Recent Advances in Developing Small Molecules Targeting Nucleic Acid

International Journal of Molecular Sciences

Review Recent Advances in Developing Small Molecules Targeting Nucleic Acid

Maolin Wang 1,2,3, Yuanyuan Yu 1,2,3, Chao Liang 1,2,3, Aiping Lu 1,2,3,* and Ge Zhang 1,2,3,* 1 Institute of Integrated Bioinfomedicine and Translational Science (IBTS), School of Chinese Medicine, Hong Kong Baptist University, Hong Kong 999077, China; [email protected] (M.W.); [email protected] (Y.Y.); [email protected] (C.L.) 2 Shenzhen Lab of Combinatorial Compounds and Targeted Drug Delivery, HKBU Institute of Research and Continuing Education, Shenzhen 518000, China 3 Institute for Advancing Translational Medicine in Bone and Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong 999077, China * Correspondence: [email protected] (A.L.); [email protected] (G.Z.); Tel.: +852-3411-2456 (A.L.); +852-3411-2958 (G.Z.)

Academic Editor: Mateus Webba da Silva Received: 2 March 2016; Accepted: 9 May 2016; Published: 30 May 2016 Abstract: Nucleic acids participate in a large number of biological processes. However, current approaches for small molecules targeting protein are incompatible with nucleic acids. On the other hand, the lack of crystallization of nucleic acid is the limiting factor for nucleic acid drug design. Because of the improvements in crystallization in recent years, a great many structures of nucleic acids have been reported, providing basic information for nucleic acid drug discovery. This review focuses on the discovery and development of small molecules targeting nucleic acids.

Keywords: nucleic acids; nucleic acids targeting; small molecules; DNA drug discovery; RNA drug discovery

1. Introduction Nucleic acids play significant roles in variety kinds of biological processes [1–5]. According to the differences on sugar scaffold, nucleic acids can be classified into two categories. DNAs and RNAs participate in gene storage, replication, transcription and other important biological activities. Thus, targeting nucleic acids can regulate a large range of biological processes, especially genetic diseases. Nucleic acids play significant roles in anticancer [6] and antiviral [7] processes. Therefore, the small molecules targeting nucleic acids are desirable and have become a topic issue in recent years. However, small molecules targeting nucleic acids are more difficult to discover than targeting protein, which result from various reasons. Nucleic acid lacks spatial structure information from X-ray crystallization, nuclear magnetic resonance (NMR) or other imaging methods. Current approaches for small molecules targeting protein are incompatible with nucleic acids. Most of the docking methods, for example, do not regard RNA and DNA receptors as flexible bodies [8–10], which constrains the conformational changes of nucleic acids. Fortunately, the areas of structural biology and computational chemistry have improved dramatically. A large number of structures of nucleic acids have been reported. Several computational methods and force fields have been developed and modified [11,12]. These improvements provide a new horizon to discovery and design novel small molecules targeting nucleic acids. Depending on the differences on sugar scaffold of nucleic acids, small molecules targeting nucleic acids can be separated into two main categories: small molecules targeting DNA and small molecules targeting RNA (in which DNA and RNA can form different conformations). This review focuses on the recent discovery and development of small molecules targeting DNA and RNA.

Int. J. Mol. Sci. 2016, 17, 779; doi:10.3390/ijms17060779 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 779 2 of 24

2. SmallInt. J. MoleculesMol. Sci. 2016, 17 Targeting, 779 DNA 2 of 23 Int. J. Mol. Sci. 2016, 17, 779 2 of 23 2.1. Small2. Small Molecules Molecules Targeting Targeting DNA DNA Duplex 2. Small Molecules Targeting DNA 2.1.1.2.1. Small Small Molecules Molecules FormingTargeting DNA Covalent Duplex Bonds with DNA Duplex 2.1. Small Molecules Targeting DNA Duplex Psoralen2.1.1. Small or Molecules psoralene Forming has been Covalent used asBonds mutagen with DNA to treat Duplex skin diseases including psoriasis and vitiligo. Psoralen interacts with standard DNA duplex, through the 5,6 double bond in pyrimidine ring, 2.1.1.Psoralen Small Molecules or psoralene Forming has beenCovalent used Bonds as mutagen with DNA to treat Duplex skin diseases including psoriasis and which can stop replication and transcription. 41-(Hydroxymethyl)-4,51,8-trimethylpsoralen, short for vitiligo.Psoralen Psoralen or psoralene interacts withhas been standard used DNAas mutagen duplex, to through treat skin the diseases 5,6 double including bond inpsoriasis pyrimidine and HMT,ring,vitiligo. has reasonablewhich Psoralen can waterinteractsstop replication solubility with standard andand hightranscription. DNA DNA duplex, binding 4 ′through-(Hydroxymethyl)-4,5 afﬁnity, the 5,6 as double shown′,8-trimethylpsoralen, bond in Figure in pyrimidine1A. Figure 2B showsshortring, the which crystalfor HMT, can structure hasstop reasonable replication of a short water and DNA transcription.solubility complex and 4 with ′high-(Hydroxymethyl)-4,5 HMTDNA reportedbinding af′ by,8-trimethylpsoralen,finity, Spielmann as shownet in al. [13]. The HMTFigureshort structurefor 1A. HMT, Figure crosseshas 2B reasonableshows both the the crystalwater minor structuresolubility and majorofand a short high grooves DNA DNA complex throughbinding with af thefinity, HMT 5,6 doubleas reported shown bonds byin of pyrimidineSpielmannFigure ring1A. etFigure and al. [13]. two 2B The thymines.shows HMT the structure crystal There structure arecrosses apparent both of a theshort stacking minor DNA and interactions complex major grooveswith from HMT through the reported ring the system 5,6by of HMTdoubleSpielmann and the bonds bases et al. of nearby. [13].pyrimidi The The HMTne ring high structure and binding two crosses thymines. afﬁnity both ofThere the HMT minor are and apparent and duplex major stacking resultsgrooves interactions fromthrough aromatic the from 5,6 rings stackingthedouble andring bonds hydrophobicsystem of of pyrimidi HMT interactions. andne thering bases and The twonearby. crystal thymines. The study high There ofbinding the are complexapparent affinity of stacking HMT DNA and andinteractions duplex HMT results showed from the from aromatic rings stacking and hydrophobic interactions. The crystal study of the complex of DNA Hollidaythe ring junction system was of HMT formed and in the the bases adduct nearby. of DNA-HMT.The high binding The affinity interaction of HMT between and duplex DNA results and HMT andfrom HMT aromatic showed rings the stacking Holliday and junctionhydrophobic was formedinteractions. in the The adduct crystal of study DNA-HMT. of the complex The interaction of DNA is related with sequence, which may play a role in repairing the psoralen damage in chemotherapy betweenand HMT DNA showed and theHMT Holliday is related junction with sequence,was formed which in the may adduct play aof role DNA-HMT. in repairing The the interaction psoralen treatment.damagebetween The inDNA conformationchemotherapy and HMT is oftreatment. related DNA iswith The extremely sequence,conformati twisted whichon of mayDNA at the play is bases extremely a role of thyminein twistedrepairing connectedat thethe psoralenbases with of the hexatomicthyminedamage pyrone inconnected chemotherapy of the with molecule. the treatment. hexatomic The pyrone conformati of theon molecule. of DNA is extremely twisted at the bases of thymine connected with the hexatomic pyrone of the molecule.

Figure 1. (A) Chemical structure of HMT; and (B) NMR structure of a short DNA complex with HMT Figure 1. (A) Chemical structure of HMT; and (B) NMR structure of a short DNA complex with HMT (RCSBFigure Protein1. (A) Chemical Data Bank structure (PDB) code: of HMT; 204D). and The (B) oranNMRge structure structure of represents a short DNA the smallcomplex molecule. with HMT The (RCSB Protein Data Bank (PDB) code: 204D). The orange structure represents the small molecule. purple(RCSB Proteinstructure Data represents Bank (PDB) the code:backbone 204D). of The nucleic oran geacid; structure the blue represents and red theatoms small in molecule.the molecule The The purple structure represents the backbone of nucleic acid; the blue and red atoms in the molecule representpurple structure nitrogen represents and oxygen the atoms, backbone respectively. of nucleic acid; the blue and red atoms in the molecule representrepresent nitrogen nitrogen and and oxygen oxygen atoms, atoms, respectively. respectively.

