Novel dimeric capsid assembly modulators as a unique class of highly potent anti-HBV agents

Abstract

Approximately 250 million people are chronic HBV carriers and are at high risk of developing hepatitis, cirrhosis, and hepatocellular carcinoma. Several drugs are currently approved, but because they do not cure, lifelong therapies are the norm. HBV Capsid Assembly Modulators (CAMs) have emerged as a promising option, as they lower several key markers of HBV replication. Novel dimeric structures (D-CAMs) were designed and evaluated. Their potency and mechanism of action were compared to those of monomeric D-CAMs, such as GLP-26. Among them, D-CAM-14 exhibited improved potency over GLP-26 and a unique effect on capsid morphology and kinetic assembly.

Graphical Abstract

Introduction

Hepatitis B virus (HBV) is still a major public health problem worldwide, with approximately 250 million individuals chronically infected, and the prevalence can be much higher in specific populations.¹ To date, nucleoside analogs such as tenofovir alafenamide, entecavir, tenofovir disoproxil fumarate, lamivudine, adefovir dipivoxil or telbivudine are the major treatment options in the clinic. However, none of the existing treatments are curative, and lifelong continued therapy is still required.² Because of this limitation, the global effort now focuses on developing new curative therapeutics targeting other viral proteins involved in HBV replication, including the HBV nucleocapsid. The HBV capsid is an icosahedral core that plays a crucial role in the HBV replication cycle. It is involved in the co-encapsidation of viral pregenomic RNA (pgRNA) with the viral polymerase; it enables reverse transcription of the encapsidated pgRNA into relaxed circular DNA (rcDNA), signals for capsid envelopment with the surface proteins and secretion of mature virions. Structurally, the most common form of the HBV capsid (T4) comprises 240 core capsid protein monomers that first dimerize before forming pentamers and hexamers on the capsid surface. Over the years, several small molecules interfering with the formation of HBV capsids have been identified. These compounds also known as HBV capsid assembly modulators (CAMs) bind at the dimer-dimer interface of the hepatitis B core protein (HBc) and interfere with assembly of new HBV cores by accelerating their formation, leading to the assembly of nonfunctional capsids, either grossly misshapen (CAM-A - Aberrant capsids) or T4-like but without nucleic acid internalized.^3,4 GLP-26 (Figure 1) remains one of the most potent and promising CAM-E (Capsid Assembly Modulator - Empty) antivirals. It inhibits HBV rcDNA neosynthesis (leading to a very strong inhibition of virion production), HBeAg formation, and cccDNA establishment when used at low nanomolar concentrations without displaying any significant cytotoxicity in a panel of cell lines. Strikingly, long term combination treatment with GLP-26 and entecavir in a humanized mouse model induced a decrease in viral loads and viral antigens that was sustained for up to 12 weeks after treatment cessation.^5,6 Notably, further optimization of GLP-26 structure led to the discovery of ALG-001075 and its water-soluble prodrug ALG-000184, which is currently in advanced clinical trials.⁷

Figure 1.

GLP-26 and general structures of the targeted GLP-dimers/D-CAMs.

Computational docking of GLP-26 using the reported crystal structure coordinates of HBV core protein dimer (PDBID:5T2P) shows that the aniline, the pyrrole, and the diketo- portions of GLP-26 display key interactions with the core proteins. At the same time, the alkyne chain remains in the solvent-exposed part of the dimer, and by extension, of the completely assembled capsid.⁵ Based on this feature, we designed novel dimeric structures by linking two GLP-26 moieties (or “Warheads”) and hypothesized that they would have a unique effect on HBV capsids (Figure 1). We considered that a dimeric CAM (D-CAM) such as a GLP-26 dimer could A) display no interaction with the HBV capsid leading to inactive compounds; B) form a single interaction with the HBV capsid, acting like a “standard” CAM; C) interact with two or more different capsids, therefore acting as an inter-capsid tether; or D) interact internally within the same capsid (intra-capsid staple) (Figure 2). The latter two hypotheses represent a completely unique and novel binding mode for an HBV CAM. Herein, we wish to report on the synthesis of a series of GLP-dimers (D-CAM), describe their antiviral activities, and discuss the unique mode of action of our most potent member in this series.

Figure 2.

Potential interaction modes of D-CAMs: A) no interaction with the HBV capsid leading to inactive compounds; B) Single interaction with the HBV capsid. Resulting mode of action would be similar to that of GLP-26; C) Interaction with multiple different capsids (two are shown in this example); D) dual internal interaction within the same capsid.

Results and discussion

Chemistry

The key diazido linkers 2a-e and 5a-b required to synthesize the targeted D-CAMs were prepared according to the chemistry described in Scheme 1. Thus, linkers 2a-e were prepared by reacting the corresponding commercially available dibromo alkyl derivatives 1a-e with NaN₃ in DMF at 100 °C, while longer linkers 5a-b were prepared from diols 3a-b by first, activation with tosyl chloride followed by substitution with NaN₃. Coupling of the different linkers 2a-e and 5a-b with GLP-26 using a standard copper-catalyzed [3+2] cycloaddition “click” protocol afforded the desired D-CAMs 6a-g (Scheme 2).

