In vitro anti-HIV-1 virucidal activity of tyrosine-conjugated tri- and dihydroxy bile salt derivatives

A. A. Al-Jabria,*, M. D. Wiggb, E. Eliasc, R. Lambkina, C. O. Millsc and J. S. Oxforda

a Department of Medical Microbiology and Retroscreen Virology, St Bartholomew's and The Royal London School of Medicine and Dentistry, 64 Turner Street, London, UK; b Departmento de Virologia, Universidade Federal do Rio de Janeiro, 21 941-590 Rio de Janeiro, Brasil; c The Queen Elizabeth Hospital, Department of Medicine, Liver Research Laboratories, Edgbaston, Birmingham, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cellular toxicity and anti-human immunodeficiency virus type 1 (HIV-1) virucidal activity of four synthesized tyrosine-conjugated bile salt derivatives with high surfactant activities, namely di-iodo-deoxycholyltyrosine (DIDCT), di-iodo-chenodeoxycholyltyrosine (DICDCT), di-iodo-cholylglycyltyrosine (DICGT) and deoxycholyltyrosine (DCT), were evaluated and compared with either sodium deoxycholate or nonoxynol-9. DIDCT, DICDCT and DCT but not DICGT showed virucidal activity against three different laboratory-adapted strains of HIV-1 (RF, IIIB and MN). All the bile salt derivatives tested excluding DICGT were virucidal at a concentration as low as 10 ng/mL. DCT had the highest anti-HIV-1 virucidal potency, suggesting that monopeptide 7{alpha},12{alpha} dihydroxy bile salt derivatives have the most potent antiviral activity. Complexing of iodine to the bile salt derivative (as in DICGT) decreases virucidal potency.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Virucides destroy virus particles on contact. They differ from virustatic drugs in that they act directly and rapidly by lysing viral membranes on contact1,2 or by binding to virus coat proteins. Attention has been directed towards the possible use of these agents for preventing the sexual transmission of human immunodeficiency virus (HIV) infection.37 The spermicide nonoxynol-9, a non-ionic surfactant, also possesses anti-HIV-1 activity3,89 and has been used experimentally to reduce the risk of viral transmission.10 However, it damages the epithelial lining of the female genital tract, and may even enhance susceptibility to, rather than prevent, HIV infection.11 Therefore, new agents that are virucidal without being cytotoxic to human cells are required.

Certain bile salt derivatives have surfactant activity with low cellular toxicity. Within the human intestinal lumen, bile salts can reach a concentration of 350 mg/L without any harmful effect on the individual12 and, in patients suffering from biliary obstruction, blood bile salt concentration can increase 100-fold without serious toxic effects.13 Furthermore, the steroidal ring of bile salts can be modified chemically to yield derivatives with low, moderate or high surfactant activity.

In the present study, four bile salt derivatives with high surfactant activities were examined for their cellular toxicity and then compared with sodium deoxycholate and nonoxynol-9 for anti-HIV-1 virucidal potency.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Synthesis of compounds

Materials.
L-Tyrosine methyl ester hydrochloride (crystalline), chenodeoxycholic acid (99% pure as judged by TLC and GLC), cholylglycine (99% pure as judged by TLC), N,N-dicyclohexylcarbodiimide, N,N' dimethylformamide, triethylamine, 1-hydroxybenzotriazole and Lipidex 5000 were purchased from Sigma Chemical Co., Poole, UK. Chloramine-T was supplied by Hopkin and Williams, Chadwell Heath, UK. Amberlite XAD-2, silica gel 60 F254 TLC plates (‘Merck’ 5554) 20 x 20 cm, ethyl acetate (Analar), chloroform (Analar), glacial acetic acid (99% pure) and potassium iodide were obtained from BDH Chemicals Ltd, Poole, UK. Methanol (99% pure) and ethanol (99% pure) were purchased from Fisons Scientific Apparatus, Loughborough, UK. Sep-Pak C18 cartridges were purchased from Waters Associates Ltd, Hertford, UK.

