Polymorphic Nucleic Acid Binding of Bioactive Isoquinoline Alkaloids and Their Role in Cancer

24 Pages·2014·4.36 MB·English

SAGE-HindawiAccesstoResearch
JournalofNucleicAcids
Volume2010,ArticleID593408,23pages
doi:10.4061/2010/593408
Review Article
Polymorphic Nucleic Acid Binding of Bioactive Isoquinoline
Alkaloids and Their Role in Cancer
MotilalMaitiandGopinathaSureshKumar
BiophysicalChemistryLaboratory,IndianInstituteofChemicalBiology(CSIR),Kolkata700032,India
CorrespondenceshouldbeaddressedtoMotilalMaiti,[email protected]
Received25June2009;Revised11September2009;Accepted14September2009
AcademicEditor:HiroshiSugiyama
Copyright©2010M.MaitiandG.S.Kumar.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Bioactive alkaloids occupy an important position in applied chemistry and play an indispensable role in medicinal chemistry.
Amongst them, isoquinoline alkaloids like berberine, palmatine and coralyne of protoberberine group, sanguinarine of the
benzophenanthridinegroup,andtheirderivativesrepresentanimportantclassofmoleculesfortheirbroadrangeofclinicaland
pharmacologicalutility.Inviewoftheirextensiveoccurrenceinvariousplantspeciesandsignificantlylowtoxicities,prospective
developmentanduseofthesealkaloidsaseffectiveanticanceragentsaremattersofgreatcurrentinterest.Thisreviewhasfocused
ontheinteractionofthesealkaloidswithpolymorphicnucleicacidstructures(B-form,A-form,Z-form,HL-form,triplehelical
form,quadruplexform)andtheirtopoisomeraseinhibitoryactivityreportedbyseveralresearchgroupsusingvariousbiophysical
techniques like spectrophotometry, spectrofluorimetry, thermal melting, circular dichroism, NMR spectroscopy, electrospray
ionizationmassspectroscopy,viscosity,isothermaltitrationcalorimetry,differentialscanningcalorimetry,molecularmodeling
studies,andsoforth,toelucidatetheirmodeandmechanismofactionforstructure-activityrelationships.TheDNAbindingof
theplanarsanguinarineandcoralynearefoundtobestrongerandthermodynamicallymorefavouredcomparedtothebuckled
structureofberberineandpalmatineandcorrelatewellwiththeintercalativemechanismofsanguinarineandcoralyneandthe
partialintercalationbyberberineandpalmatine.Nucleicacidbindingpropertiesarealsointerpretedinrelationtotheiranticancer
activity.
1.Introduction properties that are exerted through highly specific but
noncovalent and reversible interactions with nucleic acid
The mode of action of many drugs in clinical use for the structures [9–13]. The noncovalent interactions are further
treatment of cancer, genetic disorders, and viral diseases is classified in terms of intercalation and groove binding.
believed to be based on their binding to nucleic acids and Intercalation occurs when planar aromatic molecules are
subsequent modification of the genetic material [1, 2]. The sandwiched between two adjacent base pairs of nucleic
discoveryofthedoublehelicalstructureofdeoxyribonucleic acids. Nonintercalative binding occurs in the major or
acid (DNA) by Watson and Crick in 1953 [3] and the minor grooves of nucleic acids without inserting any part
subsequent revelations relating to the structural features to of the binding molecule between base pairs. The physical
the basic functional properties of DNA such as replication and molecular basis of binding of natural alkaloids to
and transcription have proven the utility of DNA as a nucleic acids has been a subject of extensive study in the
potential target for the development of biologically active recentpast[14–17].Elucidationofthenature,strengthand
agents[4,5].Ribonucleicacid(RNA)alsoplayessentialrole energetics of these interactions may reveal the molecular
in normal biological process as well as in the progression basis of many antitumor and antiviral natural products
of many diseases that led to growing interests in exploiting and provide helpful guidance for rational drug design of
in designing of new class of therapeutics [6–8]. A number new potential chemotherapeutic agents. The intercalating
of small organic molecules are endowed with cytotoxic ligandsconstituteoneofthemostwidelystudiedgroupsas
2 JournalofNucleicAcids
OCH
O 3
2 O OCH
3
1 1
3 3
A A
12 13 12 13
4 4
11 11
D C B D C B
10 N+ 5 10 N+ 5
HCO HCO
3 9 8 6 3 9 8 6
OCH OCH
3 3
Berberine Palmatine
(a) (b)
OCH
3
2 OCH 12 1
3
1 3 11 2 O
A
12 13 4 10
H3CO 11 9 3 O
4
D C B
N+ 5 8 N+
H3CO 10 9 8 6 O 7 6 5 CH3
CH O
3
Coralyne Sanguinarine
(c) (d)
Figure1:Chemicalstructuresofisoquinolinealkaloids.
theyformimportantclassofcompoundsforcancertherapy investigated as potential antitumor agents, among which
[18]. In this context, considerable attention has been paid berberine,palmatine,andcoralyne(asyntheticanalogueof
to naturally occurring protoberberine and benzophenan- berberine)representthemostintensivelystudiedmolecules.
