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

2 3

*

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.) Shenzhen Lab of Combinatorial Compounds and Targeted Drug Delivery, HKBU Institute of Research and Continuing Education, Shenzhen 518000, China 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

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2. Small Targeting DNA Int. J. Molecules Mol. Sci. 2016, 17, 779 Int. J. Mol. Sci. 2016, 17, 779

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2. Small Molecules Targeting 2.1. Small Molecules Targeting DNADNA Duplex 2. Small Molecules Targeting DNA Small MoleculesForming Targeting DNA Duplex 2.1.1. 2.1. Small Molecules Covalent Bonds with DNA Duplex 2.1. Small Molecules Targeting DNA Duplex

Psoralen orMolecules psoraleneForming has been used as mutagen to treat skin diseases including psoriasis and 2.1.1. Small Covalent Bonds with DNA Duplex vitiligo. Psoralen interacts Forming with standard DNA duplex, through the 5,6 double bond in pyrimidine ring, 2.1.1. Small Molecules Covalent with DNA Duplex Psoralen or psoralene has been usedBonds as mutagen to treat skin diseases including psoriasis and 1 -(Hydroxymethyl)-4,5 1 ,8-trimethylpsoralen, whichvitiligo. can stop replication and transcription. 4 short for Psoralen interacts with standard DNA duplex, through the 5,6 double bond in pyrimidine Psoralen or psoralene has been used as mutagen to treat skin diseases including psoriasis and HMT,ring, has reasonable solubility and DNA binding affinity, as double shownbond in Figure 1A. Figure 2B which can water stop replication and high transcription. 4′-(Hydroxymethyl)-4,5′,8-trimethylpsoralen, vitiligo. Psoralen interacts with standard DNA duplex, through the 5,6 in pyrimidine showsshort the crystal structure of a short DNA complex with HMT reported by Spielmann et in al. [13]. for HMT, has reasonable water solubility and high DNA binding affinity, as shown ring, which can stop replication and transcription. 4′-(Hydroxymethyl)-4,5′,8-trimethylpsoralen, The HMT structure crosses both the minor and major grooves through the 5,6 double bonds of Figure 1A. Figure 2B shows the crystal structure of a short DNA complex with HMT reported by short for HMT, has reasonable water solubility and high DNA binding affinity, as shown in Spielmann et al. [13]. The HMT structure crosses both the minor and major grooves through the 5,6 Figure ring 1A. Figure 2B thymines. shows the crystal of a short DNAinteractions complex with HMT by pyrimidine and two There structure are apparent stacking from thereported ring system of ofnearby. pyrimidine ring and twocrosses thymines. There areand apparent stacking interactions from Spielmann et al. [13]. TheThe HMT structure bothof the minor and major grooves through the 5,6 rings HMT double and thebonds bases high binding affinity HMT duplex results from aromatic the ringbonds systemofofpyrimidine HMT and the nearby. The high binding affinity of HMT duplex double ringbases andThe two thymines. There arecomplex apparent stacking interactions from the stacking and hydrophobic interactions. crystal study of the of DNAand and HMT results showed from aromatic rings stacking and hydrophobic interactions. The crystal study of the complex DNA the ring system of HMT and the bases nearby. The high binding affinity of HMT and duplexofresults Holliday junction was formed in the adduct of DNA-HMT. The interaction between DNA and HMT and showed Holliday was formed in theThe adduct ofstudy DNA-HMT. The interaction fromHMT aromatic ringsthe stacking andjunction hydrophobic interactions. crystal of the complex of DNA is related withDNA sequence, which may play a role in repairing the psoralen damage in chemotherapy between and HMT is related with sequence, which may play a role in repairing psoralen and HMT showed the Holliday junction was formed in the adduct of DNA-HMT. Thethe interaction treatment. The conformation of DNA is extremely twisted at the of thymine connected damage in chemotherapy treatment. The conformation of may DNA isbases extremely at the the psoralen baseswith of the between DNA and HMT is related with sequence, which play a role in twisted repairing hexatomic pyrone of the molecule. thymine connected with the hexatomic pyrone of the molecule. damage in chemotherapy treatment. The conformation 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 (RCSB Data Bank (PDB) code: 204D). structureofrepresents the small molecule. The Figure Protein 1. (A) Chemical structure of HMT; andThe (B)orange NMR structure a short DNA complex with HMT (RCSBpurple Protein Data Bank (PDB) code: 204D). The orange structure represents the small molecule. structure represents the code: backbone ofThe nucleic acid; the blue and red the atoms inmolecule. the molecule (RCSB Protein Data Bank (PDB) 204D). orange structure represents small The The purple structure the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogenrepresents and oxygen respectively. purple structure represents theatoms, backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen andand oxygen atoms, respectively. represent nitrogen oxygen atoms, respectively.

Figure 2. (A) Chemical structure of Aflatoxin B1 adduct; and (B) NMR structure of a DNA duplex complex Aflatoxinstructure B1 adduct (PDB code: structure represents theduplex small Figure 2. with (A) Chemical of Aflatoxin B12KH3). adduct;The andorange (B) NMR structure of a DNA Figure 2. (A) Chemical structure of Aflatoxin B1 adduct; and (B) NMR structure of a DNA duplex molecule; the purple structure represents backbone nucleic acid;structure the blue represents and red atoms the complex with Aflatoxin B1 adduct (PDBthe code: 2KH3).ofThe orange the in small complex with Aflatoxin B1 adduct (PDB atoms, code: respectively. 2KH3). The orange structure represents the small molecule and oxygen molecule;represent the purplenitrogen structure represents the backbone 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.