Synthetic methods.
Deoxycholyltyrosine (DCT), chenodeoxycholyltyrosine and cholylglycyltyrosine were synthesized following procedures of Spenney et al.14 (with a slight modification), Tserng et al.15 and Mills et al.16 The di-iodinated derivatives of these compounds were then synthesized. Tyrosine-conjugated bile salt (0.5 mmol) in 5 mL phosphate buffer was added to 1.0 mmol potassium iodide followed by the addition of 0.5 mL of chloramine-T solution (10 mg/mL in 10 mM phosphate buffer pH 7.4) with incubation for 30 min. The rest of the non-radioactive iodination procedure including Amberlite XAD-2 purification of the di-iodinated compound was as described elsewhere.17 Further purification was by silica gel PLC employing ethyl acetate/methanol/acetic acid (70:20:10 by volume). The di-iodo derivatives of deoxycholyltyrosine, chenodeoxycholyltyrosine or cholylglycyltyrosine were isolated from the PLC plates by extraction with methanol and then dried by evaporation in a rotary evaporator at 40°C.

The synthesized compounds, di-iodo-deoxycholyltyrosine (DIDCT), di-iodo-chenodeoxycholyltyrosine (DICDCT), di-iodo-cholylglycyltyrosine (DICGT) and DCT (Figure 1Go), and sodium deoxycholate as a positive control, were dissolved separately in RPMI 1640 (Sigma), filter sterilized using Millipore 0.22 µm filters, divided into aliquots and stored at –20°C. Nonoxynol-9 was diluted to 1% (v/v) in RPMI 1640 and filter sterilized using a 0.22 µm filter.



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Figure 1. Chemical structure of tyrosine-conjugated bile acids. Tyrosine (1.4 equiv.) and either free bile acid or glycine-conjugated bile acid (1 equiv.) were reacted via dicylclohexylcarbodiimide (1.4 equiv.) to give tyrosine-conjugated bile acid analogues. Iodination using KI with chloramine-T oxidation yielded the di-iodo derivative of tyrosine-conjugated bile acid analogues. Elemental analysis determined the chemical formulae of the tyrosine-conjugated bile acids. These bile acids were judged to be pure by TLC. (a) Deoxycholyltyrosine (DCT): R = R2 = {alpha}-OH; R1 = R3 = R4 = H; di-iodo-deoxycholyltyrosine (DIDCT): R = R2 = {alpha}-OH; R1=H; R3 = R4 = I; di-iodochenodeoxycholyltyrosine (DICDCT): R = R1= {alpha}-OH; R2 = H, R3 = R4= I. (b) Di-iodocholylglycyltyrosine (DICGT): R= {alpha}-OH; R1= {alpha}-OH; R2= {alpha}-OH, R3 = R4 = I.

 
Cell lines and viral strains

H9 and C8166 cell lines were kindly provided by Dr Harvey Holmes through the Medical Research Council's AIDS Directed Programme, UK. Three different laboratory-adapted strains of HIV-1 were used to evaluate the virucidal activity of the compounds: RF, with an infectivity titre of 106 TCID50/mL for H9 cells; IIIB, with an infectivity titre of 105.4 TCID50/mL for H9 cells; and MN, with an infectivity titre of 107 TCID50/mL for C8166 cells.

Evaluation of cellular toxicity

H9 and C8166 cells were prepared at a density of 2 x 105/mL in growth medium [RPMI 1640 medium with 2 mM l-glutamine, 50 IU/mL penicillin, 50 mg/L streptomycin, 25 mM HEPES buffer and 10% fetal calf serum (Sigma, Poole, UK)]. A volume of 180 µL of cell suspension was dispensed into wells of a flat-bottomed 96-well plate. Two-fold dilutions of each virucidal compound were made in growth medium and then 20 µL of each dilution was added to the first six wells and ten-fold dilutions were made along the plate. The last row of the plate was left as a drug-free control for cell viability.18

Plates were incubated at 37°C in 5% CO2 and cell viability was checked using the Trypan Blue exclusion method after 24 h and 3, 5 and 7 days (using two columns of the plate at each time point). The percentage of viable cells in each well was calculated and the mean for each concentration at each time point was calculated and compared with cell-free drug controls.