thridine alkaloids for their low toxicity and wide range of The physicochemical properties of protoberberine alka-
biologicalactivitiesthatincludeantimicrobial,antileukemic, loids presented in Table1 provide an understanding of
anticancer,andtopoisomeraseinhibitoryactivities[19–26]. the similarity and differences in their chemical structures
Thisreviewfocusesonthecomplexformationoftwogroups (Figure1) and properties. The basic skeleton of the qua-
of isoquinoline alkaloids with various polymorphic nucleic ternary protoberberine moiety is 5,6-dihydro[a,g] quino-
acid structures in relation to their anticancer activities. lizinium [29]. In berberine, there is an addition of 2,3-
Thesealkaloids(Figure1)includeberberine,palmatine,and methylenedioxy and 9,10 dimethoxy group to the above
coralyne of the protoberberine group, sanguinarine of the basicstructureandthusthestructureofberberinebecomes
benzophenanthridinegroup,andsomeoftheirderivatives. 7,8,13,13a-tetrahydro-9,10-dimethoxy and 2,3 methylene-
dioxy berberinium. On the other hand, palmatine has
thesametetracyclicstructure(7,8,13,13a-tetradehydro-9,10-
2.NucleicAcidBindingAspectsof dimethoxy-2,3, methylene dioxy berberinium) as berberine
BioactiveIsoquinolineAlkaloids but differs by the nature of the substituents at position
2,3 on the benzo ring; being dimethoxy for palmatine.
It is generally recognized that the most sensitive target Similarly coralyne has the basic structure with a methyl
for the inhibition of rapidly proliferating cancer cells are groupattachedtotheC8position.Notably,whileberberine
those molecules involved in aspects of DNA function, andpalmatinehavebuckledstructures,coralynehasaplanar
especially DNA itself. It is, therefore, not surprising that structure. In aqueous condition, the absorption spectra of
most clinically useful anticancer compounds act against berberine and palmatine are similar while that of coralyne
such targets. In this context, significant attention has been is remarkably different. Both berberine and palmatine have
focused on the DNA binding properties of protoberberine weak intrinsic fluorescence emission spectra while coralyne
group of alkaloids that constitute an important class of has a strong fluorescence emission spectrum in aqueous
isoquinoline alkaloids widely present as active constituents and alcoholic media. Berberine, palmatine, and coralyne
of folk medicines used world over for centuries. Several are achiral molecules as evidenced from circular dichroism
protoberberine alkaloids and their derivatives have been spectroscopy.Sanguinarineisthemostimportantmemberof
JournalofNucleicAcids 3
Table1:Somephysicochemicalpropertiesofisoquinolinealkaloids[15,16,27,28].
Sanguinarine
Properties Berberine Palmatine Coralyne
(iminiumform)
Empiricalformulae C H O N+ C H O N+ C H O N+
20 18 4 21 22 4 22 22 4
C H O N+(13-
7,8,13,13a- 20 14 4
7,8,13,13a-tetradehydro- 5,6,7,8,13,13a- methyl
tetradehydro-
9,10-dimethoxy-2,3- hexadehydro-8-methyl- [1,3]benzodioxolo
Chemicalname 2,3,9,10-
methylenedioxy 2,3,10,11-tetramethoxy [5,6-c]-1,3-
tetramethoxy-
berberinium berberinium dioxolo[4,5-i]-
berberinium
phenanthridium(1+)
Chloridesaltsare
Crystalcolour Yellow Canaryyellow Darkyellow
orangered
Saltsaresolublein
Solubility Water Water Ethanol
water
Molecularweight(ion) 336.36 352.50 364.42 332.33
Meltingpoint(◦C) 210(chloridesalt) 221(chloridesalt) 250–252(chloridesalt) 277–280◦C
219,231,300,311326,
232,268,344.5and 360,405,and424(in30% 273,327,400and
Peakpositionofabsorption 230,267,344and420
420(inaqueous ethanol)219,231,300,311 475nm(inaqueous
spectrum(nm) (inaqueousbuffer)
buffer) 326,360,405,and420(in bufferofpH5.5)
aqueousbuffer)
14,500at420nm(in
Molarextinctioncoefficient aqueousbuffer)and 30,700M−1cm−1at
22,500at344nm 25,000at344nm
(ε)(M−1cm−1) 17,500at424nm(30% 327nmin0.1NHCl
alcohol)
Strong,at470nm(bothin
Peakpositionoffluorescence Veryweak,at530nm Veryweak,at530nm at580nm(λ 327
aqueousandalcoholic ex
emissionspectrum (λ 350nm) (λ 350nm) or475nm)
ex ex media)(λ at424nm)
ex
0◦(inH Oand30%
Opticalrotation[α] (solvent) 0◦(H O) 0◦(H O) 2 0◦(zerodegree)
D 2 2 ethanol)
IC value(inmice) 27.5mg/Kg 65mg/Kg 40mg/Kg 19.4mg/Kg
50
thebenzophenanthridinegroup.ItexhibitedpHdependent the binding of individual alkaloid to DNA substrate, that
structural equilibrium between the iminium (charged) and is, no aggregation prevails [35]. Moreover, such red shift
the alkanolamine (neutral) forms with a pKa value of 7.4 presumablyarisesfromthestrongintermolecularinteraction
[15]. Sanguinarine existed almost exclusively as iminium involvingtheeffectiveoverlappingoftheelectroniccloudof
and alkanolamine forms in the pH rang 1.0–6.0 and 8.5– the base pairs of DNA with that of the alkaloid. Again, the
11.0, respectively. The characteristic uv-vis absorption and less polar environment of the bound ligand can cause the
fluorescence spectra of each form were studied [15]. One hypochromic effect on the free absorption spectrum of the
interesting point to be noted is that the fluorescence of ligandandintercalativepositionsarehydrophobicinnature.
alkanolamine form is more intense than that of iminium Alargenumberofstudiesreportedthecomplexationof
form. The iminium form is unsaturated and completely berberine with calf thymus DNA possibly by a mechanism
planar while alkanolamine form has buckled structure. of partial intercalation [15, 16] and references therein.
