Electrochemical, Spectroscopic and Molecular

R. Qureshi et al / International Research Journal of Pharmaceuticals (2011), Vol. 01, Issue 01

Research Paper

Electrochemical, Spectroscopic and Molecular Docking Studies of Anticancer Organogermalactones Fouzia Perveen, Rumana Qureshi*, Afzal Shah, Safeer Ahmed, Farzana Latif Ansari, Saima Kalsoom and Sumera Mehboob *Department of Chemistry, Quaid-i-Azam University, 45320, Islamabad, Pakistan; E-Mail: [email protected]

Abstract Cyclic voltammetry, UV-Vis spectroscopic and molecular docking methods have been used to predict the effect of biologically potent compounds on the DNA proliferation under physiological conditions (pH 7.4 and 4.7). The interaction of organogermanium compounds namely β-o-flourophenyl-γ-bis (8-quinolinoxy) germa-γ-lactone (GLf) and β-methyl-γ-bis (8-quinolinoxy) germa-γ-lactone (GLm) with DNA was investigated at human body temperature. The diffusion coefficients and heterogeneous electron transfer rate constants have been calculated and discussed. The comparison of the voltammetric signals of GLf and GLm revealed that the reduction potential of the reducing moiety, lactone can be modulated by changing the electronic properties of the attached substituents. For the investigation of the DNA binding affinity of germa-γ-lactones, the formation constants were calculated from the decrease in peak current and increase in absorbance. The experimental results were supported by molecular docking study. The current study is expected to provide useful insights into the design of anticancer drugs and understanding the mechanism by which such drugs interact with DNA and exert their biochemical action. Keywords: Germa lactones; DNA; Cyclic voltammetry; UV-Vis spectroscopy; Molecular docking; Binding Affinity

1. Introduction Organogermaniun compounds such as, germatranes, spirogermanium, 2-carboxyethyl germanium sesquioxide, carboxyethyl germanium sesquisulfide and germa-clactones have been reported for a variety of activities including their possible use in suppression of the growth of certain tumors, proliferation of the normal marrow cells in the tumor bearing animals, pain relief, immunomodulation, induction of interferon, regulation of plant growth, hepatic cirrhosis, cardiovascular function, motor activity and stimulation of red blood cells production (Ho et al., 1990, Khusainov et al., 1991). The 8-quinolinol (one of the lactone substituted compounds) and its derivatives are widely used as antiamoebic and antiseptic agents (Mambury, 1979). Due to immunomodulatory effects, inorganic germanium salts and organogermanium compounds find extensive use as nutritional supplements (Pronai and Arimori, 1991). Small molecules bind to DNA by three predominant binding modes, intercalation,

groove-binding and electrostatic interaction with negatively charged nucleic acid sugar phosphate structure. Intercalators bind to DNA by insertion of a planar aromatic substituent between the base pairs, simultaneously lengthening and unwinding the helix. Intercalators vary in the extent to which they unwind DNA, but all lengthen DNA to about the same extent (Waring, 1970). In groovebinding, the crescent-shaped ligand fits into the minor groove with little steric hinderance and slight perturbation of the DNA structure (Chaires, 2006). Besides these, small biologically important molecules also interact with DNA via interstrand and intrastrand crosslinking. Computer docking techniques play an important role in drug design and elucidation of mechanism (Amtul et al., 2007). The flexible docking programs, DOCK (Kontoyianni, et al., 2004). AutoDock and molecular operating environment (MOE) help in predicting favorable protein-ligand complex structures with a reasonable accuracy and speed (Ewing, at al., 2001). These docking programs, when used prior to experimental screening, can

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be considered as powerful computational filters to reduce labour and cost needed for the development of effective medicinal compounds. When used after experimental screening, they can help in better understanding of bioactivity mechanisms. MOE offers a reasonable result in comparison with other popular docking programs and undoubtedly continue to play an important role in drug discovery (Morris, at al., 1998). Several drugs that were designed by intensive use of computational methods are currently under investigation for clinical trials (Hu, at al., 2004). Spurred by the advancement of computer docking programs in drug designing we utilized MOE 2010 for the interpretation of the probable mechanism of antiproliferative action of the selected novel germa-γ-lactones.

water and stored at 4°C. The concentration of the stock solution was determined from UV absorbance at 260 nm using the molar extinction coefficient (ε) of 6600 M−1 cm−1. A ratio of absorbance at 260 and 280 nm of (A260/A280) > 1.8 indicated that DNA was sufficiently pure and free from protein (Shah et al., 2009). 1m M stock solutions of the germa lactones were prepared in distilled water and diluted with 10% aqueous DMSO using 0.1 M buffer as supporting electrolytes (NaH2PO4 + Na2HPO4 (pH 7.4) and HAcO + NaAcO (pH 4.7)). 2.3.