Scheme 1.

Reagents and conditions: (a) NaN₃, DMF/H₂O, 90 °C-100 °C, overnight, 75%–86%; (b) TsCl, Et₃N, CHCl₃, 8 h; (c) NaN₃, DMF, 90 °C-100 °C, overnight, 50%–70% over 2 steps.

Scheme 2.

Reagents and conditions: (a) 2a-e or 5a-b, CuSO₄.5H₂O, Na ascorbate, CH₃CN/H₂O, 100 °C, 8 h, 40–60%.

For comparison purposes and to better understand how these dimers work, the triazolo analog of GLP-26 (7) and the elongated GLP derivative 10 were prepared. Reaction of GLP-26 with NaN₃ in presence of copper sulfate and sodium ascorbate afforded 7 in 82% yield (Scheme 3). Compound 10 was synthesized by “clicking” GLP-26 with dodecyl azide 9, which was obtained from dodecyl bromide 8 by treatment with NaN₃ (Scheme 4).

Scheme 3.

Reagents and conditions: (a) NaN₃, CuSO₄.5H₂O, Na ascorbate, CH₃CN/H₂O, 90 °C, 8 h, 82%.

Scheme 4.

Reagents and conditions: (a) NaN₃, DMF/H₂O, 90 °C, overnight, 88%; (b) GLP-26, CuSO₄.5H₂O, Na ascorbate, CH₃CN/H₂O, 90 °C, 8 h, 73%.

Structure activity relationship.

The anti-HBV activity (HBV DNA production) of all synthesized compounds tested in HepAD38 cells^5,8 and cytotoxicity profile measured in human lymphocytes (PBM), T lymphoblast cells (CEM), African Green Monkey cells (Vero), and human hepatoma cells (HepG2) are summarized in Table 1. Notably, a correlation between the length of the linker and the anti-HBV activity was observed: compounds with shorter linkers (6a and 6b; C₆ and C₈ linkers, D-CAM-6 and D-CAM-8) display low anti-HBV activity with EC₅₀s of 285 and 485 μM, respectively. Increasing the length of the linker, resulted in a clear increase in potency, with the best dimers 6d (D-CAM-12) and 6e (D-CAM-14) (C₁₂ and C₁₄ linkers) being 80 times more potent than reference compound GLP-26 (EC₅₀ = 0.0001 μM for both 6d and 6e). Further increase of the linker length led to a clear decrease in potency with compound 6f and 6g being 2–3 times less potent than GLP-26. Interestingly, neither the triazolo- monomer 7 nor compound 10, bearing a triazole ring and a C₁₂ alkyl chain, were as active as GLP-26. This result highlights that the dimeric structure is a key feature of our compounds. Therefore, based on its high potency and favorable cytotoxicity profile, dimer 6e/D-CAM-14 was selected for further characterization.

Table 1.

Anti-HBV activity (HBV DNA production) in HepAD38 cells and cytotoxicity profile of compounds 6a-g, 7 and 10.

Compound	Anti-HBV activity(nM)		Cytotoxicity, CC50 (μM)				SIa
	EC50	EC90	PBM	CEM	Vero	HepG2
6a D-CAM-6	285.0 ± 21	>10,000	>100	>100	>100	>100	>350
6b D-CAM-8	438 ± 211	5,520 ± 1,520	>100	>100	>100	>100	>228
6c D-CAM-10	4 ± 1	140 ± 110	>100	>100	>100	>100	>25,000
6d D-CAM-12	0.1 ± 0.4	8 ± 1	63.4 ± 40.6	42.8 ± 0.6	>100	>100	>1,000,000
6e D-CAM-14	0.1 ± 0.5	9 ± 0.2	>100	>100	>100	>100	>1,000,000
6f D-CAM-16	18 ± 11	130 ± 60	>100	>100	>100	>100	>5555
6g D-CAM-18	25 ± 24	380 ± 10	>100	>100	>100	>100	>4000
7	47 ± 42	580 ± 30	74±14	67.8±19.6	>100	>100	>2128
10	307 ± 153	970 ± 130	1.6 ± 0.6	9.9 ± 4.6	10.43 ± 9	30.5 ± 25.6	>99
GLP-26	8 ± 1	70 ± 20	>100	>100	30.5±6.7	>100	>12500

^aSI: Selectivity index (toxicity in HepG2 / activity in HepAD38)

Effect of 6e/D-CAM-14 on HBc and HBeAg in HBV-infected HepG2-NTCP cells - Pre- and Post-infection Data

Hepatitis B e antigen (HBeAg) is a secretory protein associated with HBV replication and infectivity exhibiting a positive correlation with total HBV DNA.^9,10 In HepG2-NTCP cells, a hepatoma cell line that overexpresses the HBV entry receptor sodium taurocholate cotransporting polypeptide (NTCP),¹¹ treatment with 6e/D-CAM-14 leads to significantly greater suppression of HBeAg compared to GLP-26 or GLS-4 (CAM-A), with 6e/D-CAM-14 exhibiting an EC₅₀ that is nearly eight-fold lower (Table 2). Pre-treatment of HepG2-NTCP cells with compounds before HBV infection reveals that 6e/D-CAM-14 is also more potent in reducing hepatitis B core antigen (HBcAg) levels compared to GLP-26 or GLS-4, and is comparable to IFN-α treatment, one of the current standards of care for chronic HBV patients (Table 1).¹² Given that HepG2-NTCP cells do not support HBV spread, a reduction in HBcAg suggests diminished establishment of HBV infection.¹³

Table 2.