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Aflatoxin B1 (AFB) is a common contaminant in a variety of foods including peanuts, cottonseed meal, corn, and other grains well as animal feeds.inAflatoxin B1foods is considered most toxic aflatoxin Aflatoxin B1 (AFB) is aascommon contaminant a variety of includingthe peanuts, cottonseed andmeal, it is highly implicated in hepatocellular carcinoma (HCC) in humans [14]. The epoxide metabolite corn, and other grains as well as animal feeds. Aflatoxin B1 is considered the most toxic of AFB can react duplex DNA to a cationic adduct (Figure 2A). An[14]. NMR aflatoxin and itwith is highly implicated inproduce hepatocellular carcinoma (HCC) in humans Thestructure epoxide of AFB resolved can react and withreported duplex DNA to produce cationicinadduct 2A). An structure NMR thismetabolite adduct hasofbeen by Stone [15], asashown Figure(Figure 2B. This NMR structure of this adduct has been resolved and reported by Stone [15], as shown in Figure 2B. This shows that Aflatoxin B1 substructure inserts into DNA duplex strand. The adduct acts as a covalent NMR structure shows that B1 substructure inserts into DNA duplex strand. adduct binder, which can crosslink to Aflatoxin duplex DNA conformation inducing the modification andThe change in the acts as a covalent binder, which can crosslink to duplex DNA conformation inducing the modification DNA conformation. The changed DNA will stop interacting with related proteins, which can block change in the DNA conformation. The changed DNA will stop interacting with related proteins, the and process of replication or transcription. which can block the process of replication or transcription. Another DNA duplex adduct, a synthetic N44C–ethyl–N44 C (Figure 3A), was discovered to interact Another DNA duplex adduct, a synthetic N C–ethyl–N C (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 structuresolution. solution.However, However, the ethyl linker does not remarkably change the B-form DNA the ethyl linker does not remarkably change the B-form DNA conformation [17], as shown in Figure conformation [17], as shown in Figure3B. 3B.

Figure 3. (A) Chemical structure of N4C–ethyl–N4C adduct; and (B) one strand of NMR structure of a

4 C adduct; and (B) one strand of NMR structure Figure 3. (A) Chemical structure 4of N4 C–ethyl–N 4C adduct (PDB code: 2OKS). The orange structure DNA duplex complex with N C–ethyl–N 4 4 of a DNA duplex complex with N C–ethyl–N C adduct (PDB code: 2OKS). The orange structure represents the small molecule; the purple structure represents the backbone of nucleic acid; the blue represents small molecule; purplenitrogen structure represents the backbone of nucleic acid; the blue and red the atoms in the moleculethe represent and oxygen atoms, respectively. 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 duplex conformation Two DNA duplexes structures were resolved and submitted to Protein Similar interactions[18]. were reported in the trimethylene related structures (Figure 4A) and DNA Data Bank. Although the stacking between the linked bases and neighbor bases was different, duplex conformation [18]. Two DNA duplexes structures were resolved and submitted to both Protein ofBank. the two structuresthe indicated that the duplexes couldbases maintain hydrogen bonds the B-form Data Although stacking between the linked and the neighbor bases wasand different, both of geometry (Figure 4B,C). the two structures indicated 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 (Figure 4B,C). are extracted from natural products isolated from streptomycetes. The molecule is used as Mitomycin C (Figure 5A) 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 natural products isolated from streptomycetes. The molecule is used as The crystal complex of a short DNA with mitomycin C was reported by a chemotherapy drug for cancer. Mitomycin C alkylates the guanine in C–G pairs of the Patel et al. [20]. Unlike other standard DNA duplexes, the mitomycin alkylated DNAbase duplex shows DNA conformation [19]. The of a short DNA mitomycin reported A-form base pair stacking andcrystal B-formcomplex sugar puckers. The ring of with mitomycin locatesCinwas the minor by Patel et al. [20]. standard forms DNA aduplexes, mitomycin alkylated DNA5B. duplex shows groove. And theUnlike ring of other indoloquinone 45° to thethe helix axis, as shown in Figure A-form As base pair stacking B-form sugar Thechange ring ofthe mitomycin locates in the minor mentioned, theseand small molecules canpuckers. modify and overall conformation of the ˝ conformation by cross-linking to DNA duplexes. changed conformations will stop groove. And the ring of indoloquinone forms a 45 toThe the helix axis,DNA as shown in Figure 5B. interacting with thethese corresponding biologicalcan partners, thus thechange transcription or replication process of As mentioned, small molecules modify and the overall conformation be blocked. by Thiscross-linking is how this kind of molecules works in the chemotherapeutics treatment. These the will conformation to DNA duplexes. The changed DNA conformations will stop molecules showed high toxicity in normal cells, just because of its low selectivity. To decrease interacting with the corresponding biological partners, thus the transcription or replication process and promote the comprehension the interaction mechanism betweentreatment. DNA willresistance be blocked. This is selectivity, how this kind of molecules of works in the chemotherapeutics and drugs will be of significant importance.

These molecules showed high toxicity 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.