Bothformsareachiral.Somephysicochemicalpropertiesof Maiti et al. [30, 36–38] have first demonstrated the base
sanguinarinearegiveninTable1. and sequence specificity of berberine from studies with
several naturally occurring and synthetic DNAs of differ-
2.1.Alkaloid-DNAInteraction. Themostconvenienttoolfor ing base composition and sequences as evidenced from
detectingthecomplexformationbetweennucleicacidsand hypochromismandbathochromismintheabsorptionspec-
smallmoleculesisabsorbancespectra.Likemanyothersmall trum (Figure2), remarkable enhancement of steady-state
moleculesthatbindnucleicacids,berberinehasbyvirtueof fluorescenceemissionintensity,stabilizationofDNAagainst
heterocyclic structure visible absorption band where DNA thermal denaturation, and perturbation in the circular
does not contribute and hence is convenient to monitor dichroismspectrum.Berberineincreasedthecontourlength
the binding. The complex formation essentially involves of sonicated rod like duplex B-DNA depending on base
bathochromic and hypochromic effect on its absorption compositionandinducedtheunwinding-rewindingprocess
spectrum with clear isosbestic points indicating the onset of covalently closed superhelical DNA with an unwinding
ofequilibriumbinding.Thebathochromicshiftofthespec- angle of 13◦. Using high-fieldNMR, Saran et al. [39] first
trumofthefreealkaloidoninteractionwithDNAindicates proposedthatberberinebindstodoublehelicalcalfthymus
4 JournalofNucleicAcids
0.2 hexamine cobalt chloride, and millimolar concentration of
polyamines. In order to analyze the binding of berberine
1 to Z-form DNA, Kumar et al. [49] reported that Z-DNA
structureremainedinvariantinthepresenceofberberineup
toanucleotidephosphate/alkaloidmolarratioof∼0.8and
suggestedthatberberineneitherboundtoZ-formDNAnor
7.8 converted the Z-DNA to the bound B-form structure like
e
nc otherintercalators[15].
a
orb 0.1 ThemoleculararchitectureoftheHL-fromDNAappears
bs to be unique, stable left-handed structure with Hoogsteen
A
base pairs, is distinctly different from the right-handed B-
DNA and left-handed Z-DNA [15, 16]. The binding of
1 berberine to HL-DNA was found to be non-cooperative
resultinginperturbationofthecirculardichroismspectrum
and generation of extrinsic circular dichroism bands with
oppositesignandmagnitudecomparedtotheB-formDNA
7.8
0 [49].Itwassuggestedthatberberinecouldbeusedasaprobe
300 350 450 550
to detect the alternation of structural handedness due to
Wavelength(nm) protonationandmaypotentiateitsuseinregulatoryrolesfor
biologicalfunctions.
Figure 2: Absorption spectrum (1) of berberine (8μM) with
increasing concentration up to 117.6μM (7) and 125.0μM (8) of The formation of intermolecular triplex offers a means
calfthymusDNA.ReprintedfromthePh.D.ThesisofBhadra[28]. of achieving sequence specific recognition of duplex DNA
forming specific hydrogen bonds. Although the third
strand oligonucleotide possesses specific sequence recog-
nition properties, the binding is not so strong which
DNA by a mechanism of partial intercalation in which the results in a relatively low stability of the triplex structure.
planar isoquinoline moiety remained intercalated between The instability of triple helical structures under normal
basepairs.Thepartialintercalationmodelwasfurthersub- physiologicalconditionsisacriticallimitationthatrestricts
stantiated from several analytical techniques that included theirusesinvivo.Severalintercalatingligandshavenowbeen
absorption, fluorescence, isothermal titration calorimetry, reported that can preferentially stabilize triplex structures
differential scanning calorimetry, electrospray ionization [50–55]. Ren and Chaires [50] have shown that berberine
massspectroscopy,incombinationwithcomputermolecular preferentially and very strongly bound to TxA.T triplex
modeling studies [27, 40–43]. However, a fluorescence compared to AT duplex using novel competition dialysis
spectralstudyofberberinewithcalfthymusDNA[44]and and thermal melting experiments. Recently Das et al. [48]
a high-resolution 1HNMR and UV spectroscopy [45] in demonstratedthatberberinestabilizedtheHoogsteenbase-
conjunction with molecular modeling studies on berberine paired third strand of TxA.T triplex without affecting the
bindingtovariousshortoligonucleotideduplexes(B-form) Watson-Crick strands as revealed from thermal melting
favoured a groove binding geometry. This suggested that (Figure3(a))andcirculardichroicstudies(Figure3(b))and
berberine is located in the minor groove with a preference proposed that it binds to the third strand presumably by
for AT sequences, lying with the convex side on the helix mechanismofintercalation.Thermodynamicsofberberine-
groove where the positively charged nitrogen atom is close triplex complexation revealed that the binding process was
to the negative ionic surface of the oligonucleotide helices. exothermicandentropydrivenfavouringatightbindingat
Zhu et al. [46] supported the interaction of berberine with theintercalationsite[48].