Figure 2. (A) Chemical structure of Aflatoxin B1 adduct; and (B) NMR structure of a DNA duplex complex with Aflatoxin B1 adduct (PDB code: 2KH3). The orange structure represents the small FigureFigure 2. (A 2.) Chemical(A) Chemical structure structure of of Aﬂatoxin Aflatoxin B1B1 adduct;adduct; and and (B ()B NMR) NMR structure structure of a ofDNA a DNA duplex duplex molecule;complex with the purple Aflatoxin structure B1 adduct represents (PDB the code: backbone 2KH3). of The nucleic orange acid; structure the blue representsand red atoms the insmall the complex with Aﬂatoxin B1 adduct (PDB code: 2KH3). The orange structure represents the small moleculemolecule; representthe purple nitrogen structure and represents oxygen atoms, the backbone respectively. of nucleic acid; the blue and red atoms in the molecule; the purple structure represents the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. molecule represent nitrogen and oxygen atoms, respectively. Int. J. Mol. Sci. 2016, 17, 779 3 of 24

Int. J. Mol. Sci. 2016, 17, 779 3 of 23 Aflatoxin B1 (AFB) is a common contaminant in a variety of foods including peanuts, cottonseed meal, corn,Aflatoxin and other B1 (AFB) grains is a as common well as animalcontaminant feeds. in Aflatoxin a variety of B1 foods is considered including the peanuts, most toxiccottonseed aflatoxin andmeal, it is highlycorn, and implicated other grains in hepatocellular as well as animal carcinoma feeds. (HCC) Aflatoxin in humans B1 is considered [14]. The epoxide the most metabolite toxic of AFBaflatoxin can reactand it with is highly duplex implicated DNA to in producehepatocellular a cationic carcinoma adduct (HCC) (Figure in humans2A). An [14]. NMR The structure epoxide of thismetabolite adduct has of beenAFB resolvedcan react andwith reported duplex DNA by Stone to produce [15], as a showncationic in adduct Figure (Figure2B. This 2A). NMR An structureNMR showsstructure that Aflatoxin of this adduct B1 substructure has been resolved inserts and into repo DNArted duplex by Stone strand. [15], as The shown adduct in Figure acts as 2B. a covalent This binder,NMR which structure can crosslinkshows that to Aflatoxin duplex DNA B1 substructu conformationre inserts inducing into DNA the modification duplex strand. and The change adduct in the DNAacts conformation. as a covalent binder, The changed which can DNA crosslink will to stop duplex interacting DNA conformation with related inducing proteins, the which modification can block theand process change of replicationin the DNA orconformation. transcription. The changed DNA will stop interacting with related proteins, which can block the process of replication or transcription. Another DNA duplex adduct, a synthetic N4C–ethyl–N4C (Figure3A), was discovered to interact Another DNA duplex adduct, a synthetic N4C–ethyl–N4C (Figure 3A), was discovered to interact with DNA by Miller [16]. The DNA study indicated that the ethyl cross-linked the base pairs in with DNA by Miller [16]. The DNA study indicated that the ethyl cross-linked the base pairs in the the structure solution. However, the ethyl linker does not remarkably change the B-form DNA structure solution. However, the ethyl linker does not remarkably change the B-form DNA conformationconformation [17 [17],], as as shown shown in in Figure Figure3 B.3B.

Figure 3. (A) Chemical structure of N4C–ethyl–N4C adduct; and (B) one strand of NMR structure of a Figure 3. (A) Chemical structure of N4C–ethyl–N4C adduct; and (B) one strand of NMR structure DNA duplex complex with N4C–ethyl–N4C adduct (PDB code: 2OKS). The orange structure of a DNA duplex complex with N4C–ethyl–N4C adduct (PDB code: 2OKS). The orange structure represents the small molecule; the purple structure represents the backbone of nucleic acid; the blue representsand red theatoms small in the molecule; molecule the represent purple nitrogen structure and represents oxygen atoms, the backbone respectively. of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. Similar interactions were reported in the trimethylene related structures (Figure 4A) and DNA duplexSimilar conformation interactions [18]. were Two reported DNA duplexes in the trimethylene structures were related resolved structures and submitted (Figure4 A)to andProtein DNA duplexData conformationBank. Although [18 the]. stacking Two DNA between duplexes the linke structuresd bases wereand neighbor resolved bases and was submitted different, to both Protein Dataof Bank.the two Although structures the indicated stacking that between the duplexes the linked could bases maintain and the neighbor hydrogen bases bonds was and different, the B-form both of thegeometry two structures (Figure indicated 4B,C). that the duplexes could maintain the hydrogen bonds and the B-form Mitomycin C (Figure 5A) is a member of mitomycins, which contains aziridine substructure and geometry (Figure4B,C). are extracted from natural products isolated from streptomycetes. The molecule is used as Mitomycin C (Figure5A) is a member of mitomycins, which contains aziridine substructure a chemotherapy drug for cancer. Mitomycin C alkylates the guanine in C–G base pairs of the DNA andconformation are extracted [19]. from The natural crystal products complex isolatedof a short from DNA streptomycetes. with mitomycin The C moleculewas reported is used by as a chemotherapyPatel et al. [20].drug Unlike for other cancer. standard Mitomycin DNA duplexes, C alkylates the mitomycin the guanine alkylated in C–G DNA base duplex pairs shows of the DNAA-form conformation base pair stacking [19]. The and crystal B-form complex sugar puck ofers. a short The ring DNA of withmitomycin mitomycin locates Cin wasthe minor reported by Patelgroove.et al.And[20 the]. Unlikering of otherindoloquinone standard forms DNA a duplexes, 45° to the thehelix mitomycin axis, as shown alkylated in Figure DNA 5B. duplex shows A-formAs base mentioned, pair stacking these andsmall B-form molecules sugar can puckers. modify and The change ring of the mitomycin overall conformation locates in the of the minor groove.conformation And the ringby cross-linking of indoloquinone to DNA forms duplexes. a 45˝ to The the helixchanged axis, DNA as shown conformations in Figure 5willB. stop interactingAs mentioned, with the these corresponding small molecules biological can partne modifyrs, thus and the changetranscription the overall or replication conformation process of thewill conformation be blocked. byThis cross-linking is how this kind to DNAof molecules duplexes. works The in the changed chemotherapeutics DNA conformations treatment. These will stop interactingmolecules with showed the corresponding high toxicity in biological normal cells, partners, just because thus the of transcription its low selectivity. or replication To decrease process willresistance be blocked. and promote This is selectivity, how this kindthe comprehension of molecules of works the interaction in the chemotherapeutics mechanism between treatment. DNA Theseand molecules drugs willshowed be of significant high toxicity importance. in normal cells, just because of its low selectivity. To decrease resistance and promote selectivity, the comprehension of the interaction mechanism between DNA and drugs will be of signiﬁcant importance. Int. J. Mol. Sci. 2016, 17, 779 4 of 24 Int. J. Mol. Sci. 2016, 17, 779 4 of 23

Int. J. Mol. Sci. 2016, 17, 779 4 of 23

Figure 4. (A) Chemical structure of N2G–trimethylene–N2G; (B) crystal complex of a DNA with N2G– Figure 4. (A) Chemical structure of N2G–trimethylene–N2G; (B) crystal complex of a DNA with trimethylene–N2G cross-link (PDB code: 2KNK); and (C) crystal complex of a DNA with N2G– 2 2 N G–trimethylene–N G cross-link (PDB2 code: 2KNK);2 and (C) crystal complex of a DNA2 with trimethylene–NFigure 4. (A) Chemical2G cross-link structure (PDB of code: N G–trimethylene–N 2KNL). The orangeG; (structureB) crystal represents complex of the a DNA small with molecule; N G– 2 2 2 2 N G–trimethylene–Nthetrimethylene–N purple structureG cross-link representsG cross-link (PDB the (PDBbackbone code: code:2KNK); of nu 2KNL). cleicand acid;(C The) crystalthe orange blue complex and structure red ofatoms a represents DNA in the with molecule the N smallG– 2 molecule;representtrimethylene–N the nitrogen purpleG andcross-link structure oxygen (PDB represents atoms, code: respectively. 2KNL). the backbone The orange of nucleic structure acid; represents the blue the and small red atomsmolecule; in the moleculethe purple represent structure nitrogen represents and oxygenthe backbone atoms, ofrespectively. nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively.

