Psilocybin Investigator's Brochure March-April 2007 Psilocybin: Investigator's Brochure Table of Contents 1. Drug Substance and Formulation. 3 2. Pharmacological and toxicological effects . 3 Psilocybin and Psilocin Actions on Neurotransmitter Systems . 3 Overview. 3 Psilocybin or psilocin and 5HT2A and 5HT2C Receptors . 4 Psilocybin or Psilocin and other Serotonin Receptors . 4 Psilocybin or Psilocin and other Receptor Systems. 5 Human Neuropharmacological Studies . 6 Psilocybin and Gene Expression. 6
Doi:10.1016/s0167-4781(03)00135-0Biochimica et Biophysica Acta 1628 (2003) 111 – 122 Characterization of ciprofloxacin binding to the linear single- and double-stranded DNA Igor D. Vilfana,b,1, Petra Drevensˇeka, Iztok Turela, Natasˇa Poklar Ulriha,c,* a Faculty of Chemistry and Chemical Technology, University of Ljubljana, Asˇkercˇeva 5, 1000 Ljubljana, Slovenia b School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA c Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Received 18 February 2003; received in revised form 6 June 2003; accepted 19 June 2003 The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na+, no sequencepreferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mMNa+, a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength ofsolution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermalstability (e.g. DTmc10 jC for poly[d(AT)]poly[d(AT)]), and the most pronounced red shift in the position of the maximum of thefluorescence emission spectrum (kmax). Similar red shift in the position of kmax is also observed for ciprofloxacin binding to dodecamericduplex containing five successive alternating AT base pairs in the row. On the other hand, ciprofloxacin binding to poly[d(GC)]poly[d(GC)],calf thymus DNA and dodecameric duplex containing a mixed sequence is accompanied by the largest fluorescence intensity quenching.
Addition of NaCl does not completely displace ciprofloxacin bound to DNA, indicating the binding is not entirely electrostatic in origin. Theintrinsic viscosity data suggest some degree of ciprofloxacin intercalation into duplex.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Fluoroquinolone; DNA-binding; Mixed mode of binding; Sequence selectivity; Spectroscopic and hydrodynamic technique Binding of fluoroquinolones to DNA is relatively weak, thus it is unlikely that their binding to DNA triggers the Ciprofloxacin belongs to the family of fluoro- formation of gyrase –DNA complex Likewise, bind- quinolone antibacterial agents that also include enoxacin, ing of fluoroquinolone to gyrase is weak, even though the norfloxacin, ofloxacin and some other molecules. These presence of mutated gyrase alleles in resistant bacteria fluoroquinolone antibacterial agents are synthetic deriva- clearly implicate gyrase in the interactions Numer- tives of 6-fluoro-4-oxo-quinoline-3-carboxylic acid. They ous studies have shown that drug binding to DNA are fluorinated at position 6 and mostly bear a piperazinyl and gyrase is enhanced in the presence of Mg2+ ions moiety at position 7. Ciprofloxacin is one of the most potent and that Mg2+ is essential for antibacterial efficiency of quinolone derivatives in clinical use with a very broad drug – DNA interaction. Fluoroquinolone binding to the spectrum of antibacterial activity and is often used as an gyrase – DNA complex may prevent the religation step antibacterial agent of last resort The mechanism of fluoroquinolone's inhibitionof religation and the role of DNA in drug binding remains tobe resolved. Understanding the interactions between fluo- * Corresponding author. Biotechnical Faculty, University of Ljubljana, roquinolone and DNA may help to elucidate the mechanism Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Tel.: +386-1-423-1161; fax: of action of this important class of antibacterial agents, and may ultimately lead to the design of better, more potent E-mail address: firstname.lastname@example.org (N. Poklar Ulrih).
antibacterial agents with less side effects.
The author received the Presˇeren's Student Award of the University of Ljubljana for his work on the field of ciprofloxacin – DNA interactions, To investigate the type of DNA –fluoroquinolone inter- Ljubljana, 2000.
actions in the absence of Mg2+ ions, several aspects of DNA 0167-4781/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0167-4781(03)00135-0 I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 Before use they were thoroughly dialysed against cor-responding buffer solution. Thermally denatured calf thymusDNA was prepared by heating the sample up to 95 jC andcooling it down to the room temperature. The concentrationsof double-stranded polynucleotides were determined spec-trophotometrically at 25 jC using the following molarextinction coefficients expressed in molar concentration ofbase pairs: poly[d(AT)]poly[d(AT)], e260=13,300 M1cm1; poly[d(A)]poly[d(T)], e260=12,000 M1 cm1; Fig. 1. Ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-pi- poly[d(GC)]poly[d(GC)], e254=16,800 M1 cm1; calf thymus DNA, e259=12,800 M1 cm1. These values wereeither provided by the manufacturer or taken from theliterature For the fluorescence titration experiments binding were examined. To this aim, we have investigated the polymeric DNA concentration was f500 AM (stock) in the binding of ciprofloxacin to a series of natural and base pairs, while for UV-spectroscopic experiments the DNA synthetic polynucleotides at different solution conditions concentration was 15 AM in base pairs. For viscometry the using a combination of spectroscopic (fluorescence and concentration of poly[d(AT)]poly[d(AT)] and sonicated calf UV-spectroscopy) and hydrodynamic techniques (viscome- thymus DNA was 200 AM in base pairs. Unless otherwise try). Our results show that ciprofloxacin's apparent mode of stated, the buffer solution (pH 7.0) used in our experiments binding, structure and sequence preferences significantly with polynucleotides consisted of 2 or 10 mM cacodylate depend on solution conditions.
containing 1.7 or 108.6 mM Na+ and 0.1 mM Na2EDTA.
