Betulinic acid binding to human serum albumin: A study of protein conformation and binding affinity

Journal of Photochemistry and Photobiology B: Biology 94 (2009) 8–12

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Betulinic acid binding to human serum albumin: A study of protein conformation and binding affinity Rajagopal Subramanyam a,*, Anilkishor Gollapudi a, Persis Bonigala a, Madhurarekha Chinnaboina a, Damu G. Amooru b a b

Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India Department of Chemistry, Yogi Vemana University, Kadapa, Andhrapradesh 516003, India

a r t i c l e

i n f o

Article history: Received 14 August 2008 Received in revised form 3 September 2008 Accepted 8 September 2008 Available online 16 September 2008 Keywords: Betulinic acid Binding constants Circular dichroism Human serum albumin Micro TOF-Q mass spectrometry Tephrosia calophylla

a b s t r a c t Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be KBA = 1.685 ± 0.01  106 M1, indicating a strong binding affinity. The secondary structure of the HSA–BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA + drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction Human serum albumin (HSA) is an important plasma protein responsible for the binding and transport of many endogenous and exogenous substances such as hormones and fatty acids, as well as foreign molecules such as drugs [1–10]. HSA is a widely studied protein because its primary structure is well known and its tertiary structure has been determined by X-ray crystallography [1]. HSA is synthesized in and secreted from liver cells, and is the most abundant protein among plasma, transport, and storage proteins. It is also important for maintaining normal osmolarity in plasma as well as in interstitial fluid. HSA is a 67 kDa single chain, non glycosylated polypeptide that folds into a heart-shaped structure containing approximately 67% a-helix [1,7]. The exceptional ability of HSA to interact with many organic and inorganic molecules and function as an important regulator of intercellular fluxes, as well as the pharmacokinetic behavior of many drugs [1–10], may be attributed to the presence of multiple binding sites on its surface. Albumin binding sites on HSA may be responsible for certain drug interactions observed in patients during therapy [11,12]. For example, patients with various liver and kidney diseases may experience altered albumin binding of drugs; * Corresponding author. Tel.: +91 40 23134572; fax: +91 40 23010120. E-mail addresses: [email protected], [email protected] (R. Subramanyam). 1011-1344/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2008.09.002

such that the distribution, metabolism, elimination, and pharmacological effects of the drugs are substantially changed [13,14]. Most drugs are reversibly bound to plasma proteins, but the extent and nature of the binding varies with the specific drug. Most acidic, neutral, and basic drugs bind to HSA, although a few basic drugs bind almost exclusively to a1-acid glycoprotein [7,15]. The degree of binding between a drug and plasma proteins can govern its distribution into tissues, affect its elimination from the body, and, consequently, affect its therapeutic or toxic effects. It is generally believed that only the unbound form of a drug interacts with its receptor to produce a pharmacological effect. Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid (Fig. 1) possessing anti-retroviral, anti-malarial, and antiinflammatory properties. It is noteworthy that, through its inhibition of topoisomerase, BA shows also potential as an anticancer agent [16–20]. Studies of BA utilizing various microorganisms have predicted potential mammalian metabolites [21–23]. In addition, molecular modeling experiments have predicted that BA may be a substrate for cytochrome P450 [24]. In other reports, BA derivatives acylated on the C-3-hydroxyl group inhibited HIV-1 replication by interfering with HIV-1 maturation [25,26]. Multiple studies on HSA structure and its interactions with different ligands exist in literature [8–11,27–30]. Ghuman and coworkers have shown that the distribution, free concentration, and the metabolism of various drugs can be significantly altered as a result of binding to HSA [7]. Thus, interaction with plasma

9

R. Subramanyam et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 8–12

1

H d

0.75

HO

H O

Fig. 1. Structure of betulinic acid. The molecular mass of this molecule is 456.7 and its molecular formula is C30H48O3.

proteins, especially HSA, is an important factor to consider in drug development [1–10]. However, to our knowledge, there are no existing reports on the BA binding affinity of HSA and its effect on protein conformation. Moreover, there have been no studies of HSA-ligand binding using quadrupole time-of-flight (micro TOF-Q) mass spectrometry. We report here, for the first time, the binding at nanomolar concentration (5 nM) of BA to HSA, as determined by sensitive mass spectrometry (micro TOF-Q). We also present the conformational changes, protein-drug complex and binding studies of HSA with BA.