Electrochemical Measurements

2. Experimental

Cyclic voltammetric experiments were carried out using conventional three-electrode system at 37°C. The temperature was maintained using a circulating water bath. A three-electrode electrochemical cell (Model K64 PARC) that consisted of Ag/AgCl electrode (sentek/UK Company Cat # 924005) as reference electrode, a thin Pt wire as a counter electrode and hanging mercury drop electrode (HMDE) of 2.0 mm diameter as the working electrode was employed. All experimental solutions were degassed for 10 min with high-purity nitrogen gas, before every electrochemical experiment. Cyclic voltammetry was performed using EG and G Princeton Applied Research (PAR) electrochemical system 370.

2.1.

2.4.

The purpose of the present study is to add β-oflourophenyl-γ-bis (8-quinolinoxy) germa-γ-lactone (GLf) and β-methyl-γ-bis (8-quinolinoxy) germa-γ-lactone (GLm) to the arsenal of weapons used against cancer. These compounds are known to have antitumor activity (Wang, et al., 2003) but literature survey reveals that such an activity of germa-γ-lactones by electrochemical, UV-Vis spectroscopic and molecular docking study is an unexplored matter.

Materials and Reagents

Organogermalactones namely β-o-flourophenyl-γ-bis (8quinolinoxy) germa-γ-lactone and β-methyl-γ-bis (8quinolinoxy) germa-γ-lactone (Scheme 1) were synthesized by the method reported in literature (Amtul et al., 2007). All supporting electrolyte solutions were prepared using analytical grade reagents and doubly distilled water. All experiments were done at human body temperature.

Absorption spectra were recorded on Shimadzu 1601 spectrophotometer equipped with a Julabo F-34 thermostat (±0.1oC) using a pair of 1 cm path length quartz cuvette. First the absorbance of drug solutions in 10% queous DMSO at 310 K was studied and then the procedure was carried out in the presence of 5 ppm solution of ds. DNA by keeping the concentration and volume of the drug solution constant. 2.5.

O N O

Ge N

O

(a)

Ge N

O

O

O

(b)

Scheme 1. Molecular structures of (a) β-o-flourophenyl-γ-bis(8quinolinoxy)-germa-γ-lactone (GLf) and (b) β-methyl-γ-bis(8quinolinoxy)germa-γ-lactone (GLm).

2.2.

Molecular Docking

CH3

F O N O

UV-Vis Spectroscopic Measurements

DNA Extraction

Calf blood double stranded DNA (ds-DNA) was extracted by falcon method (Jackson and Opin, 1995). Its stock solution of 2.40 × 10-4 M was prepared in doubly distilled

MOE-dock by Chemical Computing Group Inc. was used. All docking studies were carried out on a Pentium1.6 GHz workstation, 512 MB memory with windows operating system. The ligand was drawn in the MOE 2008. The geometry was optimized through the molecular mechanics method with AMBER force field and further semiempirical method PM3. The DNA duplex receptor structure was obtained from protein data bank (PDB ID 2X0O) with 12 base pairs running in 3’-5’ direction. The base pair sequence was CCATAATTTACC: CCTATGAAATCC. The edited structure was imported into MOE workstation and all hydrogen atoms were added to the structure with their standard geometry followed by energy optimization tool using the MOPAC 7.0. The resulting model was subjected to systematic conformational search at default parameters with RMS gradient of 0.01 kcal/mol using site finder. To get the


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highly accurate final binding position 80 cycles were used for calculation. The best conformation was selected on the base of energies (Patrick, 2009). The docking procedure was carried out and the maximum negative final docking energy (∆G) was calculated. 2.6.