2.1.2. Oligonucleotides 2. Materials and methods DNA dodecameric DNA duplexes were prepared by mixing the corresponding single strands synthesized using the standard cyanoethylphosphoramide chemistry Themolar extinction coefficients at 260 nm for single-stranded oligomers, e260, were determined by phosphate analysis oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid) using enzyme degradation and colorimetric detection of free was purchased from Sigma Chemical, Ltd. (St. Louis, USA) phosphate The following dodecameric oligonucleoti- and used without further purification. The drug was dried des with the corresponding molar extinction coefficients overnight at 130 jC Ten milligrams of ciprofloxacin expressed in molar concentration of single strand at 25 jC was precisely weighted on high precision balance (Sartorius Analytic A 210P, Sartorius GmbH, Germany), dissolved indimethylsulfoxide (DMSO) and diluted with triply distilled G1 5V-GTTAGTACTTGG-3V, e260=107,000 M1 cm1; water to reduce the concentration of DMSO to 2% (v/v).
C1 5V-CCAAGTACTAAC-3V, e260=101,100 M1 cm1; The molar extinction coefficient of ciprofloxacin was deter- G2 5V-GTTAGTATATGG-3V, e260=106,900 M1 cm1; mined spectrophotometrically at 275 nm. From the slope of C2 5V-CCATATACTAAC-3V, e260=101,700 M1 cm1.
the line, A275 vs. concentration (Beer's law), the molarextinction coefficient of ciprofloxacin at 275 nm, e275, was For all fluorescence measurements the oligomeric DNA determined to be 35,900F500 M1 cm1 in 2% (v/v) concentration was between 0 and 500 AM (per single DMSO solution at 25 jC.
strand). Unless otherwise stated, the buffer solution (pH Netropsin – HCl (Net) from Boehringer Mannheim 7.0) used in our experiments with oligonucleotides consisted GmbH (Germany) and ethidium bromide (EtBr) from Sigma of 10 mM cacodylate containing 28.6 mM Na+ and 0.1 mM were used without further purification. The concentration of the netropsin and ethidium bromide in solution was deter-mined spectrophotometrically using extinction coefficients 2.2. Fluorescence measurements of e296 (25 jC)=21,500 M1 cm1 and e480 (25 jC)=5600M1 cm1, respectively.
Intrinsic fluorescence emission spectra of ciprofloxacin (titrated by HCl, NaOH, NaCl, or polymeric or oligomeric 2.1.1. Polynucleotides single- or double-stranded DNA) or at different ratio of Natural genomic DNA (calf thymus DNA) and three ciprofloxacin to DNA (R) were performed at 20 jC either in synthetic DNA polymers, poly[d(AT)]poly[d(AT)], poly a Perkin-Elmer Model LS-50 Luminescence spectrometer or [d(A)]poly[d(T)] and poly[d(GC)]poly[d(GC)], were pur- in a Jasco FP-750 Fluorimeter equipped with a water chased from Pharmacia Biotech (Uppsala, Sweden). These thermostated cell holder using 1-cm path length quartz polymers were of the highest grade commercially available.
cuvette. The excitation wavelength used was 330 nm and I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 the emission spectra were recorded in the range from 350 to solutions at different ligand to DNA ratio (R=0 to 0.2) with a 625 nm. Fluorescence titrations profiles were measured by constant concentration of DNA (200 AM per base pair) were incrementally adding aliquots of reagent (HCl, NaOH, NaCl prepared. The viscosity of each sample was measured inde- or corresponding DNA) in a cuvette containing a known and pendently. The relative viscosity, grel, of the DNA solution always constant concentration of ciprofloxacin (1 mM). The was calculated as grel=g/go=qt/qoto, where t and to are the emission spectra of ciprofloxacin, corrected for the solvent flow times for the DNA or drug–DNA and solvent solution, blank, were multiplied for dilution factor and corrected for respectively, q and qo are densities of the DNA or drug–DNA PM-tube response using a fluorescence spectrum of quinine and solvent, respectively. The specific viscosity was calcu- sulfate (c=2.5107 M) in 0.1 M perchloric acid as a lated as gsp=grel1. Reduced viscosities (gsp/CN) were eval- uated at one concentration only (for each value of R), andintrinsic viscosities, [g], were calculated using a value of 0.53 2.3. Determination of the equilibrium constants by Stern – for Huggins' coefficient k in Eq. (1) gsp ¼ ½gð1 þ k½gCNÞ: Son et al. used the Stern–Volmer method to estimate the equilibrium constants of norfloxacin binding tovarious synthetic polynucleotides. Norfloxacin, in compar- The concentration CN refers to the mole of nucleotide per ison to ciprofloxacin, contains an ethyl- instead of cyclo- liter, irrespective of the amount of drug bound. If DNA is propyl—group attached to N1 nitrogen atom of quinolone approximated with a rodlike molecule and assuming negli- ring system In order to make the results compara- gible changes in its axial ratio upon ligand binding the ble, we used the same procedure to estimate the equilibrium ratio of the intrinsic viscosities ([g]/[g]o) depends on the constants of ciprofloxacin binding to various synthetic relative ratio of the contour lengths (L/Lo) and is given by 2.4. UV spectrophotometry UV-absorbance measurements were conducted using a Cary 1 UV –VIS spectrophotometer (Varian, Australia) and where Lo and [g]o denote the apparent molecular length and a matched set of 1-cm path length quartz cuvettes. The intrinsic viscosity of DNA in the absence of ligand.
spectrophotometer was equipped with a thermoelectricallycontrolled cell holder. Absorbance versus temperature pro- 2.6. High precision densimetry files (UV-melting curves) were measured at 260 nm. Theheating rate was 1.0 jC min1. For each optically detected The densities of all samples used for viscometry were transition, the melting temperature (Tm) of DNA was deter- measured at 20 jC with a precision of F1.5106 g cm3 mined as the transition midpoint. Melting experiments of using a vibrating tube densimeter (DMA-60/602, Anton polymeric DNA at different ratios of drug to DNA (R from 0 Paar, Austria).
to 1) were performed at the same buffer conditions asdescribed above. To correct for the contribution of cipro- 2.7. pH measurements floxacin to the absorbance spectrum of DNA, the referencecuvette was filled with the solution of ciprofloxacin at the The pH values of all solutions were measured separately same concentration and buffer as in the sample cuvette.
for each experiment using Iskra model MA 5740 pH-meter(Slovenia) and Ag/AgCl combination microelectrode (Met- tler Toledo, Switzerland). Absolute error in our pH measure-ments was F0.01 pH unit.
