup. Selain itu, BTC f, ADG,
W. Wang, et al. BBA - Review on Cancer 1874 (2020) 188410
dua binaphthyl amine dan 4b, tetapi tidak 9CI, secara signifikan dapat
menekan ekspresi MYC melalui menstabilkan struktur MYC G4 [68-72].
Secara signifikan, 9CI menunjukkan spesifisitas, afinitas, dan fluoresensi
yang lebih tinggi daripada probe lainnya. Oleh karena itu, studi lebih lanjut
diperlukan untuk mengembangkan probe fluoresen molekuler kecil dengan
peningkatan spesifisitas, afinitas dan fluoresensi, serta kemampuan
menstabilkan struktur MYC G4, untuk aplikasi yang efektif dalam diagnosis
dan pengobatan kanker.
Secara keseluruhan, bukti yang cukup menunjukkan bahwa elemen
NHE III1 dalam promotor MYC membentuk struktur G4 paralel intramolekul
stabil utama baik in vitro maupun dalam sel hidup.
3. Peran fungsional MYC G4 dalam mengaturtranskripsi
motifG4 di promotor atau sekitar situs inisiasi transkripsi (TSS)
mengatur kemajuan transkripsi berbagai gen baik secara positif atau
negatif (Gbr. 3) [21,31,76,77]. Pertama, struktur G4 di hulu TSS gen dapat
memblokir transkripsi jika mereka mengganggu pengikatan RNA
polimerase II atau faktor transkripsi, tetapi mereka juga dapat mendorong
transkripsi dengan bertindak sebagai situs pengikatan untuk faktor
transkripsi tertentu atau protein pengikat G4, atau mempertahankan
keterbukaan. Konformasi DNA untai yang berlawanan dengan G4 (Gbr. 3A
dan B) [31,63-65,76,78]. Kedua, struktur G4 di hilir TSS untai pengkodean
gen umumnya meningkatkan transkripsi karena perannya dalam
mendorong inisiasi ulang transkripsi. Sebaliknya bila ditempatkan
Gambar 3. Pembentukan struktur G4 dalam promotor dapat mempengaruhi transkripsi gen. Setelah inisiasi transkripsi, gelembung transkripsi yang terbentuk secara
instan dapat memungkinkan segmen untai tunggal membentuk G-quadruplexes. Struktur A dan B. G4 yang terbentuk di hulu TSS dalam untai pengkodean atau
templat dapat mengarah ke transkripsi yang diaktifkan atau ditekan. C dan D. Selama pemanjangan transkripsi, struktur G4 yang terbentuk di hilir TSS dalam untai
pengkodean atau templat meningkatkan inisiasi ulang transkripsi atau blok pengenalan RNA polimerase II. TSS: situs inisiasi transkripsi; TF: faktor transkripsi; panah
merah: transkripsi ditekan; panah hijau: transkripsi yang diaktifkan.

Gambar 4. Diagram skematis dari efek protein pengikat MYC G4. A, B dan C. Protein mempromosikan, menstabilkan dan melepaskan struktur MYC G4, masing-
masing.

hilir TSS dalam untai template, motif G4 umumnya bertindak sebagai 5

penekan transkripsi melalui faktor transkripsi yang menghalangi yang
mengikat dsDNA atau memblokir pengenalan RNA polimerase II (Gbr. 3C
dan D) [31,76,78,79] . Dengan demikian, efek keseluruhan struktur G4
pada ekspresi gen bergantung pada fungsi protein pengikat, seperti faktor
transkripsi, RNA polimerase II dan komponen lain dalam mesin transkripsi.
Motif G4 pada untai cetakan dalam wilayah NHE III 1 terbentuk di hulu
TSS gen MYC, yang menunjukkan perannya dalam mengatur transkripsi
MYC. Studi sebelumnya menunjukkan bahwa beberapa
W. Wang, et al. BBA - ReviewCancer 1874 (2020) 188410
faktor protein dapat mengaktifkan transkripsi MYC melalui pengikatan
langsung ke wilayah NHE III 1 , termasuk spesifisitas protein 1 (SP1),
protein pengikat asam nukleat jari seng tipe CCHC (CNBP) dan memiliki
ribonukleoprotein nuklir yang erogenous K ( hnRNP K) [51,54]. Sebagai
faktor transkripsi yang penting, SP1 mengenali dan mengikat situs
pengikatannya di NHE IIIuntai gandatranskripsi 1, dan mengaktifkan
skripgen MYC [51,80]. Baik CNBP dan hnRNP K juga mempromosikan
ekspresi gen MYC melalui pengikatan pada untai kaya G dari NHE III 1 dan
untai kaya C komplementernya, masing-masing [51,81,82]. Berdasarkan
pengamatan tersebut, struktur G4 di wilayah NHE III 1 merepresi MYC
Transkripsi Genmelalui pemblokiran pengikatan faktor transkripsi tersebut.
Secara konsisten, Siddiqui-Jain et al. mendemonstrasikan bahwa mutasi
basis tunggal G-ke-A dalam wilayah Pu27 dapat menyebabkan
peningkatan aktivitas promotor MYC 3 kali lipat dalam garis sel tumor
melalui desta bilizing motif G4 [19]. Sebenarnya, mutasi individu dari
semua 14 guanine yang terlibat dalam pembentukan struktur MYC G4
dapat secara signifikan merangsang aktivitas transkripsi promotor MYC
[83]. Sejalan dengan pengamatan ini, pengobatan garis sel tumor dengan
ligan kecil yang menstabilkan G4 dapat secara signifikan menurunkan
transkripsi MYC [19,84]. Data ini sangat menyarankan bahwa struktur G4
spesifik yang dibentuk di wilayah NHE III 1 dari promotor MYC berfungsi
sebagai peredam transkripsi.
4. Regulasi oleh faktor protein yang mengikat struktur MYC G4 Struktur
G4 saja tidak dapat mengatur proses seluler tetapi membutuhkan
protein yang terkait [21,85,86]. Dalam beberapa dekade terakhir, banyak
protein telah diidentifikasi untuk menengahi transkripsi gen MYC melalui
pengikatan, promosi, stabilisasi, atau pelepasan motif G4-nya. Pada
bagian berikut, kita akan membahas mekanisme molekuler protein
pengikat MYC G4 dalam mengatur transkripsi gen MYC. Protein ini dapat
dibagi menjadi tiga kelompok berdasarkan fungsinya, termasuk mendorong
pembentukan G4, menstabilkan, atau melepaskan struktur G4 (Gbr. 4 dan
Tabel 1).
