Prevention of Human Immunodeficiency Virus Type 1 Trans- mission by Pharmaceuticals Targeted to Host Proteins Re- quired for Virus Infection? Consideration of Farnesyl Thiosalicylic Acid, a Ras Inhibitor

Recent success in defining the human immunodeficiency virus type 1 (HIV-1) – host cell protein interaction network has provided an opportunity for development of novel antiviral therapeutics targeted to host proteins required for virus infection. This expanded earlier successful development of antagonists for the cellular receptors (CD4) and co-receptors (CCR5 or CXCR4) involved in virus attachment. Induction of the G-alpha q signaling cascade by the HIV-1 envelope is required for virus entry, and it’s blocking prevented HIV-1-mediated membrane fusion and initiation of infection. One of the blockers, the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS), was reported to interfere with HIV-1 infection. Since FTS appears to have an established safety record and is being evaluated (as Salirasib, oral) in phase II human clinical trials for treatment of lung cancer, it was of interest to evaluate the potential of FTS as a topical microbicide for prevention of sexual transmission of HIV-1. Data shown here indicated that this compound did not meet the criteria of an established screening algorithm for evaluation of topical microbicides. Nevertheless, the possibility remains to be explored that FTS (especially when used in combination with other anti-HIV drugs) might be useful in sustained pre-exposure prophylaxis to prevent HIV-1 transmission. Prevention of Human Immunodeficiency Virus Type 1 Transmission by Pharmaceuticals Targeted to Host Proteins Required for Virus Infection? Consideration of Farnesyl Thiosalicylic Acid, a Ras Inhibitor A Robert Neurath*, Carol Lackman-Smith Virotech, 1496 Hemlock Farms, Hawley, PA 18428, USA Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701, USA *Corresponding author: A Robert Neurath, Virotech, 1496 Hemlock Farms, Hawley, PA 18428, USA, Tel: (570) 775 7877 or (212) 529 4584; E-mail: arneurath@att.net Received November 25, 2009; Accepted December 26, 2009; Published December 26, 2009 Citation: Neurath AR, Lackman-Smith C (2009) Prevention of Human Immunodeficiency Virus Type 1 Transmission by Pharmaceuticals Targeted to Host Proteins Required for Virus Infection? Consideration of Farnesyl Thiosalicylic Acid, a Ras Inhibitor. J Antivir Antiretrovir 1: 072075. doi:10.4172/jaa.1000010 Copyright: © 2009 Neurath AR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
The global AIDS epidemic has proceeded without abatement for about 30 years with about 36 million people having chronic HIV-1 infections and about 36 million who already succumbed to AIDS. Most new infections have been acquired by the mucosal route, heterosexual transmission playing the major (~80%) role. Anti-HIV-1 vaccines applicable to global immunization programs are not expected to become available for many years. Therefore, other prevention strategies are needed, i.e. mechanical or chemical barrier methods. The latter correspond to microbicides, topical formulations expected to block HIV-1 infection (and possibly also transmission of other sexually trans-mitted pathogens) when applied vaginally or rectally before intercourse. Several large-scale phase III efficacy trials of candidate microbicide formulations failed to demonstrate efficacy. Therefore, additional research and development in the microbicide field is needed (Balzarini and Van Damme, 2007;Cutler and Justman, 2008;Hendrix et al., 2009). Formulations of several anti-retroviral drugs targeted to HIV-1 proteins or to cell receptors (CD4) or co-receptors (CCR5) for the virus have been considered for this purpose (Schols, 2004;Moore et al., 2004;Vermeire et al., 2006). Since HIV-1 with tropism for CCR5 (= R5 viruses) are preferentially transmitted by the mucosal route, CCR5 antagonists have been primarily considered for development of microbicides.
Application of anti-HIV-1 pharmaceuticals targeted to HIV-1 proteins may lead to the emergence of drug-resistant virus variants. Recent success in defining the HIV-1-host cell interaction network has provided an opportunity for development of novel antiviral therapeutics targeted to host proteins (in addition to CD4, CCR5 and CXCR4) required for virus infection (Kellam, 2006 The promise of this new approach has been supported by the finding that inhibitors of the G-alpha q signaling cascade, required for HIV-1 entry into cells, interfere with virus-mediated membrane fusion and infection (Harmon and Ratner, 2008). Among the thirteen identified active compounds, targeted to distinct host

Cells and virus
MAGI-CCR5 cells, expressing the HIV-1 receptor CD4 and co-receptor CCR5 and beta-galactosidase under the control of the HIV-1 long terminal repeat (LTR), and HIV-1 BaL were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Program (Germantown, MD 20874).

Pharmaceuticals
The CXCR4 and CCR5 antagonists AMD3100 and TAK -779, respectively, were obtained from the NIH AIDS Research and Reference Program, and handled as described (Lackman-Smith et al., 2008). S-trans, trans-Farnesyl-Thiosalicylic Acid was obtained from Cayman Chemical Company, 1180 E. Ellsworth Road, Ann Arbor, MI 48108.

