Molecular Recognition of the Hybrid-Type G-Quadruplexes in Human Telomeres

molecules

Review Molecular Recognition of the Hybrid-Type G-Quadruplexes in Human Telomeres

Guanhui Wu 1 , Luying Chen 1, Wenting Liu 1 and Danzhou Yang 1,2,3,*

1 Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, 575 W Stadium Ave, West Lafayette, IN 47907, USA; [email protected] (G.W.); [email protected] (L.C.); [email protected] (W.L.) 2 Purdue Center for Cancer Research, 201 S University St, West Lafayette, IN 47906, USA 3 Purdue Institute for Drug Discovery, 720 Clinic Dr, West Lafayette, IN 47907, USA * Correspondence: [email protected]; Tel.: +765-494-8148

Academic Editor: Ramon Eritja Received: 23 March 2019; Accepted: 19 April 2019; Published: 22 April 2019

Abstract: G-quadruplex (G4) DNA secondary structures formed in human telomeres have been shown to inhibit cancer-speciﬁc telomerase and alternative lengthening of telomere (ALT) pathways. Thus, human telomeric G-quadruplexes are considered attractive targets for anticancer drugs. Human telomeric G-quadruplexes are structurally polymorphic and predominantly form two hybrid-type G-quadruplexes, namely hybrid-1 and hybrid-2, under physiologically relevant solution conditions. To date, only a handful solution structures are available for drug complexes of human telomeric G-quadruplexes. In this review, we will describe two recent solution structural studies from our labs. We use NMR spectroscopy to elucidate the solution structure of a 1:1 complex between a small molecule epiberberine and the hybrid-2 telomeric G-quadruplex, and the structures of 1:1 and 4:2 complexes between a small molecule Pt-tripod and the hybrid-1 telomeric G-quadruplex. Structural information of small molecule complexes can provide important information for understanding small molecule recognition of human telomeric G-quadruplexes and for structure-based rational drug design targeting human telomeric G-quadruplexes.

Keywords: human telomeres; G-quadruplex; G4; anticancer drug; solution structure; molecular recognition; rational drug design; hybrid-2; hybrid-1; epi-berberine; platinum-tripod

1. Introduction Human telomeres are essential DNA-nucleoprotein complexes capping the terminus of chromosomes to protect the chromosomes from end-to-end fusion and degradation [1,2]. Telomeres play an important role in genomic stability [3], aging [4], and cancers [5]. Human telomeric DNA consists of TTAGGG tandem repeats 5–20 kb in length, terminating in a 30–500 nucleotide single-stranded 30 overhang [6]. In human somatic cells, telomeric DNA undergoes a progressive shortening with every cell division because of the end-replication problem [7]. When the telomere length reaches a critical limit, the cell undergoes apoptosis or senescence. However, as one of the hallmarks of cancer, cancer cells counteract the progressive loss of the telomere length to achieve limitless replication potential [5,8]. One telomere length maintenance mechanism is provided by a reverse transcriptase called telomerase (Figure1), which is activated in 85–90% of cancer cells [ 9–11]. Another mechanism is the alternative lengthening of telomeres (ALT) pathway (Figure1), which maintains telomere integrity in 10%–15% of cancer cells that lack detectable telomerase activity [11–15].

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Figure 1.1.Biological Biological implications implications of targeting of targeting telomeric telomeric G-quadruplexes G-quadruplexes using G-quadruplex-interactive using G-quadruplex- ligands.interactive In ligands. human In tumors,human tumors, the free the single-stranded free single-stranded telomere telomere G-overhang G-overhang can becan extended be extended by telomerase,by telomerase, which which reverse reverse transcribes transcribes the template the temp regionlate ofregion its RNA of its subunit RNA (hTR), subunit or by(hTR), alternative or by lengtheningalternative lengthening of telomeres of (ALT), telomeres which (ALT), copies which telomeric copies template telomeric DNA template via homologous DNA via recombination. homologous G-quadruplex-interactiverecombination. G-quadruplex-interactive ligands can promote ligands the formation can promote of and the stabilize formation the telomeric of and G-quadruplex stabilize the structures,telomeric G-quadruplex thus inhibiting structures, telomere extension thus inhibiting processes telomere which extension are required processes for the indefinitewhich are growth required of humanfor the indefinite tumors. growth of human tumors. G-quadruplexes have been found to form in human telomeres and G-quadruplex formation inhibits G-quadruplexes have been found to form in human telomeres and G-quadruplex formation the activity of telomerase [16]. G-quadruplex formation is suggested to be related to replication stress inhibits the activity of telomerase [16]. G-quadruplex formation is suggested to be related to caused by oncogenic stimulation of hyperproliferation, or by genome instability, and helicases have replication stress caused by oncogenic stimulation of hyperproliferation, or by genome instability, been shown to play an important role in resolving G-quadruplex structures and G-quadruplex-induced and helicases have been shown to play an important role in resolving G-quadruplex structures and genome instability [17–19]. DNA G-quadruplexes are non-canonical four-stranded helical structures, G-quadruplex-induced genome instability [17–19]. DNA G-quadruplexes are non-canonical four- formed in DNA sequences, that are rich in guanine nucleotides [20]. They are assembled by stacked stranded helical structures, formed in DNA sequences, that are rich in guanine nucleotides [20]. They guanine tetrad planes (G-tetrad, Figure2a) in which four guanines are held together by Hoogsteen are assembled by stacked guanine tetrad planes (G-tetrad, Figure 2a) in which four guanines are held hydrogen bonds. Physiologically relevant monovalent cations, especially K+, are required to stabilize together by Hoogsteen hydrogen bonds. Physiologically relevant monovalent cations, especially K+, G-quadruplex structures by coordinating and forming electrostatic interactions with the eight guanine are required to stabilize G-quadruplex structures by coordinating and forming electrostatic carbonyl oxygen atoms of the two adjacent G-tetrads [21–23]. The therapeutic possibilities of interactions with the eight guanine carbonyl oxygen atoms of the two adjacent G-tetrads [21–23]. The targeting telomeric G-quadruplexes to inhibit telomerase were first reported in 1997 [24] and have therapeutic possibilities of targeting telomeric G-quadruplexes to inhibit telomerase were first been actively pursued [25–28]. For example, in this special issue [29], multiple reports are on reported in 1997 [24] and have been actively pursued [25–28]. For example, in this special issue [29], telomeric G-quadruplex-interactive ligands [30–37]. G-quadruplex-interactive ligands have been multiple reports are on telomeric G-quadruplex-interactive ligands [30–37]. G-quadruplex- shown to inhibit telomerase (Figure1) and induce apoptosis in cancer cells [ 38]. In addition, interactive ligands have been shown to inhibit telomerase (Figure 1) and induce apoptosis in cancer G-quadruplex-interactive ligands were also shown to inhibit the alternative lengthening of telomeres cells [38]. In addition, G-quadruplex-interactive ligands were also shown to inhibit the alternative (ALT) pathway which maintains telomere stability in a telomerase-independent manner [39–43], lengthening of telomeres (ALT) pathway which maintains telomere stability in a telomerase- and can thus avoid drug resistance of direct telomerase inhibitors through activating the ALT pathway independent manner [39–43], and can thus avoid drug resistance of direct telomerase inhibitors (Figure1)[ 44]. Therefore, human telomeric G-quadruplexes are considered as attractive cancer-specific through activating the ALT pathway (Figure 1) [44]. Therefore, human telomeric G-quadruplexes are drug targets. considered as attractive cancer-specific drug targets.