4.1. Protein yang mendorong pembentukan MYC G4
Di antara protein yang mendorong pembentukan struktur G4, nukleolin
(NCL) adalah salah satu yang paling banyak dipelajari. Seperti dijelaskan
di atas, NCL dapat secara selektif mengikat MYC G4 baik secara in vitro
maupun dalam sel. Gonza´lez dkk. pertama kali menunjukkan bahwa NCL
dapat mempromosikan pembentukan MYC G4 dan menstabilkan struktur
[66]. Mereka juga membuktikan RNA binding domain (RBDs) 3 dan 4, dan
domain arginine-glycine-glycine (RGG), bertanggung jawab untuk mengikat
dan menstabilkan MYC G4 [67]. NCL panjang penuh yang diekspresikan
secara ektopik atau wilayah minimalnya menghambat transkripsi yang
dimediasi oleh promotor MYC [66,67]. Secara mekanis, NCL dapat
mempromosikan pembentukan MYC G4 di wilayah NHE III 1 dan menekan
transkripsi MYC dengan menutupi situs pengikatan aktivator transkripsi,
seperti SP1.
Tidak seperti peran NCL yang dikarakterisasi dengan baik, regulasi
MYC G4 oleh CNBP tampak membingungkan. CNBP pertama kali dikenali
sebagai protein pengikat ssDNA yang kaya G dan pengatur potensial dari
elemen pengatur sterol [87,88]. Pengikatan in vitro ke ssRNA yang kaya G
juga dilaporkan [87,89]. Jadi, CNBP mengikat untai kaya-G dari NHE III 1
dan mengaktifkan transkripsi gen MYC. Namun, me chanisme molekuler
yang mendasari peraturan ini tidak diketahui. Borgognone dkk. melaporkan
bahwa ortolog CNBP di C. arenarum dapat mengikat ssDNA dari NHE III 1,
mendorong pelipatannya menjadi G4 paralel, dan kemudian terlepas dari
struktur. Dalam hal ini, pengikatan CNBP dapat membuat struktur DNA
yang relatif terbuka untuk memungkinkan akses faktor transkripsi dan
Jenis protein pengikat G4 Protein domain pengikat G4 Referensi
Protein mempromosikan pembentukan G4 NCL RBD 3 dan 4 dan domain RGG di wilayah C-terminal [66,67] CNBP Kotak RGG di wilayah terminal N dasar [87,90]
Protein mengikat dan menstabilkan struktur G4
ADAR1 The Z-DNA mengikat domain [109] P53 mutan Wilayah C-terminal (aa 320-393) [119,120]
Protein melepaskan struktur G4 PIF1 Domain helikase yang dilestarikan (aa 206-620, PIF1-HD) [129–131] BLM Wilayah sayap-β dari domain RQC [135]
W. Wang, et al. BBA - Reviews on Cancer 1874 (2020) 188410
addition, P53 has high affinity for various noncanonical DNA struc tures,
including G4 DNA, Holliday junctions, cruciforms, triplex DNA, T loops,
stem-loops, DNA bulges and hemicatenated DNA [113,114]. In these
processes, P53 interacts with multiple key regulators, including
transcription factors [115,116]. P53 mutants mostly deficient in DNA
Tabel 1
Ringkasan protein pengikat MYC G4.
mempromosikan re-inisiasi transkripsi, dengan efek keseluruhan dari
aktivasi gen MYC [87]. Chen et al. juga menunjukkan bahwa CNBP
manusia terikat pada NHE III 1 dan mengkatalisis transformasi topologi dari
ssDNA menjadi struktur G4. Akibatnya, CNBP dapat menyebabkan
penurunan sementara dan kemudian aktivasi transkripsi gen MYC dalam
sel [90]. Yang penting, pengikatan CNBP ke NM23-H2, protein dengan
aktivitas pelepasan G4, diperlukan untuk aktivasi transkripsional dari gen
MYC [90]. Secara mekanis, formasi G4 paralel yang dipromosikan CNBP
di wilayah NHE III1 dapat menjelaskan transkripsi yang direpresi
sementara, sedangkan NM23-H2 yang direkrut CNBP menyelesaikan
struktur G4 dan menyebabkan aktivasi promotor MYC [90].
Protein pemacu G4 lainnya adalah nukleofosmin (NPM1), sebuah
fosfoprotein bolak-balik cleocytoplasmic nu. NPM1 terutama terlokalisasi di
nukleoli, dan terlibat dalam pematangan dan ekspor ribosom, sentro
beberapa duplikasi, dan respons terhadap rangsangan stres [91-93].
Mutasi pada NPM1 C-terminal merupakan lesi genetik yang paling sering
pada leukemia myeloid akut, dan menyebabkan translokasi dari nukleus ke
sitoplasma karena sinyal ekspor nuklir yang dihasilkan oleh domain C-
terminal yang tidak distabilkan [92,94-96]. Domain C-terminal dari NPM1
berikatan dengan ssDNA dan ssRNA secara istimewa di atas dsDNA,
dengan urutannya masing-masing [97,98]. Terutama, 70 residu terakhirnya
(NPM1-C70) secara istimewa mengikat DNA G4, dan mendorong
pembentukan MYC G4 [99-101]. Data ini menunjukkan bahwa NPM1
menekan ekspresi gen MYC melalui pengikatan ke ssDNA NHE III 1 dan
mendorong pembentukan G4.
4.2. Protein mengikat dan menstabilkan struktur MYC G4
Di antara protein dalam kelompok ini, adenosin deaminase yang
bekerja pada RNA 1 (ADAR1) adalah enzim pengedit RNA untai ganda,
dan terlibat dalam berbagai proses biologis, termasuk deamina adenosin
menjadi inosin, Interferensi RNA dan pematangan mikroRNA [102-104].
Selain itu, ADAR1 juga merupakan protein pengikat Z-DNA klasik dengan
domain pengikatan Z-DNA yang sangat terkonservasi dan afinitas selektif
terhadap Z-DNA, yang merupakan DNA untai ganda kidal yang dibentuk
dalam urutan kaya G dan pirimidin / pirimidin / purin berulang, dan dapat
diinduksi olehnegatif [supercoiling105-108]. Kang et al. melaporkan bahwa
domain pengikatan DNA Z ADAR1 manusia dapat langsung mengikat MYC
G4, dan menstabilkan konformasi untai paralelnya, yang menyebabkan
ekspresi gen MYC tertekan [109]. Studi ini memberikan wawasan baru
tentang peran biologis protein pengikat Z-DNA dalam mengatur proses
yang dimediasi G4, seperti replikasi DNA, transkripsi, dan terjemahan.