Viscosity measurements were performed using an Ubbe- lodhe Micro-Viscometers (Schott Glaswerke, Mainz, Ger-many) submerged in a water bath maintained at 20.0 (F0.1) 3. Results and discussion jC. Flow times were measured with a stopwatch to anaccuracy of F0.2 s. Viscosity studies with sonicated calf 3.1. Intrinsic fluorescence properties of ciprofloxacin thymus DNA and poly[d(AT)]poly[d(AT)] were con-ducted in 2 mM cacodylate buffer (pH 7.0). Aliquots of 1 mM shows the fluorescence emission spectrum of Net or EtBr were titrated into viscometer containing 2.5 ml of ciprofloxacin at pH 7.0; pH-dependent changes in the 200 AM in base pair polynucleotide solution, and flow times fluorescence intensity of ciprofloxacin at 413 nm between in the range of 100 –140 s were measured after each addition.
pH 1 and 13, with the maximal intensity observed at pH 7.5 Due to the low solubility of ciprofloxacin, the direct titration It has been reported that ciprofloxacin exists of polynucleotides by drug was not possible. A series of as a cation below pH 5, as a mixture of anions, cations and I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 3.2. Fluorimetric characterization of ciprofloxacin binding shows the fluorescence emission spectra of ciprofloxacin in the presence of different concentrationsof calf thymus DNA and poly[d(AT)]poly[d(AT)] at 20 jCin 2 mM cacodylate buffer (pH 7.0), respectively. Asignificant decrease in the fluorescence intensity of cipro-floxacin in the presence of calf thymus DNA has beenobserved with the shape and kmax of the spectra remainingunaffected increasing ratio of base pairs to ligand, R1.
Similar results were obtained in the presence of poly[d(GC)]poly[d(GC)]. In contrast, as shown in anoticeable change in the shape of ciprofloxacin fluorescent Fig. 2. The intrinsic fluorescence emission spectra of ciprofloxacin at pH spectra accompanied by a shift in the position of kmax from 7.0. The pH dependency of ciprofloxacin single-wavelength fluorescence 413 nm (R1=0) to 436 (F1) nm (R1=47.9) is observed intensity, FI (.), at 413 nm (inset). kex=330 nm, Ccf=1 AM, T=25 jC. a.u.
upon titration with poly[d(AT)]poly[d(AT)]. The appear- stands for arbitrary units.
ance of an isosbestic point at 431 nm in the ciprofloxacinfluorescent spectra suggests the presence of two forms ofciprofloxacin, e.g. free and bound. Recently, it has been zwitterions at pH between 5 and 10, and as an anion at pH shown that the fluorescence emission spectrum of norflox- higher than 10 The molar fluorescence intensity acin undergoes a red shift in the presence of poly[d(AT)] and kmax in the fluorescence emission spectra of ciproflox- poly[d(AT)], and a small decrease in its fluorescence in- acin do not change with its concentration in the concentra- tensity, while in the presence of poly[d(GC)]poly[d(GC)] tion range from 0 to 1.1 AM in 2 or 10 mM cacodylate and calf thymus DNA a strong decrease in fluorescence buffer (both pH 7.0). Also, no significant changes in the intensity was observed fluorescence emission properties of ciprofloxacin were shows the fluorescence titration curves of observed with increasing NaCl concentration up to 1 M at ciprofloxacin at two different solvent conditions titrated by the same solution conditions (data not shown). To summa- various DNAs at 413 nm expressed as a relative fluorescence rize, under experimental solution conditions applied in emission intensity of ciprofloxacin (FI/FIj)413 versus molar fluorimetric measurements, ciprofloxacin does not self-as- ratio of DNA base pairs to drug, R1, where FIj and FI stand sociate or form a complex with sodium ions, and it is for fluorescence emission intensity of ciprofloxacin in the present in several different charged forms absence and presence of DNA, respectively. In 2 mM Fig. 3. Fluorescence emission spectra of ciprofloxacin in the presence of calf thymus DNA (A) and poly[d(AT)]poly[d(AT)] (B) at different DNA base pairs todrug ratio, R1, as marked. kex=330 nm, Ccf=1 AM, T=20 jC.
I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 experimental conditions, KSV values in the presence ofpoly[d(A)]poly[d(T)] and poly[d(AT)]poly[d(AT)] couldhave been underestimated due to the changes in fluores-cence emission spectra of free versus bound ciprofloxacin.
Similarly, a study of norfloxacin binding to polynucleotidesyield the same order of KSV's The KSV values fornorfloxacin slightly differ from our values due to differentsolution conditions applied in the norfloxacin study The similar behaviour of ciprofloxacin and norfloxacin isnot surprising since these two representatives of fluoroqui-nolone family differ only in the group attached to nitrogenatom of quinolone ring system.
The qualitative difference in the observed fluorescence emission spectra of ciprofloxacin in the presence of poly[d(AT)]poly[d(AT)] compared to poly[d(GC)]poly[d(GC)]or calf thymus DNA is likely to indicate the different way ofinteraction of ciprofloxacin with these polynucleotides. It ispossible that ciprofloxacin fits better in the narrow groove ofAT sequences, allowing for little or no rotation of the drugand thus smaller nonradiative deactivation of the excitedstate occurs The stronger quenching of ciprofloxacinfluorescence without significant shift in kmax by GC sequen-ces, compared to AT, could originate in guanine. Theoxidation potential of guanine is the lowest in nucleobases.
