The present study indicates that when EGCG is oxidized to produce dimers with two gallate groups, the effective antiviral pH range is expanded from the neutral into the acidic range. EGCG inactivates HSV-1 and HSV-2 as effectively as digallate dimers at pH 8.0 but has minimal or no anti-HSV activity at pH 5.7 or below, while the dimers inactivate HSV from pH 4.0 to 8.0. EGCG dimers are also more effective against other enveloped viruses with class I, II, and III fusion proteins at neutral to slightly alkaline pH. Both digallate and monogallate dimers reduced the titer of VSV, which, along with HSV has a class III fusion protein, by 3 log10 more than EGCG at pH 8.0. Also at neutral pH, EGCG dimers reduced the titers of viruses with class I and class II fusion proteins more effectively than EGCG. MV (class I) was not inactivated by EGCG at neutral pH but had its titer reduced by the dimer by ≥3 log10 at the same pH. At pH 5.7, the monogallate dimers and EGCG had similarly low antiviral activities against HSV, indicating that it is the presence of two gallates in addition to the dimerization that is needed for antiviral activity as the pH becomes more acidic. The importance of gallate groups for antimicrobial activity has also been shown when tea catechins and theaflavins were used as bactericidal agents (7, 9) or against human cancer cells (8). As gallate ester groups were added to the theaflavin molecule, antibacterial and antitumor activities increased. Our results suggest that the digallate dimers of EGCG (theasinensin A, P2, and TF-3), with their strong inactivation of HSV between pHs 4.0 and 8.0, would potentially function well as active ingredients in a topical microbicide. This is especially important in order for a microbicide to reduce vaginal transmission of HSV from male to female and female to male, because the vaginal pH can range from pH 3.6 to 4.5 in most women, from pH 5.0 to 6.5 in women with bacterial vaginosis, and from pH 6.0 to 7.0 when semen temporarily neutralizes vaginal pH (2, 21, 27).
TF-3 and theasinensin A were not toxic to Vero cells in the present study. In other studies utilizing TF-3, the growth of all human cancer cell lines and normal human liver cells was inhibited but the growth of normal human lung cells was unaffected (8). In a clinical study, tea polyphenols appeared to lack toxicity following human consumption (29). These in vitro and in vivo toxicity studies, when considered with the fact that cells in the female genital tract are protected by a mucosal barrier, suggest that antiviral EGCG dimers have the potential for use in a microbicide.
The results in this study suggest that inactivation of viruses by theasinensin A and TF-3 can involve interaction with viral proteins. The decrease in titers of the nonenveloped coxsackie A9 virus by EGCG dimers can only be due to interaction with the viral capsid. The variability in sensitivity to dimer inactivation between the five enteroviruses tested suggests that TF-3 and the other dimers do not bind equally as well to even closely related viruses. The ability of EGCG dimers to inactivate enveloped viruses from all three fusion classes suggests that the dimers may bind directly to all fusion proteins or, more likely, that following hydrogen binding to viral envelopes (25), the dimers may be able to move to essential glycoproteins and prevent them from functioning. It is also possible that the hydrogen binding itself between these catechins and envelope lipid bilayers may alter the function of fusion-required glycoproteins without direct interaction between the catechin and the glycoprotein. It has been shown that TF-3 inhibits HIV-1 entry into target cells by preventing envelope glycoprotein-mediated membrane fusion (15). This TF-3-dependent anti-HIV activity was correlated with the ability to block formation of the gp41 six-helix bundle. The use of TF-3 and other digallate dimers could provide direct inactivation of HSV and HIV, thereby reducing the titer of both pathogens involved in the synergistic relationship between HSV and HIV (6, 31). It is essential to prevent initial HSV infection, since once the virus is established, HIV-1 receptor-positive cells can persist at the sites of herpes virus infection for months after healing, even with acyclovir therapy (35). This may explain the failure of acyclovir treatment to reduce HIV-1 acquisition (3). Our results clearly indicate that digallate dimers of EGCG have the capability to reduce the titer of HSV-1 and HSV-2 virions by 3 to 5 log10, thereby reducing HSV transmission and consequently HIV transmission.
