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1 Synergistic Interactions between Hepatitis B Virus Ribonuclease H Antagonists 1 and Other Inhibitors 2 Elena Lomonosova a , Adam Zlotnick b , John E. Tavis a# 3 4 Department of Molecular Microbiology and Immunology 5 and 6 Saint Louis University Liver Center, St. Louis, MO, USA a ; 7 Molecular and Cellular Biology Department, Indiana University, Bloomington, IN, USA b 8 9 Running title: Anti-HBV synergy with RNaseH inhibitors 10 11 #Corresponding Author 12 Saint Louis University School of Medicine 13 1100 S. Grand Blvd. 14 Saint Louis, MO 63104 15 [email protected] 16 314-977-8893 17 18 19 AAC Accepted Manuscript Posted Online 12 December 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.02441-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved. on September 12, 2017 by guest http://aac.asm.org/ Downloaded from
Transcript of ^Çv P] ] /v ]}v Á v, ] ] s] µ Z] }vµ o ,v P}v] v K Z /vZ] ] } · ð òõ Çv Z ] }( }µ o r v...
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1
Synergistic Interactions between Hepatitis B Virus Ribonuclease H Antagonists 1
and Other Inhibitors 2
Elena Lomonosovaa, Adam Zlotnickb, John E. Tavisa# 3
4
Department of Molecular Microbiology and Immunology 5
and 6
Saint Louis University Liver Center, St. Louis, MO, USAa; 7
Molecular and Cellular Biology Department, Indiana University, Bloomington, IN, USAb 8
9
Running title: Anti-HBV synergy with RNaseH inhibitors 10
11
#Corresponding Author 12
Saint Louis University School of Medicine 13
1100 S. Grand Blvd. 14
Saint Louis, MO 63104 15
314-977-8893 17
18
19
AAC Accepted Manuscript Posted Online 12 December 2016Antimicrob. Agents Chemother. doi:10.1128/AAC.02441-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Abstract 20
Combination therapies are standard for management of human immunodeficiency virus (HIV) and 21 hepatitis C virus (HCV) infections, however no such therapies are established for human hepatitis B virus 22 (HBV). Recently we identified several promising inhibitors of HBV ribonuclease H (RNaseH) activity that 23 have significant activity against viral replication in vitro. Here, we investigated in vitro antiviral efficacy of 24 combinations of two RNaseH inhibitors with the current anti-HBV drug nucleoside analog lamivudine, 25 with HAP12, an experimental core protein allosteric modulator, and with each other. Anti-HBV activities 26 of the compounds were tested in a HepG2-derived cell line by monitoring intracellular core particle DNA 27 levels, and cytotoxicity was assessed by MTS assay. The antiviral efficiencies of the drug combinations 28 were evaluated using the median-effect equation derived from the mass-action law principle and 29 combination index theorem of Chou and Talalay. We found that combinations of two RNaseH inhibitors 30 from different chemical classes were synergistic with lamivudine against HBV DNA synthesis. Significant 31 synergism was also observed for combination of the two RNaseH inhibitors. Combinations of RNaseH 32 inhibitors with HAP12 had additive antiviral effects. Enhanced cytotoxicity was not observed in the 33 combination experiments. Because of these synergistic and additive effects, the antiviral activity of 34 combination of RNaseH inhibitors with drugs that act by two different mechanisms and with each other 35 can be achieved by administering the compounds in combination at doses below the respective single 36 drug doses. 37
38
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Introduction 39
Hepatitis B virus (HBV) is one of the main causes of chronic liver disease worldwide. 240 to 350 40
million people worldwide are chronically infected with HBV (1, 2) and are at risk for developing end-41
stage liver disease and hepatocellular carcinoma (3). 42
HBV is hepatotropic enveloped DNA virus that replicates by reverse transcription via an RNA 43
intermediate (4). The viral genome consists of a 3.2 kb partially double-stranded relaxed circular DNA 44
(rcDNA). The rcDNA is repaired to a nuclear episomal covalently closed circular DNA (cccDNA) that is the 45
template for viral transcription. Reverse transcription from viral pregenomic RNA (pgRNA) template 46
occurs inside of viral core particles, leading to formation of minus-strand HBV DNA covalently linked to 47
the viral polymerase, concomitant degradation of pgRNA by the viral ribonuclease H (RNAseH) activity of 48
the viral polymerase, and then synthesis of plus-strand DNA. Mature core particles are then either 49
transported back into the nucleus where the rcDNA is converted to cccDNA, or they are enveloped to 50
form virions and secreted from the cell non-cytolytically. 51
The most common antiviral regimen for HBV is treatment with nucleos(t)ide analogs (NAs). Five 52
NAs are approved for treatment chronic HBV infection: lamivudine (LAM), telbivudine, entecavir, 53
adefovir, and tenofovir (5). They inhibit reverse transcription of HBV DNA because their incorporation 54
into the elongating DNA strand leads to chain termination. Potent inhibitors such as entecavir and 55