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Figure 2. SchematicSchematic illustration of G-tetrad and the foldin foldingg pattern of hybrid-2 and hybrid-1 telomeric G-quadruplexes. ( (aa)) Schematic Schematic representation representation of of G-tetrad. G-tetrad. Monovalent Monovalent cations cations (M (M+), such+), such as K as+ or K Na+ or+, Naare+ required, are required for the for G-tetrad the G-tetrad formation. formation. (b) Human (b) Human telomeric telomeric sequences sequences predominantly predominantly fold into foldtwo intohybrid-type two hybrid-type G-quadruplexes G-quadruplexes with an equilibrium with an equilibrium between hybrid-1 between hybrid-1(PDB ID: (PDB2HY9) ID: and 2HY9) hybrid-2 and hybrid-2(PDB ID: (PDB2JPZ) ID: forms. 2JPZ) The forms. human The humantelomeric telomeric sequen sequencesces used to used determine to determine hybrid-2 hybrid-2 (wtTel26) (wtTel26) and andhybrid-1 hybrid-1 (Tel26) (Tel26) structures structures are shown are shown on the on top. the St top.rand Strandpolarities polarities are indica areted indicated as the black as the arrows. black arrows.Glycosidic Glycosidic conformations conformations of guanine of guanine nucleotides nucleotides are marked are marked as asfollows: follows: synsyn-pink-pink and and antianti-red.-red. DiDifferentﬀerent nucleotidesnucleotides are representedrepresented asas follows:follows: Thymine, blue sphere; guanine, red sphere; and adenine, green sphere.

The formation formation of of G-quadruplexes G-quadruplexes in in the the single-stranded single-stranded 3′-overhang 30-overhang at the at thehuman human telomere telomere end endmost most likely likely involves involves intramolecular intramolecular G-quadrupl G-quadruplexex structures. structures. However, However, intramolecular intramolecular human telomeric G-quadruplexes are structurally polymorphic and and may may adopt different different conformations, + including two two equilibrating equilibrating hybrid-type hybrid-type structures structures [45–51] [45–51 in] inK+ Ksolutionsolution and andin cells in cells[52–54]. [52 –The54]. Thestructure structure polymorphism polymorphism appears appears to be to bean anintrin intrinsicsic property property of of the the highly highly conserved telomeric sequence in higher eukaryotes with a TTATTA looploop sequencesequence [[55].55]. Interconversion between didifferentfferent human telomeric G-quadruplexes appearsappears to be kinetically slow, albeit with small energy differences differences between didifferentfferent conformations,conformations, indicatingindicating aa high-energyhigh-energy intermediate(s)intermediate(s) [[45,56–59].45,56–59]. Our previous studies showed the humanhuman telomerictelomeric G-overhangG-overhang predominantly forms two + hybrid-type G-quadruplex structures (Figure(Figure2 2b)b) in in physiologically physiologically relevant relevant K K+-containing-containing solutions, solutions, + named hybrid-1 [[45,47,49]45,47,49] andand hybrid-2hybrid-2 [[46,50].46,50]. These two structures coexistcoexist inin KK+ solution in an equilibrium [[55].55]. Their Their st structuresructures are are unique in strand orientation, G-tetrad arrangements, loop arrangements, as well as 50′- and 30′-capping, compared compared to to the parallel structures formed in the crystalline form [[38]38] andand thosethose predominantlypredominantly foundfound inin thethe oncogeneoncogene promoterspromoters [[20].20]. The 26-mer wtTel26 sequence,sequence, whichwhich waswas thethe wild-typewild-type four-repeatsfour-repeats humanhuman telomerictelomeric sequence with flankingflanking segments at both ends, adopts hybrid-2hybrid-2 conformation (Figure(Figure2 2b,b, left) left) [ [46].46]. The first, first, third, and fourth G-tracts go in one direction and the second G-tract in thethe oppositeopposite direction. In addition, the the first, first, second, andand thirdthird G-tracts G-tracts are are connected connected with with two two TTA TTA lateral lateral loops, loops, while while the thirdthe third and fourthand fourth G-tracts G- aretracts linked are linked with a with TTA sidea TTA loop, side i.e., loop, a lateral-lateral-side i.e., a lateral-lateral-side loop arrangement. loop arrangement. The glycosidic The glycosidic bonds of guaninebonds of nucleotides guanine nucleotides in a G-tetrad in a can G-tetrad adopt can either adoptsyn oreitheranti synconformations. or anti conformations. In the hybrid-2 In the structure, hybrid- 2 structure, the top G-tetrad has reversed glycosidic conformations (syn:anti:syn:syn) from those of the bottom two G-tetrads (anti:syn:anti:anti) (Figure 2b, left). On the other hand, the 26-mer Tel26

