Inhibition of HIV-1 Reverse Transcriptase and HIV-1 Replication by Calophyllum Coumarins and Xanthones

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

As part of our continuing study on the Calophyllum species, a number of coumarins, xanthones and chromene acids from different Calophyllum species of Sri Lanka were tested for inhibitory activity against the HIV-1 and its virally-encoded reverse transcriptase (RT). These compounds were found to be inactive in both the HIV-1 RT and whole virus systems. In contrast, cordatolide A and B demonstrated IC₅₀ values of 19.3 and 11.7 μM, respectively, against HIV-1 replication in a novel green fluorescent protein (GFP)-based reporter cell assay (HOG.R5).

In our previous investigations, we have reported the isolation of a variety of natural products from the extracts of Calophyllum species of Sri Lanka [1]. The biological evaluation of these compounds as inhibitors of HIV-1 and the HIV-1 RT was undertaken as a part of our ongoing investigation. In addition, we recently reported on the HIV-1 RT inhibitory activity of cordatolide A (1) and B (2) [2], unique constituents of C. cordato-oblongum Thw. These compounds demonstrated HIV-1 RT inhibitory activity of 12.3 μM and 19.0 μM, respectively. Our studies also revealed that not only is the stereochemistry at positions 10, 11 and 12 of pyranocoumarins important for HIV-1 RT inhibitory activity, but the substituent located at position 4 was found to play a significant role as well. It was, therefore, of interest to ascertain if this enzyme inhibitory activity could translate into an overall effect on viral replication. To that end, the anti-HIV activity of cordatolide A (1) and B (2) were evaluated in the HOG.R5 reporter cell system characterized in our laboratory [3]. Calanolide A (3) was included as a positive control and it demonstrated an IC₅₀ value of 0.067 μM, in agreement with values reported in the literature [4]. Cordatolide A and B exhibited IC₅₀ values of 19.3 and 11.7 μM, respectively (Table [1]).[*]

Although cordatolide B (2), was previously shown to be active [2], cordatolide B-OMe (4) has exhibited neither HIV-1 RT nor HIV-1 inhibitory activity in the present study (Table [1]), probably due to the absence of a free hydroxy group at position 12 of the coumarin skeleton. Oblongulide (5), a pyranocoumarin biosynthetically related to the cordatolides, showed no HIV-1 RT inhibitory activity (Tab. [1]) when compared to cordatolide A (1) and B (2) [2]. The inactivity of 5 may be attributable to the disruption of the critical hetero ring with three chiral centers and the hydroxy group at position 12 that are characteristic of the cordatolides and other HIV-1 RT inhibitory pyranocoumarins [5]. This compound also lacked any significant effects on viral infectivity in HOG.R5 cells at nontoxic concentrations.

All the anti-HIV-1 RT Calophyllum coumarins were observed to possess the 2,2-dimethylpyran ring system [5], as do the pyranoxanthones, as exemplified by thwaitesixanthone (6), calothwaitesixanthone (7) and calozeyloxanthone (8). However, the latter three pyranoxanthones were observed to be inactive as HIV-1 RT inhibitors (Tab. [1]), suggesting that the 2,2-dimethylpyran ring functionality by itself is biologically insignificant in conferring HIV-1 RT inhibitory activity. Furthermore, while thwaitesixanthone (6) was inactive in the HOG.R5 reporter cell assay, the cytotoxicity of calothwaitesixanthone (7) (CC₅₀= 6 μg/ml) and calozeyloxanthone (8) (CC₅₀ = 10 μg/ml) precluded their evaluation in this cell-based system.

Cordato-oblongic acid (9), cordato-oblongic acid methyl ester (10), isocordato-oblongic acid methylester (11) and inophyllum A (12) were all shown to be inactive against HIV-1 RT (Table [1]). These compounds, and others reported inactive against the viral polymerase in the present study were all confirmed to be inactive against the whole virus. In addition, based on the diminished basal fluorescence output of GFP signals in uninfected HOG.R5 cells [3] exposed to the test compounds, the toxicities of inophyllum A (12) (CC₅₀=20 μg/ml), calothwaitesixanthone (7) (CC₅₀= 6 μg/ml), calozeyloxanthone (7) (CC₅₀=10 μg/ml) and oblongulide (5) (CC₅₀=6 μg/ml) to the parental human osteosarcoma (HOS) cells became evident during the course of antiviral evaluation (Table [1]).

Materials and Methods

Calophyllum species (Guttiferae) were identified and collected from the Kanneliya forest in the Southern Province of Sri Lanka by Mr. Shantha Ekanayake (Institute of Fundamental Studies) and the plant specimens were compared with the herbarium specimens at the Royal Botanic Gardens, Peradeniya, Sri Lanka (C. cordato-oblongum, 24 771: C. moonii, 24 994). The methodology for the HIV-1 RT assay was as previously describe [6].