Dengan demikian, menarik untuk mencari protein pengikat Z-DNA
tambahan yang mengikat ke struktur G4, karena kesamaan dalam urutan
pembentuk struktur dan fungsi biologis dari struktur ini [108,109].
Protein penstabil G4 lainnya adalah mutan P53. P53 adalah penekan
tumor yang paling sering bermutasi pada kanker manusia, dan
inaktivasinya memainkan peran penting dalam transformasi keganasan
[110,111]. Sebagai faktor transkripsi, transkripsi gen yang diatur oleh P53
bergantung pada hubungannya dengan elemen pengikatnya pada
promotor target [110,112]. Dalam
NPM1 70-aa NPM1 C-terminal domain [99-101]
LARK Domain RBM [121]
WRN Dua kunci aa (T1024 and T1086) within RQC domain [136]
DDX5 Not described [142]
NM23-H2 Not described [151,153,154]
PARP-1 The first zinc finger domain [155]
6
binding could impact gene transcription through associating with other
transcription factors, such as nuclear factor Y (NF-Y), P63 and P73
[113,117,118]. Petr et al. reported that the C-terminal region (amino acids
320-393) of P53 was responsible for binding to the MYC G4 structure with
high affinity [119]. P53 mutations were mostly identi fied in its DNA binding
domain, located in the middle region of the protein. Thus, most P53
mutants still retain their affinity for G4 structures. Quante et al. reported
that a P53 mutant could bind and stabilize MYC G4 in vitro [120]. The data
indicated that mutated P53 in cancer cells may still regulate gene
expression through modulating G4 structure stability.
Niu et al. recently identified LARK, an RNA recognition motif (RRM,
also known as RBM)-containing protein, as a protein to bind and sta bilize
the MYC G4 structure in vitro [121]. However, whether LARK keeps these
properties in a cellular environment was not tested. Im portantly, the
authors demonstrated that the RBM domain was indis pensable for the G4
binding activity of LARK, implicating the G4- binding potential of other
proteins containing this conserved domain.
4.3. Proteins unwinding the MYC G4 structure
Most G4 structures exhibit inhibitory effects on DNA replication,
transcription and translation. Thus, proteins with G4-resolving activ ities can
promote these processes [21,22]. Helicases are molecular motors that
open dsDNA during replication and transcription. Multiple helicases have
been reported to preferentially resolve noncanonical secondary structures,
including G4 motifs, Holliday junctions and tri plexes [78,122–124].
Helicases are divided into six superfamilies, SF1 to SF6, based on protein
sequence homology [78]. According to moving directions on DNA,
helicases can also be classified into type A (3′ to 5′) and type B (5′ to 3′)
[78]. All G4-helicases require a single-stranded tail at either the 3′ or 5′ end
to ensure their loading onto DNA substrates. Moreover, the G4 helicases
act catalytically, and require hydrolysis of nucleotide triphosphates,
normally ATP, and Mg2+ ions [78,122].
PIF1 is an ATP-dependent 5′ to 3′ DNA SF1 helicase and a member of
a highly conserved protein family. It contributes to telomere elon gation,
Okazaki fragment processing and genome stability maintenance [125–
127]. Many studies demonstrated the activity of PIF1 in un winding dsDNA,
double DNA/RNA hybrid strands and other secondary structures
[85,127,128]. Sanders first reported that human PIF1 could bind and
unwind the tetramolecular parallel G4 DNA structures in vitro, dependent
on its conserved helicase domain [129]. Noticeably, PIF1 showed higher
affinity for the G4 motif than that for ssDNA and dsDNA. In addition, prior to
unwinding G4 DNA structures, PIF1 needed to bind to an extended (> 10
nucleotides) 5′ ssDNA tail but not G4 motifs [129]. Other groups reported
that PIF1 unfolds an in tramolecular parallel G4 structure, such as MYC G4
[130,131]. Im portantly, PIF1-catalyzed unfolding of G4 DNA is highly
dependent on both substrate sequences and reaction conditions [131].
Another class of G4-helicases is the RecQ subfamily, comprising 3′ to
5′ DNA SF2 helicases conserved from prokaryotes to eukaryotes, with five
members, including RECQ1, RECQ4, RECQ5, Bloom syndrome protein
(BLM), and Werner syndrome protein (WRN) [78,132,133]. Among these
helicases, BLM and WRN have all three conserved RecQ domains,
including a helicase domain, a RecQ C-terminal (RQC) do main, and a
helicase-and-RNaseD C-terminal (HRDC) domain [78,134]. Among them,
the RQC domain is required for the G4-specific un winding activities of
these helicases [78,135,136]. Strikingly, if the intramolecular G4 is followed
by a single stranded region as short as 5- 10 nucleotides, BLM and WRN
helicases can unwind G4 in an ATP independent fashion, with cooperative
binding of the HRDC domain to
W. Wang, et al. BBA - Reviews on Cancer 1874 (2020) 188410
binding affinity to intramolecular G4 structures in the KRAS and c-KIT
promoters, and also increases its catalytic activity when binding to G4
structures [162,163]. Additionally, Feketa et al. showed that PARP1 could
bind to the MYC G4 structure and convert it into a tran scriptionally active
B-DNA form [155].
5. Ligands targeting the MYC G4 structure as clinical anticancer
therapeutics
G4 structures are attractive molecular targets in developing antic ancer
7
the 3′ ssDNA and the RQC domain to the G4 structure [78,137–139]. In
human cells, BLM, WRN and RECQ4 are of special interest due to their
involvement in genetic disorders, including cancers and premature ageing
[140]. As the first discovered G4 helicase, BLM can unwind both
intermolecular and intramolecular G4 structures [78,141]. Lee dkk. reported
the interaction of BLM's RQC domain with MYC G4, and re vealed the
importance of the RQC β-wing region in binding and un winding the G4
motif [135]. Another important member of this sub family is WRN, exhibiting
similar functions to BLM. Ketkar et al. reported that the RQC domain of
WRN could impart a 2-fold binding preference to G4 compared to non-G4
DNA substrates [136]. Using the NMR approach, they also identified two
key amino acids, T1024 and T1086, within the RQC domain of WRN,
involved in G4 binding and unwinding activities [136].