Molecules 2019, 24, 1578 4 of 15 Molecules 2019, 24, x 4 of 15 thesequence top G-tetrad with modified has reversed 3′- and glycosidic 5′-flanking conformations segments adopts (syn:anti:syn:syn hybrid-1 conformation) from those (Figure of the 2b, bottom right) two[47]. G-tetradsThe first, second, (anti:syn:anti:anti and fourth) (Figure G-tracts2b, go left). in one On direction the other and hand, the third the 26-mer G-tract Tel26 in the sequence opposite withdirection, modified with 3a0 -side-lateral-lateral and 50-flanking segments loop arrangement. adopts hybrid-1 The top conformation G-tetrad has (Figure reversed2b, right)glycosidic [ 47]. Theconformations first, second, (syn:syn:anti:syn and fourth G-tracts) relative go in to one the direction other two and theG-tetrads third G-tract (anti:anti:syn:anti in the opposite) (Figure direction, 2b, withright). a side-lateral-lateral loop arrangement. The top G-tetrad has reversed glycosidic conformations (syn:syn:anti:synThe two hybrid-type) relative to telomeric the other G-quadruplexes two G-tetrads ( anti:anti:syn:antihave unique capping) (Figure structures,2b, right). determined by the flankingThe two and hybrid-type loop sequences telomeric together G-quadruplexes with the folding have topology. unique capping In the hybrid-2 structures, structure determined (Figure by the2b, flankingleft), the and5′-flanking loop sequences residues, together the second with TTA the folding lateral topology. loop, and In the the A21 hybrid-2 residue structure of the third (Figure TTA2b, left),reversal the loop 50-flanking are above residues, the top the G-tetrad, second TTA but are lateral not loop, well structured. and the A21 In residue contrast, of the a well-defined third TTA reversal T:A:T looptriad areis formed above theat the top 3 G-tetrad,′-end of hybrid-2 but are nottelomeric well structured. G-quadruplex In contrast, by the interactions a well-defined between T:A:T triadT8 and is ′ ′ formedA9 of the at thefirst 3 0TTA-end lateral of hybrid-2 loop, telomeric as well as G-quadruplex the T25 of the by 3 the-end interactions flanking betweensegment. T8 The and 3 A9-capping of the firststructure TTA lateralis specific loop, to asthe well hybrid-2 as the structure T25 of the and 30-end it is flankingnot possible segment. to form The in 3the0-capping hybrid-1 structure structure. is specificAdditionally, to the hybrid-2this capping structure structure and it isis not important possible tofor form the in stability the hybrid-1 for structure.the hybrid-2 Additionally,this structure, as cappingdemonstrated structure by ismutational important foranalysis the stability [46]. In for th thee hybrid-1 hybrid-2 structure structure, (Figure as demonstrated 2b, right), by both mutational 5′-end ′ ′ analysisand 3 -end [46 capping]. In the hybrid-1structures structure are well-defined. (Figure2b, The right), 5 -end both capping 5 0-end structure, and 30-end an capping adenine-triple, structures is ′ areformed well-defined. by the A3 Theresidue 50-end of the capping 5 -flanking structure, residues, an adenine-triple, the A9 residue is formedof the first by theTTA A3 strand-reversal residue of the ′ 5side0-flanking loop, and residues, the A21 the residue A9 residue of the ofthird the TTA first TTAlateral strand-reversal loop. The 3 capping side loop, structure, and the A:T A21 base residue pair, ′ ofis formed the third by TTA the lateralA25 of loop.3 -end The flanking 30 capping segment structure, and the A:T T14 base of the pair, second is formed lateral by loop the A25form. of 30-end flankingStructural segment data and of the small-molecule T14 of the second complexes lateral loop of form.the human telomeric G-quadruplexes can provideStructural important data information of small-molecule for understanding complexes sp ofecific the recognition human telomeric by small G-quadruplexes molecules and canfor providestructure-based important rational information drug design for understanding targeting human specific telomeric recognition G-quadru by smallplexes. molecules Only a handful and for structure-basedsolution structures rational are available drug design of ligand targeting comp humanlexes of telomeric human telomeric G-quadruplexes. G-quadruplexes Only a handful[60–63]. solutionThis review structures will focus are availableon the two of recent ligand soluti complexeson structural of human studies telomeric from G-quadruplexesour labs. We use [60 NMR–63]. Thisspectroscopy review will to elucidate focus on thethe twosolution recent structur solutiones structuralof a 1:1 complex studies frombetween our a labs. medicinal We use natural NMR spectroscopyproduct epiberberine to elucidate (Figure the solution 3a) and structures the hybrid-2 of a 1:1 telomeric complex G-quadruplex between a medicinal [63], and natural 1:1 productand 4:2 epiberberinecomplexes between (Figure 3aa) Pt-containing and the hybrid-2 small telomeric molecule G-quadruplex (Figure 3b) [and63], the and hybrid-1 1:1 and 4:2telomeric complexes G- betweenquadruplex a Pt-containing [62]. small molecule (Figure3b) and the hybrid-1 telomeric G-quadruplex [62].

Figure 3. The chemical structures of epiberberine (a) and Pt-tripod (b). Figure 3. The chemical structures of epiberberine (a) and Pt-tripod (b). 2. Molecular Recognition of Human Telomeric Hybrid-2 G-Quadruplex by Epiberberine 2. Molecular Recognition of Human Telomeric Hybrid-2 G-Quadruplex by Epiberberine Protoberberines are a class of isoquinoline alkaloids with antitumor activities [64,65]. Notably,Protoberberines berberine and are some a class of its of derivativesisoquinoline have alkaloids been shown with antitumor to stabilize activities telomeric [64,65]. G-quadruplexes Notably, andberberine inhibit and telomerase some of its activity derivatives [66]. have Epiberberine been shown (EPI) to was stabilize found telomeric to exhibit G-quadruplexes great fluorescence and enhancementinhibit telomerase induced activity by human [66]. telomericEpiberberine sequences (EPI) inwas K+ solutionfound to [ 67exhibit]. Our datagreat show fluorescence that EPI specificallyenhancement binds induced the hybrid-2 by human human telomeric telomeric sequences G-quadruplex in K+ solution structure [67]. and Our we data have show determined that EPI thespecifically solution binds structure the hybrid-2 of the 1:1 human complex telomeric of EPI G-quadruplex and hybrid-2 humanstructure telomeric and we G-quadruplexhave determined by NMRthe solution [63]. The structure NMR solutionof the 1:1 structure complex of of the EPI complex and hybrid-2 shows human that the telomeric EPI molecule G-quadruplex (Figure3a) by forms NMR a well-defined[63]. The NMR 1:1 solution complex structure with the hybrid-2 of the complex human telomericshows that G-quadruplex. the EPI molecule This complex(Figure is3a) stabilized forms a bywell-defined extensive hydrogen-bonding,1:1 complex with base-stackingthe hybrid-2 andhuman electrostatic telomeric interactions G-quadruplex. (Figure This4a). complex In the free is stabilized by extensive hydrogen-bonding, base-stacking and electrostatic interactions (Figure 4a). In the free telomeric hybrid-2 structure, the flanking segment and the T13-T14-A15 lateral loop at the 5′