Also, guanine can be an effective quencher of fluorescencethrough electron transfer from DNA to photo-excited fluo-roquinolone Furthermore, due to the difference in Fig. 4. Relative fluorescence emission intensity (FI/FIj)413 of ciprofloxacin the local surface properties of the studied polynucleotides, derived from multiple titration experiments at two different solution condi- factors such as differences in hydration, structural features tions: (A) 2 mM cacodylate (pH=7.0), 1.7 mM Na+ and 0.1 mM Na2EDTA,at 20 jC; (B) 10 mM cacodylate (pH=7.0) and 108.6 mM Na+, at 25 jC.
and surface charge density can affect ciprofloxacin ds-DNA (n), thermally denatured calf thymus ss-DNA (5), poly[d(AT)] binding. Surface pH may also play a role, as it has been poly[d(AT)] (E), poly[d(GC)]poly[d(GC)] (D) and poly[d(A)]poly[d(T)] shown that the fluorescence emission intensity of ciproflox- (o) at 413 nm. FIj and FI are fluorescence emission intensity of acin is pH-sensitive ciprofloxacin in the absence and presence of DNA, respectively. R1 is themolar ratio of DNA (per base pairs) to drug. kex=330 nm, Ccf=1 AM.
3.3. Electrostatic contribution to the binding of ciproflox- acin to polynucleotides cacodylate buffer the magnitude of fluorescence quenchingof double-stranded DNA follows the same order as that The quenching of the fluorescence emission intensity of observed at higher ionic strength (10 mM cacodylate buffer ciprofloxacin was more efficient at lower salt concentra- containing 108.6 mM Na+) with the exception of single-stranded calf thymus DNA poly[d(GC)]poly[d(GC)] > calf thymus DNA>poly[d(AT)]poly[d(AT)]> poly[d(A)]poly[d(T)]. Note, however, that fluorescence Equilibrium constants for ciprofloxacin polynucleotide complex formationcalculated with the Stern – Volmer method quenching by single-stranded calf thymus DNA (ss-DNA) is lower than by double-stranded calf thymus DNA (ds-DNA) at lower ionic strength (in 2 mM cacodylate buffer) and vice 2 mM Cacodylate buffer, 1.7 mM Na+, 0.1 mM Na2EDTA, pH 7.0Single-stranded calf thymus DNA versa at higher ionic strength (10 mM cacodylate buffer Double-stranded calf thymus DNA containing 108.6 mM Na+) The Stern–Volmer equilibrium constant (KSV) for the formation of ciprofloxacin –polynucleotides complex can be obtained by plotting (FIj/FI)413 versus the concentration of 10 mM Cacodylate buffer, 108.6 mM Na+, 0.1 mM Na polynucleotides. The K SV's of ciprofloxacin – DNA complex Single-stranded calf thymus DNA formation in the presence of single-stranded DNA, calf Double-stranded calf thymus DNA thymus DNA, and poly[d(GC)]poly[d(GC)] were larger than those in the presence of poly[d(A)]poly[d(T)] and poly[d(AT)]poly[d(AT)] However, at the applied I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 tions, indicating that binding of ciprofloxacin is strongly quenched in the presence of calf thymus DNA, while about salt-dependent To determine the contribu- 25% in the presence of poly[d(AT)]poly[d(AT)]. Since the tion of nonspecific (electrostatic) interactions to the binding, fluorescence emission intensity of ciprofloxacin in the the ciprofloxacin –polynucleotides complex (R1i50) base absence of DNA does not change significantly with increas- pairs) was back-titrated with NaCl. shows the relative ing NaCl concentration (data not shown), it appears that at fluorescence emission intensities, FI/FIj, of ciprofloxacin at higher NaCl concentrations (>10 mM) some fraction of 413 nm and the changes in the maximum wavelength, kmax, ciprofloxacin remains bound to all polynucleotides indepen- of ciprofloxacin emission spectra after titration by calf dent of salt concentration. This observation suggests that the thymus DNA and poly[d(AT)]poly[d(AT)] (A, B) and after electrostatic contribution to ciprofloxacin binding to DNA is titration of the final ciprofloxacin–duplex complex by NaCl significant. The majority of ciprofloxacin bound to double- (C, D). The fluorescence emission intensity of ciproflox- stranded calf thymus DNA, poly[d(GC)]poly[d(GC)] and acin – duplex complexes increases with increasing NaCl poly[d(A)]poly[d(T)] (data not shown) can be removed by concentration. At higher concentrations of NaCl (>10 increasing NaCl concentration to a value of less than 10 mM), only about 5% of the total fluorescence signal remains mM; however, the same concentration of NaCl removes Fig. 5. The changes in the relative fluorescence emission intensity (FI/FIj)413 and changes in maximum wavelength, kmax, of the ciprofloxacin emission spectraafter titration with native ds-DNA calf thymus DNA (o) (A and B), and poly[d(AT)]poly[d(AT)] (n) and after NaCl back-titration of the final ciprofloxacin –DNA complex (C and D). Solution conditions were 2 mM cacodylate (pH=7.0), 1.7 mM Na+ and 0.1 mM Na2EDTA at 20 jC. Ccf=1 AM and kex=330 nm.
I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 only a small fraction of ciprofloxacin bound to poly[d(AT)] consecutive AT base pairs, corresponding to the apparent poly[d(AT)], suggesting a qualitatively different kind of binding site size of ciprofloxacin binding to poly[d(AT)] interaction of ciprofloxacin with poly[d(AT)]poly[d(AT)] poly[d(AT)]. The fluorescence emission spectra of cipro- compared to the rest of the studied polynucleotides.
floxacin in the presence of selected dodecameric DNA To summarize, these results suggest that ciprofloxacin duplexes were measured at pH 7.0 in 10 mM cacodylate has at least two different modes of binding to double- buffer (8.6 mM Na+), 0.1 mM Na2EDTA and 20 mM NaCl.
stranded DNA: (1) a nonspecific binding to all double- The experiments could not be performed at the same ionic stranded DNA molecules which is electrostatically driven strength conditions used for poly[d(AT)]poly[d(AT)] due to (e.g. ciprofloxacin interactions with negatively charged instability of dodecameric duplexes Melting experi- phosphate groups and electrostatic stacking interactions on ments (data not shown) demonstrate that duplexes D#1 and the helix exterior), and (2) a specific nonelectrostatically D#2 are fully formed at 20 jC in the selected buffer at controlled binding (e.g. intercalation, minor or major groove concentrations higher than 40 AM per single strand.