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Figure 4. (A) Chemical structure of N2G–trimethylene–N2G; (B) crystal complex of a DNA with N2G–

Figure 4. (A) Chemical structure of N2 G–trimethylene–N2 G; (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 structure cross-link (PDB code: 2KNK);2G; and (C) crystal complex of a DNA with Figure 4. (A) Chemical ofcode: N2G–trimethylene–N (B) crystal complex ofthe a DNA N2G– 2G cross-link trimethylene–N (PDB 2KNL). The orange structure represents smallwith molecule; 2 G cross-link (PDB code: 2KNL). The orange structure represents the 2small 2G cross-link N2 G–trimethylene–N trimethylene–N (PDB code: 2KNK); and (C) crystal complex of a DNA with N G– the purple structure represents the backbone of nucleic acid; the blue and red atoms in the molecule 2G cross-link molecule; thenitrogen purple structure represents the backbone of nucleic acid; the blue the andsmall red atoms in the trimethylene–N (PDB code: 2KNL). The orange structure represents molecule; represent and oxygen atoms, respectively. the purple structure represents the backbone nucleic acid; the blue and red atoms in the molecule molecule represent nitrogen and oxygen atoms,ofrespectively. 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 Figure 5.represents Chemical structure of ofnucleic mitomycin C; and (B) crystal complex of a short DNA with structure thestructure backbone acid. C; Figure 5. (A)(A) Chemical mitomycin and (B) crystal complex of a short DNA 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 representsTargeting the backbone of DNA nucleicDuplex acid. in Minor Groove 2.1.2.structure Small Molecules with

structure represents the backbone of nucleic acid.

affect the gene Targeting expression, the DNA smallDuplex molecules can not only insert into the DNA strand, but 2.1.2.To Small Molecules with in Minor Groove

2.1.2. Targeting DNAofDuplex in Minor Groove alsoSmall bind Molecules to the major or minorwith grooves high binding affinity. For minor groove, typical small

To affect the gene expression, the small molecules can not netropsin, only insertberenil, into theand DNA strand, but molecules are heterocylic dications and polyamides, including pentamidine, To bind affectto the gene expression, the small molecules canaffinity. not onlyFor insert into the DNA strand, also the major or minor grooves of high binding minor groove, typical smallbut as shown in Figure 6. Originally, these molecules were found to bind AT-rich areas preferentially. alsomolecules bind to the major or minor grooves of high binding affinity. For minor groove, typical small heterocylic dications polyamides, including netropsin, berenil, and pentamidine, Then, theseare molecules appeared nearand G–C and C–G base pairs [21,22]. The molecules targeting minor molecules are heterocylic dications and polyamides, including netropsin, berenil, and pentamidine, as shown in Figure 6. Originally, theseofmolecules were found to bindanti-tumor AT-rich areas groove have been studied as a series therapeutics with anti-viral, and preferentially. anti-bacterial as shown in Figure 6. Originally, these molecules found to bind areas preferentially. Then, these molecules appeared near G–C and C–G were base pairs [21,22]. TheAT-rich molecules targeting minor Then, thesehave molecules appeared G–C C–G base pairs [21,22]. anti-tumor The molecules minor groove been studied as anear series of and therapeutics with anti-viral, and targeting anti-bacterial

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5 of 23 groove have been studied as a series of therapeutics with anti-viral, anti-tumor and anti-bacterial activities[23–25]. [23–25].Some Somestructural structuralstudies studieshave havebeen beeninvestigated investigatedas asthe thebasis basisof ofthe thedesign designof of small small activities activities [23–25]. Some structural studies have been investigated as the basis of the design of small molecules[26–30]. [26–30].The The crystal complex of DNA duplex Hoechst 33528 is shown in Figure molecules crystal complex of DNA duplex andand Hoechst 33528 is shown in Figure 7. The7. molecules [26–30]. The crystal complex of DNA duplex and Hoechst 33528 is shown in Figure 7. The The Hoechst 33528 is clamped in the minor groove of the DNA structure. Hoechst 33528 is clamped in the minor groove of the DNA structure. Hoechst 33528 is clamped in the minor groove of the DNA structure.

Figure of small molecules for DNA minor groove. Figure6.6. 6.Typical Typicalstructures structures Figure Typical structures of of small small molecules molecules for for DNA DNA minor minor groove. groove.

Figure 7. Crystal complex of DNA duplex and Hoechst 33528 (PDB code: 8BNA). The orange structure Figure 7. 7. Crystal complex of duplex and 33528 code: The structure Figure Crystal complex of DNA DNA duplex structure and Hoechst Hoechst 33528 (PDB (PDB code: 8BNA). 8BNA). The orange orange represents the small molecule; the purple represents the backbone of nucleic acid; structure the blue represents the small molecule; the purple structure represents the backbone of nucleic acid; the blue blue represents the small molecule; the purple structure represents the backbone of nucleic acid; the and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. and red atoms in the molecule represent nitrogen and oxygen atoms, 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 Except for the some unusual compounds were that also itidentified in the minor groove ofdrug-like DNA. Anmolecules, 8 ring molecule (Figure 8A) was reported can bindto tolocate a DNA the minor groove of DNA. An 8 ring molecule (Figure 8A) was reported that it can bind to a DNA the minor groove of DNA. structure An 8 ring indicates molecule that (Figure wasconformation reported thathas it can bind to a DNA duplex [31]. The resolved the 8A) DNA been remarkably duplex [31]. The resolved structure indicates that the DNA conformation has been remarkably duplex [31]. resolved structure indicates thatand the the DNA conformation has been remarkably changed. changed. TheThe minor groove has been widened, major groove has been narrowed. The minor changed. The minor groove has been widened, and the major groove has been narrowed. The minor The minor groove has been widened, and the major groove has been narrowed. The minor and major and major grooves are both approximate 4 Å (Figure 8B). Additionally, the helix center direction and major grooves are both approximate 4 Å (Figure 8B). Additionally, the helix center direction aremore both approximate Å (Figure 8B). Additionally, the helix center twisted more isgrooves twisted than 18° to 4the major groove, in comparison with thedirection inherentiscounterparts. is twisted more than 18° to the major groove, in comparison with the inherent counterparts. ˝ to the than 18 major groove, in comparison with the inherent counterparts. The distortion provides The distortion provides a fundamental element for supporting the concept of allosteric change of the The distortion provides a fundamental element for supporting the concept of allosteric change of the a fundamental element for supporting the concept of allosteric change of the DNA conformation with DNA conformation with inhibitors locating in minor grooves. 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.