Recently, Wu et al. reported an RNA helicase named Asp-Glu-Ala Asp
(DEAD)-box polypeptide 5 (DDX5, also called p68) that could be extremely
proficient at unwinding the MYC G4 structure [142]. As a prototypical
member of the large ATP-dependent RNA helicases family, DDX5
participates in a broad spectrum of biological processes, such as
transcription, RNA processing (eg alternative splicing and RNA transport),
DNA replication, and ribosome and miRNA biogenesis [143–145]. The
DDX5 protein has a core helicase domain that is composed of two RecA-
like domains known as domain 1 (D1) and D2. D1 can bind to ATP, and D2
can recognize dsRNA structures [146,147]. DDX5 could also effectively
unwind the MYC G4 and other in tramolecular G4 structures. However, its
unwinding activity did not need a ssDNA tail nor direct participation of ATP
hydrolysis [142], indicating a distinct mechanism among known G4
helicases so far. In MCF-7 cells, DDX5 was enriched at the MYC promoter
and induced its transcription, which could be repressed by a G4-stabilizing
compound, TMPyP4 [142].
These data suggest that the helicases PIF1, BLM, WRN and DDX5 can
efficiently unwind MYC G4 structures in a catalytic fashion. In addition,
multiple proteins can destabilize G4 structures in a non-cat alytic fashion,
with unwinding processes independent of ATP hydro lysis. Unlike the
aforementioned helicases, these proteins resolving different types of G4
DNA or RNA structures have not been extensively investigated so far.
One of the proteins to disrupt G4 structures is NM23-H2, belonging to a
class of proteins involved in transcriptional activation, and ori ginally
identified as nucleotide diphosphate kinases [148,149]. In hu mans, NM23-
H2 was first identified as a cancer metastasis inhibitor and has been
substantially studied in recent years due to its role in tran scriptionally
activating the MYC gene through binding the NHE III 1 element in vitro and
in a cellular environment [148–150]. However, the exact mechanism of
NM23-H2 in promoting MYC gene transcription remains controversial. To
date, ample evidence has demonstrated that NM23-H2 could preferentially
bind the G- and C-rich single strands of NHE III 1, but not the dsDNA
[151,152]. However, Thakur et al. re ported the activity of NM23-H2 in
binding to MYC G4 and then un winding to ssDNA [153], which was
confirmed by two other studies [151,154]. These data strongly suggested a
functional role of NM23-H2 in promoting MYC gene transcription through
disrupting the G4 motif in NHE III 1. Thus, MYC G4 and NM23-H2
interaction may be a potential intervening point to suppress MYC
expression in cancer therapies.
PARP1, poly(ADP-ribose)polymerase-1, also regulates MYC gene
transcription through its interaction with the G4 structure [155]. PARP1
protein is abundant in the chromatin of eukaryotic cells and has a high
affinity for damaged DNA. It becomes catalytically active upon binding to
DNA breaks, and then cleaves nicotinamide adenine dinu cleotide (NAD +)
into nicotinamide and ADP-ribose, the latter of which can be conjugated to
nuclear acceptor proteins, including X-ray repair cross complementing 1
(XRCC1), histones, and PARP1 itself for auto regulation [156–159]. The
enzyme activity of PARP1 is affected by its interaction with DNA, single-
and double-stranded breaks, non-B-DNA structures and others
[156,160,161]. PARP1 exhibits high in vitro
drugs, since it is discretely globular in nature and structurally different from
dsDNA. As the MYC G4 structure functions as a silencer element of
transcription, ligands binding and stabilizing G4 can po tentially be used as
therapeutics to reduce MYC expression in cancer cells. Moreover, the
detailed NMR spectroscopic and crystallographic studies together with
computational analyses offered a clear under standing of the MYC G4
structure [20,62]. Using these approaches, many research groups
discovered a great variety of molecules with activities to bind and stabilize
the MYC G4 structure, including quin dolines [164–167], cationic
porphyrins [19,84,168–171], imidazoles [35,172], quinoxalines [36],
carbazoles [173–175], berberine deriva tives [176,177], isaindigotone
derivatives [154], bisaryldiketene deri vatives [178], cyclic naphthalene
diimide derivatives [179], methylene blue derivatives [180], amido
phthalocyanines [181], and others [182–187].
With the help of computational analysis and chem-biological assays,
four basic models of MYC G4 binding to its ligands were proposed,
including external stacking, intercalating, groove/loop/backbone binding,
and central channel binding (Fig. 5) [188–190]. In the external stacking
model, ligands with an extended planar aromatic system containing
delocalized π-electrons can stack on the 5′ and/or 3′ term inal G-quartet(s)
mainly through π-π interactions, since the extended planar aromatic
system is similar to a G-quartet in size and shape, and it is a rigid flat or
twisted surface (Fig. 5A). In the intercalating model, ligands can insert into
the interspace of two G-quartets (Fig. 5B). In the groove/loop/backbone
model, the side chains of ligands interact with the grooves or loops through
hydrogen bonds, which also promote water solubility of the ligands. The
positively charged amino groups of the ligands increase their binding
affinity for the grooves and nega tively charged backbone phosphates
through electrostatic interactions (Fig. 5C). In the central channel binding 8
model, small ligands with long, positively charged side chains lying at the less popular than the other three models. Most reported ligands are prone
negative electrostatic center of a G-quartet can stabilize the G4 structure, to binding to MYC G4 in an external stacking-manner. In the following
similar to the role of monovalent cations, including K +, Na+ and NH4+ (Fig. sections, we discuss the anticancer activities and mechanisms of several
5D). For any ligand, it would be difficult for it to insert between two G- typical G4-stabilizing ligands, including small molecular drugs and
quartets of a G4 structure due to its high stability and rigidity, but relatively peptides, that have been classified according to their binding patterns with
easy to bind to the G-quartets at its two ends. Thus, the intercalator binding MYC G4 (Table 2 and Fig. 6). Furthermore, we assess the potential of
is these ligands as novel anticancer therapeutics.

5.1. Small molecular drugs

In the past decade, many small molecular drugs, both natural and
synthetic compounds, have been identified to bind and stabilize the MYC
G4 structure. To date, a number of natural compounds have been reported
to repress MYC expression through stabilizing its G4 structure, such as
Fonsecin B, alkaloids, piperine and flavonoid Quercetin [182,191–193].