binding), which could depend on several different factors Upon titration with duplex D#2 containing TATAT se- (e.g. DNA sequence, the geometry of minor or major quence, the shape and the kmax of ciprofloxacin fluorescence groove, the extent of hydration base stacking inter- emission spectra change while no significant actions, and not at least on the binding site size). From changes were observed in the presence of duplex D#1 5B the apparent stoichiometry of ciprofloxacin binding to containing a random base sequence at the same position, poly[d(AT)]poly[d(AT)] can be determined. It equals a TACTT The magnitude of quenching is higher ciprofloxacin molecule per five base pairs, while for all with duplex D#1 containing the mixed base sequence. These the other polynucleotides the stoichiometry of ciprofloxacin results are in qualitative agreement with the results obtained binding to DNA could not be accurately determined.
for poly[d(AT)]poly[d(AT)] and calf thymus DNA de-scribed above. The quenching of ciprofloxacin fluorescence 3.4. Ciprofloxacin binding to various single- and double- emission intensity by oligonucleotides follows the same stranded oligomeric DNAs pattern as previously observed for polynucleotides: (i) thefluorescence quenching is higher by double-stranded DNA In order to examine the origin of different behaviour of than by single-stranded DNA; (ii) the fluorescence quench- ciprofloxacin in the presence of poly[d(AT)]poly[d(AT)], ing is becoming more pronounced with increasing amount calf thymus DNA and poly[d(GC)]poly[d(GC)], we studied of GC base pairs (Supplementary material, Fig. S1A and B) the ciprofloxacin binding to two dodecameric DNA and (iii) the red shift in the position of kmax in the duplexes. Duplex D#1 (G1+C1) has a random sequence fluorescence emission spectra of ciprofloxacin is observed while duplex D#2 (G2+C2) contains a sequence of five only for DNA containing AT base sequences.
Fig. 6. Fluorescence emission spectra of ciprofloxacin in the presence of two dodecameric duplexes, (A) D#1 (G1+C1) and (B) D#2 (G2+C2), at differentDNA (per single strand) to drug ratio, R1, as marked. Solution conditions were 10 mM cacodylate (pH=7.0), 28.6 mM Na+ and 0.1 mM Na2EDTA at 20 jC.
Ccf=1 AM and kex=330 nm.
I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 3.5. Electrostatic contribution to the binding of ciproflox- strand. Interestingly, our data reveal that even at the highest acin to oligonucleotides R (=1) value, the complete saturation of binding of cipro-floxacin to poly[d(AT)]poly[d(AT)] was not achieved shows the changes in fluorescence emission 8). These results further support our fluorescence results that intensity of ciprofloxacin upon titration by D#1 and D#2, at low ionic strength, ciprofloxacin binds specifically to while the corresponding changes in kmax are shown in double-stranded DNA and shows higher sequence preferen- 7B. Additionally, the corresponding changes in ciproflox- ces for alternating AT base pairs.
acin fluorescence emission intensity and kmax upon back- It should be mentioned that in the buffer containing 10 titration of the ciprofloxacin–D#1 and ciprofloxacin–D#2 mM cacodylate, 8.6 mM Na+ and 0.1 mM Na2EDTA, complexes with NaCl are shown in Increas- insignificant thermal stabilization of poly[d(AT)]poly ing NaCl concentration does not completely displace cipro- [d(AT)] by ciprofloxacin and destabilization of calf thymus floxacin from ciprofloxacin –D#1 and –D#2 complexes. At DNA are observed. In contrast, in high ionic strength buffer higher concentrations of NaCl (>100 mM), only 10% of the (10 mM cacodylate, 108.6 mM Na+, 0.1 mM Na2EDTA) total fluorescence signal remains quenched in the presence ciprofloxacin shifts the melting temperature, Tm, of both of D#1 and f20% in the presence of D#2 containing five native and different synthetic DNAs to lower values AT base pairs Almost complete restoration of 2). The most pronounced decrease in Tm (DTm=3.5 (F1) ciprofloxacin fluorescence was observed after NaCl titration jC) was observed for calf thymus DNA compared to DTm of each single-stranded oligonucleotide separately, except value of 1.7 (F1) jC for poly[d(AT)]poly[d(AT)] G1 (Supplementary material, Fig. S1C and D). The stronger 2). The decrease in thermal stability of the different double- quenching of the ciprofloxacin fluorescence by double- stranded DNAs upon addition of ciprofloxacin would suggest stranded DNA compared to single-stranded DNA could be that the ligand preferentially interacts with single-stranded due to the electrostatically enhanced outside binding of DNA rather than double-stranded DNA. At these experimen- ciprofloxacin to double-stranded DNA. After back-titration tal conditions, poly[d(GC)]poly[d(GC)] is too stable to melt of ciprofloxacin –D#2 complex with NaCl, the observed in the examined temperature range.
shift in kmax to its initial value suggests that most of At low ionic strength of solution drug stabilizes duplex ciprofloxacin dissociated from dodecameric duplex contain- rather than single-stranded DNA The increase in ing five consecutive AT base pairs Slightly Tm is expected due to the outside stacking and electrostatic different behaviour observed for ciprofloxacin binding to interactions between DNA and ciprofloxacin. At neutral pH oligomeric DNA duplex containing five consecutive AT and in the absence of salt, protons may promote ciprofloxacin base pairs compared to poly[d(AT)]poly[d(AT)] binding to DNA by neutralizing the negative charge on the D) suggests that the site size and/or other structural param- carboxylate group of different drug species The observed eters are important for binding.