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Molecules 2019, 24, x 5 of 15 telomeric hybrid-2 structure, the flanking segment and the T13-T14-A15 lateral loop at the 50 end areend disordered.are disordered. Upon binding,Upon binding, EPI stacks EPI on thestacks 50-external on the G-tetrad5′-external and extensiveG-tetrad rearrangementand extensive occursrearrangement in the 5 0occurs-end flanking in the 5 residues′-end flanking and lateral residues loop and to formlateral a multi-layerloop to form drug-binding a multi-layer pocket. drug- Thebinding A3 basepocket. from The the A3 50 -flankingbase from strand the 5′-flanking is recruited strand by EPI is recruited to form aby ‘quasi-triad EPI to form plane’ a ‘quasi-triad which is intercalatedplane’ which between is intercalated the 50-G-tetrad between andthe two5′-G-tetrad additional and capping two additional layers (Figure capping4a). layers Specifically, (Figure EPI4a). stacksSpecifically, over the EPI tetrad stacks G12 over and the G16, tetrad with G12 its N7and centered G16, with over its theN7 5centered0-tetrad (Figureover the4b), 5′ and-tetrad A3 (Figure covers the4b), centerand A3 of thecovers G4–G22. the center Importantly, of the G4–G22. a hydrogen Importantly, bond is formed a hydrogen between bond A3/NH6 is formed and the between oxygen ofA3/NH6 the methylenedioxy and the oxygen ring of the of themethylenedioxy EPI, which stabilizes ring of the the EPI, EPI-A3 which plane. stabilizes The positively the EPI-A3 charged plane. N7The atompositively (Figure charged3a) of N7 the atom EPI is(Figure in proximity 3a) of the to EPI the is O6 in proximity atoms of theto the 5 0 O6external atoms tetradof the 5 guanines,′ external + likelytetrad stabilizingguanines, likely the complex stabilizing analogous the complex to the analogous K cations to withinthe K+ cations the central within channel the central of the channel G-core. Aboveof the G-core. the EPI:A3 Above plane, the a EPI: T:T:AA3 triad plane, is formeda T:T:A from triad T2 is offormed the 50 flankingfrom T2 strandof the 5 and′ flanking T13 and strand A15 from and theT13 lateraland A15 loop. from The the triad lateral is stabilizedloop. The triad by a hydrogen-bondingis stabilized by a hydrogen-bonding network, with two network, hydrogen-bond with two interactionshydrogen-bond between interactions T13 and between A15 to T13 form and a A15 reversed to form Watson–Crick a reversed Watson–Crick base pair (Figure base4 pairc) and (Figure one hydrogen4c) and one bond hydrogen between bond T2 between and T13. T2 Inand fact, T13. the In stablefact, the formation stable formation of the T:T:A of the triad T:T:A results triad results in the appearancein the appearance of a signature of a signature NMR imino NMR proton imino peak proton of T13 peak arisen of fromT13 thearisen unique from triad the conformationunique triad ofconformation the specific of EPI-binding the specific pocket EPI-binding within pocket the hybrid-2 within the telomeric hybrid-2 G-quadruplex. telomeric G-quadruplex. Finally, this Finally, triad is cappedthis triad by is a capped hydrogen-bonded by a hydrogen-bonded T1:T14 base pair,T1:T14 which base stacks pair, which over the stacks triad over and the further triad stabilizes and further the overallstabilizes complex the overall (Figure complex4d). (Figure 4d).

Figure 4. TheThe specific speciﬁc recognition recognition of of human human hybrid-2 hybrid-2 telomeric telomeric G-quadruplex G-quadruplex by EPI (PDB ID: ID: 6CCW) 6CCW) (a) Cartoon representation of 1:1 EPIEPI/hybrid-2/hybrid-2 complex.complex. Different Diﬀerent nucleotides are marked as follows: Thymine, cyan; adenine, pink; and guanine, green. The EPI EPI molecule molecule is is shown shown as as the the yellow yellow spheres. spheres. ((bb–dd)) Top viewview ofof thethe EPI:A3EPI:A3 quasi-triadquasi-triad plane ( b), T2:T13:A15 triad triad plane plane ( (cc),), and and T1:T14 T1:T14 pair pair ( (dd).). Potential hydrogen bonds are shown in black dasheddashed lines.lines.

The optimal recognition of EPI is specific to the hybrid-2 folding topology and the human telomeric The optimal recognition of EPI is specific to the hybrid-2 folding topology and the human DNA loop sequence TTA. In the EPI-hybrid-2 complex, the second TTA loop adopts a lateral loop telomeric DNA loop sequence TTA. In the EPI-hybrid-2 complex, the second TTA loop adopts a conformation above the 5 -tetrad, which provides ideal orientation for the pairing interaction with lateral loop conformation0 above the 5′-tetrad, which provides ideal orientation for the pairing the 5 -flanking segment to form the highly stable capping structures of the T:T:A triad and T:T pair interaction0 with the 5′-flanking segment to form the highly stable capping structures of the T:T:A (Figure5a,b). In the hybrid-1 structure, the third TTA loop at the 5 -end (Figure5c) is o ffset by 90 triad and T:T pair (Figure 5a,b). In the hybrid-1 structure, the third TTA0 loop at the 5′-end (Figure 5c)◦ relative to the second lateral loop at the 5 -end of the hybrid-2 structure and, thus, cannot form the is offset by 90° relative to the second lateral0 loop at the 5′-end of the hybrid-2 structure and, thus, cannot form the optimal stable capping structures for the EPI binding pocket. In the basket-type telomeric G-quadruplex structure, the diagonal loop and the 3′-flanking segments are both at the same end of the 5′-flanking (Figure 5d), sterically hindering the EPI binding. Human telomeric G-