Absorbance

OH H

a 0.5

0.25

0 250

270

290 Wavelength, nm

310

Fig. 2. UV-absorption spectra of free HSA (0.025 mM) and different concentrations of BA (0.01, 0.05 and 0.1 mM) with HSA in the region of 250–320 nm. Here, a to d are from free HSA to different concentrations of BA.

and CD spectroscopy. It was found that 30 min produces maximum binding. Thus, incubation time of BA with HSA was fixed at 30 min in all studies.

2. Materials and methods 2.3. UV/Visible and fluorescence spectroscopy 2.1. Isolation of betulinic acid and identification Betulinic acid was extracted into methanol from dried and powdered roots (6 kg) of Tephrosia calophylla. The resultant methanol extract was concentrated in vacuo to give a dark, syrupy residue (352 g), which was suspended in 2 L of water and then partitioned successively with CHCl3 and n-butanol. The CHCl3-soluble part was subjected to silica gel column chromatography, under conditions of gradient elution, using a mixture of ethyl acetate in n-hexane. Fractions were further purified by repeated silica gel column chromatography using a gradient elution with hexane/ethyl acetate followed by ODS column chromatography with 80% methanol to give 493 mg of betulinic acid. The purified compound was identified by direct comparison of spectral properties, MS, 1H-NMR and 13 C-NMR and 2D NMR with those of the authentic compounds in the literature [16–18]. The purity of the compound (BA) was checked by micro TOF-Q mass spectrometry and it showed as a single peak which attributes to BA molecular weight of 456.7 Da (data not shown). All reagents other than BA and HSA were from Sigma Aldrich.

The UV/Visible spectra were recorded on Shimadzu, UV-160 spectrophotometer. The fluorescence emission spectra were recorded on a Jobin–Yvon, FluoroMax-3, with excitation at 285 nm. BA was diluted to final concentration of 0.01, 0.025, 0.05, 0.075 and 0.1 mM with 0.1 M phosphate buffer pH 7.2 and the HSA concentration was fixed 0.025 mM. The binding constant was calculated using the fluorescence maximum at 362 nm and Eq. (1). Three independent experiments produced identical results. 2.4. CD spectroscopy Circular dichroism (CD) spectra of HSA and HSA–BA were recorded with a Jasco J-810 spectropolarimeter using a quartz cell with a path length of 0.02 cm. Three scans were accumulated at a scan speed of 50 nm min1, with data being collected every 1 nm from 190 to 300 nm. For CD studies, the final concentration of HSA was 0.025 mM and BA concentrations were 0.01, 0.05 and 0.1 mM. Secondary structure determination was done using CDNN 2.1, web based software.

2.2. Preparation of stock solutions

2.5. Electrospray ionization mass spectrometry (micro TOF-Q)

A pure sample of fat free human serum albumin (a kind gift from Virchow Biotech Pvt. Ltd., Hyderabad) was dissolved in 0.1 M phosphate buffer pH 7.2 at a final concentration of 1.5 mM protein. Non-fluorescent/absorption 2 mM BA was prepared in a 20:80% ethanol:water mixture. Twenty percent ethanol does not affect the absorption spectrum of HSA or its structure, which is similar to that shown for free HSA in Fig. 2. HSA was then dissolved in solutions with different pH (6–8) values to determine the optimum physiological pH as indicated by maximal absorbance. The maximum absorption was observed at pH 7.2. Thus, for characterization, 0.1 M phosphate buffer at a pH of 7.2 was used as a physiological buffer. The time required for BA to bind to HSA was examined using UV/Visible absorption, fluorescence emission,

Positive ion mode mass spectra were recorded on a micro TOFQ (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization source. For these measurements, the HSA concentration was reduced to 0.15 nM and the BA concentration to 5 nM. Free HSA and HSA–BA were prepared in 0.1% formic acid in water/ acetonitrile (1:1 (v/v)) and introduced into the mass spectrometer source with a syringe pump (KD Scientifics Inc, Hilliston, MA) at 3 lL/min. Electrospray was performed by setting the spray voltage at 4.5 kV. The Time-of-Flight (TOF) pressure was maintained at less than 3  107 Torr. Scanning was performed over an m/z range from 50 to 3000, with collision energy of 10 eV, and data were averaged for 2 min and then smoothed using the Gaussian algorithm in the Bruker Data Analysis 3.4 software program. The