increase in thermal stability of poly[d(AT)]poly[d(AT)] of9.8 (F1) jC is too high to be explained only by electrostatic 3.6. UV-melting profiles of polymeric double-stranded DNA interactions. The salt-dependent UV-melting, fluorescence in the presence of ciprofloxacin NaCl back-titration and viscometry data (see below) suggestthat ciprofloxacin exhibits another mode of binding in addi- Melting experiments of various double-stranded DNA at tion to electrostatically driven outside binding of the drug to different drug to DNA ratios were performed at pH 7.0 at DNA that occurs under conditions of high drug loading and is different buffer conditions (2 mM cacodylate, 1.7 mM Na+ enhanced at low ionic strength. Such a binding mode, in fact, and 0.1 mM Na2EDTA or 10 mM cacodylate, 108.6 mM Na+ is common among DNA intercalating ligands Many and 0.1 mM Na2EDTA) Ciprofloxacin shifts studies using the linear dichroism have shown that the the melting temperature, Tm, of all studied double-stranded molecular plane of norfloxacin was near parallel to the DNA to higher values at low ionic strength of the buffer DNA bases (perpendicular to the DNA helix axis) in the (2 mM cacodylate, 1.7 mM Na+ and 0.1 mM Na2EDTA) binding complex and would support the intercalative The most pronounced increase in Tm of 9.8 binding mode. However, the observed identical Tm of DNAs (F1) jC was observed at ciprofloxacin to DNA base pairs in the absence and presence of norfloxacin and the negligible ratio (R) of 1 for poly[d(AT)]poly[d(AT)] The unwinding of supercoiled DNA by norfloxacin at higher ionic observed thermal stabilization, DTm (=TmTmj), for calf strength did not classify the norfloxacin as a classical inter- thymus DNA at R=1 is 3.3 (F1) and 2.3 (F1) jC for calator or a minor groove binder poly[d(GC)]poly[d(GC)]. To test the reversibility of cipro-floxacin binding to synthetic double-stranded DNA, we 3.7. Viscometry results show that ciprofloxacin has some cooled the samples down and reheated the same sample the properties of an intercalative binder second time. The melting curves obtained upon reheatingwere completely superimposable onto the first heating scans The primary mode of binding by which a ligand interacts (data not shown), suggesting that ciprofloxacin does not with a polymeric host nucleic acid structure may be inves- inhibit reannealing by associating irreversibly with the single tigated by viscometry. In general, intercalators cause an I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 Fig. 7. The changes in the relative fluorescence emission intensity (FI/FIj)413 (A) and in the position of the maximum wavelength, kmax (B) in the ciprofloxacinemission spectra after titration by dodecameric DNA duplexes D#1 (G1+C1) (o) and D#2 (G2+C2) (n) and NaCl back-titration of the final ciprofloxacin –dodecameric duplex complex (C and D). Solution conditions were 2 mM cacodylate (pH=7.0), 1.7 mM Na+ and 0.1 mM Na2EDTA at 20 jC. Ccf=1 AM andkex=330 nm.
increase in intrinsic viscosity due to an increase in apparent increase in DNA contour length in the presence of cipro- molecular length of the DNA helix. Ligand interaction floxacin is lower than by ethidium bromide but much higher between stacked bases within a linear host duplex (interca- than by netropsin under the same experimental conditions, lation) is associated with the lengthening of the nucleic acid.
suggesting some degree intercalation of ciprofloxacin into Thus, a ligand-induced increase in the viscosity of a duplex double-stranded DNA under solution conditions employed nucleic acid solution is consistent with (but does not Several factors may account for the quantitative absolutely prove) an intercalative mode of binding differences in apparent contour length between ciproflox- shows the effect of ciprofloxacin, ethidium bro- acin and ethidium bromide binding to two different mide (a typical intercalator) and netropsin (a typical minor duplexes. First, competing nonintercalative-binding modes groove binder) binding on the apparent relative contour of ciprofloxacin, such as groove binding and electrostati- lengths of two host duplexes. Inspection of reveals cally facilitated stacking at the helix exterior, may reduce that upon addition of ciprofloxacin to calf thymus DNA and the amount of intercalative binding and therefore the extent poly[d(AT)]poly[d(AT)] duplexes, each undergoes substan- of helix lengthening. Second, structural differences between tial increase in apparent contour length. The extent of the intercalated complexes (e.g., degree of helix unwinding) I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 Fig. 8. Absorbance versus temperature profiles of poly[d(AT)]poly[d(AT)] and their ciprofloxacin complexes (at indicated values of R) at 260 nm. Solutionconditions were: (A) 2 mM cacodylate (pH=7.0), 1.7 mM Na+ and 0.1 mM Na2EDTA; (B) 10 mM cacodylate (pH=7.0), 108.6 mM Na+ and 0.1 mMNa2EDTA. CDNA=15 AM in base pairs. The arrows indicate the melting curves of poly[d(AT)]poly[d(AT)] in the absence (R=0) and presence (R=1) ofciprofloxacin, respectively.
of ciprofloxacin with DNA and ethidium with DNA to the differential effect we observed could be ciprofloxacin- which differentially alter the effective hydrodynamic length induced conformational changes in the target duplexes and stiffness of the target duplex, may contribute to the independent of intercalation. In general, such an effect could observed difference apparent contour length. Third, differ- cause a single binding mode to reflect properties character- ences in binding site size and binding affinity between istic of multiple binding modes Due to these undeter- ciprofloxacin and ethidium bromide should affect helix mined factors, the observed change in intrinsic viscosity lengthening. Fourth, as our results suggest, there are qual- does not provide absolute proof of intercalative binding.
itative differences between ciprofloxacin and ethidium bro- However, the similarity between the effect of ethidium mide binding to poly[d(AT)]poly[d(AT)] and random bromide and ciprofloxacin on the change in intrinsic vis- sequence genomic DNA. Thus, the binding-induced hydro- cosity compared to the effect of netropsin, for example, at dynamic differences, we observed between duplexes, might least strongly suggests that ciprofloxacin is also an inter- reflect the presence of some partially intercalated drug calator at conditions applied here.