Approximately half of the drugs currently in clinical use are of natural product origin (19). Many of the most promising natural lead compounds are available only in extremely small amounts; however, this is not the situation with theaflavin and catechin derivatives, which are the major polyphenols in black tea and green tea, respectively. Catechins account for 6 to 16% of the dry weight of green tea leaves and theaflavins for about 2% of the dried water extract of black tea (10). It has been suggested (13) that natural products such as TF-3 and EGCG possess superior specificity and potency compared to artificially designed molecules due to evolutionary selection. As importantly, the structures of the biological targets of natural products, e.g., protein binding sites, are often well conserved among proteins of markedly different genetic sequences (18). This is evidenced by the structural homology found between the class III fusion proteins gB from HSV-1 and glycoprotein G from VSV (11, 23). Both gB and G are viral envelope proteins that are essential for viral fusion with cell membranes, and their X-ray structures indicate that they are similar in domain organization despite having different primary sequences (11).
A number of studies have shown that compounds with anti-HSV activity function by binding to gB from either HSV-1 or HSV-2. Among 22 human cytokines examined, MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 had anti-HSV-1 activity and were shown to strongly bind to HSV-1 virions via envelope gB (17). The θ defensin retrocyclin 2, which protects cells from infection by inhibiting viral adhesion and entry, was shown by surface plasmon resonance studies to strongly bind to gB from HSV-2 (34). Also, a number of sulfated or sulfonated polysaccharides which inhibit the cell-to-cell spread of HSV bind to gB (4). It is, therefore, possible that the interaction of EGCG dimers with gB has a similar antiviral effect on HSV infectivity and that the inactivation of all the susceptible enveloped and nonenveloped viruses examined in this study is the result of an alteration of the structure of essential fusion proteins by EGCG dimers. EGCG dimers are lower-molecular-weight and inexpensive alternatives to polyanions, chemokines, and defensins and can potentially be utilized to inactivate HSV and HIV in a topical microbicide.
Эпигаллокатехин галлат инактивирует клинических изолятов herpes simplex virus.
В отсутствие полностью эффективной herpes simplex virus (HSV) вакцины, топические бактерицидные средства представляют собой важную стратегию для предотвращения передачи ВПГ. (-)-Эпигаллокатехин галлат (EGCG) (молекулярный вес, 458.4) является основной катехин в зеленом чае. Настоящее исследование показывает, что EGCG обладает большей anti-HSV деятельности, чем другие катехины зеленого чая и инактивирует несколько клинических изолятов вируса простого герпеса типа 1 (HSV-1 и HSV-2. EGCG снижение ВПГ-2 титры >или=1000 раз в 10-20 мин и снижение HSV-1 титры на ту же сумму в 30-40 мин. Anti-HSV активность EGCG является следствием прямого влияния на вириона, и инкубации Vero и клеток CV1 с EGCG за 48 ч до инфицирования ВПГ-1 и ВПГ-2, соответственно, не уменьшает ВПГ производства. Электронно-микроскопических (ЭМ) исследования показали, что очищенные вирионы воздействию EGCG были повреждены, и EM электронной маркировки конверта гликопротеины gB и gD был значительно снижен в EGCG лечения, в то время как белок капсида маркировка была неизменной. Когда очищенный HSV-1 конверт гликопротеины gB и gD инкубировали с EGCG и затем осмотрен в присутствии додецилсульфата натрия-полиакриламидный гель-электрофорез, Нижний молекулярной массой gB и gD полос уменьшилась и новых более высокой молекулярной массой полосы появились, с указанием EGCG производства, зависящие от макромолекулярных комплексов. gB и gD необходимы для ВПГ инфекции, и эти результаты позволяют предположить, что EGCG может инактивации вирионов ВПГ путем привязки к gB, gD, или другой конверт гликопротеина. EGCG стабилен в диапазоне рН нашли во влагалище и, как представляется перспективным кандидатом для использования в бактерицидных средств для снижения передачи ВПГ.
Antimicrob Agents Chemother. 2008 Mar;52(3):962-70. doi: 10.1128/AAC.00825-07. Epub 2008 Jan 14.