R. Subramanyam et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 8–12

instrument was calibrated using ES Tuning Mix (Agilent Technologies, part No. G2421-60001) diluted 1:60 (v/v) times with 95% acetonitrile, injected by a divert valve just before sample application. The data were analyzed with the Data Analysis 3.4. 3. Results and discussion 3.1. UV/Visible spectroscopy studies UV-visible spectroscopy was used to study drug binding interactions. HSA has a UV absorption peak at 280 nm. It is known that binding to HSA can be observed easily through a change in absorbance at this wavelength [8,9]. Fig. 2 shows the UV spectra of free HSA and its complexes with various concentrations of BA (0.01– 0.1 mM), after incubation for 30 min. A slight increase in intensity was also seen, as a result of complexation with the BA. This increase in the intensity was insignificant with 0.01 mM BA, but easily seen with 0.05 and 0.1 mM. Similar results have been observed for the binding other drug compounds to HSA [27,28].

45

A f

Rel. Fluorescence Units

10

30

a

15

0 300

350

400 Wavelength, nm

500

0.75

B

2

3.2. Fluorescence studies

Here, DF = Fx-Fo and DFmax = F1-Fo where Fo, Fx and F1 are the fluorescence intensities of HSA in the absence of BA, at an intermediate concentration of BA, and at the saturation of interaction, respectively. K is the binding constant and [Q], the drug concentration. The linearity in a plot of 1/(FFo) against 1/[Q] confirms a one-toone interaction between the ligand and HSA (Fig. 3B). The binding constant of BA is 1.685 ± 0.01  106 M1, which indicates strong binding of BA to HSA. The binding constant calculated here shows a strong ligand protein interaction, comparable to other strong ligand-protein complexes such as monoclonal antibodies, which show binding constant values between 107 M1 and 1010 M1 [32]. As a reference, flavonoids display binding constants in the range 1–15  104 M1 [33]. A recent report binding with resveratrol hexadecanoic and resveratrol have binding constants of 6.70  106 M1 and 1.64  105 M1 respectively [34].

KBA=1.685 + 0.01 x106 M1-

0.5

0.25

0 0

25

75

100

Fig. 3. Fluorescence emission spectra of HSA–BA in 0.1 M phosphate buffer pH 7.2, kex = 285 nm, temperature = 25 ± 1 °C. (A) Free HSA (0.025 mM) and free HSA with different concentration of BA (0.01, 0.025, 0.05, 0.075 and 0.1 mM), here, a to f are from free HSA to different concentrations of BA. (B) Plot of 1/(FF0) against 1/[Q]. kex = 285 nm, kem = 362 nm. For further details see materials and methods.

45

25

5 195

220

-15 3.3. CD spectroscopy studies CD spectroscopy is a method commonly used to characterize conformational changes in proteins. Here, when BA was added to free HSA and incubated for 30 min, the negative band intensities at 208 and 218 nm decreased (Fig. 4). This clearly indicated changes in the protein secondary structure, with some apparent loss of helical stability. This may be the result of the formation of a complex between HSA and BA. Secondary structural elements were calculated by using the CDNN program. Acquired data suggest that the secondary structure of free HSA consists of 57.9% of a-helix, 25% of b-sheets and 17% of random coils, which is an agreement with a previous

50

1/[Q]

Mol. CD

ð1Þ

1/(F-Fo)

R = 0.9756

Fluorescence emission spectroscopy was used to identify the binding constant of BA to HSA. Our results show that, with increasing concentration of drug (0.01–0.1 mM) and a fixed concentration of HSA (0.025 mM), the intensity of HSA fluorescence emission maximum at 362 nm increased upon binding of BA (Fig. 3A). Similar fluorescence results were reported for the guaiacol-HSA interaction [28]. Thus, the increase in absorption and fluorescence indicates that the BA binding to the HSA causes microenvironment changes in HSA and the formation of HSA–BA complexes. The binding constant for the interaction between the BA ligand and HSA was determined from the fluorescence intensity using the following equation, as developed by Bhattacharya et al. [31].