Figure 5. (A) Chemical structure of mitomycin C; and (B) crystal complex of a short DNA with

mitomycin C (PDB code: 199D). The orange structure represents the small molecule; the purple FigurestructureFigure 5. 5.(A represents ()A Chemical) Chemical the structure backbonestructure ofof nucleic mitomycinmitomycin acid. C; and ( (BB)) crystal crystal complex complex of of a ashort short DNA DNA with with mitomycin C (PDB code: 199D). The orange structure represents the small molecule; the purple mitomycin C (PDB code: 199D). The orange structure represents the small molecule; the purple structure represents the backbone of nucleic acid. 2.1.2.structure Small represents Molecules the Targeting backbone with of nucleic DNA Duplex acid. in Minor Groove 2.1.2.To Small affect Molecules the gene Targetingexpression, with the DNAsmall Duplexmolecules in Minor can not Groove only insert into the DNA strand, but 2.1.2.also Small bind Moleculesto the major Targeting or minor with grooves DNA of Duplex high binding in Minor affinity. Groove For minor groove, typical small moleculesTo affect are theheterocylic gene expression, dications theand small polyamides, molecules including can not netropsin,only insert berenil, into the and DNA pentamidine, strand, but alsoTo bind affect to the the gene major expression, or minor grooves the small of moleculeshigh binding can affinity. not only For insert minor into groove, the DNA typical strand, small but alsoas bindshown to in the Figure major 6. or Originally, minor grooves these molecules of highbinding were found afﬁnity. to bind For AT-rich minor areas groove, preferentially. typical small Then,molecules these are molecules heterocylic appeared dications near and G–C polyamides, and C–G base including pairs [21,22]. netropsin, The moleculesberenil, and targeting pentamidine, minor molecules are heterocylic dications and polyamides, including netropsin, berenil, and pentamidine, grooveas shown have in Figurebeen studied 6. Originally, as a series these of molecules therapeutics were with found anti-viral, to bind anti-tumor AT-rich areas and preferentially.anti-bacterial as shownThen, these in Figure molecules6. Originally, appeared near these G–C molecules and C–G were base pairs found [21,22]. to bind The AT-rich molecules areas targeting preferentially. minor Then, groove these have molecules been studied appeared as a near series G–C of andtherapeutics C–G base with pairs anti-viral, [21,22]. anti-tumor The molecules and targetinganti-bacterial minor

Int. J. Mol. Sci. 2016, 17, 779 5 of 24

Int. J. Mol. Sci. 2016, 17, 779 5 of 23 grooveInt. J. Mol. have Sci. 2016 been, 17, studied779 as a series of therapeutics with anti-viral, anti-tumor and anti-bacterial5 of 23 activitiesactivities [23–25]. [23–25]. Some Some structural structural studies studies have have been been investigated investigated as as the the basis basis of of the the design design of of small small activities [23–25]. Some structural studies have been investigated as the basis of the design of small moleculesmolecules [26–30]. [26–30]. The The crystal crystal complex complex of DNA of DNA duplex duplex and and Hoechst Hoechst 33528 33528 is shown is shown in Figure in Figure 7. The7. molecules [26–30]. The crystal complex of DNA duplex and Hoechst 33528 is shown in Figure 7. The HoechstThe Hoechst 33528 33528 is clamped is clamped in the in minor the minor groove groove of the of DNA the DNA structure. structure. Hoechst 33528 is clamped in the minor groove of the DNA structure.

Figure 6. Typical structures of small molecules for DNA minor groove. Figure 6. Typical structures of small molecules forfor DNADNA minorminor groove.groove.

Figure 7. Crystal complex of DNA duplex and Hoechst 33528 (PDB code: 8BNA). The orange structure Figure 7. Crystal complex of DNA duplex and Hoechst 33528 (PDB code: 8BNA). The orange structure representsCrystal the small complex molecule; of DNA the purple duplex structure and Hoechst represents 33528 (PDB the ba code:ckbone 8BNA). of nucleic The orange acid; structurethe blue represents thethe smallsmall molecule;molecule; thethe purple structure represents the backbonebackbone of nucleicnucleic acid;acid; thethe blueblue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. and red atomsatoms inin thethe moleculemolecule representrepresent nitrogennitrogen andand oxygenoxygen atoms,atoms, respectively.respectively. Except for the drug-like molecules, some unusual compounds were also identified to locate in Except for the drug-like molecules, some unusual compounds were also identified to locate in the minorExcept groove for the ofdrug-like DNA. An molecules, 8 ring molecule some unusual(Figure 8A) compounds was reported were that also it identiﬁed can bind toto locatea DNA in the minor groove of DNA. An 8 ring molecule (Figure 8A) was reported that it can bind to a DNA duplexthe minor [31]. groove The resolved of DNA. structure An 8 ring indicates molecule th (Figureat the8 A)DNA was conformation reported that has it can been bind remarkably to a DNA duplex [31]. The resolved structure indicates that the DNA conformation has been remarkably changed.duplex [31 The]. The minor resolved groove structure has been indicates widened, that and the the DNA major conformation groove has has been been narrowed. remarkably The changed. minor changed. The minor groove has been widened, and the major groove has been narrowed. The minor andThe major minor grooves groove has are been both widened, approximate and the4 Å major (Figure groove 8B). hasAdditionally, been narrowed. the helix The minorcenter anddirection major and major grooves are both approximate 4 Å (Figure 8B). Additionally, the helix center direction isgrooves twisted are more both approximatethan 18° to 4the Å (Figuremajor 8groove,B). Additionally, in comparison the helix with center the direction inherent is counterparts. twisted more is twisted˝ more than 18° to the major groove, in comparison with the inherent counterparts. Thethan distortion 18 to the provides major groove, a fundamental in comparison element with for thesupporting inherent the counterparts. concept of Theallosteric distortion change provides of the The distortion provides a fundamental element for supporting the concept of allosteric change of the DNAa fundamental conformationelement with for inhibitors supporting locating the concept in minor of grooves. allosteric change of the DNA conformation with DNA conformation with inhibitors locating in minor grooves. inhibitors locating in minor grooves.

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Figure 8. (A) Chemical structure of 8 ringmolecule; and (B) complex of a DNA with 8 ring Figure 8. (A) Chemical structure of 8 ringmolecule; and (B) complex of a DNA with 8 ring molecule (PDB molecule (PDB code: 3I5L). The blue and red atoms in the molecule represent nitrogen and oxygen code: 3I5L). The blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. atoms, respectively.

TheThe small small molecules molecules can can bind bind to to nonstandard nonstandard DNADNA duplex as as well. well. Hoogsteen Hoogsteen base base pairs pairs may may occuroccur in alternating in alternating A–T A–T sequences, sequences, which which are are abundant abundant in in eukaryotic eukaryotic animals. animals. BecauseBecause of the lack lack of structuralof structural information, information, however, however, there isthere little is analysis little analysis in this ﬁeld.in this Recently, field. Recently, the complex the complex of Hoogsteen of DNAHoogsteen duplex and DNA pentamidine duplex and (Figure pentamidine9A) was (Figure determined 9A) was and determined reported [and32]. reported The X-ray [32]. DNA The structureX-ray presentsDNA a structure mixture ofpresents Watson–Crick a mixture and of Hoogsteen Watson–Cri patternck and (Figure Hoogsteen9B). Unlike pattern previous (Figure minor9B). Unlike binders, pentamidineprevious minor does not binders, bind topentamidine the minor groovedoes not totally. bind to Only the minor the center groove of thetotally. pentamidine Only the center is bound of to the pentamidine is bound to the minor groove, leaving the positively charged ends detached from the minor groove, leaving the positively charged ends detached from the DNA and free to interact the DNA and free to interact with phosphate groups from adjacent duplexes in the crystal. This new with phosphate groups from adjacent duplexes in the crystal. This new binding pattern has potent binding pattern has potent inspiration for antibacterial design of pentamidine. inspiration for antibacterial design of pentamidine.

