key: cord-0920591-3oiivkn2
authors: Tanner, Julian A.; Zheng, Bo-Jian; Zhou, Jie; Watt, Rory M.; Jiang, Jie-Qing; Wong, Kin-Ling; Lin, Yong-Ping; Lu, Lin-Yu; He, Ming-Liang; Kung, Hsiang-Fu; Kesel, Andreas J.; Huang, Jian-Dong
title: The Adamantane-Derived Bananins Are Potent Inhibitors of the Helicase Activities and Replication of SARS Coronavirus
date: 2005-03-25
journal: Chem Biol
DOI: 10.1016/j.chembiol.2005.01.006
sha: 79ba46fafc5a6279ea9cc27ff3a5d5dddaacdf96
doc_id: 920591
cord_uid: 3oiivkn2

Bananins are a class of antiviral compounds with a unique structural signature incorporating a trioxa-adamantane moiety covalently bound to a pyridoxal derivative. Six members of this class of compounds: bananin, iodobananin, vanillinbananin, ansabananin, eubananin, and adeninobananin were synthesized and tested as inhibitors of the SARS Coronavirus (SCV) helicase. Bananin, iodobananin, vanillinbananin, and eubananin were effective inhibitors of the ATPase activity of the SCV helicase with IC(50) values in the range 0.5–3 μM. A similar trend, though at slightly higher inhibitor concentrations, was observed for inhibition of the helicase activities, using a FRET-based fluorescent assay. In a cell culture system of SCV, bananin exhibited an EC(50) of less than 10 μM and a CC(50) of over 300 μM. Kinetics of inhibition are consistent with bananin inhibiting an intracellular process or processes involved in SCV replication.

Further cell culture studies suggest that bananin inhib-our knowledge, an array of completely new adamantane derivatives, which may be easily diversified by re-its intracellular activities mechanistically involved with key viral processes, as opposed to the viral entry step.

acting various aromatic aldehydes with phloroglucinol. These results are consistent with this class of drugs targeting the SCV helicase within the cell.