1=DF ¼ 1=DF max þ ð1=K½QÞ=1=DF max :

450

c

245

d

a b

-35

Wavelength, nm

Fig. 4. Circular dichroism of the free HSA and its BA analogue complexes in aqueous solution with a protein concentration of 0.025 mM (a) and BA concentrations of 0.01 (b), 0.05 (c), and 0.1 mM (d).

report [10]. The secondary structure of HSA remained unchanged at low concentration, but addition of 0.1 mM of BA, produced a modifications in protein conformation. At this concentration, the a-helical content was decreased to 53%, while b-sheets and

R. Subramanyam et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 8–12 Table 1 Secondary structural analysis for the free HSA and its interaction with BA

a-Helix (%) Anti parallel (%) Parallel (%) b-turn (%) Random coil (%)

HSA

HSA + 0.01 mM BA

HSA + 0.05 mM BA

HSA + 0.1 mM BA

57.90 ± 2.5 6.00 ± 0.4

57.10 ± 2.5 6.1 ± 0.4

54.6 ± 2 6.5 ± 0.3

53.10 ± 2.6 6.8 ± 0.5

6.10 ± 0.4 13.30 ± 1 16.70 ± 1

6.5 ± 0.35 13.4 ± 0.75 16.9 ± 1

6.9 ± 0.4 13.2 ± 0.75 18.8 ± 1.4

7.2 ± 0.45 13.70 ± 1 19.20 ± 1.2

The experiment was carried out in 0.1 M phosphate buffer pH 7.2. Based on the Fig. 4, the data analyzed by web based software CDNN 2.1.

random coils were increased to 28% and 20%, respectively (Table 1). These results indicated that the secondary structure of HSA became partially disordered due to HSA–BA complex formation. Similar results were observed on binding of other ligands to HSA [8–10,27–30]. 3.4. Micro TOF-Q analysis Drug/ligand binding to HSA at nanomolar levels was demonstrated using micro TOF-Q mass spectrometry. Due to high sensi-

11

tivity of micro TOF-Q technology, nanomolar concentrations of free HSA as low as 0.15 nM were detectable. Low capillary temperatures and collision energy were critical for detection of the HSA– BA complex. Higher concentrations of HSA masked the HSA–BA complex, which was only detected after dilution by at least 100fold as compared to concentrations required for the other methods (CD, fluorescence, etc). Thus, it was deduced that BA could be identified at high concentrations (0.1 mM), while its complex with HSA could be detected only at nanomolar levels (BA concentration, 5 nM). Previous studies have shown that drug binding to HSA is detectable in the 101–105 mM concentration range [1], consistent with our results. Fig. 5A and B depict the mass spectra of free HSA and HSA complexes. The numbers on the dark vertical lines indicate the matched charge states of HSA or HSA plus BA complexes. Deconvolution of the multiply charged states resulted in the mass determinations of HSA and HSA–BA complexes. When BA bound to free HSA, the molecular mass increased from 65199 Da to 65643 Da, which suggested that BA was, indeed, binding to HSA. Since the molecular weight of BA is 456.7 Da, this is a clear indication that the additional mass on HSA originated from the BA ligand. The micro TOF-Q results supported the data acquired via absorption, fluorescence and CD experiments. Thus, micro TOF-Q mass spectrometry was deemed to be an accurate

Fig. 5. (A) Micro TOF-Q mass spectra of free HSA (B) HSA along with BA. The concentration of free HSA and BA were 0.15 nM, and 5 nM, respectively. m/z, m is mass and z is charge of the molecule. For further details see materials and methods.