tenofovir also target the priming step of reverse transcription and can lower viremia to undetectable 56
levels (6). However, NA monotherapies very rarely eradicate the virus (7, 8). In addition, the newer, 57
more potent NAs are extremely costly, prohibiting their use in many parts of the world with high HBV 58
endemicity, such as much of Asia and sub-Saharan Africa (9). 59
Anti-HBV monotherapy is unlikely to be sufficient for the eradication of HBV infection in the 60
majority of patients. Combination therapy has been successful against human immunodeficiency virus 61
(HIV) and hepatitis C virus (HCV) (10, 11). Drugs used in combination therapy should have additive or 62
synergistic activity to increase efficacy and delay or prevent the development of drug resistance, and 63
have no added toxicity (12). However, clinical studies addressing NAs combination therapy for HBV have 64
provided little evidence for clinical benefit of this approach for HBV so far (13, 14). 65
The multifunctional HBV polymerase is the only enzyme encoded by the virus. Its reverse 66
transcriptase (RT) domain synthesizes the viral DNA, and its ribonuclease H (RNaseH) domain hydrolyzes 67
the RNA strand within the RNA/DNA hybrids that are generated during reverse transcription to enable 68
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synthesis of double-stranded DNA. Both activities are necessary for viral replication, however, the 69
clinically available direct-acting anti-HBV drugs target the HBV RT, whereas RNaseH inhibitors are yet to 70
be developed. Therefore, RNaseH is an attractive target for new anti-HBV drugs that may be used in 71
combination with the NAs to increase effectiveness of treatment (15, 16). Recently, we identified 72
several promising compounds that inhibit both HBV RNaseH activity and DNA synthesis (17-20). In 73
addition to RNaseH inhibitors, several other new agents to inhibit viral replication are being developed 74
that target different critical steps in the HBV replication cycle. These agents include host-targeted 75
antivirals (HTAs) and direct-acting antivirals (DAAs) (21). Among DAAs, multiple core protein allosteric 76
modulators (CPAM) (22) are being developed including heteroaryldihidropyridimines (HAPs). CPAMs 77
affect HBV production by inappropriately activating core protein assembly in vitro and in vivo to yield 78
empty or aberrant capsid (23-26). 79
New anti-HBV agents should not only inhibit HBV replication at non-toxic concentrations, but 80
also work in combination with the existing anti-HBV drugs. Here, we tested two novel RNaseH inhibitors 81
from different chemotypes (Fig. 1A) in combination with an existing anti-HBV drug, an experimental 82
CPAM, and each other compound to evaluate whether HBV RNaseH combination treatment results in 83
improved efficiency against HBV replication. Lamivudine (LAM) was selected as a representative NA 84
because it employs the same chain-terminating mechanism as the other approved NAs. The 85
developmental CPAM HAP12 was chosen because it is known to enhance the rate of core protein 86
assembly and preferentially stabilizes non-capsid polymers of core protein (24, 27). We also assessed 87
the effects of the combinations on cellular toxicity to guide the development of novel combination 88
therapies. 89
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Material and Methods 90
Compounds. Compounds (Fig. 1A) were acquired commercially or were synthesized as described 91
previously. LAM ([(-)-β-L-2’-3’ dideoxythiacytidine]) was purchased from Sigma, compound #1 (2-92
Hydroxyisoquinoline-1,3(2H,4H)-dione) was obtained from Totonto Research Chemicals, and #46 (β-93
thujaplicinol) was acquired from the NCI Developmental Therapeutics Program. HAP12 was synthesized 94
as previously described (24). All compounds were dissolved in DMSO (Sigma) at 10 mM and stored at -95
80° C. 96
Cell Culture. HepDES19 is a HepG2-derived stably transformed cell line that supports the tetracycline-97
inducible (tet-off) replication of HBV genome (28). HepDES19 cells were grown at 37°C with 5% CO2 in 98
DMEM/F12 medium (Fisher) supplemented with 100 U of penicillin/mL, 10 μg of streptomycin/mL, and 99
10% fetal bovine serum (Fisher) in the presence of 1 μg/mL of tetracycline. 100
Determination of EC50 for Individual Compounds. The 50% effective inhibitory concentration (EC50) for 101
the individual compounds was determined by plating HepDES19 cells at 0.3 x 106 cells/well in 12-well 102
plates in medium without tetracycline to induce DNA replication. 48 hours later cells were treated with 103
medium containing antiviral compounds or DMSO as a vehicle control and without tetracycline. Two 104
days later cells were lyzed and supernatants were collected and treated for 60 min at 37°C with 105
Micrococcal Nuclease (New England Biolabs) (2 U/μL, final concentration) in the presence of 10 mM 106
CaCl2 to digest non-encapsidated nucleic acids (18). Encapsidated HBV DNA was then extracted using the 107