molecules, which manifest additional interactions withinthe groove(s). Not, at least, another potential contribution 4. Concluding remarks Table 2The melting temperatures of the nucleic acid duplexes at pH 7.0 (at The present study of ciprofloxacin binding to various different solution conditions) in the absence and presence of ciprofloxacin polymeric and oligomeric DNAs clearly shows that cipro- Nucleic acid duplex floxacin can bind to single- and double-stranded DNA. At 2 mM Cacodylate buffer, 1.7 mM Na+, 0.1 mM Na2EDTA low ionic strength, ciprofloxacin binds specifically to dou- ble-stranded DNA and shows higher sequence preferences for alternating base sequences. Similar results were pub- lished before for norfloxacin, another representative offluoroquinolone family, which is structurally very similar 10 mM Cacodylate buffer, 8.6 mM Na+, 0.1 mM Na2EDTACalf thymus DNA to ciprofloxacin and differs only in the ethyl-group attached to N1 nitrogen atom of quinolone ring system Furthermore, our results highlight the importance of solvent 10 mM Cacodylate buffer, 108.6 mM Na+, 0.1 mM Na2EDTA conditions in determining ciprofloxacin–DNA interactions and contribute to the broad-based effort to define the molecular recognition patterns that control the affinities Tm is the melting temperature of the nucleic acid duplex in the absence ofciprofloxacin. T and specificities of nucleic-acid-binding fluoroquinolone.
m is the melting temperature of the ciprofloxacin – DNA complex at the R value of 1.0. DT=T In addition, we have observed, for the first time, the slightly I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122 different behaviour of ciprofloxacin binding to oligomeric and for rechecking the values of our extinction coefficients DNA duplex containing five consecutive AT base pairs of the dodecameric single strands, and to Prof. dr. Kenneth compared to poly[d(AT)]poly[d(AT)], suggesting that the J. Breslauer in whose laboratory the oligonucleotides were size and/or other structural parameters are important for synthesised and purified (Rutgers, The State University of binding. The reversibility of ciprofloxacin binding to dou- New Jersey, Piscataway, NJ) during N.P. postdoctorial ble-stranded DNA, as observed from UV-melting curves, appointment. This work was supported by the Slovenian suggests that ciprofloxacin does not inhibit the reannealing Ministry of Education, Science and Sport.
by associating irreversibly with the single strands. Further-more, the experimental observations from UV-meltingcurves, fluorescence emission spectra and fluorescence NaCl back-titration of ciprofloxacin–DNA complexes sug-gest that ciprofloxacin has at least two different binding  D.C. Hooper, Quinolone mode of action—new aspects, Drugs 45 modes: a nonspecific binding to single- and double-stranded (1993) 8 – 14.
 K. Drlica, X. Zhao, DNA gyrase, topoisomerase IV, and the 4-quino- DNA molecules, which is electrostatically driven, and a lones, Microbiol. Mol. Biol. Rev. 61 (1997) 377 – 392.
specific non-electrostatically controlled binding. The effect  D.C. Hooper, Clinical applications of quinolones, Biochim. Biophys.
of ciprofloxacin on the change in intrinsic viscosity strongly Acta 1400 (1998) 45 – 61.
suggests that ciprofloxacin has the properties of an inter-  K. Drlica, Mechanism of fluoroquinolone action, Curr. Opin. Micro- calative binder.
biol. 2 (1999) 504 – 508.
 L.L. Shen, A.G. Pernet, Mechanism of inhibition of DNA gyrase by analogs of nalidixic-acid: the target of the drugs is DNA, Proc. Natl.
Acad. Sci. U. S. A. 82 (1985) 307 – 311.
 C. Bailly, P. Colson, C. Houssier, The orientation of norfloxacin bound to double stranded DNA, Biochem. Biophys. Res. Commun.
We are especially grateful to Prof. dr. Jens Vo¨lker for 243 (1998) 844 – 848.
critical reading of the manuscript, many helpful suggestions  G.S. Son, J.A. Yeo, J.M. Kim, S.K. Kim, H.R. Moon, W. Nam, Base specific complex with DNA, Biophys. Chemist. 74 (1998) 225 – 236.
 G.S. Son, J.A. Yeo, J.M. Kim, S.K. Kim, A. Holme´n, B. Akerman, B.
Norde´n, Binding mode of norfloxacin to calf thymus DNA, J. Am.
Chem. Soc. 120 (1998) 6451 – 6457.
 L.M. Fisher, J.M. Lawrence, I.C. Jostly, R. Hopewell, E.E. Margerri- son, M.E. Cullen, Ciprofloxacin and fluoroquinolones. New conceptson mechanism of action and resistance, Am. J. Med. 87 (1989) 2S – 8S.
 J. Fung-Tomc, B. Kolek, D.P. Bonner, Ciprofloxacin-induced, low- level resistance to structurally unrelated antibiotics in Pseudomonasaeruginosa and methicillin-resistant Staphylococcus aureus, Antimi-crob. Agents Chemother. 37 (1993) 1289 – 1298.
 P. Heisig, B. Kratz, E. Halle, Y. Graser, M. Altwegg, W. Rabsch, J.P.
Faber, Identification of DNA gyrase A mutations in ciprofloxacin-resistant isolates of Salmonella typhimurium from men and cattle inGermany, Microb. Drug Resist. 1 (1995) 211 – 218.
 D. Niccolai, L. Tarsi, R.J. Thomas, The renewed challenge of anti- bacterial chemotherapy, Chem. Commun. 1997 (1997) 2333 – 2342.
 T.R. Hammonds, S.R. Foster, A. Maxwell, Increased sensitivity to quinolone antibacterial can be engineered in human topoisomeraseII by selective mutagenesis, J. Mol. Biol. 300 (2000) 481 – 491.
 J.-Y. Fan, D. Sun, H. Yu, S.M. Kerwin, L.H. Hurley, Self-assembly of a quinobenzoxazine – Mg2+ complex on DNA: a new paradigm forstructure of a drug – DNA complex and implications for the structureof the quinolone bacterial gyrase – DNA complex, J. Med. Chem. 38(1995) 408 – 424.
 G. Palu , S. Valisena, G. Ciarrocchi, B. Gatto, M. Palumbo, Quinolone binding to DNA is mediated by magnesium ions, Proc. Natl. Acad.
Sci. U. S. A. 89 (1992) 9671 – 9675.
 C. Sissi, M. Andreolli, V. Cecchetti, A. Fravolini, B. Gatto, M. Pal- umbo, Mg2+-mediated binding of 6-substituted quinolones to DNA:relevance to biological activity, Bioorg. Med. Chem. 6 (1998)1555 – 1561.