small molecules can bindtotononstandard nonstandard DNA DNA duplex duplex as base pairs may TheThe small molecules can bind aswell. well.Hoogsteen Hoogsteen base pairs may occur in alternating A–T sequences, which abundantinineukaryotic eukaryoticanimals. animals. Because Because of occur in alternating A–T sequences, which areare abundant of the the lack lack of of structural information, however, little analysis in this field. Recently, the complex of structural information, however, there isthere little is analysis in this field. Recently, the complex of Hoogsteen Hoogsteen DNA duplex and pentamidine (Figure 9A) was determined and reported [32]. The X-ray DNA duplex and pentamidine (Figure 9A) was determined and reported [32]. The X-ray DNA structure DNAa structure a mixture Watson–Crick and(Figure Hoogsteen patternprevious (Figure minor 9B). Unlike presents mixture ofpresents Watson–Crick andofHoogsteen pattern 9B). Unlike binders, previous minor binders, pentamidine does not bind to the minor groove totally. Only the center of pentamidine does not bind to the minor groove totally. Only the center of the pentamidine is bound 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 structureofrepresents theand small purple structure represents Figure 9. (A) Chemical pentamidine; (B) molecule; complex ofthe DNA with pentamidine (PDB the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen code: 3EY0). The orange structure represents the small molecule; the purple structure represents the atoms, respectively. backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively.

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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 For major groove studies, there are less reports of small molecule targeting information. Due to the requirement of larger compounds, few complexes of molecules and DNA major grooves 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 showshow binding affinity to DNA major groove. The reason for this major groove binding is the large size binding affinity to DNA major groove. The reason for this major groove binding is the large of carbohydrates and theirand hydrophilic and hydrophobic substructures. Some classic major groove size of carbohydrates their hydrophilic and hydrophobic substructures. Some classic major binders are listed inare Figure groove binders listed10 in [33]. Figure 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]. This kind of molecule can form a complex damageagents DNAfor bydecades hydrogen Neocarzinostatin and related derivatives have with been protein used as and anti-tumor [34,35]. abstraction [36]. Incan order to aunderstand the mechanism interaction DNA duplex and [36]. This kind of molecule form complex with protein andand damage DNAbetween by hydrogen abstraction molecule, the complex the DNA duplex and neocarzinostatin wereduplex reported. In order to understand the of mechanism and interaction between DNA andNeocarzinostatin-gb molecule, the complex and neocarzinostatin-glu were used as ligand to bind to the DNA duplex. As shown in Figure 11, of the DNA duplex and neocarzinostatin were reported. Neocarzinostatin-gb and neocarzinostatin-glu 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. structures are observed [37,38]. The majority of the neocarzinostatin-gb binds major groove. However, the neocarzinostatin-glu binds to the minor groove. The differences of to thethe two binding The two rings of the molecule are close to each other in the structure. However, the neocarzinostatin-glu modes provide significant inspiration into small molecule-DNA specific targeting. binds to the minor groove. The differences of the two binding modes provide significant inspiration into small molecule-DNA specific targeting.

Figure 11. Cont.

Figure 11. Cont.

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Figure 11. (A) Chemical structure of neocarzinostatin-gb; (B) complex of a DNA with neocarzinostatin-gb (PDB code: 1KVH); (C) chemical structure of neocarzinostatin-glu; and (D) 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 1 clinical trial [44]. 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

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give inspiration inspirationon on approximate 15° to minorgroove grooveofofDNA. DNA.The Thementioned mentioned structural structural features features give approximate 15° to minor groove of DNA. The mentioned structural features give inspiration on further research of optimizing this kind of molecules. further research of optimizing this kind of molecules. further research of optimizing this kind of molecules.

Figure 12. (A) Chemical structure of daunomycin; (B) one strand a DNA duplex complex Figure 12.12. (A)(A) Chemical structure of of daunomycin; (B)(B) oneone strand of aofof DNA duplex complex with Figure Chemical structure daunomycin; strand a DNA duplex complex with daunomycin (PDB code: 1D10); (C) chemical structure of adriamycin; and (D) one strand of daunomycin (PDB code: 1D10); (C) chemical structure of adriamycin; and (D) one strand of a DNA with daunomycin (PDB code: 1D10); (C) chemical structure of adriamycin; and (D) one strand of a DNA duplex complex with adriamycin (PDB code: 1D12). The orange structure represents duplex complex with adriamycin code: 1D12). orange structure represents the small molecule; a DNA duplex complex with (PDB adriamycin (PDBThe code: 1D12). The orange structure represents the small molecule; the purple structureofrepresents thethe backbone of nucleic the acid; the molecules; red dots thethe purple structure represents the backbone nucleic acid; red dots represent water small molecule; the purple structure represents the backbone of nucleic acid; the red dots represent the water molecules; the blue and red atoms in the molecule represent nitrogen and therepresent blue andthe redwater atomsmolecules; in the molecule represent nitrogen oxygen atoms, respectively. The and green the blue and red atoms and in the molecule represent nitrogen oxygen atoms, respectively. The green square represents the water molecule that interacts with the square represents the water molecule that interacts with the molecule. oxygen atoms, respectively. The green square represents thesmall water molecule that interacts with the small molecule. small molecule.