Figure 9. (A) Chemical structure of pentamidine; and (B) complex of DNA with pentamidine (PDB code: 3EY0). The orange structure represents the small molecule; the purple structure represents Figure 9. (A) Chemical structure of pentamidine; and (B) complex of DNA with pentamidine (PDB the backbonecode: 3EY0). of The nucleic orange acid; structure the blue represents and red the atoms small in molecule; the molecule the purple represent structure nitrogen represents and oxygen the atoms,backbone respectively. of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively.

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Int. J. Mol. Sci. 2016, 17, 779 7 of 23 2.1.3. Small Molecules Targeting with DNA Duplex in Major Groove 2.1.3. Small Molecules Targeting with DNA Duplex in Major Groove For major groove studies, there are less reports of small molecule targeting information. Due to the requirementFor major of groove larger studies, compounds, there are few less complexesreports of small of moleculesmolecule targeting and DNA information. major grooves Due to are the requirement of larger compounds, few complexes of molecules and DNA major grooves are determined. Although majority of carbohydrates bind to the DNA minor grooves, some of them can determined. Although majority of carbohydrates bind to the DNA minor grooves, some of them can show binding afﬁnity to DNA major groove. The reason for this major groove binding is the large size show binding affinity to DNA major groove. The reason for this major groove binding is the large of carbohydratessize of carbohydrates and their and hydrophilic their hydrophilic and hydrophobic and hydrophobic substructures. substructures. Some Some classic classic major major groove bindersgroove are listedbinders in are Figure listed 10 in[ 33Figure]. 10 [33].

Figure 10. Typical structures of small molecules for DNA major groove. (A) The structure of Figure 10. Typical structures of small molecules for DNA major groove. (A) The structure of neomycin-grove binder; (B) The structure of nogalamycin; (C) The structure of neocarzinostatin. neomycin-grove binder; (B) The structure of nogalamycin; (C) The structure of neocarzinostatin. Neocarzinostatin and related derivatives have been used as anti-tumor agents for decades [34,35]. NeocarzinostatinThis kind of molecule and related can form derivatives a complex have with been protein used as and anti-tumor damage agents DNA forby decadeshydrogen [34 ,35]. This kindabstraction of molecule [36]. In can order form to aunderstand complexwith the me proteinchanism and and damage interaction DNA between by hydrogen DNA duplex abstraction and [36]. In ordermolecule, to understand the complex the of mechanism the DNA duplex and interaction and neocarzinostatin between DNA were duplex reported. and Neocarzinostatin-gb molecule, the complex of theand DNA neocarzinostatin-glu duplex and neocarzinostatin were used as were ligand reported. to bind to Neocarzinostatin-gb the DNA duplex. As and shown neocarzinostatin-glu in Figure 11, both of the two complex structures are observed [37,38]. The majority of the neocarzinostatin-gb were used as ligand to bind to the DNA duplex. As shown in Figure 11, both of the two complex binds to the major groove. The two rings of the molecule are close to each other in the structure. structuresHowever, are observedthe neocarzinostatin-glu [37,38]. The majoritybinds to the of theminor neocarzinostatin-gb groove. The differences binds of tothe the two major binding groove. The twomodesrings provide of the significant molecule areinspiration close to into each small other molecule-DNA in the structure. specific However, targeting. the neocarzinostatin-glu binds to the minor groove. The differences of the two binding modes provide signiﬁcant inspiration into small molecule-DNA speciﬁc targeting.

Figure 11. Cont.

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Figure 11. (A) Chemical structure of neocarzinostatin-gb; (B) complex of a DNA with neocarzinostatin-gb C D (PDB code: 1KVH); ( ) chemical structure of neocarzinostatin-glu; and ( ) complex of DNA with neocarzinostatin-glu (PDB code: 1MP7). The orange structure represents the small molecule; the purple structure represents the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively.

2.1.4. Small Molecules Intercalating into DNA Duplex Inserting a molecule between the base pairs of DNA is another binding pattern of small molecules targeting DNA duplexes. Inserting to the base pairs can interrupt DNA replication and transcription. The insertion results from hydrophobic, hydrogen bonding and van der Waals forces [39]. The insertion lengthens the length of DNA and reduces the helical twist [40,41]. In previous study, only molecules with fused ring systems were found to insert into the base pairs of DNA duplexes. Later, molecules with electrophilic or cationic groups behaved the same insertion pattern. However, the ring systems were not required [42,43]. Various compounds have been identiﬁed as intercalators, including ditercalinium, dactinomycin, daunomycin and adriamycine. Intercalators can be used as anti-fungal, anti-tumor, and anti-neoplastic agents. However, due to the toxicity, most of the compounds were failed during the clinical trial [44]. 1 Daunomycin (trade name Cerubidine) is used to treat various cancers as chemotherapy drug [45].

Speciﬁcally, the most common use is treating some types of leukemia. Daunomycin (Figure 12A) consists of a planar ring, an amino sugar structure and a fused cyclohexane ring system. A lot of structural studies have been investigated to understand the interaction between DNA duplex and molecule [28,46–51]. Most of the structures indicate that two daunomycin molecules insert into the G–C steps with the sugar moiety in duplex, as shown in Figure 12B. Through sequence selectivity investigation by different biophysical methods, it was revealed that this ligand prefers a purine-pyrimidine step and the DNA duplexes are usually elongated or twisted after binding to the small molecule. Adriamycin (Figure 12C) contains an extra hydroxyl group, compared to daunomycin. Although, the daunomycin and adriamyc in have nearly the same structure, their functions are remarkably diverse. Adriamycin is used to treat the tumor, while daunomycin is an effective drug in leukemias [47]. The complex structures of DNA duplex and compounds have been determined. In order to understand the differences between the two compounds, the complexes of the two molecules with a same DNA were resolved. The two complex structures showed a little differences between each other. Moreover, the hydroxyl group of the adriamycin interacts with a water molecule in the complex (Figure 12D). Ditercalinium, as shown in Figure 13A, is regarded as a DNA intercalator in treatment of cancer. The structure of ditercalinium is a dimer of pyridocarbozole. Thus, the molecule can bind to DNA duplex by bis-intercalation and then induce DNA repair in eukaryotic or prokaryotic cell [52–54]. Afterwards, pyridocarbozole derivatives and related bioactivity have been studied [55–57]. The structure of DNA duplex and ditercalinium showed that the dimer of the molecule inserted to two G–C steps of the conformation (Figure 13B). The duplex of the DNA maintains a right-handed helix and has a binding site in major groove. The nitrogens with positive charge of the molecule make the charge interaction toward major groove of DNA [58]. In the complex structure, the helix is twisted at Int. J. Mol. Sci. 2016, 17, 779 9 of 24 Int. J. Mol. Sci. 2016, 17, 779 9 of 23 Int. J. Mol. Sci. 2016, 17, 779 9 of 23 ˝ approximateapproximate 15 15°to to minor minor groove groove of of DNA. DNA. The The mentionedmentioned structuralstructural features give give inspiration inspiration on on approximate 15° to minor groove of DNA. The mentioned structural features give inspiration on furtherfurther research research of of optimizing optimizing this this kind kind of of molecules. molecules. further research of optimizing this kind of molecules.

Figure 12. (A) Chemical structure of daunomycin; (B) one strand of a DNA duplex complex FigureFigure 12. 12.(A ()A Chemical) Chemical structure structure of of daunomycin; daunomycin; (B )(B one) one strand strand of aof DNA a DNA duplex duplex complex complex with with daunomycin (PDB code: 1D10); (C) chemical structure of adriamycin; and (D) one strand of daunomycinwith daunomycin (PDB code:(PDB 1D10);code: 1D10); (C) chemical (C) chemical structure structure of adriamycin; of adriamycin; and ( Dand) one (D strand) one strand of a DNA of a DNA duplex complex with adriamycin (PDB code: 1D12). The orange structure represents duplexa DNA complex duplex with complex adriamycin with (PDBadriamycin code: 1D12). (PDB Thecode: orange 1D12). structure The orange represents structure the small represents molecule; the small molecule; the purple structure represents the backbone of nucleic acid; the red dots thethe purple small structure molecule; represents the purple the backbonestructure ofrepresen nucleicts acid; the thebackbone red dots of represent nucleic acid; the water the red molecules; dots represent the water molecules; the blue and red atoms in the molecule represent nitrogen and therepresent blue and the red water atoms molecules; in the molecule the blue represent and red nitrogen atoms andin the oxygen molecule atoms, represent respectively. nitrogen The and green oxygen atoms, respectively. The green square represents the water molecule that interacts with the squareoxygen represents atoms, respectively. the water molecule The green that square interacts repres withents the the small water molecule. molecule that interacts with the small molecule. small molecule.