12

R. Subramanyam et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 8–12

and sensitive method for detecting binding of the drug or ligand to HSA. 4. Conclusion We have determined that betulinic acid (BA) binds to human serum albumin (HSA) with an association constant of KBA = 1.685 ± 0.01  106 M1. At low BA concentrations, no major protein conformational changes occur, whereas with increasing BA concentration, a decrease in protein a-helical content and increased amounts of b-sheets and random coil structures can be observed. In addition, a low concentration of the drug (5 nM) can bind to the HSA, as we have proved here, using micro TOF-Q mass spectrometry. This indicates stable alteration of protein secondary structure at moderate concentrations of BA. BA is comparatively small, and is therefore able to penetrate to the HSA protein interior. It is likely that, once there, it binds to the polar side chains of HSA through hydrogen bonding or poly-functional interactions. A less likely scenario would involve BA binding to patches of nonpolar, solvent-exposed side chains, since the BA has a hydrophobic hydrocarbon skeleton (Fig. 1). Regardless of the mode of binding, it is likely that a part of the hydration shell of water is removed from the HSA molecule when binding occurs. This may also result in altered surface tension at the protein surface. Details of these changes must be verified by appropriate physical methods in future experiments. Acknowledgments We thank Dr. N. Sreepad, Virchow Biotech, Hyderabad, India for the kind gift of pure HSA samples and Prof. Abani Buyan, Department of Chemistry, University Hyderabad, India for invaluable discussion. The authors are grateful to Dr. Daniel C. Brune, Dr. DeRuyter Yana Bukhman Arizona State University, USA for critical reading of this manuscript and also thank David Joly, University of Quebec at Trois-rivieres, Canada for helping in calculating the binding constant. We greatly acknowledge the School of Life Sciences, UOH-CREBB for providing the facility of micro TOF-Q mass spectrometry at University of Hyderabad. References [1] H.M. He, D.C. Carter, Atomic structure and chemistry of human serum albumin, Nature 358 (1992) 209–215. [2] S. Curry, H. Mandelkow, P. Brick, N.P. Franks, Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites, Nature Struct. Biol. 5 (1998) 827–835. [3] S. Curry, P. Brick, N.P. Frank, Atomic structure and chemistry of human serum albumin, Biochim. Biophys. Acta 1441 (1999) 131–140. [4] S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 Å resolution, Protein Eng. 12 (1999) 439– 446. [5] A.A. Bhattacharya, T. GruÈne, S. Curry, Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin, J. Mol. Biol. 303 (2000) 721–732. [6] I. Petitpas, T. Grune, A.A. Battacharya, S. Curry, Crystal structure of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids, J. Mol. Biol. 314 (2001) 955–960. [7] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry, Structural basis of the drug-binding specificity of human serum albumin, J. Mol. Biol. 353 (2005) 38–52. [8] R. Beauchemin, C.N. N’soukpoe-Kossi, T.J. Thomas, T. Thomas, R. Carpentier, H.A. Tajmir-Riahi, Polyamine analogues bind human serum albumin, Biomacromolecules 8 (2007) 3177–3183. [9] C.D. Kanakis, P.A. Tarantilis, H.A. Tajmir-Riahi, M.G. Polissiou, Crocetin, dimethylcrocetin, and safranal bind human serum albumin: stability and antioxidative properties, J. Agric. Food Chem. 55 (2007) 970–977.