We have previously developed a colorimetric assay to measure the NTPase activity of the SCV helicase in 96-Results well plates, in a high throughput format [26, 32, 33] . In this discontinuous colorimetric assay using malachite Synthesis of Bananin and Its Derivatives The bananins were synthesized by the reaction of green and ammonium molybdate, released phosphate is quantified after a 5 min reaction period, observed at phloroglucinol (most likely in its triketo tautomeric form) with aromatic aldehydes, catalyzed by hydrochloric a wavelength of 630 nm. Oligo-dT 24 was included in the assay at a saturating concentration of 200 nM, to mimic acid or sodium hydroxide in aqueous solution [9] . Generally, acidic catalysis was used due to the degradation the nucleic acid-stimulated NTPase activity of the SCV helicase. Potential inhibitors of the ATPase reaction of pyridoxal under highly basic conditions. Alkaline catalysis was used for reaction with aromatic aldehydes would be expected to reduce the amount of phosphate released during the reaction, reflected in a decrease in such as vanillin. Bananin synthesis is driven by the creation of the highly symmetric trioxa-adamantane-triol the measured absorbance at 630 nm. We first checked whether the bananin compounds (TAT) cage system. The prototypical compound of the TAT series, the vitamin B 6 -derived bananin (BAN) or were able to inhibit the dT 24 -stimulated ATPase activity of the SCV helicase. Controls were carried out to en-1-[3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridinyl]-2,8,9-trioxaadamantane-3,5,7-triol, can be iodinated sure that the bananin compounds themselves did not affect the phosphate measurement assay. Reactions with subsequent oxidation to iodobananin (6#-iodobananin 5#-carboxylic acid, IBN). The iodine in IBN can be were carried out in the presence of various concentrations of the six bananin derivatives and the results were replaced by various substituents as exemplified by the synthesis of adeninobananin (6#-adeninobananin 5#-plotted and fitted to a simple model (Figure 2A) . We also checked that under our reaction conditions, we carboxylic acid hydrochloride, ADN) using an activated adenine nucleobase derivative. Interestingly, BAN is were making measurements within the linear region ( Figure S1 ). susceptible to Michael addition with the natural product eugenol, isolated from the essential oil of cloves Our results showed that the parent compound bananin inhibited the ATPase activity of the SCV helicase (Syzygium aromaticum). This NaOH-catalyzed addition leads to eugenolbananin (eubananin, EUB), which can with an ATPase IC 50 value of 2.3 M (IC 50 values shown in Figure 4C ). Iodobananin and vanillinbananin exhib-be transformed by cyclic hemiketal condensation into the ansa-compound ansabananin (ABN), inspired by ited the strongest inhibition, with ATPase IC 50 values of 0.54 and 0.68 M, respectively. Inhibition by vanillinba-ansamycins such as rifamycin and geldanamycin [31] . In the aromatic aldehyde series, vanillin was reacted nanin indicates that the presence of a six-membered nitrogen heterocycle is not absolutely essential for in-with phloroglucinol to yield vanillinbananin (VBN) or 1-(4-hydroxy-3-methoxyphenyl)-2,8,9-trioxaadaman-hibitory activity. Eubananin showed similar inhibitory activity to bananin itself with an ATPase IC 50 of 2.8 M. tane-3,5,7-triol. It is expected that numerous naturally occurring aldehydes can be introduced to form the cor-Interestingly, ansabananin was a weak inhibitor, with an ATPase IC 50 of 51 M, while adeninobananin did not show responding TATs with phloroglucinol in 3.33 M aqueous NaOH. The bananin group of compounds represents, to any inhibitory activity at all. These results suggest that are the double reciprocal Lineweaver Burke plots for the data in Figures 3A and 3C . In both cases, as the V max was significantly decreased in the presence of the inhibitor, but the K M changed little, this indicated that bananin was acting as a noncompetitive inhibitor of the ATPase activity of the SCV helicase with respect to both ATP and nucleic acid. This suggests that bananin inhibits by binding at a site distinct from the ATP and nucleic acid binding sites. Building on this foundation, we next tested the antihelicase activities of these compounds. We used a newly developed fluorimetric assay based on the very strong fluorescence resonance energy transfer (FRET) from the fluorophore Cy3 to the quencher Black Hole Quencher 2 (BHQ2). A similar approach has been outlined very recently in assaying the hepatitis C virus (HCV) helicase, a 3# to 5# helicase [34] . However, as we have recently shown that the SCV helicase holds strict 5# to 3# directionality [26] , we designed a system with a 5#-oligo(dT) overhang. The principle behind this new assay is outlined in Figure 4A . There is a Cy3 fluorophore at the 3# end of one of the oligomers of the duplex, in close proximity to a BHQ2 quencher at the 5# end of the other oligomer. When the two oligomers are in very close proximity (i.e., when the two oligomers are annealed), then the Cy3 fluorescence is strongly quenched by the FRET effect. After the duplex has been unwound by the SCV helicase, then the Cy3 fluorescence is no longer quenched, and a dramatic increase in the fluorescence may be observed. To ensure that the primers do not reanneal, a second capture primer is included in the reaction. This is identical to the BHQ2 primer but does not contain the BHQ2 quenching group, therefore the annealing process has little effect on the fluorescence of Cy3. We optimized reaction conditions to ensure that all measurements were carried out in the linear region ( Figure S2 ). As a one minute time-point was in the linear region, we then probed the larger) than the ATPase IC 50 values, it was observed that the inhibitors followed the same general trends as those observed for the ATPase data. Bananin, iodobabulky side groups on the six-membered ring of this nanin, vanillinbananin, and eubananin were effective inclass of compounds reduce their inhibitory activity hibitors of helicase activity, while ansabanin and adenagainst the SCV helicase.

inobananin barely inhibit the reaction. We also checked whether bananin would inhibit the We also performed a final control to check whether unstimulated basal ATPase activity in the absence of bananin acted as a general helicase inhibitor or not. We dT 24 of the SCV helicase ( Figure 2B ). It is clear that cloned and purified the E. coli DnaB helicase, which is bananin is not an effective inhibitor of the unstimulated a well characterized helicase with 5# to 3# polarity of ATPase activity, although slight inhibition was observed unwinding [35] . The purity of DnaB may be observed at 100 µM.

by SDS-PAGE in Figure S3A . We found that 250 µM We then tested the mechanism of inhibition of the bananin did not inhibit DnaB in our FRET-based assay ATPase activity by bananin, and checked competition ( Figure S3B ). These results suggest that bananin does with respect to both ATP (Figures 3A and 3B ) and with respect to dT 24 (Figures 3C and 3D) . Figures 3B and 3D not act as a general helicase inhibitor. 