Figure 13. (A) Chemical structure ofdimer dimer ofditercalinium; ditercalinium; and and (B) (B) complex complex of a DNA duplex with Figure 13.13. (A) DNAduplex duplex with Figure (A)Chemical Chemicalstructure structureof of dimer of of ditercalinium; and (B) complex ofofaaDNA with ditercalinium (PDB code: 1D32). The orange structure represents the small molecule; the purple ditercalinium structure represents representsthe thesmall smallmolecule; molecule;the thepurple purple ditercalinium(PDB (PDBcode: code: 1D32). 1D32). The The orange orange structure structure represents the backboneofofnucleic nucleicacid; acid;the theblue blue and and red red atoms atoms in the molecule represent structure represents the backbone in the molecule represent structure represents the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively. nitrogen and nitrogen andoxygen oxygenatoms, atoms,respectively. respectively.

Actually, the specificity of DNA intercalators is the reason for drug toxicity. Molecules can bind Actually, thespecificity specificityofofDNA DNAintercalators intercalators is the the reason drug Molecules can bind the reason for for drugtoxicity. toxicity. can bind to Actually, unexpected DNA sequence for new treatment.isCryptolepine (Figure 14A) is aMolecules good example for to unexpected DNA sequence for new treatment. Cryptolepine (Figure 14A) is a good example for to unexpected DNA sequence for new treatment. Cryptolepine (Figure 14A) is a good example for occasional drug discovery. Cryptolepine is isolated from Cryptolepis triangular and used as antioccasional drug discovery. Cryptolepine is isolated from Cryptolepis triangular and as used as antioccasional drug discovery. Cryptolepine is isolated from Cryptolepis triangular and used anti-malarial malarial and cytotoxic agent [59]. This compound can insert with C–G rich sequences [60]. The crystal malarial and cytotoxic agent [59].compound This compoundinsert can insert C–G richsequences sequences[60]. [60]. The crystal and cytotoxic agent [59]. This withwith C–G rich The crystal complex structure indicates that the drug can interacts with the duplex in a base stacking insertion complex structure indicates that the drug interacts with the duplex in a base stacking insertion

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complex structure indicates that the drug interacts with the duplex in a base stacking insertion pattern J. Mol. Sci. 2016, 17, 779 10 of 23 (Figure 14B).Int. The molecule and two consecutive C–G base pairs form a sandwich conformation 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 mildly twisted And at about 6.8˝ between rings. Besides, the withwas two C–G rich sequences. the hexatomic ring stackstwo in thearomatic middle of cytosine and guanine. Structurally,the cryptolepine wasof mildly at about 6.8°the between two aromatic rings. charged nitrogen can improve stability the twisted complex with interaction between molecule Besides, the charged nitrogen can improve the stability of the complex with the interaction between and DNA duplex. The insertion into base pair steps and the molecular dissymmetry were important molecule and DNA duplex. The insertion into base pair steps and the molecular dissymmetry were elements for important 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. (A) Chemical structure of code: dimer of cryptolepine; and (B) one of a DNA duplex complex with cryptolepine (PDB 1K9G). The orange structure represents thestrand small molecule; thecryptolepine purple structure (PDB represents the backbone nucleic acid; thestructure blue and red atoms in the the molecule complex with code: 1K9G).ofThe orange represents small molecule; represent nitrogen and oxygen atoms, respectively. the purple structure represents the backbone of nucleic acid; the blue and red atoms in the molecule represent2.1.5. nitrogen and oxygen atoms, respectively. Small Molecules Targeting with DNA Duplex through Multiple Binding Patterns Small molecules can not only bind to DNA duplex in a single mode, but also interact with DNA duplex in multiple bindingwith patterns, which will make the complex more stable [61,62].Patterns PBD-BIMZ 2.1.5. Small Molecules Targeting DNA Duplex through Multiple Binding (Figure 15A) binds to a DNA duplex in a covalent link to a guanine base. The complex shows that the

Small molecules can orients not only DNA duplex in aAlthough single the mode, butbinding also interact hybrid molecule in thebind minorto groove (Figure 15B) [63]. covalent distorts with DNA the DNA helix, the overall duplex still maintains the standard B-form conformation. It is revealed duplex in multiple binding patterns, which will make the complex more stable [61,62]. PBD-BIMZ that covalent binding site in local region possesses specific twisted helix. By comparison, the (Figure 15A)piperazine binds toring a DNA in a covalent to a guanine base. complex shows aduplex high flexibility with variouslink conformations. Later, the sameThe group reported shows that the hybrid molecule orients the (Figure 15B) [63]. Although thebinds covalent binding another ligand (Figurein 15C) in minor complex groove with the same DNA duplex. This hybrid compound to a guanine in a covalent link and the naphthalimide substructure embeds to (A–T) 2 steps, as shown in distorts the DNA helix, the overall duplex still maintains the standard B-form conformation. It is Figure 15D [64]. The covalent binding will remarkably make the DNA and ligand stable. Moreover, revealed thatbecause covalent local region helix. manybinding molecules site havein low specificity for possesses sequence, thespecific multiple twisted patterns can makeBy the comparison, the piperazine ring shows a high flexibility with various conformations. Later, the same group molecules sequence targetable. 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.