Virucidal test

‘Virus/virucidal mix’ (200 µL of high-titre virus and 200 µL of each test compound) and ‘virus/growth medium mix’ (200 µL of high-titre virus and 200 µL of growth medium) were prepared and incubated for 15 min at 37°C in 5% CO2. For 96-well tissue culture plates, 180 µL of H9 (when using RF and IIIB strains) or C8166 (when using the MN strain) cells at a density of 2 x 105 cells/mL were added to each well. Twenty microlitres of ‘virus/virucidal mix’ was added to the first six wells of the first row and ‘virus/growth medium mix’ (used as positive control for virus-induced syncytium formation) added to the next six wells of the first row. Ten-fold dilutions were made starting from the first row downwards and keeping the last row as cell control. On days 3–7 after infection, all wells were examined under the microscope and scored for the presence of virus-induced syncytia18 according to the following scoring system: 3, >60 syncytia; 2, between ten and 60 syncytia; 1, between two and ten syncytia, 0.5, one or two syncytia; 0, no syncytia observed in the entire field. The wells containing virus and drug were scored and compared with the wells containing virus but no drug.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Relatively low cellular toxicity was observed for the four bile salts tested (Figure 2Go). Three of them showed minimal cellular toxicity (>80% cell viability) at concentrations up to 500 mg/L when assayed using the cell line H9, while with DCT at 500 mg/L there was >50% cell viability. Similar results were obtained with C8166 cells (data not shown), which are more sensitive indicators of HIV replication. Sodium deoxycholate, used as a positive virucidal control, showed detectable cellular toxicity at a concentration of 500 mg/L (Figure 2Go) and nonoxynol-9 was toxic to cells at a concentration of 0.1% (v/v) with H9 cells (data not presented).



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Figure 2. Evaluation of the cellular toxicity of the bile salt derivatives. {diamondsuit}, DIDCT; {blacksquare}, DICDCT; {blacktriangleup}, DICGT; {circ}, DCT; *, sodium deoxycholate.

 
Nonoxynol-9 was virucidal for three different isolates of HIV-1 (RF, IIIB and MN) at a concentration of >=0.1% (v/v) with the cell lines H9 and C8166, in agreement with data reported by others.3,8,9 Sodium deoxycholate was virucidal at a concentration of 10 mg/L (Figure 3Go), confirming the results of Oxford et al.2



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Figure 3. Virucidal effects of the bile salts DIDCT, DICDCT, DICGT and DCT and of sodium deoxycholate (SDC) against HIV-1 isolate IIIB, with a high titre (approximately 105.4 TCID50/mL) and cell line H9, presented as the syncytia score during the culture period. A DICGT concentration of <=100 mg/L failed to inhibit formation of syncytia during the culture period. Syncytia were scored as indicated in Materials and methods.

 
Three of the bile salt compounds, DIDCT, DICDCT and DCT, were virucidal at concentrations of 0.1 mg/L, producing a >4 log10 reduction in HIV-1. In contrast, DICGT, a dipeptide bile salt derivative, was not virucidal at concentrations up to 100 mg/L (Figure 3Go). DIDCT and DICDCT had virucidal effects at concentrations as low as 0.01 mg/L with the HIV-1 IIIB isolate and the H9 cell line (Figure 3Go). However, with the C8166 cell line and the MN strain, DICDCT inhibited the formation of syncytia only at a concentration of 0.1 mg/L (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is a need for effective, safe, anti-HIV virucidal agents that can be used as topical applications before sexual contact. Such agents would need to have low toxicity to human cells.7,19 Bile salt derivatives with high surfactant activities have low cellular toxicity to human tissues12,13 and we show in the present study that certain bile salt derivatives possess anti-HIV-1 virucidal activity at concentrations that have no detectable destructive effects on H9 and C8166 cells. However, we have not tested the drugs for potential toxicity on vaginal cells or peripheral blood mononuclear cells.

In contrast to the bile salt derivatives, nonoxynol-9 did damage cells, and so it might increase the risk of HIV infection rather than preventing it. Its use against the sexual transmission of HIV is thus unacceptable.