Molecules 2019, 24, 1578 6 of 15 optimal stable capping structures for the EPI binding pocket. In the basket-type telomeric G-quadruplex structure,Molecules 2019 the, 24 diagonal, x loop and the 30-flanking segments are both at the same end of the 50-flanking6 of 15 (Figure5d), sterically hindering the EPI binding. Human telomeric G-quadruplexes bear inherent structurequadruplexes polymorphism, bear inherent predominantly structure withpolymo tworphism, hybrid-type predominantly G-quadruplexes with in two equilibrium hybrid-type between G- hybrid-2quadruplexes and hybrid-1 in equili structuresbrium between under physiologically hybrid-2 and relevant hybrid-1 solution structures conditions. under Remarkably, physiologically EPI is ablerelevant to convert solution hybrid-1, conditions. basket, Remarkably, and unfolded EPI telomeric is ableG-quadruplex to convert hybrid-1, structures basket, to the hybrid-2and unfolded form, independenttelomeric G-quadruplex of available solutionstructures cations to the (Figure hybrid-25). Thisform, is independen the only sucht of compound available reportedsolution tocations date. The(Figure highly 5). This specific is the and only selective such compound binding of reported EPI to the to hybrid-2date. The structure highly specific likely shiftsand selective the equilibrium binding betweenof EPI to di thefferent hybrid-2 forms instructure solution, likely resulting shifts in th thee overallequilibrium conversion between of other different telomeric forms G-quadruplex in solution, structuresresulting in to the the overall hybrid-2 conversion form. of other telomeric G-quadruplex structures to the hybrid-2 form.

Figure 5. Conversion of human telomeric G-quadruplex structures to the hybrid-2 form induced by EPI. Figure 5. Conversion of human telomeric G-quadruplex structures to the hybrid-2 form induced by (a–b) EPI binding induces extensive rearrangement of previously disordered 5 -flanking and lateral EPI. (a–b) EPI binding induces extensive rearrangement of previously disordered0 5′-flanking and loop segments (b) to form a well-defined four-layer binding pocket (a) specific to hybrid-2 telomeric lateral loop segments (b) to form a well-defined four-layer binding pocket (a) specific to hybrid-2 G-quadruplex. (c–e) Other human telomeric G-quadruplex forms, including hybrid-1 (c) and basket telomeric G-quadruplex. (c–e) Other human telomeric G-quadruplex forms, including hybrid-1 (c) (d), or the free telomeric sequence in the absence of salt (e) can be converted to the hybrid-2 form as the and basket (d), or the free telomeric sequence in the absence of salt (e) can be converted to the hybrid- addition of EPI molecules. The specific recognition of human hybrid-2 telomeric G-quadruplex by EPI 2 form as the addition of EPI molecules. The specific recognition of human hybrid-2 telomeric G- (PDB ID: 6CCW). quadruplex by EPI (PDB ID: 6CCW). The EPI-hybrid-2 complex structure reveals several features which enable specific recognition of The EPI-hybrid-2 complex structure reveals several features which enable specific recognition the hybrid-2 human telomeric G-quadruplex. EPI contains a crescent-shaped asymmetric stacking of the hybrid-2 human telomeric G-quadruplex. EPI contains a crescent-shaped asymmetric stacking moiety that can only stack over two tetrad guanines, allowing the recruitment of a flanking base moiety that can only stack over two tetrad guanines, allowing the recruitment of a flanking base partner to co-stack over the 50-G-tetrad and lock the EPI position. The pairing with the recruited partner to co-stack over the 5′-G-tetrad and lock the EPI position. The pairing with the recruited adenine and the central location of positively charged N7 together anchor the position and orientation adenine and the central location of positively charged N7 together anchor the position and orientation of EPI above the 50 external G-tetrad. In addition, the appropriately positioned hydrogen-bond of EPI above the 5′ external G-tetrad. In addition, the appropriately positioned hydrogen-bond acceptors in the methylenedioxy ring E of EPI enable it to optimally hydrogen-bond with the recruited acceptors in the methylenedioxy ring E of EPI enable it to optimally hydrogen-bond with the adenine (Figures3a and4b). This is highlighted by the observation that the structurally similar recruited adenine (Figures 3a and 4b). This is highlighted by the observation that the structurally berberine alkaloids berberine, palmatine, and coptisine, which only differ in the positions of the similar berberine alkaloids berberine, palmatine, and coptisine, which only differ in the positions of methylenedioxy ring and methoxy groups, are unable to form a well-defined complex with the the methylenedioxy ring and methoxy groups, are unable to form a well-defined complex with the telomeric G-quadruplex. This molecular-level recognition information can only be obtained from telomeric G-quadruplex. This molecular-level recognition information can only be obtained from detailed structural study and is important for rational drug design of improved analogs targeting the hybrid-2 human telomeric G-quadruplex.

3. Molecular Recognition of Human Telomeric Hybrid-1 G-Quadruplex by Pt-Tripod in Monomeric and Dimeric Complexes

Molecules 2019, 24, 1578 7 of 15 detailed structural study and is important for rational drug design of improved analogs targeting the hybrid-2 human telomeric G-quadruplex.