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 and oxygen atoms, respectively. 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 purpleTargeting structure represents the backbone of nucleic acid; the blue and red atoms in the molecule 2.2. Small Molecules DNA Triplex represent nitrogen and oxygen atoms, respectively.

Single strand DNA can bind to a DNA duplex structure to form a triplex conformation DNA [65]. 2.2. Small Molecules Targeting DNA Triplex The interactions between strands are Hoogsteen or reverse Hoogsteen base pairs. Small molecules can Single strand DNA can bind to a DNA duplex structure to form a triplex conformation DNA [65]. bind to triplex DNA groove, blocking the access of other DNA natural substrate [66]. The DNA triplex The interactions between strands are Hoogsteen or reverse Hoogsteen base pairs. Small molecules can can display bind twotoforms, intermolecular triplexes and intramolecular triplexes [67]. Intermolecular triplex triplex DNA groove, blocking the access of other DNA natural substrate [66]. The DNA triplex is generatedcanfrom a DNA chainintermolecular of an extratriplexes DNA structure. Generally, the[67]. intermolecular display two forms, and intramolecular triplexes Intermolecular triplex has triplex is due generated from a DNA treatment chain of an extra DNA structure. intermolecular attracted attention to the possible for various cancersGenerally, or otherthediseases. In fact, most of triplex has attracted attention due to the possible treatment for various cancers or other diseases. In the DNA duplex intercalators can insert to the triplex as well [68]. fact, most of the DNA duplex intercalators can insert to the triplex as well [68]. As mentioned above,above, neomycin is is a atypical majorDNA DNA groove binder. triplex system, As mentioned neomycin typical major groove binder. In triplexInsystem, neomycin canthe make the mixed base mixed DNAtriplexes triplexes and triplex stable. stable. The computational neomycin can make base DNA andT–A–T T–A–T triplex The computational docking conformation the triplex and neomycin isisshown in Figure 16. The16. computational and docking conformation [69] of [69] theoftriplex and neomycin shown in Figure The computational and biophysical researches both indicate that this Watson–Hoogsteen complementarity leads to biophysical the researches both indicate that this Watson–Hoogsteen complementarity leads to the selectivity. selectivity.

Figure 16. Computer generated model of neomycin bound to a DNA triplex. Ring I of neomycin

Figure 16. locates Computer generated model of and neomycin to abridging DNAthe triplex. Ring I of neomycin in the middle of the DNA groove, Ring II andbound IV facilitate pyrimidine strands; themiddle orange, green andDNA purplegroove, structuresand represent three IV chains of the DNA; the bluethe andpyrimidine red atoms locates in the of the Ringthe II and facilitate bridging strands; in the molecule represent nitrogen and oxygen atoms, respectively. the orange, green and purple structures represent the three chains of the DNA; the blue and red atoms in the molecule represent nitrogen and oxygen atoms, respectively.


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Similar binders, neomycin conjugates are also found to bind DNA Similar to tomajor majorgroove groove binders, neomycin conjugates are also found to with bindthe with thetriplex DNA structures. Computational modeling of pyrene–neomycin indicates pyrene will insert into the triplex structures. Computational modeling of pyrene–neomycin indicates pyrene will insert intobase the pairs whenwhen neomycin bind with groove [70]. [70]. base pairs neomycin bind Watson–Hoogsteen with Watson–Hoogsteen groove 2.3. Small Molecules Molecules Targeting 2.3. Small Targeting DNA DNA Quadruplex Quadruplex Telomere as aa significant significant potential potential strategy strategy for for cancer cancer therapy. therapy. Telomere regulation regulation has has been been regarded regarded as Guanosine Guanosine rich rich nucleic nucleic acids acids are are regarded regarded as as aa new new class class of of non-antisense non-antisense nucleic nucleic acids acids with with active active G-quartet conformation [71]. Except for telomerase, G-quadruplex-forming sequences have G-quartet conformation [71]. Except for telomerase, G-quadruplex-forming sequences have also also been been found in the promoter regions of many other genes. Small molecules can stabilize G-quartet structure, found in the promoter regions of many other genes. Small molecules can stabilize G-quartet structure, causing causing the the telomerase telomerase low low activity activity [72–74]. [72–74]. Therefore, Therefore, the the G-quadruplex G-quadruplex structures structures have have attracted attracted more attention for drug design. more attention for drug design. Because consist of Because G-quartet G-quartet conformations conformations consist of planar planar stacked stacked guanine guanine bases, bases, numbers numbers of of small small molecules with electron deficient aromatic rings are identified to bind [75]. As mentioned above, molecules with electron deficient aromatic rings are identified to bind [75]. As mentioned above, distamycin typical intercalators for DNA duplex. In quadruplex systems, these distamycin AAand anddaunomycin daunomycinareare typical intercalators for DNA duplex. In quadruplex systems, compounds are able bind DNA quadruplex structures. The first of G-quadruplex and these compounds aretoable to to bind to DNA quadruplex structures. The complex first complex of G-quadruplex daunomycin was determined and reported by Neidle [76]. Figure 17 shows the four parallel strands and daunomycin was determined and reported by Neidle [76]. Figure 17 shows the four parallel and three daunomycin molecules at the end the complex. This complex that the structure strands and three daunomycin molecules atof the end of the complex. This indicates complex indicates that the shows high stability with the molecules stacking on the end. structure shows high stability with the molecules stacking on the end.

