Discovery of acylphloroglucinol-based meroterpenoid enantiomers as KSHV inhibitors from Hypericum japonicum

Kaposi's sarcoma associated herpesvirus (KSHV) has gained considerable attention as a type of carcinogenic pathogen. Recent research suggests that KSHV has participated in the pathogenesis of Kaposi's sarcoma-related malignant neoplastic diseases. Viral lytic infection might be pivotal for the etiopathogenesis of KSHV-induced diseases; however, most clinical KSHV lytic replication inhibitors like ganciclovir, nelfinavir, or cidofovir do not restrain virus replication effectively enough to achieve clinical efficacy. In our continued pharmaceutical studies on Chinese herbal medicines, new acylphloroglucinol-based meroterpenoid enantiomers have been discovered from Hypericum japonicum. Most of these metabolites have potential inhibitory activities that target KSHV lytic replication. Amongst these analogues, compounds 1a and 1b possess an unreported ring system cyclopenta[b]chromene. Compounds 1a with 4a exhibit stronger inhibitory activities towards the lytic replication of KSHV in Vero cells. In addition, 1a and 4a have IC50 values of 8.30 and 4.90 μM and selectivity indexes of 23.49 and 25.70, respectively. Qualitative and quantitative SAR and molecular docking studies for acylphloroglucinol-based meroterpenoids with regard to anti-KSHV activity were conducted. An explanation for the variation in the activity and selectivity indexes was proposed in accordance with the predicted binding pose found with molecular docking to a putative target, thymidylate synthase (kTS). Compounds 1a and 4a have potential for further development and optimization of their anti-KSHV activities which could lead to new candidate drugs.


Introduction
Kaposi's sarcoma-associated herpesvirus (KSHV), containing double-stranded DNA, is in the family of Herpesviridae and within the subfamily of gamma-herpesvirus. 1 It is thought to participate in the pathogenesis of Kaposi's sarcoma-related malignancies, such as multicentric Castleman's disease, AIDSrelated multicentric Castleman's disease, and primary effusion lymphoma.1a,2 Like other herpesviruses, KSHV has two life cycles of latency and lytic replication, and both contribute to virus-associated disease.2a Generally, during the latent infection, KSHV is established as episomes via a certain amount of latent genes expressed to support viral latent replication.While in the lytic replication, viral genes, comprised of immediate early, early, and late phases, are expressed, leading to the production of mature progeny virions and ultimate lysis of cells.2b Since viral lytic infection might be pivotal for the etiopathogenesis of KSHV-induced diseases, most clinical agents, including ganciclovir, nelnavir, or cidofovir, target the inhibition of KSHV lytic replication. 3However, most of these antiviral drugs do not restrain virus replication effectively enough to achieve clinical efficacy. 3,4Therefore, additional antiviral regimens are needed which concentrate on developing candidate drugs or lead compounds which inhibit on KSHV lytic replication.
Natural products continue to arouse the interest of pharmacochemistry scientists due to their therapeutic applications and their use as an arsenal of sources for exploiting lead compounds. 5Our on-going course of systematic study on the genus Hypericum has led to an enormous amount of phloroglucinol derivatives that exhibit anti-virus activities. 6Amongst this genus, Hypericum japonicum Thunb (Guttiferae), a perennial or annual herb, is extensively distributed throughout North America, Oceania, and Asia. 7Extracts of the entire herb, also named "Tianjihuang" in Chinese, have been manufactured to be used by injection.Some metabolites isolated from H. japonicum have been demonstrated to be lead compounds with bioactivities such as anti-cancer, antimalarial, antibacterial, and anti-oxidative stress. 8With the intent of nding bioactive lead compounds from Chinese herbal medicine, our research team has investigated acylphloroglucinol-based compounds from H. japonicum and has discovered that most compounds exhibit potential anti-KSHV activities.6c Our present study of this herb-medicine led to analogue enantiomer pairs 1a/1b-4a/ 4b, of which compound 1a/1b contains the novel ring system of a cyclopenta[b]chromene, and compounds 1a and 4a exhibit stronger inhibitory activities towards the lytic replication of KSHV in Vero cells, with IC 50 values of 8.30 and 4.90 mM, and selectivity indexes of 23.49 and 25.70, respectively.Furthermore, the rst qualitative and quantitative SAR and molecular docking studies for acylphloroglucinol-based meroterpenoids with regard to anti-KSHV activity were conducted.These studies suggest that 4a is able to anchor with kTS by hydrogen bonding to residues Arg199, Arg200, Pro217, Ser240 and Tyr282.An additional salt bridge between 5 0 -OH and Arg199 and a hydrophobic pocket formed by residues Phe115, Val158, Pro218 and other hydrophobic residues are also suggested.