Qiagen QIAamp cador Pathogen Mini Kit (Qiagen). Quantitative PCR (qPCR) was performed with a 108
strand-preferential assay with TaqMan reagents as described (19). The amounts of plus- and minus-109
polarity DNA strands were calculated as the percentage relative to the quantity of DNA in DMSO-treated 110
cells. The EC50 values were calculated with GraphPad Prism software using nonlinear regression 111
log(inhibitor) vs. response algorithm. 112
Combination Experiments. Each combination experiment included 24 samples, seven doses of each 113
compound alone, three DMSO-only wells and seven compound combinations. Of the seven 114
concentrations tested for each compound, the middle dose was equal to 1.1 times the EC50, two higher 115
doses (corresponding to 1.9 and 3.2 times the EC50) and four lower doses (corresponding to 0.1, 0.2, 0.4 116
and 0.7 times of EC50) were also tested (Fig. 1B). Compound treatment, DNA extraction, and viral DNA 117
quantification were performed as described above. The data were analyzed using the method 118
developed by Chou and Talalay (29-31) that allows quantitative determination of drug interactions, 119
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where combination indexes (CI) CI<1, =1, and >1 indicate synergism, additive effect, and antagonism, 120
respectively. The dose-reduction index (DRI) was determined by comparing the ratio of the doses 121
required to reach a given degree of HBV replication inhibition for a single compound and for each 122
compound in the combination. The CompuSyn program was used to calculate CIs and DRIs 123
(http://www.combosyn.com/index.html). The weighted CI value was calculated as CIwt = (CI50 + 2*CI75 + 124
3*CI90 + 4*CI95)/10 (31). A one-sample t-test was used to determine statistical significance of the 125
calculated CI values from the expected value of 1.0. The anti-viral effects of compound combinations 126
were also assessed using Loewe additivity model with Chalice Analyser 127
(http://chalice.horizondiscovery.com/chalice-portal/documentation/analyzer/home.jsp). Activity of 128
compound combinations was numerically evaluated using Loewe excess volume, which is positive for 129
synergistic combinations and negative for antagonism, and synergy score, which is always positive and 130
provides an additional prioritization favoring combinations whose synergy occurs at high effect level 131
(http://chalice.horizondiscovery.com/chalice-portal/documentation/analyzer/analyticMethods.jsp). 132
Cytotoxicity. To assess cytotoxic effects of HBV inhibitor combinations, HepDES19 cells were seeded 133
into 96-well plates at a density of 1 x 104 cells/well and exposed to compounds with a treatment 134
schedule identical to that used for the HBV replication inhibition assays. Each RNaseH inhibitor was 135
tested at a range of concentrations alone and in combination with LAM or HAP12 at the highest doses 136
available. Following compound treatment, cytotoxicity was assessed by MTS assay. Cytotoxicity was 137
calculated as the percentage relative to cytotoxicity determined in DMSO-treated cells. 50% effective 138
cytotoxic concentration (CC50) values were calculated with GraphPad Prism software using the nonlinear 139
regression log(inhibitor) vs. response algorithm. Student’s t-test was used to compare cell viability 140
among the single- and combination-treated cultures. 141
142
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Results 143
Anti-HBV efficacy and cytotoxicity of individual compounds. Anti-HBV activity of the individual 144
compounds was determined in HepDES19 cells. HepDES19 cells are HepG2 hepatoblastoma cells with a 145
tetracycline-repressible expression cassette for a replication-competent HBV genotype D genome (28) 146
that express high levels of HBV. We determined anti-HBV activity of the compounds by treating cells 147
replicating HBV following release of tetracycline suppression with the test compounds, and quantifying 148
the amount of HBV minus- and plus-polarity DNA strands in core particles by qPCR (19, 20). Two HBV 149
RNAseH inhibitors we previously identified from different chemical scaffolds were selected for this 150
study: #1 (19) and #46 (20) (Fig. 1). Compound #1 is an N-Hydroxyisoquinolinedione and #46 is the 151
natural product β-thujaplicinol, an α-hydroxytropolone. Compound #1 had an EC50 of 4.2 ± 1.4 μM 152
against plus-polarity DNA and did not suppress minus-polarity DNA (Table 1). Compound #46 inhibited 153
plus-strand HBV DNA synthesis with an EC50 of 1.0 ± 0.6 μM. Minus-strand HBV DNA was suppressed at 154
significantly higher concentration (EC50 of 49 ± 3 μM) (Table 1). Thus, we observed the expected 155
preferential inhibition of plus-polarity DNA strand synthesis by quantitative PCR by the RNaseH 156
inhibitors. We also determined EC50 values for LAM and HAP12 (Table 1, Fig. 2) as well as their CC50s in 157
this cell system. As expected, HAP12 inhibited HBV plus- and minus-strand with similar efficiencies, with 158
EC50 values of approximately 0.3 μM (Table 1, Fig. 2) as it acts prior to DNA replication. LAM efficiently 159