However, their binding affinity and specificity to MYC G4 were much lower
than the reported synthetic compounds. Thus, we focus on discussing the
anticancer activities and underlying mechan isms of important synthetic
G4-stabilizing compounds in this section.
Interestingly, several synthetic drugs bind MYC G4 through either of
two additional models, specifically the groove/loop/backbone binding and
central channel binding models. However, as described above, external
stacking on terminal G-quartets is the primary model of MYC G4-ligand
complexes. Based on the stacking interactions of these li gands, we further
divide them into three groups, of which the ligands bind the 5′, 3′ and both
terminal G-quartets (Fig. 5A).

5.1.1. Compounds binding to both 5′ and 3′ terminal G-quartets of G4

structures
Various synthetic small molecules have been identified to bind and
stabilize the MYC G4 structure at the 5′ and 3′ terminal G-quartets of G4
structures (Fig. 6A). Brown et al. discovered GQC-05, a 9-dimethyla
minoethoxy substituted ellipticine compound, that could bind MYC G4 with
high affinity when analyzed by CD, competition dialysis and sur face
plasmon resonance (SPR) approaches. Importantly, through sta bilizing
MYC G4 to repress MYC gene transcription, GQC-05 could cause cell
death of Burkitt's lymphoma [194]. However, GQC-05 also showed low
affinity to G4 structures in the promoters of other genes, such as B cell
leukemia/lymphoma 2 (BCL2), platelet-derived growth factor A (PDGF-A)
and hypoxia-inducible factor 1-alpha (HIF-1α) [194]. It could also bind to a
G4 structure in the pre-mRNA of BCL-X to modulate its splicing process
[195]. The combinatorial use of GQC-05 with Navitoclax, a BCL2/BCL-XL
inhibitor, could cause remarkable cytotoxicity in acute myelocytic leukemia
(AML) cells, more pro nounced than that of each molecule alone, or
Navitoclax in combina tion with cytarabine or doxorubicin [196]. The data
suggest that the combination of G4-stabilizing agents with clinical
therapeutics is an

Fig. 5. Schematic diagrams of four basic binding

models of ligands with G4. A. The external stacking
model with ligands stacking to the 5′, 3′ or both
terminal G-quartets of a G4 structure. B. The inter
calating model with ligands inserted between G
quartets. C. The groove/loop binding model with
ligands binding to the groove and loop of a G4
structure. D. The central channel binding model with
the ligands staying in the center of a G4 structure.
W. Wang, et al. BBA - Reviews on Cancer 1874 (2020) 188410
Table 2
Summary of MYC G4 ligands with antitumor activity
Types of ligands Target/binding models Compounds Tumor type/cell lines Pre-clinical/clinical stages References
Small molecular drugs Binding both 5′ and 3′ terminal G-quartets GQC-05 Burkitt's lymphoma (CA46) Pre-clinical [194] SYUIO-05 Burkitt's lymphoma (Ramos and CA46) Pre-clinical
[164]
Binding to 5′ terminal G-quartet T-BFQs Lymphoma (Raji) Lung cancer (A549)
Binding to 3′ terminal G-quartet
IZCZ-3 Squamous cell Carcinoma (SiHa) Pre-clinical with in vivo efficacy [172] QN-1 Triple-
MYC G4/NCL CX-3543 Most of cancers Phase II [203,204] MYC G4/NM23-H2 Compound 37 Squamous cell Carcinoma (SiHa) Pre-clinical with in vivo efficacy
Peptides Binding both 3′ and 5′ terminal G-quartets TH3 Cervical cancer (HeLa) Pre-clinical [ 38] Binding groove/loop/backbone KR12C Breast cancer (MCF-7) Pre-clinical [39] Not
described α,ε-PLL Not described Not described [211]
optional and promising strategy in cancer therapies.
SYUIO-05, a quindoline derivative, was reported to preferentially
stabilize MYC G4 with a negligible effect on telomeric G4. Thus, SYUIO 05
could induce cell senescence and block cell proliferation through
repressing MYC gene transcription. Additionally, SYUIO-05 could dis rupt
the binding between NM23-H2 and MYC G4, leading to down regulated
MYC expression [164]. Dai et al. used NMR to demonstrated a
stereometric structure with two SYUIO-05 molecules bound to top and
bottom G-quartets of MYC G4 [166].
Later on, the same research group designed three highly specific MYC
G4-stabilizing small molecules, 7a4, 19d and 22d. Among them, 7a4 is a
disubstituted quindoline derivative by introducing a second cationic amino
side chain and 5-N-methyl group based on the structure of SYUIO-05 (Fig.
6A) [165]. Importantly, 7a4 exhibited better stabi lity, binding affinity, and
selectivity to G4 structures (over dsDNA) than SYUIO-05 in vitro [165]. In
molecular docking studies, 7a4 retained its ability to bind to the two terminal
G-quartets of MYC G4, as SYUIO-05 did; meanwhile, its crescent aromatic
core also stacked onto two gua nine residues of both G-quartets. Through
this binding, the 5-N elec tropositive center of SYUIO-05 overlapped with
the cation channel of G4, acting as a monovalent cation, such as K + to
stabilize MYC G4 [165]. Moreover, 7a4 has two amino acid side chains
forming a V-shape and stretching toward two grooves. Thus, the complex
model greatly increases the binding stability, affinity and selectivity of 7a4
to MYC G4. Functionally, 7a4 could decrease MYC gene expression
through stabilizing its G4 structure and disrupting NM23-H2/G4 interaction
in Burkitt's lymphoma cells. This anticancer activity was also verified in a
tumor xenograft mouse model [165]. In addition to quindoline deri vatives, a
series of new isaindigotone derivatives were also designed. Among them,
19d and 22d could specifically bind to MYC G4 and prevent its interaction
with NM23-H2-G4 [154]. As for the binding model, these two molecules
could bind to MYC G4 with similar end stacking interactions [154].
Moreover, the two phenyl groups of their structures were twisted, which
further induced two cationic amine side chains to bind the negative
phosphate backbone through electrostatic interactions [154]. Likewise, 19d
and 22d also exhibited significant anticancer activity in SiHa cells [154].