Results and discussion
Enantiomeric metabolites are recognized by researchers.However, they still are infrequently studied due to the abundance of secondary metabolites. 9Oen, both enantiomers have been discovered as either a scalemic or racemic mixture. 10nterestingly, another four pairs of new phloroglucinol-based terpenoid enantiomers, (AE)-japonicols E-H (1a/1b-4a/4b), possessing diverse monoterpenoid skeletons, especially in 1a/1b, with the novel carbon skeleton of a cyclopenta[b]chromene, were obtained from Hypericum japonicum.The structures and absolute congurations were conrmed via extensive NMR spectroscopic data and calculated ECD analyses.Herein, the stereochemistry elucidation, biological activity measurements, as well as the Qualitative and quantitative structure-activity relationship (SAR) and molecular docking studies for these acylphloroglucinol-based meroterpenoids with regard to anti-KSHV activity are presented in detail.
(AE)-Japonicol E (1a/1b), an off-white amorphous powder, with the molecular formula of C 20 H 26 O 4 , was inferred via HRESIMS data (m/z 331.1963 [M + H] + , calcd 331.1909).The FTIR spectrogram shows that the characteristic absorptions for hydroxy (3279 cm À1 ), carbonyl (1626 cm À1 ), and aromatic ring (1593 and 1438 cm À1 ) groups on the molecule are presented.Analyses of the 1 H and 13 C NMR, including HSQC spectrum, suggested the presence of a acylphloroglucinol base along with a carbonyl (d C 212.1), aromatic carbons (d C 163.5, 108.0, 164.1,  162.2, 104.8, and one carbon resonance of C-4 0 is not displayed in the one-dimensional NMR spectrum, but the existence of this carbon could be deduced from the HRESIMS data), two methyls (d H 1.12, d, J ¼ 6.8 Hz; 1.14, d, J ¼ 6.8 Hz), and one methine (d H 4.02, J ¼ 6.8 Hz).These analyses, together with the close carbon chemical shis of (AE)-japonicols A-D, 6c lead to the acylphloroglucinol base of the molecule.The planar structure of its monoterpene moiety was established by HMBC and 1 H-1 H COSY NMR spectra analyses (Fig. 3).The occurrence of spin systems of H-2/H-6/H-5/H-4 in the 1 H-1 H COSY spectrum, suggested the presence of a cyclopentane unit.Meanwhile, the long-range correlations in the HMBC spectrum from H 3 -10 to C-2, C-3, and C-4, and from H 3 -9 to C-6, C-7, and C-8, determined the location of Me-10 and the linkage between the isoalkyl group and C-6.Ultimately, the connection of monoterpene and acylphloroglucinol motifs was demonstrated by the pivotal correlations of H-1 with C-2, C-3, C-6, C-2 0 , and C-3 0 (HMBC), as well as H-1 with H-2 ( 1 H-1 H COSY), which also established a pyran ring between the aforementioned motifs to suit the seven degrees of unsaturation.Thus, compound 1 with a ring system of a cyclopenta[b]chromene was characterized, which is an unreported carbon skeleton discovered for natural product compounds.
The relative conguration was tackled by performance of NOESY correlation analyses (Fig. 3).The NOE responses of H 3 -10/H-2 assigned these two protons to the same face in an aorientation.Concurrently, a NOE interaction of H-6/H-8b (d H 4.80) was observed, but there was no NOE interaction between H-6 with H-2, suggesting the b-orientation of H-6 (Fig. 3).
The relative conguration of 2 was designated through analyses of NOESY experiments.The NOE interactions of H-6 with H-5b (d H 2.16), H-5b with H-4b (d H 1.80), H-4b with H-1b (d H 2.85), and H-1b with H-2 suggested that these protons could be arbitrarily assigned as a b orientation.On the contrary, NOE correlations of H 3 -10 with H-5a (d H 2.20) and no NOE crosspeak between H 3 -10 with H-2 led to the reasonable assignment of the a-oriented position for H 3 -10.
The side chain attached to C-2, viz., the geranyl group, has free rotation and the absence of one stereogenic carbon to C-2.This made it impossible to conrm the relative congurations of C-2 and C-3 merely via the NOESY signals, for instance, of harronin I and II. 11The relative congurations of the two adjacent carbons C-2 and C-3, presented with six preferential rotamers of 3 are shown in Newman projections (Fig. 5B).The major rotamer of 3 should be I as shown in Fig. 5B, since its NOESY interactions acted in accordance with the results of NOESY experiments, viz., H-2 with H-4/H-10, and H-1 with H-4 (more details showed in ESI †).Thus, the relative chiralities of C-2 and C-3 were deduced to be 2R*,3S*, which is consistent with the same type of stereogenic carbons in bonannione B and bonanniol D.  The absolute stereochemistry of C-2 was established as 2S and 2R for 3a and 3b respectively, since the ECD spectra of 3a and 3b showed similar Cotton effects with ones of peperobtusin A (negative or positive Cotton effect around 230 nm, and positive or negative Cotton effect around 275 nm, comparatively). 13onsequently, the absolute congurations of (AE)-japonicol G (3a/3b) were ascertained to be 2S,3R, and 2R,3S, respectively, which were also consistent with the matched ECD spectra between experimental and calculated ones (Fig. 4).
(AE)-Japonicol H (4a/4b) was isolated as a reddish brown oil.The only structural difference between it and japonicol G was the presence of a phenyl ring instead of an isopropyl group in the latter at C-7 0 of the acylphloroglucinol unit, which was inferred from HRESIMS data with a protonated molecular ion at m/z 383.1876 (calcd 383.1858).The enantiomeric separation procedure and 2D NMR correlations of 4 are presented in Fig. 2 and 5A, respectively.The relative conguration of 4 was conrmed due to its 13 C NMR chemical shis at C-2 and C-3 at d C 91.9 and d C 74.4, respectively, which were identical values to those presented of compound 3 (Table 1).In comparison with 3a/3b (Fig. 4), the homologous ECD Cotton curves showed that 4a/4b possessed a consistent intrinsic chirality.
Biological activities of these four pairs of enantiomers 1a/1b-4a/ 4b towards anti-KSHV activity were carried out by reported methods.2a,6c Human iSLK.219 cells (derived from iSLK cells) inserted with rKSHV.219[a sort of recombinant virus encoded with green uorescent protein (GFP) and red uorescent protein (RFP)], were utilized to assay the antiviral activity.The cytotoxicity of the compounds was measured by an AlamarBlue® Cell Viability Assay (Invitrogen), applying the protocol of the manufacturer, and was expressed as CC 50 (the 50% cytotoxic concentration value).The inhibitory effects on KSHV lytic replication, assessed by infectivity assays through the GFP expression per well in Vero cells, were expressed as IC 50 values (the 50% inhibiting concentration value).Effect of each compound demonstrated that 1a and 4a possessed encouraging inhibitory effects on KSHV lytic replication in Vero cells, with IC 50 values of 8.30 and 4.90 mM, and selectivity indexes of 23.49 and 25.70, respectively (Table 2).For subsequent SAR studies of these enantiomers, the foregoing bioactivities data were used (Table 2).With a common scaffold of the acylphloroglucinol moiety, a variety of heterocycles were incorporated into the scaffold in the 1 0 and 2 0 positions by formation of pyran, furan, or oxepine rings.Generally, with only fused rings, addition of a remote vinyl group (1a/1b-4a/4b) caused a coarse-grained increase of IC 50 when compared to 6a/6b-8a/8b with the vinyl group replaced by a tertiary hydroxyl group, suggesting that a polar group in that position is not tolerated.The occurrence of a bridged ring (5a/ 5b) caused a decrease of the IC 50 , which might indicated that the at conformation of the fused ring plays a role.Notably, given the commonness of 3a, the enhanced activity and selectivity of 4a is likely attributable to the unique phenyl group at the 7 0 position.Furthermore, it is noteworthy that the selectivity indices roughly correlate linearly with the IC 50 values.However, they are entirely uncorrelated with the CC 50 values.
Qualitative analysis was followed by quantitative SAR (QSAR) modelling utilizing the Field-Based QSAR Panel implemented in Schrödinger Suite 14 to set up CoMFA/CoMSIA models from these enantiomers.These enantiomers rst superimposed using the exible shape-based alignment method (Fig. 6).The training set was used to set up the model (Table 3), and the best model (Table 4) showed moderate predictive ability.Experimental activities of additional analogues would likely provide a better model.Moreover, correlation between the observed activities and the predicted activities was plotted in Fig. 7.
To further unravel the SAR of these enantiomers, a search for all complex assemblies relevant to KSHV lytic replication in the Protein Data Bank 15 was performed, and two targets were found, namely Thymidylate Synthase (kTS) and protease (kPr).Crystal structures of kTS and kPr complexes (PDB ID: 5H39 (ref.16) and 5V5D, 17 respectively) were retrieved and 4a was docked into the pocket of associated reference ligands for each target using Glide.The Glide GScore of 4a docking to kTS was lower than    that for kPr (Table 5), and thus, kTS was assumed to be a putative target for further Induced t docking (additionally implemented in Suite).Induced t docking allows the receptor to alter its binding site so that it more closely conforms to the shape and binding mode of the ligand, which gave a Glide GScore of À9.326 for 4a docking to kTS.As shown in Fig. 8, the acylphloroglucinol scaffold of 4a was anchored via hydrogen bonding to Arg199, Arg200, Pro217, Ser240 and Tyr282 with an additional salt bridge between 5 0 -OH and Arg199.Moreover, the remote vinyl group was inserted in a hydrophobic pocket formed by Phe115, Val158, Pro218 and other hydrophobic residues.In general, this predictive binding mode agreed roughly with the SAR previously discussed.