inhibited HBV minus-strand DNA (EC50 = 10 μM), and also plus-strand DNA with higher efficiency (EC50 = 160
0.4 μM) because the minus-strand DNA is the template for plus-strand DNA. The cytotoxicity of 161
compounds was also tested in HepDES19 cells. LAM and HAP12 showed no cytotoxic effects at the 162
highest tested concentration of 100 μM (Table 1). CC50 values for #1 and #46 were 75 μM and 25 μM, 163
respectively (Table 1). 164
Efficacy of RNaseH inhibitors plus lamivudine. For each compound pair, two to four independent 165
combination experiments (Fig. 1B) were performed, and viral DNA accumulation was quantified by 166
strand-preferential qPCR. To analyze anti-HBV efficacy of compound combinations we employed the 167
method of Chou and Talalay (30, 32). The combination index (CI) derived for mixed inhibitors was used 168
to identify additivity (CI=1), synergy (CI<1) and antagonism (CI>1). We first determined the effect on 169
HBV replication of combining of RNaseH inhibitors #46 and #1 with LAM. The results of representative 170
experiment are shown in Fig. 3A. Isobolograms were constructed for the doses of LAM and #46 with 171
50%, 75%, and 90% HBV replication inhibition efficiency, referred to as fraction affected (Fa) of 0.5, 0.75, 172
and 0.9, respectively. The combination of #46 and LAM was potently synergistic against HBV, as the 173
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experimental combination data points on the isobolograms fall to the lower left of the lines representing 174
the predicted additive effect at each different efficiency level (Fig. 3A). The quantitative parameters of 175
the combination experiments of #46 and #1 with LAM are in Table 2. In all combination experiments the 176
linear correlation coefficient (r) for the median dose-effect plots was greater than 0.92, indicating good 177
conformity to the median-effect principle of the mass-action law. The combinations of LAM with #46 or 178
#1 consistently produced average CIs of <1 at efficiency levels from 50% to 95% (Table 2), indicating 179
synergy. Average weighted CI values that emphasize values of high efficiency for LAM plus #46 and LAM 180
plus #1 were 0.7 ± 0.1 and 0.5 ± 0.3, respectively. These values are significantly different by a one-181
sample t-test from the expected CI=1.0 with P=0.018 and P=0.033 with a 0.05 significance level, 182
respectively. 183
A major objective of having synergistic drug combination is to reduce doses of the drugs used, 184
thereby reducing the toxicity while maintaining efficacy. The median-effect principle and the 185
combination index theorem can be used to predict dose reduction for drugs during combination 186
treatment (31). Therefore, we calculated the dose-reducing index (DRI), a measure of the degree the 187
dose of each agent in a synergistic combination may be reduced to achieve given effect level compared 188
with the doses of each agent alone (31). DRI >1 is beneficial, and the greater the DRI value indicates a 189
greater dose reduction. The average DRI for compound #1 was 2 and for LAM was 6 at 95% HBV 190
replication inhibition efficiency (Table 2), indicating that the concentrations of LAM and #1 necessary to 191
inhibit HBV replication by 95% could be decreased 2- and 6- fold, respectively, when the compounds are 192
used in combination. DRIs for compound #46 and LAM were similar, being 2 and 3, respectively (Table 193
2). 194
To confirm these calculated DRIs for LAM and RNaseH inhibitor combinations, we measured the 195
inhibition activity of LAM with #1 or #46 singly or in combination at the suboptimal reduced doses of 196
LAM (0.4 μM) with #1 (2.5 μM) or #46 (0.8 μM) as calculated by Chou-Talalay method to inhibit HBV 197
replication by 95% when used in combination (Fig. 3B). As expected, #1 and #46 as single agents only 198
slightly inhibited HBV DNA accumulation (14 ± 3.2 % and 30 ± 10 %, respectively). LAM alone had 199
inhibitory effect of 53 ± 2.1 %. As predicted, the combination of LAM with #1 and #46 resulted in more 200
than 90% of inhibition, 91.5 ± 0.7 % and 91.4 ± 0.5 %, respectively. Since the doses of #46, #1 and LAM 201
used in combination experiments were significantly less than their respective 50% cytotoxic 202
concentrations, cellular toxicity was not expected. However, to ensure that unpredicted combinatorial 203
toxicities did not contribute to the anti-viral synergistic effects for #46, #1 and LAM, we assayed the 204
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cytotoxic effects of #46, #1 and LAM combinations at concentrations that inhibit of HBV plus-strand DNA 205
synthesis by 95% (Fig. 3C). No significant cytotoxic effects were observed for any of the cultures treated 206
with one or two compounds, indicating that the observed synergistic effect of LAM and #46 and #1 on 207
HBV DNA synthesis is not due to the increased overall cellular toxicity. 208
Efficacy of RNaseH inhibitors plus HAP12. Combinations of HAP12 with #46 or with #1 were evaluated 209
by the same method. They produced CIs of approximately 1 at all indicated levels of efficiency, and the 210