Panda et al. used magnetic nanoparticle-linked MYC G4 as a tem plate
for covalently assembling potent azide-alkyne partners to gen erate high-
affinity G4 ligands. They identified a triazole compound Tz1 (Fig. 6A) with
higher affinity for and better stabilization of MYC G4 over dsDNA than other
triazole derivatives [197]. Importantly, in col orectal adenocarcinoma
HCT116 cells, Tz1 could specifically inhibit MYC gene transcription through
stabilizing its G4 structure, leading to
W. Wang, et al. BBA - Reviews on Cancer 1874 (2020) 188410
7a4 Burkitt's lymphoma (Raji) Pre-clinical with in vivo efficacy [165]
19d, 22d Squamous cell Carcinoma (SiHa) Pre-clinical [154]
TZ1 Colorectal Adenocarcinoma (HCT116) Pre-clinical [197]
DC-34 Myeloma (L363) Pre-clinical [34]
IZTZ-1 Melanoma (B16) Pre-clinical with in vivo efficacy [35]
Pre-clinical with in vivo efficacy [167]
negative breast cancer (4T1) Pre-clinical with in vivo effic acy [36] Cz-1 Cervical cancer (HeLa)
Pre-clinical [174] ANTP Cervical cancer (HeLa) Pre-clinical [37]
[205] Stauprimide Renal cancer (RXF 393) Pre-clinical with in vivo efficacy [206]
9
cell apoptosis [197]. This study provides an innovative approach based on
DNA nano-templates, which may serve as an efficient strategy to generate
target-selective ligands in drug discovery.
In 2018, Calabrese et al. identified a trifluoromethyl-substituted
compound DC-34 from a library of benzofuran derivative analogs [34,198].
DC-34 exhibited strong affinity, remarkable binding affinity to MYC G4, and
could efficiently downregulate MYC gene expression in multiple myeloma
cells [34]. Unlike other G4-stabilizing compounds, DC-34 did not bind
dsDNA and showed relatively weak affinity to the G4 structures of other
tested oncogenes [34]. Analysis by NMR spec troscopy indicated that two
DC-34 molecules could independently bind to the 5′ and 3′ terminal G-
quartets of MYC G4 [34]. Ample evidence from this group indicate that DC-
34 is a potential therapeutic in mye loma treatments.
Most recently, Wu et al. designed an imidazole-benzothiazole con
jugate, IZTZ-1, based on the structures of QN-1 and 4l, two previously
reported G4 ligands [35,36,199]. As predicted, the core imidazole
benzothiazole structure of IZTZ-1 could stack on both 5′- and 3′-term inal
G-quartets of MYC G4 [35]. Meanwhile, the two extended posi tively
charged 1-methylpiper-azine groups could increase the binding affinity of
IZTZ-1 with negatively charged phosphate backbone via electrostatic
interactions. Through these interactions, IZTZ-1 could strongly stabilize the
MYC G4 structure [35]. Consistently, IZTZ-1 could reduce the proliferation
and migration of melanoma cells, and suppress tumor growth in a
melanoma mouse model through downregulating MYC expression [35]. In
addition, IZTZ-1 showed a desirable solubility and its core structure
possessed potential drug-likeness, suggesting its great potential as a
molecular drug in clinical treatment of melanoma.
5.1.2. Compounds binding to 5′ terminal G-quartets of G4 structures
Several highly specific MYC G4 stabilizing molecules have been reported
to exclusively bind the 5′ terminal G-quartets of G4 structures (Fig. 6B). In
2017, Zeng et al. designed a series of 11-triazole benzo furoquinoline
derivatives (T-BFQs) based on the chemical structure of quindoline [167].
Three of these compounds, 5a, 5h and 5l, exhibited the highest binding
affinity to MYC G4, but could still bind to other G4 structures [167]. Notably,
these compounds could significantly pro mote the formation of parallel MYC
G4 structures in the absence of cations, indicating their strong interaction
with MYC G4. In line with the results of the biochemical studies, the three
compounds could specifically inhibit MYC in human lymphoma cells
through stabilizing MYC G4 [167]. In addition, 5l could significantly reduce
tumor growth in a lung cancer xenograft mouse model [167]. Nevertheless,
the spe cificity of these molecules to MYC G4 versus other G4 structures
has not
Fig. 6. Chemical structures of compounds that directly or indirectly target MYC G4 structure. A. Compounds binding to both 5′ and 3′ terminal G-quartets of MYC G4
structure, including GQC-05, SYUIO-05, 7a4, 19d, 22d, Tz1, DC-34 and IZTZ-1. B. Compounds binding to 5′ terminal G-quartet of MYC G4 structure, including T BFQs,
IZCZ-3 and QN-1. C. Chemical structures of compounds binding to 3′ terminal G-quartet of MYC G4 structure, including Cz-1 and ANTP. D. Compounds indirectly
targeting MYC G4 structure, including CX-3543, stauprimide and compound 37.
10
 W. Wang, et al. BBA - Reviews on Cancer 1874 (2020) 188410
been explored.
In 2018, Hu et al. synthesized a four-leaf clover-like ligand, IZCZ-3,
which was designed through structural modification of aryl-substituted
imidazole/carbazole conjugates. IZCZ-3 could preferentially bind and
stabilize MYC G4 structure in vitro [172]. The molecular docking study
indicated that IZCZ-3 could perfectly stack on the 5′ terminal G-quartet plane of
G4 via π−π interaction. Moreover, the positively charged central imidazole moiety
of IZCZ-3 was located in the ion channel of MYC G4, contributing to its strong G4
binding affinity [172]. Likewise, a line of in vitro cell-based studies also
proved that IZCZ-3 could ef fectively downregulate MYC transcription
through specifically targeting its G4 structure, leading to growth inhibition of
SiHa cells, but not normal BJ fibroblasts and primary mouse mesangial
cells [172]. Fur thermore, IZCZ-3 exhibited a strong inhibitory activity of
repressing tumor growth in nude mice xenografted with human cervical
squamous cancer cells [172]. Thus, IZCZ-3 is a promising anticancer ligand
due to its high selectivity to MYC G4 structure.