3. Molecular Recognition of Human Telomeric Hybrid-1 G-Quadruplex by Pt-Tripod in Monomeric and Dimeric Complexes Recently, a series of platinum (II) compounds was found to bind telomeric G-quadruplex and suppress telomerase activity [68,69]. Among them, a Pt-based tripod (Pt-tripod) showed strong in vitro and in vivo anticancer activity upon light irradiation. This tripod is a non-planar compound with a central tertiary amine connecting three arms, each bearing two aromatic rings and a cationic platinum complex with a three-fold symmetry (Figure3b). Interestingly, Pt-tripod binds to the intramolecular hybrid-1 human telomeric G-quadruplex formed by the Tel26 sequence and forms well-deﬁned monomeric and dimeric Pt-tripod-Tel26 complexes dependent on the drug-DNA ratio [62]. At the 1:1 ratio, Pt-tripod binds to the 50-end of the hybrid-1 Tel26 to form a well-defined 1:1 Pt-tripod–Tel26 complex (Figure6a). In the 1:1 complex, the ligand binding induces a large conformational rearrangement at the 50 end. Pt-tripod intercalates between A3 of the 50-flanking segment and G4 of the 50-tetrad and recruits A21 from the third lateral loop to form a Pt-tripod-A21 paired plane above the 50-external tetrad (Figure6c), replacing the adenine triad formed by A3:A9:A21 in the free Tel26 G-quadruplex. The Pt-tripod covers G10 and G4 by its arm 1 and arm 2, respectively, with the central tertiary amine nitrogen right above the G4 and G10 edge, while A21 stacks over the G18 and G22. Above the Pt-tripod-A21 plane, A3 and T20 form a hydrogen-bonded reversed Watson–Crick base pair, while A9 from the first side loop also positions in the same plane to cover the aromatic moiety of arm 3 (FigureMolecules6 2019b). , 24, x 7 of 15

Figure 6. Solution structure of 1:1 and 4:2 Pt-tripod/hybrid-1 G-quadruplex complexes (PDB ID: 5Z80Figure and 6. Solution 5Z8F). (a structure) Cartoon of representation 1:1 and 4:2 Pt-tripod/hy of 1:1 Pt-tripodbrid-1/hybrid-1 G-quadruplex complex. complexes Diﬀerent (PDB nucleotides ID: 5Z80 areand marked 5Z8F). as(a) follows:CartoonThymine, representation cyan; adenine,of 1:1 Pt-tripod/hy pink; andbrid-1 guanine, complex. green. TheDifferent Pt-tripod nucleotides molecule are is shownmarked as as the follows: yellow spheres.Thymine, (b cy–can;) Top adenine, views of pink; the A3:A9:T20 and guanine, triad green. plane (bThe) and Pt-tripod Pt-tripod:A21 molecule plane is shown as the yellow spheres. (b–c) Top views of the A3:A9:T20 triad plane (b) and Pt-tripod:A21 at 50 site (c). (d) Cartoon representation of 4:2 Pt-tripod/hybrid-1 complex. (e–g) Structural details of plane at 5′ site (c). (d) Cartoon representation of 4:2 Pt-tripod/hybrid-1 complex. (e–g) Structural the 30 site binding pocket. Bottom views of the Pt-tripod:A15 plane (e), T13:T14:A25 triad plane (f), anddetails the of A26:A26* the 3′ site (A26 binding fromeach pocket. hybrid-1) Bottom base views pair of (g the). Potential Pt-tripod:A15 hydrogen plane bonds (e), areT13:T14:A25 shown in blacktriad dashedplane (f lines.), and the A26:A26* (A26 from each hybrid-1) base pair (g). Potential hydrogen bonds are shown in black dashed lines.

Recently, a series of platinum (II) compounds was found to bind telomeric G-quadruplex and suppress telomerase activity [68,69]. Among them, a Pt-based tripod (Pt-tripod) showed strong in vitro and in vivo anticancer activity upon light irradiation. This tripod is a non-planar compound with a central tertiary amine connecting three arms, each bearing two aromatic rings and a cationic platinum complex with a three-fold symmetry (Figure 3b). Interestingly, Pt-tripod binds to the intramolecular hybrid-1 human telomeric G-quadruplex formed by the Tel26 sequence and forms well-defined monomeric and dimeric Pt-tripod-Tel26 complexes dependent on the drug-DNA ratio [62]. At the 1:1 ratio, Pt-tripod binds to the 5′-end of the hybrid-1 Tel26 to form a well-defined 1:1 Pt- tripod–Tel26 complex (Figure 6a). In the 1:1 complex, the ligand binding induces a large conformational rearrangement at the 5′ end. Pt-tripod intercalates between A3 of the 5′-flanking segment and G4 of the 5′-tetrad and recruits A21 from the third lateral loop to form a Pt-tripod-A21 paired plane above the 5′-external tetrad (Figure 6c), replacing the adenine triad formed by A3:A9:A21 in the free Tel26 G-quadruplex. The Pt-tripod covers G10 and G4 by its arm 1 and arm 2, respectively, with the central tertiary amine nitrogen right above the G4 and G10 edge, while A21 stacks over the G18 and G22. Above the Pt-tripod-A21 plane, A3 and T20 form a hydrogen-bonded reversed Watson–Crick base pair, while A9 from the first side loop also positions in the same plane to cover the aromatic moiety of arm 3 (Figure 6b). At the 3:1 Pt-tripod–DNA ratio, Pt-tripod binds the hybrid-1 Tel26 to predominately form a well- defined 4:2 Pt-tripod–Tel26 3′–3′ dimeric complex (Figure 6d) with a twofold symmetry. In the 4:2 Pt- tripod–Tel26 complex, Pt-tripod binds at both the 5′- and 3′-ends of Tel26 G-quadruplex. In addition to the 5′-end complex, the second ligand induces a large conformational rearrangement at the 3′-end to form a well-defined binding pocket, in which Pt-tripod intercalates between A25 and G24 of the