B-DNA by partial intercalation in which isoquinoline part In recent years, recognition that certain sequences pro-
intercalated in presence of β-cyclodextrin. However, Chen mote formation of higher-order structures of DNA has
et al. [47] studied the interaction of berberine with sev- revealed new targets for chemotherapy [58–60]. These
eraldoublestrandedoligonucleotides[d(AAGAATTCTT)2, structures can form the association of four single strands
d(AAGGATCCTT)2,andd(AAGCATGCTT)2usingelectro- containing tracts of guanine bases (tetramer), and can also
spray ionization mass (ESI-MS) and fluorescence spectro- arise within one or two strands containing several of these
scopic techniques to establish the sequence selectivity and sequences as the strands fold back upon themselves. In
preference of binding to these structures. The results from order to understand the potential of quadruplex DNA as
ESI-MS and fluorescence titration experiments indicated a chemotherapeutic target, it is important to understand
thatthesequenceselectivityofberberinewasnotsignificant the role and occurrence of telomeres in cells. Guanine rich
and remarkable AT rich DNA binding preference was not sequenceswiththeabilitytoformquadruplexstructuresare
observed. found in telomeres at the end of eukaryotic chromosomes
ItisknownthatalternatingGCpolymeranditsmethy- (TTAGGGrepeatsinhumans)aswellasinpromoterregions
latedanaloguestructuresassumesleft-handedconformation of genes and in recombination sites [61, 62]. Telomeres are
(Z-DNA)inhighmolarsalt(Na+,K+),inlowmolardivalent pieces of DNA that are extensions of linear chromosomes
cations (Ca2+,Mg2+,Ni2+), micromolar concentration of [63]. The observations that telomerase is expressed in
JournalofNucleicAcids 5
20 9
1.6
1
10
1 1
nm −1) 1
at260 1.4 dmole 0 9 9
ce 2m −10
eabsorban 1.2 −30(deg.c −20 9
v 1
ati × −30
Rel ]θ
[
−40 1
1
−50
30 40 50 60 70 80 90 210 250 300 350 400
Temperature(◦C) Wavelength(nm)
(a) (b)
Figure3:(a)Thethermalmeltingprofileofpoly(dT)·poly(dA)xpoly(dT)triplex(-•-)anditscomplexwithberberineatalkaloid/DNA
ratioof 0.1 (-O-)and 0.3 (-(cid:2)-).(b) Circular dichroic spectrum(1) of the same triplex(48.8μM) withincreasing concentrations up to
(31.05μM)ofberberine(9).ReprintedinpartfromDasetal.[48]withpermissionfrom2003AdeninePress.
Table2:ComparativebindingparametersofinteractionofvariousalkaloidswithcalfthymusDNAandcovalentlyclosedsuperhelicalDNA.
Alkaloid Ka(×105M−1) n ΔT b (◦C) βc θd References
m
12.6
Berberine 0.38 4.0 7.0 1.35 [28,30]
13.0
Palmatine 0.19 4.0 11.0 1.40 15.0 [28,31]
21.0
Coralyne 1.0 9.0 12.0 1.62 [28,32]
18.0
Sanguinarine 9.45 3.4 21.3 1.86 27.0 [33,34]
aK theintrinsicbindingconstantdeterminedusingequationr/Cf = K(1−nr)[(1−nr)/(1−(n−1)r)]n−1,whereristhenumberofalkaloidmolecules
boundpermoleofnucleotideandCfisthemolarconcentrationoffreealkaloid,wherendenotesthebindingsitesizeinbasepairs.
bΔTmistheTmoftheDNAinthepresenceofalkaloid-TmofthefreeDNA.
cβvaluewascalculatedfromtheslope,whenL/Loor(η/η0)1/3isplottedagainstrusingtheequationL/Lo = (η/η0)1/3 = 1+βr,whereLandLoarethe
contourlengthofhelixinpresenceandabsenceofligandcorrespondingvaluesofintrinsicviscosity(approximatedtothereducedviscosity)ofthesolution
andristhemoleligandbound/molenucleotides.
dunwindingangle.
malignant but not normal tissues led to the paradigm that Bhadra et al. [31] reported that addition of calf thymus
drugs that interfere with the action of telomerase could be DNA caused marked changes in the absorption spectrum
expected to cause the death of cancer cells. If interactions of palmatine (Figure4) with strong hypochromic and
of telomeric proteins with specific sequences of telomeric bathochromic shifts, with well-resolved isosbestic points,
DNA are lost, this also leads to cell death by apoptosis and with increased thermal melting temperature to form
[64]. This suggests that compounds that bind to DNA and stable complexes with B-DNA. Further from electric linear
inhibit protein binding would cause the rapid death of dichroism studies, it was interpreted that palmatine binds
tumor cells. Compounds that stabilize quadruplex DNA, eitherbyanintercalationorbyanexternalstackingparallel
presumablypreventinginteractionswithproteinshavebeen tothebasepairs.However,anearlierstudybyYuetal.[70]
shown in vitro to inhibit telomerase [65]. Recent studies proposedanonintercalativebindingofpalmatine.Recently,
show that berberine inhibits telomere elongation [66] and the author’s laboratory [56] demonstrated the base and
bindsstronglytovariousG-quadruplexDNAstructuresand sequence-dependent binding of palmatine and established
inhibits telomerase activity [50, 67]. Thus berberine may that palmatine formed a partial intercalation complex with
be used as a target to probe for G-quadruplex DNA for its all the polymers except the homo and heteropolymer of
developmentaspotentialantitumordrugs. GC, with which it formed a nonintercalative complex. The
Palmatine is structurally similar to the well-known binding of palmatine to calf thymus DNA was further
isoquinoline alkaloid berberine. To evaluate the affinity characterized by thermodynamic studies using isothermal
and mode of binding to B-DNA, Kluza et al. [69] and titrationcalorimetryandthebindingprocesswasexothermic
6 JournalofNucleicAcids
Table3:ThermodynamicparametersfortheinteractionofvariousalkaloidswithcalfthymusDNAcomplexationobtainedfromisothermal
titrationcalorimetricstudiesa.