 C. Sissi, E. Perdona, E. Domenici, A. Feriani, A.J. Howells, A. Max- Fig. 9. Viscometric titration of sonicated calf thymus DNA (open symbol) well, M. Palumbo, Ciprofloxacin affects conformational equilibria of and poly[d(AT)]poly[d(AT)] (solid symbol) with ethidium bromide (o, .), DNA gyrase in the presence of magnesium ions, J. Mol. Biol. 311 netropsin (5, n) and ciprofloxacin (D, E) at 20.0 jC. L/Lo is plotted as a (2001) 195 – 203.
function of moles of drug added per mole of DNA base pairs, R. Solution  L.L. Shen, J. Baranowski, A.G. Pernet, Mechanism of inhibition of conditions were 2 mM cacodylate (pH=7.0), 1.7 mM Na+ and 0.1 mM DNA gyrase by quinolone antibacterials: specificity and cooperativity of drug binding to DNA, Biochemistry 28 (1989) 3879 – 3885.
I.D. Vilfan et al. / Biochimica et Biophysica Acta 1628 (2003) 111–122  L.L. Shen, L.A. Mitscher, P.N. Sharma, T.J. O'Donnell, D.W.T. Chu,  I. Turel, N. Bukovec, E. Farkas, Complex formation between some C.S. Cooper, T. Rosen, A.G. Pernet, Mechanism of inhibition of DNA metals and a quinolone family member (ciprofloxacin), Polyhedron Gyrase by quinolone antibacterials: a cooperative drug – DNA binding 15 (1996) 269 – 275.
model, Biochemistry 28 (1989) 3886 – 3894.
 I. Turel, P. Bukovec, M. Quiro´s, Crystal structure of ciprofloxacin  M. Palumbo, B. Gatto, G. Zagatto, G. Palu , On the mechanism of hexahydrate and its characterization, Int. J. Pharm. 152 (1997) 59 – 65.
action of quinolone drugs, Trends Microbiol. 1 (1993) 232 – 235.
 A. Larsson, C. Carlsson, M. Jonsson, Characterization of the binding  S.E. Critchlow, A. Maxwell, DNA cleavage is not required for the of YO to [poly(dA – dT)]2 and [poly(dG – dC)]2, and of the fluorescent binding of quinolone drugs to the DNA gyrase – DNA complex, Bio- properties of YO and YOYO complexed with the polynucleotides and chemistry 35 (1996) 7387 – 7393.
double stranded DNA, Biopolymers 36 (1995) 153 – 167.
 I. Turel, P. Bukovec, Comparison of the thermal stability of ciproflox-  E.-J. Lee, J.-A. Yeo, C.-B. Cho, G.-J. Lee, S.K. Kim, Amine group of acin and its compounds, Thermochim. Acta 287 (1996) 311 – 318.
guanine enhances the binding of norfloxacin antibiotics to DNA, Eur.
 M. Riley, B. Maling, M.J. Chamberlin, Physical and chemical char- J. Biochem. 267 (2000) 6018 – 6024.
acterization of two- and three-stranded adenine – thymine and ad-  C.V. Kumar, R.S. Turner, E.H. Asuncion, Groove binding of a styr- enine – uracil homopolymer complexes, J. Mol. Biol. 20 (1966) ylcyanine dye to the dna double helix—the salt effect, J. Photochem.
118 – 124.
Photobiol., A Chem. 74 (1993) 231 – 238.
 Hamilton HPLC Application Handbook, Hamilton Co., Reno, NV,  T.V. Chalikian, J. Vo¨lker, A.R. Shrinivasan, W.K. Olson, K.J. Bresla- uer, The hydration of nucleic acid duplexes as assessed by a combi-  B.L. Griswold, F.L. Hummdler, A.R. McIntyre, Inorganic phos- nation of volumetric and structural techniques, Biopolymers 50 phates and phosphate esters in tissue extracts, Anal. Chem. 23 (1999) 459 – 471.
(1951) 192 – 194.
 L.A. Marky, K.J. Breslauer, Calculating the thermodynamic data for  J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, transitions of any molecularity from equilibrium melting curves, Bio- New York, 1983, p. 257.
ploymers 26 (1987) 1601 – 1620.
 G. Cohen, H. Eisenberg, Conformational studies on the sodium and  S. Neidle, Z. Abraham, Structural and sequence-dependent aspects of cesium salt of calf thymus deoxynucleic acid (DNA), Biopolymers 8 drug intercalation into nucleic acids, CRC Crit. Rev. Biochem. 17 (1969) 45 – 55.
(1984) 73 – 121.
 W. Mu¨ller, D.M. Crothers, Studies of the binding of actinomycin and  D.S. Pilch, M.A. Kirolos, X.L. Liu, E.G. Plum, K.J. Breslauer, Bere- related compounds to DNA, J. Mol. Biol. 35 (1968) 251 – 290.
nil[1,3-bis(4V-amidinophenyl)triazenol] binding to DNA duplexes and  V.A. Bloomfield, D.M. Crothers , I. Tinoco Jr., Physical Chemistry of to a RNA duplex: evidence for both intercalative and minor groove Nucleic Acids, Harper & Row, New York, 1974, pp. 442 – 445.
binding properties, Biochemistry 34 (1995) 9962 – 9976.
 D.-S. Lee, H.-J. Han, K. Kim, W.-B. Park, J.-K. Cho, J.-H. Kim,  M.J. Waring, N.F. Totty, Binding of drugs to supercoiled circular Dissociation and complexation of fluoroquinolone analogues, J.
DNA. Evidence for and against intercalation, Prog. Mol. Subcell.
Pharm. Biomed. Anal. 12 (1994) 157 – 164.
Biol. 2 (1971) 216 – 231.
Contraceptive Use and Discontinuation Patterns in Nepal: Norplant, IUCD, Pill, and Injectables ©2003 EngenderHealth I. Introduction The main aims of Nepal's family planning program are to assist individuals and couples to space their children, prevent unintended pregnancies, and improve their overall reproductive health.