Figure 13. (A) Chemical structure of dimer of ditercalinium; and (B) complex of a DNA duplex with FigureFigure 13. 13.(A (A) Chemical) Chemical structure structure ofof dimerdimer ofof ditercalinium;ditercalinium; and and ( (BB)) complex complex of of a aDNA DNA duplex duplex with with ditercalinium (PDB code: 1D32). The orange structure represents the small molecule; the purple ditercaliniumditercalinium (PDB (PDB code: code: 1D32).1D32). TheThe orange structure represents represents the the small small molecule; molecule; the the purple purple structure represents the backbone of nucleic acid; the blue and red atoms in the molecule represent structurestructure represents represents the the backbone backbone ofof nucleicnucleic acid; the blue and and red red atoms atoms in in the the molecule molecule represent represent nitrogen and oxygen atoms, respectively. nitrogennitrogen and and oxygen oxygen atoms, atoms, respectively. respectively. Actually, the specificity of DNA intercalators is the reason for drug toxicity. Molecules can bind Actually, the specificity of DNA intercalators is the reason for drug toxicity. Molecules can bind to Actually,unexpected the DNA speciﬁcity sequence of DNA for new intercalators treatment. is Cr theyptolepine reason for (Figure drug toxicity.14A) is a Molecules good example can bindfor to unexpected DNA sequence for new treatment. Cryptolepine (Figure 14A) is a good example for to unexpectedoccasional drug DNA discovery. sequence Cr foryptolepine new treatment. is isolated Cryptolepine from Cryptolepis (Figure triangular 14A) is aand good used example as anti- for occasional drug discovery. Cryptolepine is isolated from Cryptolepis triangular and used as anti- occasionalmalarial drugand cytotoxic discovery. agent Cryptolepine [59]. This compound is isolated fromcan insertCryptolepis with C–G triangular rich sequencesand used [60]. as anti-malarial The crystal malarial and cytotoxic agent [59]. This compound can insert with C–G rich sequences [60]. The crystal andcomplex cytotoxic structure agent [indicates59]. This that compound the drug caninteracts insert with with the C–G duplex rich sequencesin a base stacking [60]. The insertion crystal complex structure indicates that the drug interacts with the duplex in a base stacking insertion

Int. J. Mol. Sci. 2016, 17, 779 10 of 24 complex structure indicates that the drug interacts with the duplex in a base stacking insertion pattern (Figure 14B).Int. The J. Mol. molecule Sci. 2016, 17, 779 and two consecutive C–G base pairs form a sandwich conformation10 of 23 with two C–G rich sequences. And the hexatomic ring stacks in the middle of cytosine and guanine. pattern (Figure 14B). The molecule and two consecutive C–G base pairs form a sandwich ˝ Structurally,conformation cryptolepine with was two C–G mildly rich se twistedquences. And at about the hexatomic 6.8 between ring stacks two in the aromatic middle of cytosine rings. Besides, the charged nitrogenand guanine. can improveStructurally, the cryptolepine stability was of mildly the twisted complex at about with 6.8° thebetween interaction two aromatic between rings. molecule and DNA duplex.Besides, Thethe charged insertion nitrogen into can base improve pair the steps stability and of the complex molecular with the dissymmetry interaction between were important molecule and DNA duplex. The insertion into base pair steps and the molecular dissymmetry were elements forimportant this interaction. elements for this interaction.

Figure 14. (A) Chemical structure of dimer of cryptolepine; and (B) one strand of a DNA duplex Figure 14. (Acomplex) Chemical with cryptolepine structure (PDB of code: dimer 1K9G). of cryptolepine;The orange structure and represen (B) onets the strand small molecule; of a DNA duplex complex withthe cryptolepine purple structure (PDBrepresents code: the backbone 1K9G). of The nucleic orange acid; the structure blue and red represents atoms in the themolecule small molecule; the purple structurerepresent nitrogen represents and oxygen the backbone atoms, respectively. of nucleic acid; the blue and red atoms in the molecule represent2.1.5. nitrogen Small Molecules and oxygen Targeting atoms, with respectively. DNA Duplex through Multiple Binding Patterns Small molecules can not only bind to DNA duplex in a single mode, but also interact with DNA 2.1.5. Small Moleculesduplex in multiple Targeting binding with patterns, DNA which Duplex will make through the complex Multiple more stable Binding [61,62]. Patterns PBD-BIMZ (Figure 15A) binds to a DNA duplex in a covalent link to a guanine base. The complex shows that the Small moleculeshybrid molecule can orients not only in the bind minor togroove DNA (Figure duplex 15B) [63]. ina Although single the mode, covalent but binding also interactdistorts with DNA duplex in multiplethe DNA bindinghelix, the overall patterns, duplex which still maintains will make the standard the complex B-form conformation. more stable It is [revealed61,62]. PBD-BIMZ that covalent binding site in local region possesses specific twisted helix. By comparison, the (Figure 15A)piperazine binds to ring a DNAshows a duplex high flexibility in a covalentwith various link conformations. to a guanine Later, the base. same The group complex reported shows that the hybrid moleculeanother ligand orients (Figure in 15C) the in minorcomplex groovewith the same (Figure DNA duplex.15B)[ This63]. hybrid Although compound the binds covalent to binding distorts the DNAa guanine helix, in a covalent the overall link and duplexthe naphthalimide still maintains substructure the embeds standard to (A–T) B-form2 steps, as shown conformation. in It is Figure 15D [64]. The covalent binding will remarkably make the DNA and ligand stable. Moreover, revealed thatbecause covalent many binding molecules sitehave inlow local specificity region for possessessequence, the specific multiple twistedpatterns can helix. make Bythe comparison, the piperazinemolecules ring sequence shows targetable. a high flexibility with various conformations. Later, the same group reported another ligand (Figure 15C) in complex with the same DNA duplex. This hybrid compound binds to a guanine in a covalent link and the naphthalimide substructure embeds to (A–T)2 steps, as shown in Figure 15D[64]. The covalent binding will remarkably make the DNA and ligand stable. Moreover, because many molecules have low specificity for sequence, the multiple patterns can make the molecules sequence targetable.

Figure 15. Cont.

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Figure 15. (A) Chemical structure of PBD-BIMZ; (B) complex of a DNA duplex with PBD-BIMZ (PDB code: 2KTT); (C) chemical structure of PBD-naphthalimide; and (D) complex of a DNA duplex with PBD-naphthalimide (PDB code: 2KY7). The orange structure represents the small molecule; the purple