Alkaloid K(×105M−1) n ΔG◦(kcalmol−1) ΔH◦(kcalmol−1) ΔS◦(cal·deg−1mol−1) References
Berberine 0.12 3.6 −5.50 −2.01 +11.40 [28,42]
Palmatine 0.28 3.0 −5.82 −4.40 +5.00 [28,56]
Coralyne 71.5 4.0 −9.25 −7.05 +7.70 [28,42]
Sanguinarine 9.52 2.2 −8.02 −6.91 +3.70 [57]
aAverageoffourdeterminations,KandΔH◦valuesweredeterminedfromfitsoftheITCprofilestoOrigin7.0.ThevaluesofΔG◦andΔS◦weredetermined
usingtheequationsΔG◦=−(RTlnK)andΔG◦=ΔH◦−TΔS◦.respectively.TheITCprofilewasfittoamodelofsinglebindingsites,wherendenotesthe
bindingsitesize.
Table 4: Spectroscopic and thermodynamic parameters for the interaction of various alkaloids with DNA triple helical structure with
referencesa.
Complexes Ka n ΔT b ΔG◦ ΔH◦ ΔS◦ References
m
(×105M−1) (◦C)(3 → 2)atr kcalmol−1 kcalmol−1 cal·deg−1mol−1
max
Poly(dT)·poly(dA)xpoly(dT)+berberine 3.2 6.4 7.80 −7.35 −4.61 +9.52 [48]
Poly(dT)·poly(dA)xpoly(dT)+coralyne dna dna 19.0 dna dna dna [68]
Poly(dT)·poly(dA)xpoly(dT)+sanguinarine 26.0 3.3 24.5 −8.56 −4.14 +15.11 [48]
Poly(dC)·poly(dG)xpoly(dC+)+sanguinarine 16.0 2.5 7.50 −8.28 −10.73 −8.36 [48]
aK(theintrinsicbindingconstant)determinedusingequationr/Cf =K(1−nr)[(1−nr)/(1−(n−1)r)]n−1,whereristhenumberofalkaloidmolecules
boundpermoleofnucleotideandCfisthemolarconcentrationoffreealkaloid.
bΔTm(3 → 2)istheTmofthetriplexDNAinthepresenceofalkaloid-TmofthefreetriplexDNA.(3 → 2)denotesthetransitiontemperaturefromtriplex
complextoduplexcomplexformation.
∗dna:datanotavailable.
0.275 Another important alkaloid of protoberberine group is
the synthetic molecule coralyne, which was found to be
potentially capable of intercalating with B-DNA [32, 73,
0.22 1 74] and the results led to the assumption that the DNA
binding may be correlated with biological activity. It was
reported that at a lower molar ratio of coralyne to B-
DNA, an intercalating complex by π stacking along the
e 0.165
nc backbone of DNA, while at higher molar ratios, a DNA-
a
b induced aggregation occurred [73]. Although a wealth of
bsor 11.12 data[32,73,74]ispresentlyavailableregardingthecomplex
A 0.11
formationbetweencoralyneandB-DNA,theinterpretation
ofthesedataneedstobeperformedwithcautionascoralyne
exhibits a high tendency of aggregation in aqueous buffer
0.055 1 even at very low concentrations. Maiti et al. [32] first
addressed this problem and showed that aqueous solutions
containing 30% (v/v) ethanol suppressed the aggregation
11.12
0 of coralyne and allowed spectrophotometric titration of
300 400 500
a fully monomeric form with calf thymus DNA. The
Wavelength(nm) base and sequence specificity of coralyne (monomeric) on
bindingtovariousnaturallyoccurringandsyntheticB-DNA
Figure 4: Absorption spectrum (1) of palmatine (7.6μM) with
increasingconcentrationsupto151.6μM(11)and175.2μM(12) werestudiedbyspectrophotometricandspectrofluorimetric
of calf thymus DNA. Reprinted from the Ph.D. Thesis of Bhadra titrations, circular dichroic spectroscopy, thermal melting,
[28]. isothermal titration calorimetry, and viscometric studies.
The results of spectrophotometric and calorimetric studies
of a fully monomeric form of coralyne with calf thymus
and entropy-driven which favour the intercalative mode of DNA is presented in Figures 5(a) and 5(b), respectively.