Conclusions
KSHV and its viral genes are potentially associated with human malignant neoplastic diseases.Additionally, immunoassays have shown that KSHV-related antibodies remain in individuals with AIDS. 18In this research, (AE)-japonicols E-H (1a/1b-4a/4b) as racemic mixtures were studied.Compounds 1a/1b, possess an unreported ring system of a cyclopenta[b]chromene.Each pair of japonicols E-H was obtained with approximate 100% values of enantiomeric excess (ee) from Hypericum japonicum.
(AE)-Japonicols E-H (1a/1b-4a/4b), as well as (AE)-japonicols A-D (5a/5b-8a/8b), were carried out the inhibition assay on KSHV lytic replication using previously developed procedures.6c The inhibitory effects revealed that (+)-japonicols E (1a) and H (4a) presented promising anti-KSHV activity in vitro along with IC 50 values of 8.30 and 4.90 mM and selectivity indexes of 23.49 and 25.70, respectively.These aspects might gain insight into an extensive research for developing analogous molecules for the medical treatment of diverse KSHV associated diseases.These enantiomers contained a variety of heterocycles incorporated into the scaffold in the 1 0 and 2 0 positions, leading to difficulties in analysis of their structure-activity relationships.Notwithstanding, some general correlations with the variation of IC 50 and selective indices were determined.Furthermore, to nd a putative target and to predict its binding mode were undertaking using molecular docking, which represents the rst example of Qualitative and quantitative structure-activity relationship (SAR) and molecular docking studies for acylphloroglucinol-based meroterpenoids as anti-KSHV agents.This study provides a useful starting point for further development and optimization of the anti-KSHV activities of these types of compounds and for the discovery of new therapeutic candidate drugs.Future work will include the development of synthetic routes to afford 4a and its analogues, and the investigation of the molecular mechanisms involved in the inhibitory effects of these types of compounds.