weighted CIs for HAP12 were 0.9 ± 0.01 with #46 (P =0.03) and 1.01 ± 0.15 with # #1 (P=0.86) (Table 2). 211
These data imply additive anti-HBV effects for HAP12 when used with #1 and either additive or slightly 212
synergistic interactions when used with #46. 213
Efficacy of RNaseH inhibitors combination. We next tested the two RNaseH inhibitors in combination. 214
CI values of #1 and #46 at efficiency levels from 50% to 95% were all below 1 (from 0.85 ± 0.32 to 0.28 215
±0.22), and the weighted CI of 0.40 ± 0.12 was significantly less than 1.0 (P=0.04), indicating that the 216
two RNaseH inhibitors were synergistic (Table 2). The average DRI for 95% of efficiency to inhibit HBV 217
replication was 3.5 for compound #1 and 9.5 for compound #46, indicating that compounds #1 and #46 218
could be lowered three- or nine-fold when given as combination therapy. 219
Loewe additivity model. A variety of different methods have been developed for analysis of 220
combination experiments (33, 34). Therefore, the anti-viral effects of the compound combinations were 221
also analyzed by Loewe additivity models using the Chalice Analyser software (Table 3). Data analyses 222
demonstrated that each of the LAM+#1, LAM+#46, and #1+#46 combinations had positive values for 223
Loewe excess volume and synergy score, indicating synergistic interactions. The combinations of HAP12 224
with RNaseH inhibitors also had positive values for Loewe excess volume and synergy score; however 225
because of high standard deviations these data are considered insignificant, indicating additive effect. 226
Thus, analyses on the basis of the Loewe additivity model confirmed the results of analyses by Chou-227
Talalay method for all tested combinations. 228
Cytotoxic effects of combinatorial compound treatment. Combination therapy can modulate 229
cytotoxicity as well as efficiency. Therefore, we determined the effects of LAM and HAP12 on CC50 230
values of #46 and #1 by the MTS assay. LAM and HAP12 each have CC50 values above 100 μM, the 231
highest dose tested. Therefore, we measured CC50 values for #46 and #1 alone and in the presence of 232
LAM and HAP12 at 100 μM. The CC50 value for #46 alone was 47 ± 12 μM, which was not significantly 233
different from the CC50 value of 42 ± 8 μM for #46 in the presence of 100 μM LAM (P = 0.59; Student’s t-234
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test) (Fig. 4A), or from the 34 ± 8 μM CC50 value for #46 in the presence of 100 μM HAP12 (P = 0.22) (Fig. 235
4B). Similarly, the CC50 value for compound #1 was determined to be more than 100 μM in all treatment 236
conditions, alone or in combination with 100 μM LAM (Fig. 4C) or 100 μM HAP12 (not shown). These 237
data exclude the possibility that the observed anti-HBV activities of these compound combinations were 238
a result of cytotoxicity and/or cytostatic effects of the compounds. 239
To determine the cytotoxic effect of combination of two RNaseH inhibitors HepDES19 cells were 240
treated with both #1 and #46 simultaneously or independently across a range of concentrations and 241
analyzed for viability by the MTS assay (Fig. 4D). Combining #1 and #46 did not increase overall 242
cytotoxicity, with cytotoxicity being determined by more toxic compound (#46) (111 ± 3 μM total, which 243
corresponds to #46 part of being 56 ± 1 μM as #1 and #46 were used in 1:1 combination ratio). This is 244
not significantly different from the CC50 for compound #46 alone (P = 0.36). Analysis using the method of 245
Chou-Talalay (30) showed the weighted cytotoxicity CI for #1 and #46 was 1.2. Thus, #1 and #46 have 246
additive or slightly antagonistic effects on cytotoxicity. 247
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Discussion 248
Current HBV therapies attenuate chronic hepatitis B, but rarely cure chronic HBV infections. This 249
warrants a search for effective multidrug cocktails that target multiple steps in viral life cycle. 250
Combination therapy is essential to management and cure of chronic viral infections including HIV and 251
HCV, and it is expected that combination therapy will provide similar benefits for patients with hepatitis 252
B (15, 35). In addition, combination anti-HBV therapy is expected to achieve longer-term suppression of 253
HBV replication and consequently viral load with shorter treatments regiments than currently available. 254
This should reduce side-effects through reducing the dose of each drug used, and also reduce risk of 255
resistant mutants developing. 256
Currently there is no standard therapy that includes drug combination for treatment of patients 257
with HBV infection. A few controlled clinical studies addressing NAs or NA plus interferon combination 258
therapies against HBV have been undertaken, but there is little evidence of the clinical benefit of this 259
approach (14, 36). Therefore, establishing whether combination therapy with newer agents provides 260
increased benefit over monotherapy remains an urgent task. 261