Assay for the inhibition of HIV infectivity in HOG.R5 cells: Briefly, a reporter cell line for quantitating HIV-1 replication was developed using HOS (human osteosarcoma) cells rendered susceptible to HIV-1 infection by the transfection of genes for CD4 and CCR5, the coreceptor utilized by macrophage-tropic (R5) HIV-1 isolates. This microtiter assay is based on the transactivation of a stably-integrated HIV-1 LTR-green fluorescent protein (GFP) transcription unit by the viral Tat protein.

HOG.R5 cells are plated in 96-well microtiter plates at a density of 4,000 per well in DMEM (100 ml) containing 10 % FBS, 4 μM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin, and incubated at 37 °C for 24 h prior to each assay. The anti-HIV activity of pure compounds is assessed by the addition of test agents to cells in triplicate just before the addition of virus (HIV-1_IIIB, 5 ng p24/ml final inoculum concentration). Relevant controls consisting of infected cells which have not been treated with the test material are included. Infected cultures are incubated for 4 days. At the end of the assay, the media is thoroughly removed and 200 μl of 0.5 % (v/v) Nonidet P-40 in phosphate buffered saline (PBS) are added to each well. The contents are mixed by repeated pipeting, and GFP fluorescence signal is quantitated as Relative Fluorescence Units (RFUs) at excitation and emission wavelengths of 485 nm and 535 nm, respectively.

Acknowledgements

Technical assistance from Ms. Himashi Amarapala is acknowledged.

References

• 1 Dharmaratne H RW, Perera D SC, Jamie J, Marasinghe G PK. A chromene acid from Calophyllum cordato-oblongum .  Phytochemistry. 1999;  51 111-3
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• 2 Dharmaratne H RW, Wanigasekera W MAP, Mata-Greenwood E, Pezzuto J M. Inhibition of human immuno-deficiency virus type 1 reverse transcriptase activity by cordatolides isolated from Calophyllum cordato-oblongum .  Planta Medica. 1998;  64 460-1
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• 4 Kashman Y, Gustafson K R, Fuller R W, Cardellina II J H, McMahon J B, Currens M J, Buckheit Jr R W, Hughes S H, Cragg G M, Boyd M R. Journal of Medicinal Chemistry 1992 35: 2735-43
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• 5 McKee T C, Covington C D, Fuller R W, Bokesch H R, Young S, Cardellina II J H, Kadushin M R, Soejarto D D, Stevens P F, Cragg G M, Boyd M R. Pyranocoumarins from tropical species of the genus Calophyllum: a chemotaxonomic study of extracts in the National Cancer Institute collection.  Journal of Natural Products. 1998;  61 1252-6
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  CrossrefPubMedSearch in Google Scholar

Dr. H. R. W. Dharmaratne

Natural Products Propgramme

Institute of Fundamental Studies

Kandy

Sri Lanka

Email: hrwd13@hotmail.com

Fax: 94 - 8 232131

References

• 1 Dharmaratne H RW, Perera D SC, Jamie J, Marasinghe G PK. A chromene acid from Calophyllum cordato-oblongum .  Phytochemistry. 1999;  51 111-3
  CrossrefPubMedSearch in Google Scholar
• 2 Dharmaratne H RW, Wanigasekera W MAP, Mata-Greenwood E, Pezzuto J M. Inhibition of human immuno-deficiency virus type 1 reverse transcriptase activity by cordatolides isolated from Calophyllum cordato-oblongum .  Planta Medica. 1998;  64 460-1
  PubMedSearch in Google Scholar
• 3 Tan G T, Honnen W J, Kayman S C, Pinter A. A sensitive microtiter infectivity assay for macrophage-tropic and primary isolates of HIV-1 based on the transactivation of a stably integrated gene for the green fluorescent protein. The 9th National Cooperative Vaccine Development Group (NCVDG) Meeting: Advances in AIDS Pathogenesis and Preclinical Vaccine Development NIH, Bethesda, Maryland; May 4 - 7, 1997
  Search in Google Scholar
• 4 Kashman Y, Gustafson K R, Fuller R W, Cardellina II J H, McMahon J B, Currens M J, Buckheit Jr R W, Hughes S H, Cragg G M, Boyd M R. Journal of Medicinal Chemistry 1992 35: 2735-43
  Search in Google Scholar
• 5 McKee T C, Covington C D, Fuller R W, Bokesch H R, Young S, Cardellina II J H, Kadushin M R, Soejarto D D, Stevens P F, Cragg G M, Boyd M R. Pyranocoumarins from tropical species of the genus Calophyllum: a chemotaxonomic study of extracts in the National Cancer Institute collection.  Journal of Natural Products. 1998;  61 1252-6
  CrossrefPubMedSearch in Google Scholar
• 6 Tan G T, Pezzuto J M, Kinghorn A D, Hughes S H. Evaluation of natural products as inhibitors of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase.  Journal of Natural Products. 1991;  54 143-54
  CrossrefPubMedSearch in Google Scholar

Dr. H. R. W. Dharmaratne

Natural Products Propgramme

Institute of Fundamental Studies

Kandy

Sri Lanka

Email: hrwd13@hotmail.com

Fax: 94 - 8 232131