Data analysis and quality control
For each assay, IC 50 , IC 90 and TC 50 values were calculated using regression analysis. The Therapeutic Index (TI) was calculated as TC 50 /IC 50 . All assays passed internal quality standards established at the Sothern Research Institute, Frederick, MD 21701. This included quality of replicates, endpoint signal and performance of assay controls.
Solutions of the latter compound were prepared as follows:

Assays for inhibition of HIV-1 infection
The assays were performed as descried earlier (Lackman-Smith et al., 2008). Briefly, 24 hours prior to initiation of the assay, the cells were trypsinized, counted, and plated in 96-well flat bottom wells at 1 x 10 4 cells per well. Medium was removed and diluted test article or diluent controls in medium placed on the cells, and incubated for 15 min at 37ºC. Ten (10) TCID 50 of the BaL strain of HIV-1 was then added to the wells and the plates were incubated for 40 to 48 h at 37 °C. At termination of the assay, media was removed and β-galactosidase enzyme expression determined by chemiluminescence per the manufacturer's instructions (Tropix Gal-screen β, Applied Biosystems, Bedford, MA 01730). TAK-779 and AMD3100 were the positive and negative control compounds respectively for the assay. Toxicity using CellTiter96® Reagent (Promega, Fitchburg, WI 53711) was tested in parallel with tests for inhibition of HIV-1 infection. All assays were performed in triplicate and mean values were calculated.

Results
To confirm the proper performance of infectivity assays, the inhibitory effect of the CCR5 antagonist TAK 779 on infection , and TAK 779 is a specific CCR5 antagonist while AMD 3100, used as a "negative" control, is a CXCR4 antagonist. Virus infection was monitored by chemiluminometric quantitation of beta-galactosidase (relative quantity corresponding to infection in the absence of any compounds listed above = 100%). Cytotoxicity was determined using the CellTiter Reagent (Promega). Absorbance measured by the assay is directly proportional to the number of living cells in culture (=100%).  Figure 2 (bottom panel). The IC 50 and IC 90 values, calculated using regression analysis, were 2 and 20 nM, respectively. Cytotoxicity was minimal, and the calculated therapeutic index (TI) was >5,000. This conforms to already established results (Lackman-Smith et al., 2008).
FTS was even less inhibitory than AMD 3100 (Figure 2, top panel; IC 50 = >100 µM). These results were obtained using a stock solution of FTS prepared in DMSO. The medium containing DMSO only had no detectable inhibitory effects. Similar results were obtained with an FTS stock solution prepared in an ethanol-PBS mixture (data not shown).
In conclusion, the inhibitory activity of FTS on HIV-1 BaL infection was too low for further consideration of this compound for inclusion into topical microbicide formulations.

Discussion
The inhibition of HIV-1 BaL infection of the MAGI-CCR5 target cells by FTS was unimpressive (~40% inhibition at a 100 µM concentration) but significant in comparison with that attributable only to the diluent containing DMSO (Figure 2, top panel). The observed inhibition could not have been caused by cytotoxic effects of the compound. Nevertheless, these data suggest that FTS does not meet criteria for further development as a topical microbicide. These results merit further discussion, and should not discourage from additional research regarding the potential application of FTS for pre-exposure prophylaxis.
Results of earlier experiments (Harmon and Ratner, 2008) showed that FTS at a 50 µM concentration caused a 99.7 +/-2 % inhibition of infection. The R5 virus HIV YU2 and TZM-BL cells were used in these experiments. More significantly, the cells were pre-incubated with FTS for 1 hr prior to the addition of virus, as compared with 15 min in the experiments described in the Results section. An appropriate duration of pre-exposure to a compound targeted to host cell proteins before exposure to HIV-1 is likely to be required for optimal expression of antiviral activity. In this respect it should also be mentioned that inhibition of Ras activation by FTS and other compounds was performed with serum-starved cells (Harmon and Ratner, 2008). This was not so when inhibition of HIV-1 infection was measured. Pre-exposure to compound with anti-HIV-1 activity for 1 hr or longer is impractical for topical microbicides to be applied vaginally or rectally before intercourse.
The limited success of microbicide efficacy trials has led to the consideration of oral pre-exposure prophylaxis with antiretroviral drugs to prevent sexual transmission of HIV (Garcia-Lerma et al., 2008; Cohen et al., 2008). In the latter setting, pharmaceuticals targeted to host cell proteins essential for HIV-1 replication (including FTS) may find advantageous new applications. Methods for sustained continuous release (e.g. from a vaginal ring) should be considered in this regard. Continual topical FTS release would likely overcome problems due to the relatively short half-life of orally administered FTS (about 3hr; Tsimberidou et al., 2009) and the dose-dependent potential side effects (diarrhea; completely correctable with oral antidiarrheals) of the compound applied systemically (Borthakur et al., 2007).
Journal of Antivirals & Antiretrovirals -Open Access JAA/Vol.1 Issue. 2 Animal model experiments will be required in order to investigate further the potential of FTS for anti-HIV-1 prophylaxis.