structure represents the backbone of nucleic acid; the blue and red atoms in the molecule represent Figure 15. (A) Chemical structure of PBD-BIMZ; (B) complex of a DNA duplex with PBD-BIMZ (PDB nitrogen andcode: oxygen 2KTT); atoms,(C) chemical respectively. structure of PBD-naphthalimide; and (D) complex of a DNA duplex with PBD-naphthalimide (PDB code: 2KY7). The orange structure represents the small molecule; the 2.2. Small Moleculespurple Targetingstructure represents DNA Triplexthe backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. Single strand DNA can bind to a DNA duplex structure to form a triplex conformation DNA [65]. The interactions2.2. Small between Molecules strands Targeting are DNA Hoogsteen Triplex or reverse Hoogsteen base pairs. Small molecules can bind to triplex DNASingle strand groove, DNA blocking can bind to the a DNA access duplex of otherstructure DNA to form natural a triplex substrate conformation [66 DNA]. The [65]. DNA triplex The interactions between strands are Hoogsteen or reverse Hoogsteen base pairs. Small molecules can can displaybind two to forms, triplex DNA intermolecular groove, blocki triplexesng the access and of other intramolecular DNA natural substrate triplexes [66]. [67 The]. Intermolecular DNA triplex triplex is generatedcan from display a DNA two forms, chain intermolecular of an extra triplexes DNA structure. and intramolecular Generally, triplexes the [67]. intermolecular Intermolecular triplex has attracted attentiontriplex is duegenerated to the from possible a DNA treatmentchain of an extra for various DNA structure. cancers Generally, or other the diseases. intermolecular In fact, most of the DNA duplextriplex has intercalators attracted attention can insert due to the to thepossible triplex treatment as well for vari [68ous]. cancers or other diseases. In fact, most of the DNA duplex intercalators can insert to the triplex as well [68]. As mentionedAs mentioned above, above, neomycin neomycin is is a a typical typical major DNA DNA groove groove binder. binder. In triplex In system, triplex system, neomycin canneomycin make can the make base the mixedbase mixed DNA DNA triplexes triplexes and and T–A–T T–A–T triplex triplex stable. stable.The computationalThe computational docking conformationdocking conformation [69] of [69] the of triplex the triplex and and neomycin neomycin is is shown shown in Figure in Figure 16. The 16 computational. The computational and and biophysical researches both indicate that this Watson–Hoogsteen complementarity leads to biophysicalthe researches selectivity. both indicate that this Watson–Hoogsteen complementarity leads to the selectivity.

Figure 16. Computer generated model of neomycin bound to a DNA triplex. Ring I of neomycin Figure 16. locatesComputer in the middle generated of the DNA model groove, of and neomycin Ring II and bound IV facilitate to abridging DNA the triplex. pyrimidine Ring strands; I of neomycin locates in thethe middle orange, green of the and DNA purple groove, structures and represent Ring the II andthree IVchains facilitate of the DNA; bridging the blue the and pyrimidine red atoms strands; the orange,in green the molecule and purple represent structures nitrogen and represent oxygen atoms, the three respectively. chains of the DNA; the blue and red atoms

in the molecule represent nitrogen and oxygen atoms, respectively.

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Similar toto majormajor groove groove binders, binders, neomycin neomycin conjugates conjugates are alsoare also found found to bind to withbind thewith DNA thetriplex DNA structures.triplex structures. Computational Computational modeling modeling of pyrene–neomycin of pyrene–neomycin indicates indicates pyrene pyrene will insert will insert into the into base the pairsbase pairs when when neomycin neomycin bind withbind Watson–Hoogsteenwith Watson–Hoogsteen groove groove [70]. [70].

2.3. Small Molecules Targeting DNADNA QuadruplexQuadruplex Telomere regulationregulation has been regarded as a significant significant potential strategy strategy for for cancer cancer therapy. therapy. Guanosine rich nucleic acids are regarded as a newnew classclass ofof non-antisensenon-antisense nucleic acids with active G-quartet conformation [[71].71]. Except for telomerase telomerase,, G-quadruplex-forming sequences have also been found in the promoter regions of many other genes. Small Small molecules molecules can can stabilize stabilize G-quartet G-quartet structure, structure, causing the telomerase low activityactivity [[72–74].72–74]. Therefore,Therefore, the G-quadruplexG-quadruplex structures have attractedattracted more attention for drug design.design. Because G-quartet conformations consist of planar stacked guanine bases, numbers of small molecules with electron deficientdeficient aromatic rings areare identifiedidentified to bindbind [[75].75]. As mentioned above, distamycin AA andand daunomycin daunomycin are are typical typical intercalators intercalators for DNAfor DNA duplex. duplex. In quadruplex In quadruplex systems, systems, these compoundsthese compounds are able are to able bind to tobind DNA to DNA quadruplex quadrupl structures.ex structures. The firstThe complexfirst complex of G-quadruplex of G-quadruplex and daunomycinand daunomycin was determinedwas determined and reported and reported by Neidle by Neidle [76]. Figure [76]. Figure17 shows 17 theshows four the parallel four parallel strands andstrands three and daunomycin three daunomycin molecules molecules at the end at ofthe the end complex. of the complex. This complex This indicatescomplex indicates that the structure that the showsstructure high shows stability high with stab theility molecules with the molecules stacking on stacking the end. on the end.

Figure 17.17. Crystal complex of aa DNADNA quadruplexquadruplex withwith daunomycindaunomycin (PDB(PDB code:code: 1O0K). The orange structure represents the small molecule;molecule; the purple structure represents the backbone of nucleic acid; the blue and red atomsatoms inin thethe moleculemolecule representrepresent nitrogennitrogen andand oxygenoxygen atoms,atoms, respectively.respectively.

It isis knownknown thatthat distamycindistamycin AA bindsbinds withwith thethe minorminor groovegroove ofof DNADNA duplex.duplex. The structure of G-quadruplex and distamycin A was determined and reported by Randazzo [77]. The structure G-quadruplex and distamycin A was determined and reported by Randazzo [77]. The structure indicates that molecule performs the similar binding to DNA duplex minor groove binding. The indicates that molecule performs the similar binding to DNA duplex minor groove binding. dimer of the molecule binds at the converse position of the quadruplex conformation (Figure 18). The The dimer of the molecule binds at the converse position of the quadruplex conformation (Figure 18). quadruplex conformation is made up of four identical parallel sequences TGGGGT. At the top of the The quadruplex conformation is made up of four identical parallel sequences TGGGGT. At the top of structure, three stacking daunomycins with sodium ions are located. The quadruplex conformation the structure, three stacking daunomycins with sodium ions are located. The quadruplex conformation resembles the inherent counterpart. The structures of daunomycins interact to the DNA grooves by resembles the inherent counterpart. The structures of daunomycins interact to the DNA grooves forming hydrogen bonds and van der Waals forces. The complex shows the structure is more stable by forming hydrogen bonds and van der Waals forces. The complex shows the structure is more when the molecules stack at the top than intercalate, because of the large termination of the molecule stable when the molecules stack at the top than intercalate, because of the large termination of the surface. molecule surface.

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Figure 18. Complex of a DNA quadruplex with distamycin A (PDB code: 2JT7). The orange structure Figure 18. Complex of a DNA quadruplex with distamycin A (PDB code: 2JT7). The orange structure Figure 18.represents Complex the smallof a DNA molecule; quadruplex the purple with structure distamycin represents A the (PDB backbone code: of 2JT7). nucleic The acid; orange the blue structure represents the small molecule; the purple structure represents the backbone of nucleic acid; the blue representsand thered atomssmall inmolecule; the molecule the represent purple structurenitrogen and represents oxygen atoms, the respectively.backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. Compared to DNA duplex binding molecules, some novel kinds of molecules have been found. These molecules can bind specifically to DNA quadruplex structures, as shown in Figure 19. The ComparedCompared toto DNADNA duplex binding molecules, some novel kinds of molecules have been found. structural and biochemical studies displayed detailed information of the interaction between small These molecules can bind specifically to DNA quadruplex structures, as shown in Figure 19. The structural These moleculesmolecules andcan DNA bind quadruplex. specifically In to addition, DNA quadruplexit also provideda structures, novel platform as shown for further in Figure drug 19. The andstructural biochemicaldesign and targeting biochemical studies DNA displayed quadruplex. studies detaileddisplayed The progress information detailed in this informationfield of thehas been interaction reportedof the interactionbetween [78]. small between molecules small andmolecules DNA quadruplex.and DNA quadruplex. In addition, itIn also addition, provideda it also novel provideda platform novel for further platform drug for design further targeting drug DNAdesign quadruplex. targeting DNA The quadruplex. progress in this The ﬁeld progress has been in this reported field has [78 been]. reported [78].

Figure 19. Typical structures of small molecules for DNA quadruplex.