17. Crystal Crystal complex of aa DNA DNA quadruplex quadruplex with with daunomycin daunomycin (PDB (PDB code: code: 1O0K). The orange Figure 17. structure represents the small molecule; the purple structure represents the backbone of nucleic acid; in the the molecule molecule represent represent nitrogen nitrogen and and oxygen oxygenatoms, atoms,respectively. respectively. the blue and red atoms in

It is known that distamycin A binds with the minor groove of DNA duplex. The structure of It is known that distamycin A binds with the minor groove of DNA 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 Complex of quadruplex with distamycin A (PDB code: 2JT7). The orange structure Figure 18. 18. Complex of aa DNA DNA quadruplex distamycin (PDB code:of 2JT7). The orange structure represents the small molecule; the purplewith structure representsAthe backbone nucleic acid; the blue represents the small molecule; the purple structure represents the backbone of nucleic acid; the blue and the red atoms the molecule nitrogen and oxygen atoms, represents smallinmolecule; therepresent purple structure represents 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 Compared Compared to to DNA DNA duplex duplex binding binding molecules, molecules, some novel kinds of molecules have been found. structural and biochemical studies displayed detailed information of the interaction between small These can bind specifically to DNA quadruplex structures, as shown Figurein 19.Figure The structural Thesemolecules molecules can bind specifically DNA quadruplex structures, as in shown molecules and DNA quadruplex. Into addition, it also provideda novel platform for further drug 19. The and biochemical studies displayed detailed information of the interaction between small molecules structural and biochemical studies displayed detailed information of the interaction between small design targeting DNA quadruplex. The progress in this field has been reported [78].

and DNA quadruplex. In addition, itInalso provideda novel platform novel for further drug for design targeting molecules and DNA quadruplex. addition, it also provideda platform further drug DNA The quadruplex. progress in this has been reported [78]. designquadruplex. targeting DNA Thefield progress in this field has 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 ribosomal (rRNA)structures is the mostof comprehensively among various kinds of RNA FigureRNA 19. Typical small moleculesstudied for DNA quadruplex. Figure 19. structures of small target molecules forits DNA quadruplex. types. The ribosome is Typical an attractive anti-bacterial due to important 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. Small Small Molecules Molecules Targeting TargetingRibosome Ribosome 3.1. The ribosomal ribosomal RNA RNA (rRNA) (rRNA) is is the the most most comprehensively comprehensively studied studied among among various various kinds kinds of of RNA RNA The types. The due to to its its important important function function in in protein protein types. The ribosome ribosome is is an an attractive attractive anti-bacterial anti-bacterial target target due expression [79–83]. The ribosomes in prokaryotic cells mainly contain 30S and 50S subunits. The expression [79–83]. The ribosomes in prokaryotic cells mainly contain 30S and 50S subunits. The subunit

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subunit 30S is for ensuring that the tRNA can locate in the correct position. The subunit 50S is 30S is for ensuring that theconnection. tRNA can Various locate inkinds the correct position.have The subunit 50S is responsible responsible for peptide of compounds been identified to bind thefor peptide connection. kinds of compounds have been identified to bind the subunits of rRNA, subunits of rRNA,Various including pleuromutilin, oxazolidinones and aminoglycosides, as shown in Figure 20pleuromutilin, [84,85]. including 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 (Figure a member of aminoglycoside is used to inhibit replication of Amikacin various kinds of 21A), bacteria. Amikacin binds with the antibiotics, 30S subunits of rRNA, leading to the of various kinds of bacteria. Amikacin binds with the 30S subunits of rRNA, leading to the wrong reading wrong reading of message RNA and making the bacteria cell incapable of translation and growing. of The message RNA and making the bacteria cell the incapable translation and growing. The structure of structure of RNA duplex is nearly same of with the previous RNA-aminoglycoside RNA duplex is nearly the same with the previous RNA-aminoglycoside complexes [86–90]. One of complexes [86–90]. One of the aromatic rings inserts into A site helix by stacking. Another aromatic thering aromatic rings inserts into A site helix by stacking. Another aromatic ring forms four hydrogen forms four hydrogen bonds to the H-bond acceptors nearby. These common interactions make bonds to the H-bond nearby. These common interactions make the amikacin bind toand RNA the amikacin bind toacceptors RNA duplex tightly, as shown in Figure 21B [91], and help maintain A1493 A1492tightly, in the bulge structure, corresponding with themaintain “on” state in 30Sand subunit. duplex as shown in Figure 21B [91], and help A1493 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 (PDB 21. code: 4P20). The orange structure representsand the small molecule;ofthe purpleduplex structure represents Figure (A) Chemical structure of amikacin; (B) complex a RNA with amikacin the backbone of nucleic acid; the blue and red atoms in the molecule represent nitrogen and oxygen (PDB code: 4P20). The orange structure represents the small molecule; the purple structure represents 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 2 nitrogen and represents the backbone of nucleic acid; the blue and red atoms in the molecule represent 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.

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Figure 23. (A) Chemical structure of tetrahydrobiopterin; (B) complex of a RNA duplex with

Figure 23. (A) Chemical(PDB structure of tetrahydrobiopterin; complex RNA duplex with tetrahydrobiopterin code: 4LVX); (C) chemical structure of(B) pemetrexe; andof(D)a complex of a tetrahydrobiopterin (PDB code: 4LVX); (C)4LVY). chemical structure ofrepresents pemetrexe; andmolecule; (D) complex of RNA duplex with pemetrexed (PDB code: The orange structure the small the purple represents thecode: backbone of nucleic acid; thestructure blue and red atoms in the a RNA duplex withstructure pemetrexed (PDB 4LVY). The orange represents themolecule small molecule; represent nitrogen and oxygen respectively. the purple structure represents theatoms, backbone 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 riboswitch specificity, the complexesare of paromomycin (Figure and ribostamycin (Figure 24C)including Recently, some aminoglycosides identified to bind24A) with the riboswitch RNA, with neomycin riboswitch RNA were resolved. The complexes of the two structures indicate that ribostamycin and paromomycin [97].To understand the biochemical elements for thethe 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 neomycin riboswitch RNA were resolved. The complexes of the two structures indicate that substructures of the two molecules locate at a close area, near the hinge region between the upper and the lower helix the two helix structures ofstem. 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.