Computer Modeling and Calculation of The Electronic and Steric Descriptors

The structure of each drug was drawn and subjected to energy optimization. These were then imported to ds.DNA molecule for docking purpose. The resulting Drug-DNA complex was used for calculating the energy parameters using MMFF 94x force field. The software package MOE was used to generate the descriptor-based QSAR. The final structures that represented the most stable conformer were used to obtain these descriptors. Electronic descriptors (like dipole moment (µ), energy of frontier orbitals (EHOMO & ELUMO) and total energy of the most stable conformer (ETotal) and steric descriptors (such as octanol-water partition coefficient (log P), molecular refractivity (MR) and heat of formation (Ηf) of GLf and GLm were selected and calculated.

The facile reduction of GLm as indicated by its lower peak potential values may be due to weak electron donating effect of methyl group than fluorophenyl substituent. A dramatic situation was encountered at pH 4.7 when GLf and GLm registered single reduction peaks as shown in the Fig. 1b and Fig. 2b. Such a behavior can presumably be due to the hydrolysis of the reduction product. The variation in voltammetric behavior of the selected germaγ-lactones at two different pH values indicates that their redox behavior is pH dependent. The cathodic shifts in the E1/2 values of the second peak of germa lactones at pH 7.4 in the presence of ds.DNA show facile reduction of these potential anticancer compounds.

CH3

CH3

CH3

-

+

1e + 1H

1e- + 1H+

Ge

Ge

Ge O

peak 2

o

peak 1

O

O

OH

O

OH

3. Results and Discussion 3.1.

Redox Behavior of Germa- γ-Lactones

Cyclic voltammogram (Fig. 1a) of β-o-flourophenyl-γ-bis (8-quinolinoxy) germa-γ-lactone (GLf) at pH 7.4 shows two cathodic peaks at -1.04 and -1.56 V. Corresponding CV values of β-methyl-γ-bis (8-quinolinoxy) germa-γlactone (GLm) at the same pH are -0.95 and -1.35 V (Fig. 2a). The cathodic peaks are attributed to the two steps 1e-, 1H+ reduction of the lactone moiety to lactol ring. Such a conversion by chemical reduction has also been reported in literature (Abdel-Hamid, 2007).

F

F

F 1e- + 1H+

1e- + 1H+

Ge

Ge

Ge O

peak 1

O

O

peak 2

o

OH

O

Fig. 2. Cyclic voltammograms of GLm in the absence and presence of DNA at pH (a) 7.4 and (b) 4.7.

The diffusion coefficients (Do) of germa lactones (GL’s) in the presence and absence of DNA were calculated using Randles-Sevcik equation (Granqvist, et al., 1997, Sevick, 1948).

OH

1/2

Ip = -0.4463 n(α) FACo

Fig. 1. Cyclic voltammograms of GLf in the absence and and presence of DNA at pH (a) 7.4 and (b) 4.7.

*

nF1/2 1/2 1/2 RT Do ν

(1)

where Ip is peak current in ampere, Co* the bulk concentration i.e; [GL’s] in (mol/cm3), ν the scan rate in V/s, α the charge transfer coefficient, A the area of electrode in cm2. A plot of Ip vs. ν1/2 gives a straight line for a diffusion controlled reversible process as observed in the present case (see Table 1). Diffusion coefficient of the free drug (Df) and that of the drug-DNA adduct (Db) was found to have the same magnitude implying that adduct formed is not electroactive.


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Table. 1. Heterogeneous Electron Transfer Rate Constants for GLf and GLm at pH 7.4

Compound/Adducts GLf GLf + DNA GLm GLm + DNA

D / cm2s-1 5.05 ×10-7 8.95 ×10-7 2.77 ×10-6 1.06 ×10-8

ks,h / cms-1 1.22 ×10-3 1.44 ×10-3 12.60 ×10-3 12.70 ×10-2

3.2.2. Electronic Spectroscopy

To probe into the heterogeneous kinetics of redox reactions of GL’s, heterogeneous rate constant has been determined using Gileadi’s method (Bard et al, 1980).

 nFα υ c Do  = −0.48α + 0.52 + log   2.303RT 

1/ 2

ks , h

(2)

Getting the critical scan rate, υ c, ks,h can be calculated as, “ α ” is a dimensionless parameter, called transfer coefficient and Do is diffusion coefficient. The experimental ks,h values (see Table 1) for both the cathodic steps are approximately the same showing kinetic feasibility of the reduction processes. Addition of DNA does not significantly change the ks,h values because order of magnitude of ks,h before and after DNA addition is the same. 3.2. 3.2.1.

of GLf with values 3.98 × 105 and 8.57 × 105 M-1 were calculated at pH 7.4 and 4.7 according to Eq (3). For GLm the Kf with values 4.31 × 104 and 2.63 × 102 M-1 was obtained at the respective pH values. The higher Kf of GLf indicates its preferred candidature as anticancer drug.