3. Small Molecules Targeting RNA

3.1. Small Molecules Targeting Ribosome The ribosomalFigure RNA 19. Typical (rRNA) structures is the most of comprehensively small molecules studied for DNA among quadruplex. various kinds of RNA types. The ribosomeFigure 19. is Typicalan attracti structuresve anti-bacterial of small target molecules due to for its DNA important quadruplex. function in protein expression [79–83]. The ribosomes in prokaryotic cells mainly contain 30S and 50S subunits. The 3. Small Molecules Targeting RNA 3. Small Molecules Targeting RNA

3.1.3.1. Small Molecules TargetingTargeting RibosomeRibosome TheThe ribosomalribosomal RNARNA (rRNA)(rRNA) isis thethe mostmost comprehensivelycomprehensively studiedstudied amongamong variousvarious kindskinds ofof RNARNA types.types. The The ribosome ribosome is is an an attracti attractiveve anti-bacterial target due to its important function in proteinprotein expressionexpression [ 79[79–83].–83]. The The ribosomes ribosomes in prokaryoticin prokaryotic cells cells mainly mainly contain contain 30S and 30S 50S and subunits. 50S subunits. The subunit The

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subunit 30S is for ensuring that the tRNA can locate in the correct position. The subunit 50S is 30Sresponsible is for ensuring for peptide that the connection. tRNA can Various locate in kinds the correct of comp position.ounds have The subunitbeen identified 50S is responsible to bind the for peptidesubunits connection. of rRNA, Various including kinds pleuromutilin, of compounds oxazolidinones have been identiﬁed and aminoglycosides, to bind the subunits as shown of rRNA, in includingFigure 20 pleuromutilin, [84,85]. oxazolidinones and aminoglycosides, as shown in Figure 20[84,85].

Figure 20. Typical structures of small molecules for 30S or 50S subunit of rRNA. Figure 20. Typical structures of small molecules for 30S or 50S subunit of rRNA. Amikacin (Figure 21A), a member of aminoglycoside antibiotics, is used to inhibit replication of Amikacinvarious kinds (Figure of 21bacteria.A), a member Amikacin of aminoglycosidebinds with the antibiotics,30S subunits is usedof rRNA, to inhibit leading replication to the of variouswrong kinds reading of bacteria. of message Amikacin RNA and binds making with the the bacteria 30S subunits cell incapable of rRNA, of leading translation to the and wrong growing. reading ofThe message structure RNA andof RNA making duplex the bacteriais nearly cell the incapable same ofwith translation the previous and growing. RNA-aminoglycoside The structure of RNAcomplexes duplex [86–90]. is nearly One the of same the aromatic with the rings previous inserts RNA-aminoglycoside into A site helix by stacking. complexes Another [86– 90aromatic]. One of thering aromatic forms four rings hydrogen inserts into bonds A siteto the helix H-bond by stacking. acceptors Another nearby. aromaticThese common ring forms interactions four hydrogen make bondsthe amikacin to the H-bond bind to acceptors RNA duplex nearby. tightly, These as show commonn in Figure interactions 21B [91], make and help the amikacinmaintain A1493 bind to and RNA duplexA1492 tightly, in the bulge as shown structure, in Figure corresponding 21B[91], and with help the maintain “on” state A1493 in 30S and subunit. A1492 in the bulge structure, corresponding with the “on” state in 30S subunit.

Figure 21. (A) Chemical structure of amikacin; and (B) complex of a RNA duplex with amikacin Figure(PDB 21.code:(A 4P20).) Chemical The orange structure structure of amikacin; represents andthe small (B) complex molecule; of the a RNApurple duplex structure with represents amikacin (PDBthe code:backbone 4P20). of nucleic The orange acid; structurethe blue and represents red atoms the in small the molecule molecule; represent the purple nitrogen structure and representsoxygen atoms, respectively. the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively.

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Other aminoglycoside compounds have also been determined and reported. The paromomycin (Figure 22A) is also called monomycin and aminosidine. The detailed complex of two paromomycin molecules and an RNA fragment was solved. The molecule is located in the deep position of the RNA groove forming hydrogen bonds with water, as shown in Figure 22B[88]. Neomycin B (Figure 22C) is an aminoglycoside antibiotic different with paromomycin. Neomycin B contains an ammonium group not hydroxyl group in the structure. The complex structure (Figure 22D) of neomycin B and RNA duplex shows a similar interaction, compared to other aminoglycosides [86].

Figure 22. (A) Chemical structure of paromomycin; (B) complex of a RNA duplex with paromomycin (PDB code: 1J7T); (C) chemical structure of neomycin B; and (D) complex of a RNA duplex with neomycin B (PDB code: 2ET4). The orange structure represents the small molecule; the purple structure represents the backbone of nucleic acid; the blue and red atoms in the molecule represent2 nitrogen and oxygen atoms, respectively.

3.2. Small Molecules Targeting Riboswitches In molecular biology, a riboswitch is a regulatory segment of a messenger RNA (mRNA) molecule, resulting in a change in production of the proteins encoded by the mRNA. Riboswitch consists of an aptamer domain and an expression platform. In recent years, various kinds of riboswitches have been reported [92–95]. Because riboswitch can bind a ligand naturally, it is an ideal target for anti-bacterial agent. The tetrahydrofolate (THF) riboswitch can regulate the transportation and metabolism of folate through combining THF molecules. This riboswitch was identiﬁed to bind various kinds of folates, such as dihydrofolate (DHF) and tetrahydrobiopterin (BH4). Tetrahydrobiopterin (Figure 23A), for example, is an important cofactor of the hydroxylase enzymes of aromatic amino acid. The complex of BH4 and THF riboswitch (Figure 23B) indicates that the placement of the pterin ring in BH4 is identical to that of THF. Pemetrexed (Figure 23C) is a structural analog with folic acid and is also used as chemotherapeutic drug in treatment. The crystal complex of riboswitch and pemetrexed indicates that the compound binds to the RNA structure almost identically as THF, as shown in Figure 23D[96]. Both structures reveal that reduced folates are primarily recognized by the RNA through their pterin moiety. Int. J. Mol. Sci. 2016, 17, 779 16 of 24 Int. J. Mol. Sci. 2016, 17, 779 16 of 23

Figure 23. (A) Chemical structure of tetrahydrobiopterin; (B) complex of a RNA duplex with Figure 23.tetrahydrobiopterin(A) Chemical (PDB structure code: 4LVX); of tetrahydrobiopterin; (C) chemical structure of ( Bpemetrexe;) complex and of (D a) complex RNA duplexof a with tetrahydrobiopterinRNA duplex with (PDB pemetrexed code: 4LVX);(PDB code: (C 4LVY).) chemical The orange structure structure of repres pemetrexe;ents the small and molecule; (D) complex of a RNA duplexthe purple with structure pemetrexed represents (PDB the code: backbone 4LVY). of nu Thecleic orangeacid; the structureblue and red represents atoms in the the molecule small molecule; the purplerepresent structure nitrogen represents and oxygen the atoms, backbone respectively. of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. Recently, some aminoglycosides are identified to bind with the riboswitch RNA, including ribostamycin and paromomycin [97].To understand the biochemical elements for the significant Recently,riboswitch some specificity, aminoglycosides the complexes are of paromo identifiedmycin to (Figure bind 24A) with and the ribostamycin riboswitch (Figure RNA, 24C) including ribostamycinwith neomycin and paromomycin riboswitch RNA [97 were].To resolved. understand The complexes the biochemical of the two structures elements indicate for that the the significant two helix structures of the rRNA formed a consecutive A-form helical structure through stacking riboswitch specificity, the complexes of paromomycin (Figure 24A) and ribostamycin (Figure 24C) between two G–C base pairs, as shown in Figure 24B,D. In the two complex structures, the 6′ with neomycinsubstructures riboswitch of the two RNA molecules were locate resolved. at a close The area, complexes near the hinge of the regiontwo between structures the upperindicate that the two helixand the structures lower helix of stem. the rRNA formed a consecutive A-form helical structure through stacking between two G–C base pairs, as shown in Figure 24B,D. In the two complex structures, the 61 substructures of the two molecules locate at a close area, near the hinge region between the upper and the lower helix stem.

Figure 24. Cont.

