Naturally occurring Vpr inhibitors from medicinal plants of Myanmar

Human immunodeficiency virus type-1 (HIV-1) is a lentiviral family member that encodes the retroviral Gag, Pol, and Env proteins, along with six additional accessory proteins, Tat, Rev, Vpu, Vif, Nef, and Vpr. The currently approved anti-HIV drugs target the Pol and Env encoded proteins. However, these drugs are only effective in reducing viral replication. Furthermore, the drugs’ toxicities and the emergence of drug-resistant strains have become serious worldwide problems. Resistance eventually arises to all of the approved anti-HIV drugs, including the newly approved drugs that target HIV integrase (IN). Drug resistance likely emerges because of spontaneous mutations that occur during viral replication. Therefore, new drugs that effectively block other viral components must be developed to reduce the rate of resistance and suppress viral replication with little or no long-term toxicity. The accessory proteins may expand treatment options. Viral protein R (Vpr) is one of the promising drug targets among the HIV accessory proteins. However, the search for inhibitors continues in anti-HIV drug discovery. In this review, we summarize the naturally occurring compounds discovered from two Myanmar medicinal plants as well as their structure-activity relationships. A total of 49 secondary metabolites were isolated from Kaempferia pulchra rhizomes and Picrasama javanica bark, and the types of compounds were identified as isopimarane diterpenoids and picrasane quassinoids, respectively. Among the isolates, 7 diterpenoids and 15 quassinoids were found to be Vpr inhibitors lacking detectable toxicity, and their potencies varied according to their respective functionalities.


Introduction
Human immunodeficiency virus (HIV) disease has remained the fifth major worldwide killer during the past decade and the second major killer in low-income countries. According to UNAIDS, globally there were 36.7 million people living with HIV in 2015, and 1.2 million people died of AIDS that year [1]. To date, the US Food and Drug Administration (FDA) has approved 26 anti-HIV drugs, including nucleoside and non-nucleoside inhibitors of reverse transcriptase, protease inhibitors, fusion inhibitors, an entry inhibitor, and HIV integrase strand transfer inhibitors [2,3]. However, the treatment of HIV with the FDAapproved drugs is only effective in reducing viral replication, and HIV rapidly gains resistance to all known agents [4]. To circumvent this problem, combination therapy (highly active antiretroviral therapy, HAART) has proven very effective at both reducing the virus load and suppressing the emergence of resistance in a number of patients [5]. However, the patients who received HAART have The original version of this article was revised due to a retrospective Open Access order.
& Nwet Nwet Win nwetwin2012@gmail.com developed symptoms of Cushing's syndrome, with hyperglycemia, hyperlipidemia, centripetal fat distribution, and peripheral muscle wasting [6]. These adverse phenomena highlight the need for new antiviral agents, preferably targeting other viral components to reduce the rate of resistance and suppress viral replication even further. Viral protein R (Vpr) is a small basic accessory protein (14 kDa) and is well conserved in HIV-1, HIV-2, and simian immunodeficiency virus (SIV) [4,7]. Numerous investigations over the past 2 decades have shown that Vpr is a multifunctional protein. Vpr mediates many processes that aid HIV-1 infection, evasion of the immune system, and persistence in the host, thus contributing to the morbidity and mortality of AIDS. Vpr molecular functions include nuclear import of viral preintegration complex (PIC), induction of G 2 cell cycle arrest, modulation of T cell apoptosis, transcriptional coactivation of viral and host genes, and regulation of nuclear factor kappa B (NF-jB) activity [8][9][10][11]. The numerous functions of Vpr in the viral life cycle suggest that Vpr would be an attractive target for therapeutic intervention [12,13]. To date, only a few small molecule Vpr inhibitors that are derived from natural sources, such as medicinal plants and fungi, are available. Among the reported Vpr inhibitors, fumagillin is an isolate from fungal metabolites [14]. Damnacanthal and quercetin are constituents of noni and a well-known plant flavonoid, respectively [15,16]. Vipirinin is a coumarin-based synthetic compound [17]. The structures of the reported Vpr inhibitors are quite different from each other at the scaffold level, and thus the further discoveries of structurally distinct Vpr inhibitor candidates are eagerly expected from natural resources. Consequently, the anti-Vpr activities of crude extracts from Myanmar medicinal plants were evaluated to identify Vpr inhibitors. Herein, we compiled the naturally occurring Vpr inhibitors from Kaempferia pulchra rhizomes [18][19][20][21] and Picrasma javanica bark [22][23][24].