binding. It does not bind to Z-DNA nor does it convert Z- Coralyneincreasedthecontourlengthofsonicatedrodlike
form to B-form. Unlike berberine, palmatine showed very duplex B-DNA and induced the unwinding and rewinding
weak binding to HL-form. To date there is no report on process of covalently closed superhelical B-DNA with an
the binding of palmatine to higher-order DNA structures, unwinding angle of 21◦. These experiments revealed that
triplexes, and quadruplexes and study in this direction is monomeric coralyne binds to B-DNA by a mechanism
awaited. of intercalation with a relatively high specificity toward
JournalofNucleicAcids 7
Time(min)
0 20 40 60 80 100
0.16
0
0.12 1 cal/s) −0.1
μ
(
e
c
an −0.2
orb 0.08 10.11
bs
A 0
e)
mol −2
0.04 cal/
(k −4
nt
a
ct
nje −6
I
0 −8
370 400 450 500 0 0.2 0.4 0.6
Wavelength(nm) Molarratio
(a) (b)
Figure5:(a)Absorptionspectrum(1)ofcoralyne(7.09μM)withincreasingconcentrationsupto81.73μM(10)and96.28μM(11)ofcalf
thymusDNA.(b)TheITCprofilesforthetitrationofcalfthymusDNA(65μM)tocoralyne(15μM)at20◦C.Eachheatburstcurveinthe
upperpanelistheresultofa5μLinjectionofDNAintocoralyne.Thelowerpanelrepresentsthecorrespondingnormalizedheatsignals
versusmolarratio.Thedatapointsreflecttheexperimentalinjectionandthesolidlinerepresentsthebestfitofthedata.Reprintedfromthe
Ph.D.ThesisofBhadra,[28].
Table5:EffectofberberineincubatedwithS180cellsinvitroa. tionwithTxA.TandCxG.C+ containingDNAtriplexesthat
coralyne does not exhibit significant sequence selectivity
Pathway Percentcontrol
during triplex complexation [68]. A later study, however,
4μg/mL 40μg/mL
using various techniques like DNase foot printing, ther-
Glycine → [14C]Protein 2.9 0.1 maldenaturationanalysis,spectrophotometrictitrationand
Thymine → [3H]DNA 48.9 1.6 circular dichroic, as well asNMR spectroscopy, showed a
Uridine → [3H]RNA 13.4 0 preferential intercalation of coralyne into TxA.T triplexes
aDataobtainedfromW.A.Creasey[80]. [81], whereas in CxG.C+ triplexes, the coralyne molecule
wasonlypartiallyintercalatedduetotheelectronicrepulsion
between the cationic alkaloid and protonated cytosine. The
alternating GC sequences. Comparative spectroscopic and highpropensityofcoralynetostabilizetriplexDNAhasbeen
thermodynamics binding parameters for the interaction of demonstrated by Hud et al. [55, 82] from the observation
isoquinoline alkaloids with calf thymus DNA are presented thatduplexpoly(dT)·poly(dA)istransformedintoamixture
inTables2and3,respectively.Recently,ithasbeenreported of poly(dT)xpoly(dA)·poly(dT) triplex and single stranded
[28, 75] that coralyne, unlike other classical intercalators, poly(dA) in presence of coralyne at slightly elevated tem-
bindstoZ-formDNAwithoutconvertingtheboundform perature (T > 37◦C). This disproportionation reaction of
to the B-form bound structure as revealed from circular double-strandedDNAhasbeenrationalizedintermsofthe
dichroicspectroscopy.Studiesindicatedthatcoralynecould formation of a highly stable intercalation complex between
be used as a probe to detect the Z-form structure in coralyne and triplex DNA. Further, they demonstrated that
vivo [28]. Coralyne showed strong perturbation of circular coralyne may act as a template for the formation of an
dichroic spectrum of HL-form DNA (Figure6). However, antiparallelDNAtriplexfromhomoadeninesequences[83].
the mode and mechanism of binding of coralyne-HL-DNA Coralyne was found suitable for stacking interactions with
complexationcouldnotberevealedfromthesestudies. G-quadruplex[67].Inthisstudyitwasshownthatcoralyne
In addition, coralyne was shown to bind to parallel hastheabilitytoinduceandstabilizevariousG-quadruplex
triplex DNA but it does not bind to antiparallel triplex structuresincludinghumanG-quadruplexaswellasitseffi-
DNA [53]. It was concluded from early experiments using ciencyininhibitingtelomerase.Thethermalstabilizationof
thermaldenaturationanalysisandspectrophotometrictitra- a preformed intermolecular G-quadruplex structure shows
8 JournalofNucleicAcids
was proposed. Recent calorimetric studies of sanguinarine
calf thymus DNA complexation (Figure7(b)) showed the
24
bindingprocesstobeexothermicandenthalpydriven[57].
Binding and thermodynamic parameters of sanguinarine—
calf thymus DNA complexation obtained from isothermal
1 10
12 titration calorimetry are presented in Table3. A recent
)
−1 study [93] showed that sanguinarine also binds to single
e
mol pyrimidinebulgesinhairpinsequences.
d
2m 0 The interaction of sanguinarine with Z-DNA structure
g.c wasfirststudiedbyDasetal.[94].Itwasdemonstratedthat
e
d 1 onadditionofthealkaloidtotheZ-formstructure,adirect
(
−3 conversion of the Z-form toward the characteristic right-
0
×1 −12 handed bound complex without forming any intermediate
]θ 10 forms was observed. Sanguinarine bound strongly to B-
[
form in a noncooperative manner while its binding to Z-
form was highly cooperative and ∼2-3 base pairs of Z-
−24
form switched to the right-handed form for each bound
sanguinarinemolecule[94].Theinteractionofsanguinarine
215 250 300 350 400 with HL-form showed that it does not bind to HL-form
Wavelength(nm) structure,butitconvertedthestructuretotheboundright-
handed B-form [94, 95]. Here also approximately 2-3 base
Figure 6: Circular dichroic spectrum of (1) HL-DNA (20μM)
pairsoftheHL-formswitchedtotheright-handedboundB-
withincreasingconcentrationsupto(20.64μM)ofcoralyne(10).
formforeachboundmoleculeofsanguinarine.