Here, we hypothesized that combining treatment of NAs with RNaseH inhibitors could have 262
additive or synergistic effects because these antiviral agents will be independently targeted to the HBV 263
polymerase and RNaseH catalytic centers. Our results indicate that combining an approved anti-HBV NA, 264
LAM, with two different RNaseH inhibitors, #46 and #1, produces greater in vitro anti-viral effects on 265
HBV DNA synthesis than any of the agents used alone (Fig. 5). This is similar to the synergistic effect of 266
combining of β-thujaplicinol (compound #46) and calanolide A, a nonnucleoside HIV RT inhibitor, that 267
was described against the HIV RT polymerase activity in vitro (37). Another study of influenza 268
endonuclease inhibition by compound #1 demonstrated that compound #1 was competitive with RNA 269
but noncompetitive with NTPs (38), further supporting the possibility of simultaneous targeting of two 270
active sites of a viral polymerase by combination of NA and RNase H inhibitors. 271
The molecular mechanism for the observed synergy between LAM and RNaseH inhibitors is 272
unclear at present. LAM is a cytidine analog that inhibits DNA polymerase function by terminating chain 273
elongation and therefore is unlikely to have any direct inhibitory effect on the HBV RNaseH. Similar to 274
the HIV RNaseH, the active site of the HBV RNaseH is believed to contain two essential Mg2+ ions. 275
Compounds #46 and #1 represent two different scaffold types that are both known HIV RNaseH active 276
site metal binders (37, 39, 40). However, HBV RNaseH inhibitors might indirectly affect HBV polymerase 277
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activity, thus synergistically contributing to the HBV polymerase inhibition by LAM. Binding of RNaseH 278
inhibitors to the RNaseH active site may induce conformational changes that could distort HBV RT 279
polymerase catalytic site. This could cause reduced interaction of HBV polymerase with RNA/DNA 280
template and/or steric effects that reduce HBV polymerase activity. Also, binding of RNaseH inhibitors to 281
the HBV RT RNaseH domain might reduce/restrict the mobility of enzyme, thus slowing down or 282
preventing DNA translocation and thereby inhibit elongation of the nascent viral DNA. There is no 283
experimental evidence at present to support these mechanisms for HBV RNaseH inhibitors. However, 284
such phenomena are established for other drugs inhibiting divalent metal-dependent enzymes. For 285
example, nonnucleoside reverse transcriptase inhibitors of HIV RT bind to a site distinct from its catalytic 286
center and induce conformational changes that affect the polymerase activity (41-43). Furthermore, 287
mutational studies with duck hepatitis B virus (DHBV), a common model for HBV, suggest that the 288
RNaseH domain of DHBV polymerase may contribute to multiple steps of viral genome replication, such 289
as RNA encapsidation and minus-strand DNA synthesis (44, 45). Substitution mutations of the putative 290
catalytic residues (46) as well as other charged residues in the RNaseH domain (47) also affect HBV 291
polymerase activity. In addition, HBV RNaseH inhibitors might directly affect DNA strand elongation 292
activity of HBV polymerase through chelating and/or altering the coordination geometry of the divalent 293
metal cations in the HBV RT active site because HBV polymerase activity also requires divalent metal 294
ions for catalysis (48). 295
Overall, the results presented here on the synergistic action of NA and RNaseH inhibitors 296
support possibility of convergent therapy (i.e., using multiple compounds that target the same stage of 297
viral replication) for future efficient treatment of chronic HBV infections. The aim would be to fully 298
inhibit the HBV polymerase’s multiple activities by directing two or more drugs at the same viral target 299
and thus the preventing viral replication (Fig. 5). 300
We also examined the combined effect of two RNaseH inhibitors #1 and #46 on HBV DNA 301
accumulation. Surprisingly in view of the fact that both compounds appear to act on the same site on 302
the HBV RNaseH, these two compounds synergistically suppressed HBV DNA synthesis. Several possible 303
mechanisms could explain this synergism. First, the enhanced inhibition of HBV DNA accumulation by #1 304
and #46 combination may simply reflect higher levels of RNaseH inhibition with two-compound 305
regimen. Even when both RNaseH inhibitors are present, the total intracellular availability of active 306
compounds may be insufficient to saturate all HBV RNaseH active sites. It is also possible that access of 307
RNaseH inhibitors to the HBV polymerase within viral core particles might be limited. Addition of two 308
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RNaseH inhibitors simultaneously would increase the probability of RNaseH binding and inhibition by 309
these compounds. Second, synergy could result from effects other than RNaseH inhibition, although we 310
have no data supporting this possibility. A precedent for synergistic interactions for two compounds that 311