A band corresponding to n-σ* transition was observed in the UV spectrum of the investigated drugs with λmax at 242 nm for GLf and 360 nm for GLm accompanying n-π* transitions at the selected pH values. Beer-Lambert law has been used to find out the absorbance in the presence and absence of ds.DNA. By applying the law, it is possible to measure intensity of the two components A and B in a mixture. For such an analysis we can write the absorbance as: A = AA+BB Where AA is the absorbance of component A (i.e., GL) and BB is the absorbance of component B (i.e., GL-DNA adduct). The UV-Vis spectra of GLm in the presence DNA (Fig. 3) registered hyperchromism accompanied with a red shift at pH 4.7 and 7.4. The same behavior was also noticed for GLf. Such peculiar spectroscopic characteristics may be due to the overlap between the electronic clouds of lactones and nitrogenous bases of DNA.

DNA Binding Studies Cyclic Voltammetry

The change in voltammetric response of the drug in the presence of DNA is an efficient tool for the elucidation of the nature of interaction and the strength of binding. For the determination of binding parameters cyclic voltammetric titration of DNA was carried out with anticancer GLf and GLm. The decrease in peak current in the presence of ds.DNA could be used for the determination of formation constants. The interaction of germa lactones with DNA can be described using the following equation: drug + DNA

↔ drug-DNA

An equation for amperometric titrations can be deduced as (Carter, et al., 1997, Feng, et al., 1997),

Ip 1 log( ) = log( K f ) + log( o ) [DNA] Ip − Ip

(3)

Where Kf is the formation constant, Ipo and Ip are the peak currents of free guest and complex respectively. By varying the concentration of DNA while keeping the concentration of the drug constant the formation constants

Fig. 3 Comparison of UV-Vis spectra of GLm in the absence and presence of 5 ppm soln. of ds.DNA at pH (a) 7.4 and (b) 4.7.

Since absorbance varied in the presence of ds.DNA so based on variation in absorption maxima the formation constant “Kf” of drug-DNA complexation were determined according to the following Benesi-Hildebrand equation (Nie, et al., 1997).

εG εG A0 1 = + (4) A − A0 ε H −G − ε G ε H −G − ε G K f [DNA]

where A0 and A are the absorbance’s of free drug and complex respectively, εG and εH-G are the molar extinction coefficients of drug and complex respectively. The Kf is obtained from the intercept to slope ratio of the plot of A0/(A-A0) vs. 1/[DNA].


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Table. 2. Kf (expressed in M-1), ∆G (in kJ mol-1) and molar extinction coefficients for the complexes of anticancer compounds with ds.DNA from UV-Vis spectrophotometric data at both physiological pH values and 309.5K

pH

Complex

“kf”

-∆G

εG

represent the electronic interactions, a good inverse relation was observed between Kf and these parameters. Table.3. Comparison of formation constants obtained obtained from three methods.

εH-G From CV

From UV

From theory

“Kf”/M-1

“Kf”/M-1

“Kf”/M-1

GLf-DNA

3.98×105

1.710×105

6.78×107

GLm-DNA

4.31×104

4.54×103

5.31×103

Complex

GLf-DNA

1.710×105

30.70

187.62

190.24

GLm-DNA

1.572×104

24.62

198.80

218.00

GLf-DNA

4.54×103

21.45

172.36

178.90

7.4

4.7 GLm-DNA

39.10

09.34

190.40

192.29

Formation constants and free energy is a measure of stability of drug-DNA adducts. More stable adduct was formed in case of GLm. Formation constants ”Kf” calculated at pH 4.7 are 10 times smaller than those at pH 7.4 revealing that complex formed at blood pH has greater stability than that in acidic media. That is why their administration via blood veins is recommended rather than oral intake. The values of molar extinction coefficient calculated from UV-Vis data are listed in the Tables 2. The values of ε of adducts are greater than the free compounds due to greater size of the chromophore.

A direct correlation of the Kf with partition coefficient (log P) is indicative of the fact that drugs with a higher log P are expected to be form more stronger complexes with DNA. The present study showed a good proportionality of log P with the Kf of the selected anticancer compounds. Table 4. Data Set Of Selected Electronic Descriptors.