Bile salts in the unconjugated form are relatively potent physiological detergents.20 Side-chain conjugation with amino acid residues, which increases the hydrophilicity of a bile acid molecule, is associated with a predictable decrease in detergent potency.21 Iodine, which tends to increase the electron density of the side chain, also decreases detergent properties. In the present study we found that, of the tyrosine-conjugated bile salt analogues, DCT, which has the lowest critical micellar concentration,22 was the most effective compound against HIV-1, suggesting that it was the most surface active of the tyrosine bile acid conjugates tested (Figure 1Go). This anti-HIV-1 effect of DCT could be explained, in part, by a preferential uptake of DCT by surface receptors of the HIV-1 envelope but not by viable cells. This selective uptake would then concentrate DCT (a 7{alpha},12{alpha}-dihydroxy bile salt analogue) in the phospholipid-enriched bilayer of HIV lipid membranes. The reasoning is consistent with the relatively low hydrophilicity of DCT in comparison with the 3{alpha},7{alpha},12{alpha} trihydroxy or the 3{alpha},7{alpha}-dihdroxy tyrosine bile acid analogues (unpublished data).

During viral budding, HIV incorporates host-derived cellular components such as the HLA class I and II molecules that are important for in vitro HIV infectivity.23,24 The difference between the effect of bile salt derivatives on the viral envelope and on the cellular plasma membrane might result from the different proportions of such host-derived molecules in the viral envelope and the cellular plasma membrane. This needs to be investigated further.

Bile salts with high surfactant activity may be useful clinically as virucides against HIV-1. Their ability to inactivate relatively high titres of HIV-1 might enable them to be used as biological disinfectants as well as virucidal compounds.


    Acknowledgments
 
This work was supported by a grant from the British Society for Antimicrobial Chemotherapy and Retroscreen Ltd, St Bartholomew's and The Royal London School of Medicine and Dentistry. M. Wigg was a post-doctoral scholar supported by Conselho Nacional de Pesquisas (Proc. 20 1537/92–3).


    Notes
 
* Correspondence address. Department of Microbiology and Immunology, College of Medicine, SQU, Muscat, Sultanate of Oman. Tel: +968-515186; Fax: +968-513419; E-mail: aaljabri{at}squ.edu.om Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Oxford, J. S., Potter, L. W., McLaren, C. & Hardy, W. (1971). Inactivation of influenza and other viruses by a mixture of virucidal compounds. Applied Microbiology 21, 606–10.[ISI][Medline]

2 . Oxford, J. S., Zuckerman, M. A., Race, E., Dourmashkin, R., Broadhurst, K. & Sutton, P. M. (1994). Sodium deoxycholate exerts a direct destructive effect on HIV and influenza viruses in vitro and inhibits retrovirus-induced pathology in an animal model. Antiviral Chemistry and Chemotherapy 5, 176–81.[ISI]

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9 . Polsky, B., Baron, P. A., Gold, J. W. M., Smith, J. L., Jensen, R. H. & Armstrong, D. (1988). In vitro inactivation of HIV-1 by contraceptive sponge containing nonoxynol-9. Lancet i1456.

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12 . Horrobin, D. F. (1968). Medical Physiology and Biochemistry. Edward Arnold, London.

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18 . Al-Jabri, A. A., Wigg, M. D. & Oxford, J. S. (1996). Initial in vitro screening of drug candidates for their potential antiviral activities. In Virology Methods Manual (Kangro, H. & Mahy B., Eds), pp. 293–308. Academic Press. London.

19 . O'Connor, T. J. A., Kinchington, D., Kangro, H. O. & Jeffries, D. J. (1995). The activity of candidate virucidal agents, low pH and genital secretions against HIV-1 in vitro. International Journal of STD and AIDS 6, 267–72.[Medline]

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21 . Roda, A., Minutello, A., Angellotti, M. A. & Fini, A. (1990). Bile acid structure–activity relationship: evaluation of bile acid lipophilicity using 1-octanol/water partition coefficient and reverse phase HPLC. Journal of Lipid Research 31, 1433–43.[Abstract]

22 . Mills, C. O. & Elias, E. (1992). Biliary excretion of chenodeoxycholyl lysyl-rhodamine in Wistar rats: a possible role of a bile acid as a carrier for drugs. Biochimica et Biophysica Acta 1126, 35–40.[ISI][Medline]

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Received 20 July 1999; returned 13 October 1999; revised 25 November 1999; accepted 22 December 1999





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