Molecules 2019, 24, 1578 8 of 15

At the 3:1 Pt-tripod–DNA ratio, Pt-tripod binds the hybrid-1 Tel26 to predominately form a well-defined 4:2 Pt-tripod–Tel26 30–30 dimeric complex (Figure6d) with a twofold symmetry. In the 4:2 Pt-tripod–Tel26 complex, Pt-tripod binds at both the 50- and 30-ends of Tel26 G-quadruplex. In addition to the 50-end complex, the second ligand induces a large conformational rearrangement at the 30-end to form a well-defined binding pocket, in which Pt-tripod intercalates between A25 and G24 of the 30-tetrad and recruits A15 from the second lateral loop to form a Pt-tripod-A21 paired plane above the 30-external tetrad (Figure6e). Here, arm 1 and arm 2 of the Pt-tripod cover G24 and G6, respectively, with the central tertiary amine nitrogen above the G6 and G24 edge, while A15 stacks over G12 and G16. While the 4:2 dimeric complex is induced by Pt-tripod binding, the binding of Pt-tripod at the 30-end also appears to be facilitated by the 30-end interlocking interface in the dimeric complex, including a well-defined hydrogen-bonded T:A:T triad (Figure6f) and a A:A base pair (Figure6g). This stable three-layer binding pocket at the 30 dimeric interface gives rise to a less dynamic binding of Pt-tripod as compared to the 50-complex. The T14:A25 base pair observed at the 30-end in the free Tel26 G-quadruplex is completely rearranged, with the glycosidic conformation of T14 changed from anti to syn and A25 from syn to anti. It is noted that this unique 30 dimeric interface appears to be specific to the modified 30-flanking sequence of Tel26. A unique feature of the two Pt-tripod–Tel26 complexes is the anchoring of the three positively charged arms of Pt-tripod in three G-quadruplex grooves, which defines and locks the position of Pt-tripod in the complexes (Figure7). Interestingly, the anchoring of the Pt-tripod arms appears to avoid the lateral loops, which may cause potential steric hindrance. Each arm of Pt-tripod stretches into a groove without a lateral loop and thereby positions its outer platinum complexes for interactions with the loop residues and DNA backbone. These include electrostatic interactions of the positively charged platinum moiety of the three arms with the negatively charged phosphate backbone, hydrogen-bonding interactions, and π–π interactions of the three arms with loop and flanking residues. As the tertiary amine nitrogen of Pt-tripod is located above the edge of the external G-tetrad in both complexes, the aromatic moieties of arm 1 and arm 2 of both ligands stack over a guanine of the external G-tetrad, whereas the arm 3 almost completely extends into the groove without much stacking interaction with the external G-tetrads. In the 50-end complex (Figure7a), however, the arm 3 of Pt-tripod extensively interacts with the propeller side loop, with A9 stacking over its first aromatic ring and T8 hydrogen-bonded with its terminal moiety. While A21 of the third lateral loop at the 50-end is recruited to pair with Pt-tripod, T19 of the same loop interacts with arm 1 through hydrogen bonding with T20 further stacking over A21. Arm 2 is intercalated between A3 of the flanking segment and G4 of the 50-tetrad, with its platinum-moiety interacting with the phosphate backbone in the groove. In the 30-end complex (Figure7b), arm 1 and arm 2 of Pt-tripod show electrostatic and potential hydrogen bonding interactions with the phosphate backbone. MoleculesMolecules 20192019,, 2424,, x 1578 99 of of 15 15

FigureFigure 7. 7. Arm-grooveArm-groove interactions interactions of of Pt-tripod Pt-tripod at at 5 5′ 0andand 3 3′ 0sitessites in in the the 4:2 4:2 Pt-tripod/hybrid-1 Pt-tripod/hybrid-1 complex. complex. ((aa)) Top Top view of Pt-tripod at the 50′ site of hybrid-1 without capping structure (middle). The The binding binding pocketpocket surface surface is is color color coded coded according according to to the the ch charge.arge. The The interaction interaction details details between between each each arm arm and and groovegroove are are shown in enlarged viewview (corners).(corners). ((bb)) TheThe samesame information information is is shown shown for for Pt-tripod Pt-tripod at at the the 30 3site.′ site. Potential Potential hydrogen hydrogen bonds bonds are are shown shown in in black black dashed dashed lines. lines. 4. Insights Obtained from the Complex Structures 4. Insights Obtained from the Complex Structures EPI and Pt-tripod both induce extensive conformational rearrangements of flanking and loop EPI and Pt-tripod both induce extensive conformational rearrangements of flanking and loop residues after binding to the human telomeric G-quadruplexes. The EPI-hybrid-2 G-quadruplex and residues after binding to the human telomeric G-quadruplexes. The EPI-hybrid-2 G-quadruplex and 1:1 and 4:2 Pt-tripod-hybrid-1 G-quadruplex complexes all include ligand-induced well-defined 1:1 and 4:2 Pt-tripod-hybrid-1 G-quadruplex complexes all include ligand-induced well-defined multi-layer binding pockets involving the external tetrad, flanking, and loop residues. multi-layer binding pockets involving the external tetrad, flanking, and loop residues. Such Such rearrangements are more extensive than most G-quadruplex-interactive-ligand complexes, rearrangements are more extensive than most G-quadruplex-interactive-ligand complexes, particularly those observed with the parallel-stranded G-quadruplexes that lack the lateral loops.