ReprintedfromthePhDThesisofK.Bhadra,[28].
Latimer et al. [96] first demonstrated that sanguinar-
ine binds to poly(dT)·poly(dA)xpoly(dT) but only weakly
to poly(dC)·poly(dG)xpoly(dC+) more quantitatively and
that coralyne and berberine is able to stabilize rather than reported that sanguinarine binds to triplexes by inter-
to induce G-quadruplex intermolecular structure. In both calation in a noncooperative manner and it stabilized
cases,coralyneisthemostactivecomponentpossiblydueto the Hoogsteen base paired third strand of DNA com-
itsfullyaromaticcoreanditisthemostefficienttelomerase paredtotheirWatson-Crickstrands.Thermodynamicstudy
inhibitor. revealed that the process of binding of sanguinarine to
Among the benzophenanthridine alkaloids, sanguinar- poly(dC)·poly(dG)xpoly(dC+) triplex was exothermic and
ine, an isoquinoline plant alkaloid, is known to generate enthalpy-driven while that to poly(dT)·poly(dA)xpoly(dT)
a wide variety of biochemical and pharmacological effects triplex was exothermic and entropy-driven. The binding
including its use in cancer prevention. The physical and constants and thermodynamic parameters of berberine,
molecularbasisofthebindingofsanguinarinewithcanoni- coralyne,andsanguinarinecomplexationtovarioustriplexes
calB-DNAwerefirstdemonstratedbyMaitietal.[33,34,84– are presented in Table4. Recently, Bai et al. [97] reported
88] using various physicochemical techniques like absorp- that sanguinarine can form a complex with G-quadruplex
tion,fluorescence,circulardichroism,thermalmelting, vis- telomereDNAasrevealedfromDNApolymerasestopassay,
cometry,calorimetry,andNMRspectroscopy.Onbindingto uv melting, electrospray mass spectroscopy, and circular
B-DNAstructures,theabsorptionspectrumofsanguinarine dichroism studies which may potentiate a strategy for
underwentbathochromicandhypochromicchangesexhibit- structure-based anticancer drug design targeting human
ing clear isosbestic points (Figure7(a)), the steady-state telomeres.
fluorescence emission intensity quenched, thermal melting
temperature of B-DNA shifted to higher temperatures and
therewere strongperturbations of circulardichroic pattern 2.2.Alkaloid-RNAInteractions. RNAsareversatilemolecules
of B-DNA. Sanguinarine induced the unwinding process that are now known to play an essential role in normal
of covalently closed superhelical plasmid B-DNA with an biological process and in the progression of several serious
unwindingangleof27◦.Thebindingofsanguinarinetocalf humandiseaseslikeHIVandHepatitisCvirus(HCV),and
thymus DNA was also investigated [39] by 1HNMR and all soforthwhichhasledtogrowinginterestinexploitingRNA
theprotonresonancesinthemoleculewereassignedusinga as a cellular target for therapeutic drug design. The recent
combination of 2DCOSY, NOSEY,and ROSEYtechniques. discovery of a number of micro RNAs and the knowledge
AlltheseexperimentsconclusivelysuggestedthatitbindsB- of their functions further reiterated the critical and diverse
DNAbyamechanismofintercalation[39].Theintercalative roles played by RNA in cell biology [98, 99]. Ribosomal
binding was subsequently supported by several authors RNA (r-RNA) has long been known to be the receptor for
[89–92]. The sequence selective binding of sanguinarine antibiotics and antisense oligonucleotides are being used
to B-DNA was also studied by Waring group [90] using to downregulate gene expression [100]. New drugs could
DNAse foot printing technique and a mixed nucleotide potentiallybedesignedtobindtouniquestructuralregions
sequencebindingforsanguinarine-DNAcomplexformation in mRNA and thereby regulate gene expression. Virtually
JournalofNucleicAcids 9
0
0.15
1 al/s)
c
(μ −0.1
e
nc 0.1 −0.2
a
b
or
Abs e) 0
ol
m
0.05 al/ −2
6 c
k
1 (
nt −4
a
ct
e
6 Inj −6
0
300 350 450 550 0 0.5 1 1.5
Wavelength(nm) Molarratio
(a) (b)
Figure7:(a)Absorptionspectrum(1)ofsanguinarine(4.9μM)withincreasingconcentrationsupto54.8μM(10)ofcalfthymusDNA.
ReprintedfromthePhDThesisofA.Das[71].(b)TheITCprofilesforthebindingofsanguinarine(100μM)toCalfthymusDNA(15μM).
Thelowerpanelrepresentsthecorrespondingnormalizedheatsignalsversusmolarratio.Thedatapointsreflecttheexperimentalinjection
andthesolidlinerepresentsthebestfitofthedata.ReprintedfromthePh.D.ThesisofHossain[72].
all mRNAs formed in eukaryotic cells have a poly(A) tail a strong affinity of palmatine to poly(A). The energetics of
of approximately 200–250 adenosines at the 3’end. The thisstrongbindingofpalmatinetopoly(A)wasfoundtobe
long poly(A) tail is an important determinant of mRNA exothermicandenthalpy-driven[77].