Compared with IZCZ-3, a quinoline derivative, QN-1, showed rela tively
high selectivity to MYC G4 with several advantages, such as a drug-like
core structure, a new promising scaffold targeting MYC G4, and ease in
chemical synthesis [36]. After examination by different approaches,
including absorption titrations, CD analysis, NMR titrations and 2-
aminopurine (2-Ap) fluorescent titrations experiments, QN-1 was identified
as the most promising G4-stabilizing ligand with high se lectivity to MYC
G4 through stacking onto the 5′-end G-quartet, and also binding to the
grooves or loops with its two outstretched N-methyl piperazine side chains
[36]. Consistently, QN-1 showed a decent an ticancer activity against triple-
negative breast cancer (TNBC) in both cell-based and mouse model
studies. Importantly, QN-1-caused MYC repression was significantly
greater than that of other G4-mediated genes, such as BCL2, c-KIT, VEGF
and HRAS [36]. Moreover, the TNBC 4T1 cells with overexpressed MYC
were more sensitive to QN-1, as compared to normal cells. Of note, by
comparing to classical TNBC chemo therapeutics (eg doxorubicin,
paclitaxel and cisplatin) and a pan G4-binding molecule BRACO19, 2.5 μM
of QN-1 could completely repress the growth of 4T1 cells, but the other
compounds at this con centration could only eliminate less than 70% of the
cells [36]. These data indicate that QN-1 is an alternative and promising
agent in the treatment of TNBC.
5.1.3. Compounds binding to 3′ terminal G-quartets of G4 structures
Several compounds bind only to the 3′ terminal G-quartet to stabi lize the
G4 structure (Fig. 6C). Das et al. synthesized a series of car bazole
derivatives that could bind and stabilize MYC G4 [174]. Among these
compounds, Cz-1 exhibited prominent G4 versus dsDNA se lectivity,
especially to MYC G4. Cell-based experiments indicated that Cz-1 could
downregulate MYC expression through its binding and sta bilizing MYC G4,
and consequently promote HeLa cell apoptosis [174]. As a fluorescent
probe, Cz-1 is mainly localized in the nucleus with blue fluorescence [174].
When detected by the G4 antibody BG4 in an im munocytochemical study,
Cz-1 treated cells showed increased immuno positive foci compared to the
control cells, colocalized with the Cz-1 signal [27]. Cz-1 treatment showed
more BG4 foci than those in the control cells, and Cz-1 colocalized with
BG4-stained sites [174]. The results confirmed that Cz-1 could stabilize
and/or promote the forma tion of G4s in cancer cells. Therefore, this new
modulable carbazole scaffold exhibits high G4 binding affinity and
stabilizing activity with potent anticancer potential.
Another example is ANTP, an anthraquinone with a tri-N-methyl pyrrole
side chain, which was synthesized by Roy et al. through con jugating
anthraquinone pharmacophore with oligopyrrole carbox amides, and
showed remarkable binding affinity to various G4 DNA structures,
especially MYC G4 [37]. The discovery of this compound was based on
efficient π-π interaction of anthraquinone derivatives with G-quartets and
the capability of oligopyrrole moieties in binding to the grooves of dsDNA
and G4 DNA [37,200–202]. This dual recognition
W. Wang, et al. BBA - Reviews on Cancer 1874 (2020) 188410
an anticancer agent.
Also in 2018, Sengupta et al. designed a peptide, KR12C, by pruning
the G4-binding domain (FK13) of a naturally occurring human Cathelicidin
(LL37) protein [39,209,210]. KR12C could interact with MYC G4 in a
groove/loop/backbone binding model, and selectively stabilize the 5′-
propeller loop of MYC G4 via arginine-driven electro static-interactions at
the sugar-phosphate backbone [39]. The treatment of KR12C could reduce
the binding of SP1 and NM23-H2 to the NHE III 1 region, suggesting its
activity in stabilizing MYC G4. Consistently, KR12C could promote
11
ability of ANTP to G4 structures increased its potential selectivity of G4
DNA over dsDNA. Moreover, ANTP-mediated MYC G4 stabilization was
clearly verified by fluorescence titration, CD titration and melting, and Taq
polymerase stop assays [37]. Furthermore, ANTP exhibited marked
cytotoxicity in HeLa and HEK293T cells at 5.3 μM, but not in primary
NIH3T3 and HDFa cells at a concentration up to 100 μM, indicating its
excellent cancer cell selectivity [37].
Taken together, the above data from different groups suggest that
synthetic small molecules can inhibit MYC transcription by directly binding
the G4 in the MYC promoter using these three binding models, and
consequently stabilizing its structure, leading to significant antic ancer
effects in cancer cells and/or in mouse models. However, several
compounds can repress MYC transcription and inhibit cancer cell growth
through indirectly stabilizing the MYC G4 structure. As men tioned above,
NCL and NM23-H2 mediate MYC G4 folding or unfolding. Indeed, small
molecules specifically targeting the NM23-H2-G4 inter action, or inducing
NCL redistribution from nucleoli into the nucleo plasm were reported. For
example, a fluoroquinolone derivative, CX 3543, also known as quarfloxin,
is the first-in-class G4-interacting compound to reach the human phase II
clinical trial for neuroendo crine/carcinoid tumors [203]. CX-3543 could bind
to ribosomal DNA (rDNA) G4 and selectively disrupt its interaction with
NCL, thereby inducing NCL relocation into nucleoplasm, which could inhibit
DNA Polymerase I-mediated transcription and induce cancer cell apoptosis
[203]. After being relocated in nucleoplasm, NCL may also promote MYC
G4 formation and thus inhibit MYC transcription [203,204]. In addition,
several compounds repress MYC transcription through dis rupting the
interaction between NM23-H2 and MYC G4, such as a novel isaindigotone
derivative named compound 37 and stauprimide (Fig. 6D) [205,206]. The
anticancer activity of compound 37 was at tributed to its high specific
binding affinity to NM23-H2 protein, re sulting in effectively disrupting the
interaction of NM23-H2 with G4 and greatly decreasing MYC transcription
[205]. However, stauprimide could indirectly disrupt NM23-H2 and G4
interaction through blocking NM23-H2 nuclear translocation [206].
Therefore, disruption of MYC G4 interaction with its binding protein, such
as NCL and NM23-H2, re presents an alternative strategy to develop novel
anticancer ther apeutics.
5.2. Peptides
Although a large number of small molecules targeting MYC G4 were
reported, most of them have not been used in clinical applications due to
their off-target effects and narrow therapeutic windows [207]. In view of
these disadvantages, peptides can offer an alternative and promising
approach in designing G4-targeted anticancer therapeutics based on their
variability and plasticity of 3D structures, and potential in recapitulating
G4/protein interactions. These features can help re searchers to design
peptides with increased intracellular affinity and selectivity in targeting G4
structures, and relatively low off-target ef fects in regulating oncogene
expression [39]. In addition, peptides also have advantages in clinical
applications, including lower im munogenicity, minimal drug-drug
interaction, easy intra-tumoral dif fusion, fast blood clearance, and
excellent tolerability in patients [39,208]. So far, several peptides have
been reported to bind and sta bilize the MYC G4 structure.