target a single active site exists. Combinations of the NAs LAM and penciclovir are synergistic against 312
HBV in HepG2 2.2.15 cells (49), as well as against duck HBV in primary duck hepatocytes (50). A more 313
recent study of in vitro combinations of tenofovir with different NAs (LAM, entecavir, telbivudine, and 314
adefovir) in HepAD38 cells also showed additive to slight synergistic anti-HBV effects (51). 315
The challenge of combination therapy is to achieve a maximal antiviral effect with minimal 316
toxicity. We did not observe any evidence of enhanced cytotoxicity during HBV replication inhibition 317
assays at the highest tested combination doses. In addition, the cytotoxicity experiment to determine 318
the effect of highest possible concentration of LAM in our experimental conditions on CC50 of #46 and #1 319
demonstrated a lack of synergistic/additive in vitro cytotoxicity for the LAM and #46/#1 combinations. 320
Similarly, we did not find any evidence of synergistic toxicity of compounds #1 and #46. Together, our 321
results demonstrate feasibility of a strategy of combining two different of RNaseH inhibitors in anti-HBV 322
convergent combination therapy (Fig. 5). 323
Divergent therapy involves a combination of anti-viral agents targeting different stages of viral 324
replication. To evaluate the potential for the divergent therapy against HBV we focused on combination 325
of two classes of compounds – RNaseH and core protein allosteric modulators (Fig. 5). We found 326
additive anti-hepadnaviral activity of combining new developmental agents – RNaseH inhibitors and 327
core protein allosteric modulators. Thus, HBV cores which escape the action of these inhibitor(s) 328
because of incomplete effectivity could be later targeted by RNaseH inhibitor(s) at a later stage of the 329
replication cycle. As with the RNaseH inhibitors and LAM, we found no evidence for synergistic/additive 330
in vitro cytotoxicity for HAP12 plus #1 or #46. 331
HBV is a genetically diverse virus, with 8 to 10 genotypes that differ by ≥8% at the nucleotide 332
level (52). Our studies were done with a genotype D isolate, but it is probable that our conclusions will 333
apply to other genotypes as well for two reasons. First, the catalytic center of the RNaseH is likely to be 334
highly conserved due to selective pressures to maintain its essential function. Second, we recently 335
evaluated the activity of 18 variant HBV RNaseH sequences from genotypes B, C, and D (52). Although 336
the basal RNaseH activity varied substantially among the variant enzymes, they were equivalently 337
sensitive to compounds #1 and #46. 338
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In conclusion, this study provides strong evidence that targeting multiple hepatitis B viral targets 339
can improve the efficacy of molecular targeted therapies without exacerbating cytotoxicity. It therefore 340
opens one path towards developing rational combination strategies for treating chronic HBV infections.341
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Acknowledgments 342
We thank Stephen Locarnini for helpful conversations. JET is an inventor on patents covering use of HBV 343
RNaseH inhibitors against HBV. Some of this intellectual property has been licensed to Arbutus 344
Biopharma, Inc., and Arbutus Biopharma provides research support to the Tavis lab. 345
346
Funding Statement 347
This work was supported by NIH grant R01 AI104494 to JT and an ancillary study within the Hepatitis B 348
Virus Research Network (U01 DK082871, Adrian Di Bisceglie, P.I.). The funders had no role in study 349
design, data collection and interpretation, or the decision to submit the work for publication. 350
351
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Figure Legends 352
Figure 1. Compounds and study design. A. HBV inhibitors used in this study. B. Matrix of compound 353
concentrations used during two-compound anti-viral combination experiments. During each two-drug 354
combination assay, individual compounds (1 and 2) were tested alone at seven concentrations from 0.1 355
to 3.2 times their respective EC50s. To test the combination activity of compounds 1 and 2, seven 356
combination doses were administered to the cells as indicated to ensure constant compounds 357
concentration ratio though the experiment. x – addition of compounds or DMSO. 358
Figure 2. Efficacy of lamivudine (A) and HAP12 (B) against HBV replication. Inhibition of HBV replication 359
was measured relative to DMSO-treated cells. EC50 values were calculated for both the plus and minus 360
polarity DNA strands. The curves and the EC50 values are the mean ± one standard deviation from two 361
independent experiments. 362
Figure 3. Anti-HBV efficacy of compound combination. A. Representative isobolograms of an #46 and 363
LAM combination experiment. Isobolograms at efficiency doses (referred to as fraction affected (Fa)) of 364
50%, 75% and 90% are shown. The actual doses of #46 and LAM are plotted on the x- and y-axis 365