EHOMO

ELUMO

ETotal

(Kcal/mol)

(Kcal/mol)

(Kcal/mol)

GLf

-9.0

-1.48

-17.50

6.78×107

GLm

-8.97

-1.378

-16.76

5.31×103

Kf/M-1

Drug

3.2.3. Molecular Docking Studies Docking studies of GLf and GLm were also carried out for the evaluation of DNA binding parameters. Using MOE tool, binding free energy (∆G), total energy of the complex, (Etotal), electrostatic interactions (Eelec) and van der Waals energies (Evdw) between drugs and DNA were calculated on the basis of force field energy calculations. The formation constants obtained from MOE, CV and UVVis spectroscopic data have been listed in Table.3. The Kf values from the docking studies are of the same order of magnitude as those obtained from CV and UV-Vis spectroscopic investigations. A number of electric and molecular descriptors were calculated with a view of finding some possible correlations between the descriptors and observed binding strengths. The descriptors are tabulated in Table 4 and 5. It is generally true that an electron donor (D) increases EHOMO while an electron acceptor (A) decreases it. Therefore, drugs substituted with D have a higher EHOMO as shown in Table. 4. Since both EHOMO and ELUMO

Another important steric descriptor is molar refractivity (MR). A direct correlation of the binding strength with the MR was observed. An examination of Table 5 reveals that the values of Kf and MR have direct relationship. Table 5. Data Set of Selected Steric Descriptors

Hf / Drug

log P

MR

Vsurf

Kf /M-1

-1

(Kcal.mol )

GLf

2.51

13.19

749.87

-2.46

6.78×107

GLm

1.33

13.05

741.51

-2.46

5.31×103


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Figure 4 shows the presence of arene-arene interactions between the GLf and guanine base of DNA. These interactions also exist between GLm and adenine base of DNA. Guanine has an electron pair donor –NH2 group which increases electron density over the aromatic ring of GLf that may result in the effective overlap of electronic clouds of GLf and nitrogenous base pairs of DNA. GLm exhibits maximum exposure to the ligand atoms rather than base pairs with widest proximity contour. This could be the reason of higher formation constant of GLf than GLm, which is manifested from experiment and docking results.

shows interaction between the binding sites of DNA and the ligand. For docking purpose the ligand is replaced by potential drug of interest. The binding may involve hydrogen bonding, hydrophobic interactions and rarely covalent bonds between the base pairs of nucleic acid and drug moiety. The lactone antibiotics arguably interact with DNA by cross-linking of the strands.

a

Fig. 5. Docked complex of GLf (labeled white) with DNA

b

Fig. 6.

Fig. 4. Ligplot showing interactions between (a) GLf, base pairs and ligand atom and (b) GLm , base pairs and ligand atom

Most of the diseases are due to malfunctioning of the enzymes or ligands attached to DNA. The drugs used to cure such diseases interact with specific enzymes. Ligplot

Docked complex of GLm (labeled green) with DNA

Docking studies depicted in Fig. 5 and 6 predict that both GLf and GLm form an intrastrand cross link and develop arene-arene interactions between nitrogenous base pairs of DNA. Intrastrand cross-linking also results in the formation of replication nodes which limits the free proliferation of DNA. The docking data complement the experimental results.

4. Conclusion Two potential anticancer germa-γ-lactones were successfully characterized by cyclic voltammetry and


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UV-Vis spectroscopy. Both the compounds were found to reduce in two steps 1e-, 1H+ processes. GLm was found to reduce at lower potential. The reduction of germa-γlactones followed irreversible, diffusion controlled processes that occurred in pH dependent mechanism. Based upon the obtained results reduction mechanisms were proposed and the observed waves were attributed to the hydroxylation of the lactone moiety to lactol ring. The mechanisms suggested for the electro-reduction of biologically important compounds are expected to provide deep insights in to the understanding of unexplored pathways by which such compounds exert their biochemical action. The DNA binding parameters of germa-γ-lactones were also evaluated and the results obtained from methods were found in good agreement. Computational analysis supplemented the cyclic voltammetric and UV-Vis spectroscopic findings. The experimental results revealed that the selected drugs are more effective at blood pH.

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Acknowledgement Financial support from Quaid-i-Azam University and Higher Education Commission Islamabad, Pakistan, is gratefully acknowledged.

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