Molecules 2019, 24, 1578 10 of 15 particularly those observed with the parallel-stranded G-quadruplexes that lack the lateral loops. Therefore, the importance of interactions with flanking segments and various loop types in the hybrid-type human telomeric G-quadruplexes for binding specificity is highlighted. On the other hand, both EPI and Pt-tripod recruit a DNA base to form a ligand-base plane covering the external G-tetrad. Both the EPI and the Pt-tripod scaffolds cover only two guanine bases and by recruiting an adenine residue, a full tetrad coverage is achieved. Such a phenomenon has also been observed for ligand complexes of the parallel-stranded c-MYC promoter G-quadruplex [70,71]. The engagement of the target-DNA residue anchors the specific orientation of the ligand and might be the basis for the specific recognition of a ligand. In contrast to quadruplex ligands with large aromatic systems covering most of the tetrad, the asymmetric crescent shaped EPI or the tri-armed Pt-tripod facilitate this target-base recruitment. Comparing how EPI and Pt-tripod bind in the 50-complexes of the two hybrid-type human telomeric quadruplexes reveals similarities and critical differences in stacking, hydrogen bond, and electrostatic interactions (Figure8). Similarly, both drugs stack upon two guanine bases of the 50-tetrad and recruit an adenine base to form a ligand-base plane covering the 50-tetrad. However, their binding positions and interactions are different. The more compact and planar EPI intercalates between the second lateral loop and the 50-tetrad and recruits A3 from the 50-flanking segment in the hybrid-2 structure. EPI forms a clear hydrogen bond with A3 in the ligand-base plane, while the T13 and A15 of the second lateral loop provide an optimal capping covering the long axis of EPI. In contrast, the tri-armed Pt-tripod intercalates within the 50-flanking segment between A3 and the 50-tetrad, and recruits A21 from the lateral loop in the hybrid-1 structure using two arms in a more shape-complementary manner. Thereby, the EPI molecule is opposite to the 50-flanking strand anchoring towards the A3, while the Pt-tripod is opposite to and restricted by the lateral loop above the 50 external tetrad. Both ligand-base planes are covered by a triad of flanking and loop residues, each of which including a reverse Watson–Crick AT base pair. Stacking interactions are more predominant for the planar EPI in its interactions with the 50-tetrad and the capping triad than for the non-planar Pt-tripod (Figure4c). In the EPI complex, the capping triad is more stable and well-defined, as the three bases are all connected through hydrogen-bonds, and is covered by an additional layer of T:T base pair. This extensive binding pocket defines the stable complex formation of the EPI and hybrid-2 structure, which promotes the EPI-induced conversion of other human telomeric quadruplex topologies towards the hybrid-2 folding. In contrast, in the capping triad of the Pt-tripod 50-complex, the A3:T20 base pair is distant from the A9 residue, which likely stabilizes the arm 3 by stacking with its aromatic moiety (Figure6b). The positive charge of the two drugs contributes to their respective binding interactions in different ways. The positively charged nitrogen of EPI is located above the central pore close to the negatively polarized carbonyl functional groups of the 50-tetrad, analogous to the centrally coordinated cations. For Pt-tripod, the central moiety is uncharged and also moved to the tetrad edge. Instead, its long arms position the three terminal positively charged Pt(II) complexes for interactions with negatively charged phosphate backbones of three different G-quadruplex grooves. Although cationic drugs might have general binding to other nucleic acids structures, this groove-anchoring mode may be used to enable further G-quadruplex selectivity. For example, cationic side chain might be added in an EPI derivative to similarly anchor it to the grooves of the hybrid-2 quadruplex. In summary, the reviewed high-resolution complex structures of the hybrid-type human telomeric G-quadruplexes with the EPI and the Pt-tripod small molecules advance our knowledge about quadruplex-ligand interactions. Both drugs induce a previously unprecedented rearrangement of the capping residues to form extensive multi-layer binding pockets, with each drug recruiting a G-quadruplex DNA residue to form a ligand-base plane and define its respective binding position. Additional features observed for each drug are the EPI-induced conversion of alternative human telomeric quadruplex topologies towards the hybrid-2 type and the Pt-tripod-induced 30–30 Molecules 2019, 24, 1578 11 of 15 dimerization to form a 4:2 complex. The identified mechanisms of molecular recognition will provide insightsMolecules 2019 into, 24 designing, x improved cancer therapeutics targeting the human telomeric G-quadruplexes.11 of 15

Figure 8. Comparison of the 5 -binding modes between EPI and Pt-tripod with human telomeric Figure 8. Comparison of the 50′-binding modes between EPI and Pt-tripod with human telomeric hybrid-type G-quadruplexes (a) Upon binding, the 5 -flanking A3 is recruited by EPI and forms a hybrid-type G-quadruplexes (a) Upon binding, the 50′-flanking A3 is recruited by EPI and forms a quasi-triad plane (right). The multi-layer drug-binding pocket for EPI is formed by the 5 -end flanking quasi-triad plane (right). The multi-layer drug-binding pocket for EPI is formed by the 50′-end flanking residues and TTA lateral loop. (left). Potential hydrogen bond is shown as the black dashed line. residues and TTA lateral loop. (left). Potential hydrogen bond is shown as the black dashed line. (b) (b) Upon binding, A21 in the second lateral loop is recruited by Pt-tripod and forms a quasi-triad plane Upon binding, A21 in the second lateral loop is recruited by Pt-tripod and forms a quasi-triad plane (right), stacking on top of the 50-external G-tetrad and locking the position of Pt-tripod. Each arm of (right), stacking on top of the 5′-external G-tetrad and locking the position of Pt-tripod. Each arm of Pt-tripod stretches into different grooves. The Pt-tripod:A21 plane is capped by the A3:A9:T20 triad (left). Pt-tripod stretches into different grooves. The Pt-tripod:A21 plane is capped by the A3:A9:T20 triad (left). Funding: This research was supported by the National Institutes of Health (R01CA122952 (DY), R01CA177585 (DY), and P30CA023168 (Purdue Center for Cancer Research)). The positive charge of the two drugs contributes to their respective binding interactions in Acknowledgments: We are grateful to Jonathan Dickerhoff and Clement Lin for their insightful comments, editing, anddifferent proofreading ways. The of the positively manuscript. charged We thank nitrogen Kaibo Wang of EPI for is his located comments. above the central pore close to the negatively polarized carbonyl functional groups of the 5′-tetrad, analogous to the centrally Conflicts of Interest: The authors declare no conflict of interest. coordinated cations. For Pt-tripod, the central moiety is uncharged and also moved to the tetrad edge. Instead, its long arms position the three terminal positively charged Pt(II) complexes for interactions References with negatively charged phosphate backbones of three different G-quadruplex grooves. Although 1.cationicVerdun, drugs R.E.; might Karlseder, have J.general Replication binding and protectionto other nucleic of telomeres. acids Naturestructures,2007, 447this, 924–931.groove-anchoring [CrossRef] mode[PubMed may be] used to enable further G-quadruplex selectivity. For example, cationic side chain might 2.be addedO’Sullivan, in an R.J.;EPI Karlseder,derivative J. to Telomeres: similarly Protecting anchor it chromosomes to the grooves against of the genome hybrid-2 instability. quadruplex.Nat. Rev. Mol. CellIn summary, Biol. 2010, 11 the, 171–181. reviewed [CrossRef high-resolution][PubMed] complex structures of the hybrid-type human 3.telomericHackett, G-quadruplexes J.A.; Feldser, D.M.; with Greider, the EPI C.W. and Telomerethe Pt-tripod dysfunction small molecules increases mutation advance rate our and knowledge genomic instability. Cell 2001, 106, 275–286. [CrossRef] about quadruplex-ligand interactions. Both drugs induce a previously unprecedented rearrangement of the capping residues to form extensive multi-layer binding pockets, with each drug recruiting a G- quadruplex DNA residue to form a ligand-base plane and define its respective binding position. Additional features observed for each drug are the EPI-induced conversion of alternative human telomeric quadruplex topologies towards the hybrid-2 type and the Pt-tripod-induced 3′–3′ dimerization to form a 4:2 complex. The identified mechanisms of molecular recognition will provide

Molecules 2019, 24, 1578 12 of 15

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