[10] C.N. N’soukpoe-Kossi, R. Sedaghat-Herati, C. Ragi, S. Hotchandani, H.A. TajmirRiahi, Retinol and retinoic acid bind human serum albumin: stability and structural features, Int. J. Biol. Macromol. 40 (2007) 484–490. [11] E.J. Segre, M. Chaplin, E. Forchielli, R. Runkel, H. Sevelius, Naproxen-aspirin interaction in man, Clin. Pharmacol. Ther. 15 (1974) 374–379. [12] J.K. Aronson, Clinical pharmacokinetics of digoxin, Clin. Pharmacokinet. 5 (1980) 137–149. [13] P.R. Jackson, G.T. Tucker, H.F. Woods, Altered plasma drug binding in cancer: role of alpha-i-acid glycoprotein and albumin, Chin. Pharmacol. Ther. 32 (1982) 295–302. [14] A.P. Van Peer, F.M. Belpaire, M.G. Bogaert, Binding of drugs in serum, blood cells and tissues of rabbits with experimental acute renal failure, Pharmacology 22 (1981) 146–152. [15] J.J. McKichan, Influence of protein binding and use of unbound (free) drug concentrations, in: W.E. Evans, J.J. Schentag, W.J. Jusko (Eds.), Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring (Applied Therapeutics), W.A.Vancouver, 1990, pp. 5–48. [16] E. Pisha, H. Chai, I.S. Lee, T.E. Chagwedera, N.R. Farnsworth, A.C. Cordell, Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis, Nat. Med. 1 (1995) 1046–1051. [17] R.H. Cichewicz, S.A. Kouzi, Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection, Med. Res. Rev. 24 (2004) 90–114. [18] A.R. Chowdhury, S. Mandal, N. Mittra, S. Sharma, S. Mukhopadhyay, H.K. Majumder, Betulinic acid, a potent inhibitor of eukaryotic topoisomerase I: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives, Med. Sci. Monitor 8 (2002) 254– 265. [19] A. Ganguly, B. Das, A. Roy, N. Sen, S.B. Dasgupta, S. Mukhopadhayay, H.K. Majumder, Betulinic acid, a catalytic inhibitor of topoisomerase I, inhibits reactive oxygen species-mediated apoptotic topoisomerase I-DNA cleavable complex formation in prostate cancer cells but does not affect the process of cell death, Cancer Res. 67 (2007) 11848–11858. [20] P. Rajendran, M. Jaggi, M.K. Singh, R. Mukherjee, A.C. Burman, Pharmacological evaluation of C-3 modified betulinic acid derivatives with potent anticancer activity, Invest New Drugs 26 (2008) 25–34. [21] P. Chatterjee, S.A. Kouzi, J.M. Pezzuto, M.T. Hamann, Biotransformation of the antimelanoma agent betulinic acid by bacillus megaterium ATCC 13368, Appl. Environ. Microbiol. 66 (2000) 3850–3855. [22] S.A. Kouzi, P. Chatterjee, J.M. Pezzuto, M.T. Hamann, Microbial transformations of the antimelanoma agent Betulinic acid, J. Nat. Prod. 63 (2000) 1653–1657. [23] T. Akihisa, Y. Takamine, K. Yoshizumi, H. Tokuda, Y. Kimura, M. Ukiya, T. Nakahara, T. Yokochi, E. Ichiishi, H. Nishino, Microbial transformations of two lupane-type triterpenes and antitumor-promoting effects of the transformation products, J. Nat. Prod. 65 (2002) 278–282. [24] D.F.V. Lewis, M. Dickins, R.J. Weaver, P.J. Eddershaw, P.S. Goldfarb, M.H. Tarbit, Molecular modeling of human CYP2C subfamily enzymes CYP2C9 and CYP2C19: rationalization of substrate specificity and sitedirected mutagenesis experiments in the CYP2C subfamily, Xenobiotica 28 (1998) 235–268. [25] F. Li, R. Goila-Gaur, K. Salzwedel, N.R. Kilgore, M. Reddick, C. Matallana, A. Castillo, D. Zoumplis, D.E. Martin, J.M. Orenstein, G.P. Allaway, E.O. Freed, C.T. Wild, PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing, Proc. Natl. Acad. Sci. USA 100 (2003) 13555–13560. [26] J. Zhou, Y. Xiong, D. Dismuke, B.M. Forshey, C. Lundquist, K.H. Lee, C. Aiken, C.H. Chen, Pharmacologic inhibition of HIV-1 replication by a novel mechanism: specific interference with the final step of virion maturation, J. Virol. 78 (2004) 922–929. [27] B. Ahmad, S. Parveen, R.H. Khan, Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site, Biomacromolecules 7 (2006) 1350–1356. [28] W. He, Y. Li, H. Si, Y. Dong, F. Sheng, X. Yao, Z. Hu, Molecular modeling and spectroscopic studies on the binding of guaiacol to human serum albumin, J. Photochem. Photobiol. A Chem. 182 (2006) 158–167. [29] P. Sen, B. Ahmad, R.H. Khan, Formation of a molten globule like state in bovine serum albumin at alkaline, Eur. J. Biophys. 37 (2008) 1303–1308. [30] S.S. Sinha, R. Mitra, S.K. Pal, Temperature-dependent simultaneous ligand binding in human serum albumin, J. Phys. Chem. B 112 (2008) 4884–4891. [31] J. Bhattacharya, M. Bhattacharya, A. Chakraborty, U. Chowdhury, R.K. Podder, Interaction of chlorpromazine with mioglobin and hemoglobin. A comparative study, Biochem. Pharmacol. 47 (1994) 2049–2052. [32] U. Kragh-Hansen, Structure and ligand binding properties of human serum albumin, Dan. Med. Bull. 37 (1990) 57–84. [33] C. Dufour, O. Dangles, Flavonoid-serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy, Biochim. Biophys. Acta 1721 (2005) 164–173. [34] Y.L. Jiang, Design, synthesis and spectroscopic studies of resveratrol aliphatic acid ligands of human serum albumin, Bioorg. Med. Chem. 16 (2008) 6406– 6414.