General experimental procedures
All isolated metabolites were carefully screened by analytical TLC plates (Merck, Germany) under ultraviolet-visible detector with l 254 nm.Separations were performed by silica gel (200-300 mesh, Yantai Chemical Co., Ltd., China), ODS (YMC Co., Japan), and Sephadex LH-20 (Mitsubishi Chemical Co., Japan) column chromatography.Generally, all target products were puried through an analytical HPLC (Shimadzu LC-10AVP Plus) with a RP-C 18 column (5 mm, 10 Â 250 mm, Welchrom®, China) and a CHIRALPAK®IC column (5 mm, 10 Â 250 mm, Daicel, China).HRESIMS data were acquired by a Thermo Scientic LTQ-Orbitrap XL apparatus.IR spectra were measured on a Bruker Vertex 70 spectrophotometer using KBr discs.UV spectra were recorded using a Varian Cary 50 instrument.Circular dichroism chiroptical spectra were performed using a JASCO J-1700 spectrometer.The 1 H (400 MHz) and 13 C (100 MHz) NMR spectra were obtained using a Bruker AM-600 spectrometer with tetramethylsilane (TMS) as an internal standard while the chemical shis were characterized to solvent peaks (CD 3 OD, d H 3.31 ppm; d C 49.15 ppm).

Plant material
The whole herbs of H. japonicum were harvested from the DaBie Mountain area of Hubei Province, P. R. China, in October 2011, and were authenticated by Professor Jianping Wang (School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology).A voucher specimen (ID 20111011) has been stockpiled in the Hubei Key Laboratory of Biotechnology of Chinese Traditional Medicine, Tongji Medical College, Huazhong University of Science and Technology.

Fig. 6
Fig. 6 Ligands were aligned through flexible shape-based alignment, which was implemented in Schr ödinger Suite.

Fig. 7
Fig. 7 Scatter plots of observed versus predicted activities.

Table 3
Data set assignment, observed and predicted activity, and their residuals (prediction error) of training and test set

Table 4
PLS statistics of field-based QSAR model a Factors ¼ number of PLS factors in the model; R 2 ¼ correlation coefficient of experimentally observed and predicted activity; SD ¼ standard deviation of regression; F ¼ variance ratio; p ¼ statistical signicance; Q 2 ¼ value of Q 2 ; RMSE ¼ root mean square error; Pearson's r ¼ correlation coefficient of predicted and experimentally observed activity.
a b Best model.

Table 5
Docking results for 4a to kPr and kTS at SP level of precision