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 molecule represent and oxygen atoms, small molecule; the nitrogen purple structure represents 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, MicroRNAs (miRNA), a class of noncoding RNA molecules, contain ~22 length nucleotides, which function for gene expression [98–100]. In order to understand the structure of miRNA 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 compounds targeting miRNA, compared drugdiscovery discovery strategy. of compounds targeting miRNA, comparedtototraditional traditional drug strategy. Recently, Shi et al. discovered that AC1MMYR2 (Figure 25) was an efficient and selective inhibitor Recently, Shi et al. discovered that AC1MMYR2 (Figure 25) was an efficient and selective of microRNA-21 through in silicon virtual screening [101]. The pre-miRNA-21 was modeled inhibitor of microRNA-21 through in silicon virtual screening [101]. The pre-miRNA-21 was modeled by by MC-Fold/MC-Sym pipeline. Forty-eightpredicted predicted compounds compounds were selected by AutoDock. MC-Fold/MC-Sym [102] [102] pipeline. Forty-eight were selected by AutoDock. Finally, AC1MMYR2 identified inhibittumor tumorproliferation proliferation and Finally, AC1MMYR2 waswas identified to to inhibit andmigration. migration.

Figure 25.25.Chemical ofAC1MMYR2. AC1MMYR2. Figure Chemical structure structure of

3.4. Small Molecules Targeting Long Non-CodingRNA RNA 3.4. Small Molecules Targeting Long Non-Coding noncoding (lncRNA) has shown been shown play important functional roles in LongLong noncoding RNA RNA (lncRNA) has been to play to important functional roles in development development and disease processes [103–106]. Influenza A virus contains eight separated, singleand disease processes [103–106]. Influenza A virus contains eight separated, single-stranded 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 transcription process A small molecule (DPQ, Figure 26A) was found to the bind with the lncRNA [109]. The [107,108]. crystal structure showed small molecule locates in the major groove of (A–A)– lncRNA [109]. The crystal structure showed small molecule locates in the major groove of the (A–A)–U U loop (Figure 26B). The bindings of small molecule widen the major groove close the position of loop G14–C21 (Figure 26B). The bindings of small molecule widen the major groove close the position of and G13–C22 base pairs. G14–C21 and G13–C22 base pairs.


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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, including target selection, binding verification, system potent inhibitor screening, compound Nucleic acid drug discovery is asite sophisticated and comprises of and a large number of optimization. Small molecule targeting nucleic acid is still a tremendous challenge. Due to the low stages, including target selection, binding site verification, potent inhibitor screening, and compound 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. The progress oftarget nucleic drugsmall discovery needsfor theinstance development of structural as the fundamental of acid various molecules, antibiotics and biology and other related technologies. antitumor agents, have been verified. It is obvious that small molecules bind with different DNAs as theto fundamental target of various Small small molecules molecules,can forbind instance antibiotics and antitumor mechanisms different DNA conformation. to DNA duplex in various kinds of mechanisms, including covalent binding, insertion, and some Formechanisms triplex and to agents, have been verified. It is obvious that small molecules bindmultifunction. with different quadruplex, however, the intercalation or insertion is the binding mode forkinds small of molecules. different DNA conformation. Small molecules can bind to common DNA duplex in various mechanisms, In particular, majority of such and smallsome molecules contain planar aromatic systems. Such including covalentthe binding, insertion, multifunction. For triplex andring quadruplex, however, substructure can keep the complex more stable. the intercalation or insertion is the common 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 RNA attracts less attention than DNA targets. Nevertheless, recently, much progress has been RNA binders. made in discovering stargeting tocan RNA with ahigh specificity bioactivity. In summary,small small molecule molecules inhibitor targeting nucleic acids regulate large number ofand biological In silico virtual However, screeningmajor for novel inhibitors targeting RNA produced original processes. challenges and issues are yet to behas resolved. Therefore, thesmall studymolecule and of promising small molecules targeting nucleic acids strategies is ongoing. RNAdevelopment binders. In summary, small molecules targeting nucleic acids can regulate a large number of biological Acknowledgments: We thank the other academic staff members in Aiping Lu and Ge Zhang’s group at Hong processes. However, major challenges and issues are yet to be resolved. Therefore, the study and Kong Baptist University. We also thank Hong Kong Baptist University for providing critical comments and development of promising small targeting acidsResearch strategies is (HKBU ongoing. technical support. This study wasmolecules supported by the Hongnucleic Kong General Fund 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 Author Contributions: Wang Kong wrote Baptist the manuscript; Yuanyuan Yu and critical Chao Liang contributed the Baptist University. We also Maolin thank Hong University for providing comments and technical manuscript for literature research; Lu and Ge General Zhang revised and Fund approved the manuscript. support. This study was supported byAiping the Hong Kong Research (HKBU 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 manuscript for literature research; Aiping Lu and Ge Zhang revised and approved the manuscript. References Conflicts Interest: authors no conflict of interest.gene silencing in mammals. Trends Genet. 2005, 1. of Bayne, E.H.;The Allshire, R.C.declare RNA-directed transcriptional 21, 370–373.

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