Establishment of the screening system
Since Vpr function is crucial for the development of AIDS and Vpr is proposed to be a possible target molecule of anti-AIDS drugs, several groups have established assay systems to screen for Vpr inhibitors. Shimura and coworkers established a cell line [MIT (multinuclear cell induced by tetracycline)-23], in which Vpr-induced cell cycle perturbation could be manipulated by a tetracycline promoter, and identified quercetin as a Vpr inhibitor in 1999 [16]. Watanabe and co-workers established HeLaderived cell lines (MT-Vpr 1 cells and MT-Vpr 2 cells) that allow conditional expression of Vpr and examined the mechanism of cell death following Vpr expression. They identified fumagillin as an antagonist of Vpr-mediated growth inhibition in yeast cells in 2006 [14]. In 2011, Ong and co-workers identified vipirinin, which inhibits the cell cycle arrest activity of Vpr in yeast and the Vpr-dependent viral infection of human macrophages [17]. In the same year, Kamata and co-workers reported that damnacanthal, a component of noni, is a specific inhibitor of Vpr-associated cell death with no effect on cell cycle arrest [15].
On the basis of the previously reported assay systems, we have developed a tetracycline-inducible expression system that consists of two key expression vectors, pcDNA4/TO and pcDNA6/TR. Briefly, Vpr expression plasmids were first constructed and then inserted into TREx-Hela cells to establish TREx-HeLa-Vpr cells. In this assay system, the addition of tetracycline leads to the expression of Vpr, which causes the death of TREx-HeLa cells. In contrast, Vpr-induced cell death does not occur in the presence of a Vpr inhibitor. This assay system is feasible for the preliminary screening of extracts or compounds possessing anti-Vpr activity for HIV infection as compared to the use of the virus. The detailed methods are described in our previous reports [21,24]. The anti-Vpr activity is monitored by the cell proliferation (%), occurring because of the inhibitory effects of the tested compounds on the expression of Vpr.

Kaempferia pulchra and Picrasma javanica as folk medicines
Kaempferia pulchra Ridl. is a perennial herb of the Zingiberaceae family. It is cultivated in some tropical countries, including Myanmar, Indonesia, Malaysia, and Thailand. It is commonly known as ''Shan-pan-oot'' in Myanmar and has been extensively used for cough, blood stimulation, carminative, quenching heat, deodorant, urinary tract infection, diuretic, and diabetes mellitus purposes [25]. It reportedly possesses antiinflammatory and antitumor activities [26,27]. The rhizomes have been locally used for self-medication by cancer and AIDS patients. Sandaracopimaradiene diterpenoids and ethyl 4-methoxy-trans-cinnamate have been detected in extracts from the plant grown in Thailand [26,28].

Structure-activity relationships of isopimarane diterpenoids and of picrasane quassinoids
Since the isolated 7 diterpenoids and 15 quassinoids exhibited Vpr inhibitory activities without detectable toxicities, the structure-activity relationships (SARs) for each type of isolate were studied. The SARs among diterpenoids revealed that the presence of the b-OH groups at C-6 and  (7)]. Thus, among the isopimara-8(9),15-dienes, the presence of either a methoxy or carbonyl group led to a decrease in the inhibition potency. Since 17, 20, and 23 exhibited similar inhibitory effects at 1.56 lM, it can be concluded that the presence of an a-OAc or b-OAc group at either C-1 or C-7 on the 6b-hydroxy-isopimara-8(14),15diene skeleton is important for the inhibition of Vpr expression. Among the tested picrasane quassinoids, javanicin I (47) was the most potent inhibitor, with potency comparable to that of the positive control, damnacanthal [cell proliferation (%): 214 (47); 218 (damnacanthal)]. SAR studies between picrajavanicins G (38) and I (47) suggested that the presence of the methyl group at C-13 is crucial for exhibiting significant anti-Vpr activity in the 2,12,14-triene-1,11,16-trione-2,12-dimethoxy-18-norpicrasane quassinoids [cell proliferation (%): 140 (38)]. It should be noted that javanicin I (47) is the least polar compound among the isolates from the CHCl 3 extract. This finding suggested that the polarity of the C-13 side chain is related to the Vpr inhibitory effect.
Furthermore, the C-13 methylated 2,12-diene-1,11dione-2,12-dimethoxy-18-norpicrasane quassinoid, 45, is the second most potent Vpr inhibitor among the isolates [cell proliferation (%): 214 (47) [ 174 (45)]. The differences between the structures of 45 and the most potent inhibitor, 47, are the substitutions of the double bond with a single bond between C-14 and C-15 and of the carbonyl group with the hydroxy group at C-16. Hence, the presence of the hydroxy group at C-16 may serve as an effective functional group that increases the anti-Vpr activity. A comprehensive assessment of the results obtained between the tested quassinoids suggests that the presence of the methyl group at C-13 is an important factor and the substitution of the carbonyl group with a hydroxyl group at C-16 of 47 increases the anti-Vpr activity.

Antiproliferative activities of isolated isopimarane diterpenoids and picrasane quassinoids
Some of the isolates did not show any Vpr inhibitory activity. However, the rhizomes of K. pulchra and the bark of P. javanica have been locally used for self-medication by cancer patients [25]. Therefore, the antiproliferative activities of the isolated compounds (1-49) were evaluated against a panel of five human cancer cell lines, including A549 (human lung cancer), HeLa (human cervical cancer), PANC-1 and PSN-1 (human pancreatic cancer), and MDA-MB-231 (human breast cancer) using the CCK-8 assay.  Tables 3, 4). It is interesting that all of the potent anti-Vpr diterpenoids and quassinoids lacked cytotoxic effects against the tested human cancer cell lines. These results provided convincing evidence that the Vpr inhibitors from the present study might be capable of selectively targeting the expression of Vpr with no toxicity.

Future perspectives
Isopimarane diterpenoids and picrasane quassinoids are naturally occurring Vpr inhibitor candidates that are chemically quite distinct from the previously reported ones [14][15][16][17]. Further structurally interesting Vpr-inhibitors might be expected from structural modifications of the isopimarane diterpenoids and picrasane quassinoids. However, while numerous Vpr inhibitors have been identified over the past