respectively. The points on the axes are the doses of each compound necessary to generate the given Fa 366
value. The line drawn between the points on the axes corresponds to the possible combination of doses 367
that needed to generate the same Fa value, indicating the expected additive effect for the compound 368
combination. The experimental combination data points for #46 and LAM fall on the lower left of the 369
line at each efficiency level, indicating synergistic effects. A dose reduction for LAM and #46 370
combination is also apparent at all three Fa values. B. Inhibition of HBV plus-strand DNA by 371
combinations of LAM and #1 or #46. HepDES19 cells replicating HBV were treated with indicated 372
compounds at concentrations calculated to inhibit HBV replication at 95% of efficiency in combination. 373
HBV core DNA was purified, the amount of plus-strand DNA was quantified by qPCR, and the amount of 374
DNA for each treatment condition is shown as percentage to the DMSO control; error bars are ± one 375
standard deviation. C. Cytotoxic effect of LAM and #46 and #1. HepDES19 cells were exposed to the 376
compounds for three days at concentrations that inhibit HBV replication at 95% of efficiency in 377
combination. Cell viability as percentage to the DMSO control was assayed by MTS assay; error bars ± 378
one standard deviation. 379
Figure 4. Cytotoxicity associated with combining anti-HBV agents. Cell viability was assayed by MTS 380
assay. A. Effect of LAM on #46 CC50. HepDES19 cells were exposed to #46 alone or in combination with 381
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100 μM of LAM. B. Effect of HAP12 on #46 CC50. HepDES19 cells were exposed to #46 alone or in 382
combination with 100 μM HAP12. C. Effect of LAM on #1 CC50. HepDES19 cells were exposed to #1 383
alone or in combination with 100 μM of LAM. D. Combined cytotoxicity of #1 and #46. HepDES19 cells 384
were seeded into 96-well plate and exposed to #1 and #46 alone or in combination at a range of 385
equimolar concentrations. 386
Figure 5. Sites of action of anti-viral agents used in this study and their combined effects on HBV 387
replication. The NAs and RNaseH inhibitors act inside core particles and have synergistic effect on HBV 388
replication inhibition. Core particles assembly modulators (e.g., HAP12) inhibit the formation of mature 389
replication-competent HBV core particles, thus reducing the overall rate of HBV replication. 390
Combination of RNaseH inhibitors with core particles assembly modulators has additive effect on HBV 391
replication inhibition. Domains of HBV polymerase (terminal protein domain (TP), reverse transcriptase 392
domain (RT), and RNaseH domain (RH)) and the RNA (back)/DNA (gray) heteroduplex are indicated 393
inside core particles. 394
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530
531
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TABLE HBV repli atio i hi itio effi ie a d toto i it of RNaseH i hi itors, la ivudi e a d HAP
Co pou d EC µM for suppressio of a: CC µM a HBV ore + sta d DNA HBV ore - stra d DNA
# . ± . o suppressio . ± # . ± . 9 ± . ± LAM . ± . 9. ± . > HAP . ± . . ± . >
a ea ± o e sta dard deviatio .
Data fro 9 .
Data fro .
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TABLE Co i atio effe ts of RNaseH i hi ito s, la i udi e a d HAP o HBV epli atio i hi itio
Co pou d Co i atio
Co i atio Ratio
ra
CIb alues at i hi itio of:
Weighted
CI alues
DRId at % i hi itio : % % % % LAM # #
LAM + # : . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . LAM + # : . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± .# + # : . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± .HAP + # : . ± . . ± . . ± . . ± . . ± . . ± . HAP + # : . ± . . ± . . ± . . ± . . ± . . ± .
a r, li ea o elatio oeffi ie t of the edia -effe t plot. Results a e ea s ± sta da d de iatio s.
CI, o i atio i de as al ulated the CI e uatio of Chou a d Talala . CI < , CI = , a d CI > i di ate s e gis , additi e effe t, a d a tago is , espe ti el .
C The eighted CI is CI t = CI + CI + CI + CI / .
d DRI, dose- edu tio i de al ulated o pa i g the doses e ui ed to ea h % of HBV epli atio i hi itio he usi g the o pou d as si gle age t a d i o i atio .
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TABLE Loe e additi it odel a al ses of a ti-HBV o pou d o i atio s
Co pou d o i atio Loe e e ess olu ea, S erg s orea LAM + # . ± . . ± . LAM + # . ± . . ± . # + # . ± . . ± . HAP + # . ± . . ± . HAP + # . ± . . ± .
a Results are ea ± o e sta dard de iatio .
Positi e alues i di ate s ergisti o i atio s, a d egati e alues i di ate a tago is .
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Compound 1 (Fold EC50 value)
DMSO 0.1x 0.2x 0.4x 0.7x 1.1x 1.9x 3.2x
Co
mp
ou
nd
2 (
Fo
ld E
C5
0 v
alu
e)
DMSO x x x x x x x x
0.1x x x
0.2x x x
0.4x x x
0.7x x x
1.1x x x
1.9x x x
3.2x x x
Compound #1
Compound #46
Lamivudine HAP12
A
B
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HAP12
Core particle
TP RT
RH
Synergy Additive Synergy
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