The potency of these inhibitors against the SCV helicase enzymatic activities prompted us to investigate effects were observed three days after infection with the serial dilutions, thereby allowing measurement of their ability to inhibit SCV replication in a cell culture system. We chose to test bananin itself, it being the the viral titre. In this study, drugs were added either one hour before or one hour after the infection with a 0.03 most representative of the class and the parent com-

shown to be 17-19 hr (our unpublished data). Therefore, the readings at 24 hr are effectively after a single fects (CPEs): the cells appear inflamed with "ridged" cell membranes when infected with the virus. Visual in-generation. It can be seen from this data that at a concentration of 10 M bananin, the viral titre was reduced spection of cell cultures infected with SCV in the presence of 50 M bananin, revealed that CPEs were dis-by almost 50% after 24 hr (Figure 5) , and the drug was most effective when added one hour after infection tinctly reduced relative to those of a control infection (results not shown). However, levels of CPEs were diffi-compared to one hour before. When drug was added before, it was not removed on addition of the virus and cult to quantify accurately, and so an alternative procedure was pursued.

was present for the rest of the experiment. After 48 hr, the difference between addition of drug before and af-To quantify the antiviral activity of bananin, we mea- ference in SCV S-gene levels between the control and nonspecific aggregator. The ATPase inhibition results were consistent with those from the helicase assays: bananin, vanillinbananin, eubananin, and iodobananin were the best inhibitors of DNA-unwinding, while ansabanin and adeninobananin were poor inhibitors. Generally, the helicase inhibition activities measured for each compound, as determined by IC 50 values, were less than the corresponding ATPase inhibition activities, but the trends remained consistent. This appears to be a characteristic common to many helicase inhibitors [24, 25] .

Bananin, which acted as an effective inhibitor in both enzymatic assays and is the prototypical member of this class of compounds, was tested in a First, this would suggest that the compound does not helicase for several structurally unusual trioxa-adainhibit a viral entry process. Second, it suggests that mantane derivatives, trivially referred to as bananins.

bananin may be affecting other cellular pathways that We also show that bananin exhibits significant anti-SCV in the absence of viral infection may resist the protecactivity in cell culture, through the inhibition of a protive effects of the drug. The SCV helicase is one poscess occurring after viral entry into the cell. We have sible target within the cell, although at this stage we developed an extremely convenient and quick method cannot exclude the possibility that bananin may be infor testing both ATPase activities colorimetrically, and hibiting via other pathway(s). helicase activities fluorimetrically through a type of FRET assay. This combination of assays may be adapted easily for high-throughput screening of compound li-Significance braries against both NTPase and DNA-unwinding enzymatic activities, and avoids the use of radioactive 32 P, Adamantane derivatives have been used clinically for which is commonly used in many traditional helicase many years as antiviral treatments and as muscle reassays.

laxants. Here, we have demonstrated that a class of ATPase assays revealed that iodobananin and vanilpyridoxal-conjugated trioxaadamantanes, the banalinbananin were the most effective SCV helicase inhibinins, inhibit both the ATPase and helicase activities tors, with ATPase IC 50 values of 0.54 M and 0.68 M, of the SARS coronavirus helicase. Testing a number respectively. Bananin ( ATPase IC 50 = 2.3 M) and eubaof bananin derivatives, we have shown that it is imnanin ( ATPase IC 50 = 2.8 M) were also reasonable inhibiportant to reduce steric hindrance around the pyritors, but ansabananin and adeninobananin, which condoxal ring for effective inhibition of the SCV helicase. tain bulky appendages on the pyridoxal system, showed Furthermore, bananin was shown to be an effective little if any inhibition. Bananin acted as a noncompetiantiviral drug in a cell culture of the virus. The mode tive inhibitor with respect to both ATP and nucleic acid, of viral inhibition supports the hypothesis that the suggesting this class of inhibitors binds at a site dis-SCV helicase is a target of these compounds. Given tinct from the ATP and nucleic acid binding sites. Far the paucity of drugs shown to be effective in treating weaker inhibition of the unstimulated ATPase activity was observed, showing that bananin does not act as a this recently emerged disease, the bananins repre- plemented by UV/VIS spectrophotometry.

The SCV helicase domain (nsp13-pp1ab, accession number NP_ Synthesis of Eugenolbananin (Eubananin, EUB) 828870, originally denoted as nsp10) was cloned and purified as from Bananin and Eugenol previously described [26] . We used a protocol modified from that described [34] , using oligodisodium (±)-rel-[6R,10S-(R p ,R p )]-6-exo,10-exo-dioxido-6-endo,10mers suitable for a 5# to 3# helicase. Two oligomers were synthe- 2,8,9-trioxaadamantane-3,5,7-triol or 1-(4-hydroxy-3methoxyphenyl)-2,8,9-trioxatricyclo[3.3.1.1 3,7 ]decane-3,5,7-triol  (2000 cells per well) under minimum essential medium containing

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