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Figure 24. (A) Chemical structure of paromomycin; (B) complex of a neomycin riboswitch RNA

with paromomycin (PDB code: 2MXS); (C) chemical structure of ribostamycin; and (D) complex of Figure 24. (A) Chemical structure of paromomycin; (B) complex of a neomycin riboswitch RNA with a neomycin riboswitch RNA with ribostamycin (PDB code: 2N0J). The orange structure represents the paromomycin (PDB code: 2MXS); (C) chemical structure of ribostamycin; and (D) complex of a small molecule; the purple structure represents the backbone of nucleic acid; the blue and red atoms in neomycin riboswitch RNA with ribostamycin (PDB code: 2N0J). The orange structure represents the the moleculesmall molecule; represent the nitrogenpurple structure and oxygen represents atoms, the respectively.backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. 3.3. Small Molecules Targeting MicroRNA 3.3. Small Molecules Targeting MicroRNA MicroRNAs (miRNA), a class of noncoding RNA molecules, contain ~22 length nucleotides, which functionMicroRNAs for gene (miRNA), expression a class [98 of– 100noncoding]. In order RNA to understandmolecules, contain the structure ~22 length of miRNAnucleotides, and the which function for gene expression [98–100]. In order to understand the structure of miRNA and the interaction between miRNA and small molecules, computational high throughput screenings play interaction between miRNA and small molecules, computational high throughput screenings play an important role, because computers could drastically speed up the identification and optimization an important role, because computers could drastically speed up the identification and optimization of compoundsof compounds targeting targeting miRNA, miRNA, compared compared to to traditional traditional drug discovery discovery strategy. strategy. Recently,Recently, Shi et Shi al. etdiscovered al. discovered that AC1MMYR2that AC1MMYR2 (Figure (Figure 25) was25) anwas efficient an efficient and selectiveand selective inhibitor of microRNA-21inhibitor of microRNA-21 through in through silicon invirtual silicon screeningvirtual screening [101]. [101]. The The pre-miRNA-21 pre-miRNA-21 waswas modeled modeled by MC-Fold/MC-Symby MC-Fold/MC-Sym [102] [102] pipeline. pipeline. Forty-eight Forty-eight predicted predicted compounds were were selected selected by AutoDock. by AutoDock. Finally,Finally, AC1MMYR2 AC1MMYR2 was was identified identified to to inhibit inhibit tumor tumor proliferationproliferation and and migration. migration.

FigureFigure 25. 25.Chemical Chemical structurestructure of of AC1MMYR2. AC1MMYR2.

3.4. Small3.4. Small Molecules Molecules Targeting Targeting Long Long Non-Coding Non-Coding RNA RNA LongLong noncoding noncoding RNA RNA (lncRNA) (lncRNA) has been has shownbeen show to playn to important play important functional functional roles in developmentroles in and diseasedevelopment processes and disease [103–106 processes]. Inﬂuenza [103–106]. A virus Influe containsnza A virus eight contains separated, eight single-stranded separated, single- RNAs stranded RNAs that encode 13 kinds of proteins. The RNA dependent RNA polymerase (RdRp) can that encode 13 kinds of proteins. The RNA dependent RNA polymerase (RdRp) can recognize recognize a specific RNA conformation, lncRNA in particular, regulating the beginning of replication a speciﬁc RNA conformation, lncRNA in particular, regulating the beginning of replication and and transcription process [107,108]. A small molecule (DPQ, Figure 26A) was found to bind with the transcriptionlncRNA [109]. process The [crystal107,108 structure]. A small showed molecule small molecule (DPQ, Figurelocates in26 theA) major was foundgroove of to the bind (A–A)– with the lncRNAU loop [109 (Figure]. The crystal26B). Thestructure bindings showed of small small molecule molecule widen locates the major in the groove major close groove the ofposition the (A–A)–U of loopG14–C21 (Figure 26andB). G13–C22 The bindings base pairs. of small molecule widen the major groove close the position of G14–C21 and G13–C22 base pairs.

Int. J. Mol. Sci. 2016, 17, 779 18 of 24 Int. J. Mol. Sci. 2016, 17, 779 18 of 23

Figure 26. (A) Chemical structure of DPQ; and (B) structure of a RNA promoter complex with DPQ Figure 26. (A) Chemical structure of DPQ; and (B) structure of a RNA promoter complex with DPQ (PDB code: 2LWK). The orange structure represents the small molecule; the purple structure (PDB code: 2LWK). The orange structure represents the small molecule; the purple structure represents represents the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen and oxygen atoms, respectively. atoms, respectively. 4. Conclusions 4. Conclusions Nucleic acid drug discovery is a sophisticated system and comprises of a large number of stages, Nucleicincluding acid target drug selection, discovery binding is asite sophisticated verification, systempotent inhibitor and comprises screening, of and a large compound number of stages,optimization. including targetSmall molecule selection, targeting binding nucleic site verification, acid is still potenta tremendous inhibitor challenge. screening, Due and to the compound low specificity of DNA or RNA binders, high rate of the failure in clinical trials is the obstacle of nucleic optimization. Small molecule targeting nucleic acid is still a tremendous challenge. Due to the low acid drug discovery. The progress of nucleic acid drug discovery needs the development of structural specificity of DNA or RNA binders, high rate of the failure in clinical trials is the obstacle of nucleic biology and other related technologies. acid drugDNAs discovery. as the The fundamental progress of target nucleic of acidvarious drug small discovery molecules, needs for the instance development antibiotics of structuraland biologyantitumor and other agents, related have technologies. been verified. It is obvious that small molecules bind with different DNAsmechanisms as the to fundamental different DNA target conformation. of various Small small molecules molecules, can for bind instance to DNA antibiotics duplex in and various antitumor agents,kinds have of mechanisms, been verified. including It is obviouscovalent binding, that small insertion, molecules and some bind multifunction. with different For mechanismstriplex and to differentquadruplex, DNA conformation. however, the intercalation Small molecules or insertion can bind is the to common DNA duplex binding in variousmode for kinds small ofmolecules. mechanisms, includingIn particular, covalent the binding, majority insertion, of such andsmall some molecules multifunction. contain planar For triplex aromatic and ring quadruplex, systems. Such however, the intercalationsubstructure can or insertionkeep the complex is the commonmore stable. binding mode for small molecules. In particular, the RNA attracts less attention than DNA targets. Nevertheless, recently, much progress has been majority of such small molecules contain planar aromatic ring systems. Such substructure can keep made in discovering small molecule inhibitor stargeting to RNA with high specificity and bioactivity. the complex more stable. In silico virtual screening for novel inhibitors targeting RNA has produced original small molecule RNARNA binders. attracts less attention than DNA targets. Nevertheless, recently, much progress has been made in discoveringIn summary,small small moleculemolecules inhibitortargeting nucleic stargeting acids to can RNA regulate with a high large specificity number of and biological bioactivity. In silicoprocesses.virtual However, screening major for novel challenges inhibitors and issues targeting are yet RNA to be has resolved. produced Therefore, original the small study moleculeand RNAdevelopment binders. of promising small molecules targeting nucleic acids strategies is ongoing. In summary, small molecules targeting nucleic acids can regulate a large number of biological processes.Acknowledgments: However, majorWe thank challenges the other academic and issues staff members are yet in to Aiping be resolved. Lu and Ge Therefore, Zhang’s group the at study Hong and Kong Baptist University. We also thank Hong Kong Baptist University for providing critical comments and developmenttechnical support. of promising This study small was moleculessupported by targeting the Hong nucleicKong General acids Research strategies Fund is (HKBU ongoing. 478312) and HKBU Faculty Research Grant (FRG2/14-15/010). Acknowledgments: We thank the other academic staff members in Aiping Lu and Ge Zhang’s group at Hong Kong BaptistAuthor University. Contributions: We also Maolin thank HongWang Kongwrote Baptistthe manuscript; University Yuanyu for providingan Yu and criticalChao Liang comments contributed and technicalthe support.manuscript This study for literature was supported research; by Aiping the Hong Lu an Kongd Ge GeneralZhang revised Research and Fundapproved (HKBU the manuscript. 478312) and HKBU Faculty Research Grant (FRG2/14-15/010). Conflicts of Interest: The authors declare no conflict of interest. Author Contributions: Maolin Wang wrote the manuscript; Yuanyuan Yu and Chao Liang contributed the manuscriptReferences for literature research; Aiping Lu and Ge Zhang revised and approved the manuscript.

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