In 2018, Dutta et al. synthesized a crescent-shaped thiazole tripep tide,
named TH3, with selective binding affinity for MYC G4 over other
investigated G4s or dsDNA [38]. Furthermore, the NMR analysis in dicated
that the extended planar structure of TH3 could interact with MYC G4
through binding to both 5′ and 3′ terminal G-quartets [38]. The AT-rich
capping structures of two terminal G-quartets are unique in MYC G4, and
thus determine TH3's specificity for the MYC G4 structure over other G4
structures. Consistently, TH3 significantly inhibited MYC gene transcription
through a G4-dependent mechanism, and caused S phase arrest and
apoptosis of HeLa cells [38], suggesting its potential as
apoptotic signaling through the E2F1/VEGF-A/ BCL2 axis in MCF-7 cells
[39]. Overall, the KR12C peptide could spe cifically target MYC G4 and
exhibit significant anticancer activity.
In addition to these two G4-stabilizing peptides, Marzano et al. re cently
reported a synthetic analogue of ε-poly-l-lysine (ε-PLL), named as α,ε-PLL,
binding to both MYC G4 and telomeric G4 structures with high affinity [211].
The ε-PLL peptide is a cationic biopolymer isolated from the marine
bacterium Bacillus subtilis with antibacterial and anticancer activity, and is
used worldwide as a food preservative [211,212]. Im portantly, both ε-PLL
and α,ε-PLL are used in the biomedical applica tions, such as helpers of
drug and gene delivery [213,214]. Thus, de veloping more specific peptides
based on amino acid sequences of α,ε PLL peptide and evaluating its
anticancer activity in cancer cells are interesting research topics in future
studies.
Notably, multiple G4-unwinding proteins have been reported to bind
and unfold the MYC G4 structure, leading to MYC gene activation and
malignant transformation. Hence, it is a logical strategy in dis covering
anticancer peptides through mapping the shortest binding domain of a G4-
binding protein and synthesizing peptides based on its sequence to
competitively disrupt the protein-G4 complex.
6. Conclusions and future perspectives
Cancer initiation and progression are often accompanied by altered
gene expression. Significantly, the MYC gene has acquired a formidable
oncogenic reputation based on its aberrant expression through elevated
and constitutive transcription, leading to unrestricted proliferation, impaired
apoptosis, enhanced angiogenesis and metastasis, and genomic instability
in a variety of cancers. Among the regulatory ele ments in the MYC gene,
the GC-rich NHE III1 region upstream of its P1 promoter has been identified
as the most critical regulator of its ex pression. Ample biochemical, cell-
based and in vivo evidence discussed in this review suggested that the GC-
rich NHE III1 element in the MYC promoter is able to form an intramolecular
parallel G4 structure. The MYC G4 functions as a transcription repressor
motif in MYC gene ex pression. The biological role of MYC G4 depends on
its interacting proteins that may promote G4 formation, stabilize their
structures, or unwind them. Stabilization of G4 structures by specific
ligands can affect their interaction with proteins leading to reduced MYC
tran scription. These ligands are thus potential anticancer molecules.
Especially, based on well characterized MYC G4, small molecules and
peptides can be designed to target this structure and potentially used as
promising anticancer drugs through downregulating MYC gene ex pression.
Although important progress has been made in targeting MYC G4, we
propose several prospective routes for future basic and clinical in
vestigations. First, the development of additional probes with high
specificity, affinity and fluorescence to detect MYC G4 elements in living
cells will expand the experimental tools available for studying these
important structures. Second, future studies should seek to iden tify
additional proteins that bind and stabilize MYC G4; we predict that these
insights will reveal novel therapeutic targets. Third, future work should also
design novel, stabilizing ligands with high specificity and affinity for MYC G4
and/or its associated proteins. We predict these molecules will possess
significant anticancer activity and low side-ef fects in the clinic. Fourth, it is
important to screen naturally occurring small molecules as G4-binding and
oncogene silencing agents from a
W. Wang, et al. BBA - Reviews on Cancer 1874 (2020) 188410
biologically relevant G-quadruplex element in the human c-MYC promoter. Implications
for G-quadruplex stabilization, Biochemistry 44 (2005) 2048–2058. [21] D. Varshney, J.
Spiegel, K. Zyner, D. Tannahill, S. Balasubramanian, The regula tion and functions of DNA
and RNA G-quadruplexes, Nat. Rev. Mol. Biol Sel. 21 (2020) 459–474.
[22] CK Kwok, CJ Merrick, G-quadruplexes: prediction, characterization, and bio logical
application, Trends Biotechnol. 35 (2017) 997–1013.
[23] Z. Tan, YH Hao, KW Zheng, Kinetics, conformation, stability, and targeting of G
quadruplexes from a physiological perspective, Biochem. Biofis. Res. Komun. (2020),
https://doi.org/10.1016/j.bbrc.2020.04.047.
[24] A. Virgilio, A. Russo, T. Amato, G. Russo, L. Mayol, V. Esposito, A. Galeone,
Monomolecular G-quadruplex structures with inversion of polarity sites: new topologies
and potentiality, Nucleic Acids Res. 45 (2017) 8156–8166.
[25] R. Hansel-Hertsch, D. Beraldi, SV Lensing, G. Marsico, K. Zyner, A. Parry, M. Di Antonio,
J. Pike, H. Kimura, M. Narita, D. Tannahill, S. Balasubramanian, G quadruplex
12
vast library of natural compounds. Finally, we need to evaluate the
anticancer activities and side-effects of G4-stabilizing ligands in com
binatorial use with clinical chemotherapeutics to synergistically elim inate
cancer cells. Collectively, the role of G4 structures in MYC reg ulation
provides fertile questions for future research.
Author contributions
WW, DL and GS conceived and wrote the manuscript. DBS critically
read the manuscript and provided constructive suggestions. WW designed
the tables and created the figures. SH, YG and YY searched and organized
literature. Semua penulis telah membaca dan menyetujui versi naskah
yang diterbitkan.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the Fundamental Research Funds for the
Central Universities (2572017BA10) to DL, the National Natural Science
Foundation of China (81802798) to DL and (81872293 and 81672795) to
GS, the China Postdoctoral Science Foundation (2018M631897) and the
Heilongjiang Postdoctoral Fund (LBH-Z16005) to DL.
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