stability and maturation, and is essential for the initiation Xing et al. [110] showed that coralyne binds to single-
of transcription [101, 102] and the production of alternate stranded poly(A) helix and induced the formation of a
forms of proteins [103, 104]. The structure of poly(A) has self-structureinpoly(A).Dependingonsolutionconditions
beenestablishedtoexistasasingle-strandedhelixstabilized such as pH, salt and temperature, single-stranded poly(A)
bypair-wisestackinginteractionbetweenadjacentadenines can adopt a parallel double stranded conformation formed
at physiological pH and temperature [105, 106]. Poly(A) by the pairing of adenine bases at the N1 position [111,
polymerase (PAP) catalyses 3’-end poly(A) synthesis, par- 112]. The possibility of the formation of such a double
ticipates in an endonucleolytic cleavage step and is one of stranded poly(A) in eukaryotic cells was hypothesized to
the key factors in polyadenylation of the 3’end of mRNA. result in the termination of the polyadenelation process
Neo-PAP, a recently identified human PAP, is significantly [113]. Small molecules that could bind strongly to single-
overexpressed in human cancer cells in comparison to its stranded poly(A) may be useful as potential leads for
expressioninnormalorvirallytransformedcells[107,108] controllingthemRNA-poly(A)chainelongationandinturn
and may represent a tumor-specific target. Small molecules mRNA degradation as well as the conversion of single-
capable of recognizing and binding to the mRNA-poly(A) strandedpoly(A)todoublestrandedpoly(A)thatmayopen
tailmightinterferewithfullprocessingofmRNAbyPAPand up an opportunity for modulating nucleic acid structure-
wouldrepresentanewtypeofpotentialtherapeuticagent.In functions. The plant alkaloids berberine and palmatine
thiscontext,severalyearsagoMaitietal.[76]firstobserved exhibit stronger binding to single stranded poly(A), but do
a preferential binding of berberine to poly(A) structure not induce any self-structure formation in poly(A) helix
over B-DNA and tRNA and reported that the binding [109,114].Bindingofcoralynetothepoly(A)helixleading
affinityofberberinetopoly(A)wasapproximately12times to self structure formation was further supported by Giri
higher than B-DNA and approximately 100 times higher and Kumar [79] and the binding process was found to
thant-RNAmolecules.Recently,theyinvestigatedthemode, be predominantly exothermic and enthalpy-driven with a
mechanism,andenergeticsofberberinebindingtopoly(A) stoichiometryofonecoralyneperfouradeninesbases.
structure in details [109]. It was suggested that berberine Sanguinarine was also found to bind very strongly to
binds strongly to single-stranded poly(A) structure by a single stranded poly(A) [71, 78] and induce unique self-
mechanism of partial intercalation and the binding was structureinpoly(A)[78].Thealkaloidbindingprocesswas
found to be endothermic and entropy driven. Giri et al. exothermic and predominantly enthalpy-driven as revealed
[77]studiedtheinteractionofpalmatinewithpoly(A)using fromisothermaltitrationcalorimetry.Comparativecircular
variousbiophysicaltechniquesandtheirresultsalsoshowed dichroicspectrumofpoly(A)treatedwithvariousalkaloids
10 JournalofNucleicAcids
30 60
40
15
mdeg) mdeg) 20
( (
Ellipticity 0 Ellipticity 0
−15
−20
−30 −40
250 300 350 400 210 250 300 350 400
Wavelength(nm) Wavelength(nm)
(a) (b)
50
40
30
mdeg) 20 mdeg)
Ellipticity( 0 Ellipticity( 10
−10
−20
−30
−40
210 250 300 350 400 210 250 300 350 400
Wavelength(nm) Wavelength(nm)
(c) (d)
Figure8:Circulardichroicspectrumofpoly(A)helix(solidline)treatedwith(a)berberine(dashedline),(b)palmatine(dashedline),(c)
coralyne(dashedline),and(d)sanguinarine(dashedline)atalkaloid/poly(A)molarratioatsaturationineachcase.Partiallyreprintedfrom
Nandietal.[76],Girietal.[77],GiriandKumar[78],andGiriandKumar[79]withpermissionfromElsevier.
are shown in Figure8. Spectroscopic studies showed that three states help this cause. However, when transcription
amongthefouralkaloids,theaffinityofbindingofberberine, or replication occurs, DNA needs to be free and these
palmatine,andcoralynetopoly(A)helixisoneorderlower statesseriouslyhindertheprocess.DNAtoposiomerasesare
than that of sanguinarine [116]. Thus, the very strong involved in the regulation of DNA supercoiling. Topoiso-
bindingofsanguinarinetopoly(A)leadingtoselfstructure merase I (Topo I) changes the degree of supercoiling of
formation may open up a new avenue for the use of this DNAbycausingsinglestrandbreaksandreligationwhereas
natural product in modulating the gene expression and topoisomerase II (Topo II) causes knotting, catenation and
developmentofanticanceragent. decatenation of DNA by passing the double-stranded DNA
helix through a transient double-stranded break and then
resealinginthestrandbreak[118,119].Thedifferenceroles
3.TopoisomeraseActivityof ofDNATopoIandIImayindicateanopposingpairofroles
MedicinalAlkaloids in the regulation of DNA supercoiling. Both activities are
especiallycrucialduringDNAtranscriptionandreplication,
Topoisomerasesarenuclearenzymesthatactonthetopology when the DNA helix must be unwound to allow proper
of DNA. It was first discovered by Wang [117]. There are function of large enzymatic machinery. Recently, two more
three main types of DNA topology; supercoiling, knotting, topoisomerases have been discovered and these are namely
andcatenation.Whenoutsideofreplicationortranscription, topoisomerase III and topoisomerase IV. Topoisomerase
DNA needs to be kept as compact as possible and these III enzymes are implicated in regulation of recombination