Median effective concentration (EC₅₀) of compounds in HBV-infected HepG2-NTCP cells.

Compound	HBeAg EC50 (nM)	HBcAg EC50 (nM)
GLP-26	872 ± 64	1,282 ± 902
GLS-4	804 ± 45	153 ± 8
6e D-CAM-14	98 ± 12	67 ± 53
IFN-α	85 ± 17	320 ± 67

HepG2-NTCP cells received serial dilutions of compound treatments 24 hr before, during, and after HBV infection. Levels of secreted HBeAg in supernatant were assessed 7 days post-infection by chemiluminescent immunoassays. HBcAg was assessed 7 days post-infection by immunofluorescence. All conditions were performed in triplicate.

Effect of 6e/D-CAM-14 on HBV parameters in HBV-infected HepG2-NTCP cells - Post infection data

When dosed into HepG2-NTCP cells already replicating HBV (i.e., 4 days post HBV inoculation and post cccDNA establishment), which represents a more relevant context for treatment evaluation, 6e/D-CAM-14, in short-term dosing schedule (i.e., four consecutive days of treatment), had improved antiviral properties as compared to parental GLP-26 (Figure 3), with respective EC₅₀ for HBV intracellular DNA synthesis, HBeAg biogenesis, and viremia of 7.81 nM, 3,130 nM, 1.3 nM (Table 3; Figure 3A).. 6e/D-CAM-14 was 10-fold more potent than GLP-26 at inhibiting HBV virion production (i.e., for virosuppression) and HBeAg biogenesis. However, both drugs were quite inefficient at inducing HBc protein degradation within cells, when a harsh protein extraction procedure was used to prepare Western blotting samples (Figure 3B).

Figure 3. Comparative anti-HBV properties of 6e/D-CAM-14 and GLP-26 in HBV infected HepG2-NTCP cells - post infection data.

HepG2-NTCP cells were infected at a multiplicity of infection of 500 vge/cell. Four days post-infection, cells were treated with the two drugs at the indicated concentrations each day for four consecutive days, using freshly prepared drug dilutions. (A) At day-8 post infection (day-4 post onset of treatment), total DNA was extracted from cells and subjected to HBV qPCR (left panel), HBeAg was quantified by CLIA in supernatant (middle panel), HBV DNA was purified from supernatant and subjected to HBV qPCR (right panel). (B) Proteins were also extracted from cells and subjected to Western blot analysis using an anti-HBc antibody; representative blots are shown. Results are from 2 independent experiments. For intracellular analyses, six biological replicates per condition were analyzed. For extracellular analyses 12 biological repeats/condition were analyzed.

Table 3:

Effect of 6e/D-CAM-14 on intracellular HBV DNA, HBeAg and viremia HepG2-NTCP cells already replicating HBV

Compound	Intracellular HBV DNA EC50 (nM)	HBeAg EC50 (nM)	Viremia EC50 (nM)
GLP-26	26.98 (range: 8.30 – 88.03)	32,150 (range: 12,300 – 131,000)	12.67 (range: 9.471 – 16.89)
6e D-CAM-14	7.81 (range 2.71 – 23.42)	3,132 (range 2,152 – 4,808)	1.30 (range 1.06 – 1.68)

Biophysical and structural characterization of monomeric CAM GLP-26 compared to D-CAM 6e/D-CAM-14

- In vitro assembly of Cp149 dimers in the presence of GLP-26 induces moderate structural changes, similar to other CAM-E

Dimers of HBc are the building blocks of the HBV capsid. We triggered self-assembly of Cp149 dimers (subunits) by mixing them with ammonium acetate (200 mM) at a final dimer concentration of 30 μM, either without CAM or with CAM at a CAM-to-dimer molar ratio ρ of 3 (see Methods). After incubation, samples were analyzed by mass photometry, SAXS, electron microscopy and cryo-electron microscopy. Figure 4 compares the results for assembly without CAM and with GLP-26. Because of experimental constraints, mass photometry was performed after dilution to a final concentration of 0.18 μM of subunits. The mass distributions were broadly similar, with a capsid-mass peak at around 4,000 kDa (expected mass for a T = 4 Cp149 capsid: 4042 kDa) and a distribution of smaller objects peaking at 150–200 kDa. There were differences, though: With GLP-26, the capsid-sized peak was both broader and shifted towards higher molecular masses. It was also smaller (27% of capsid-sized events vs 57% without CAM), suggesting that capsid-sized objects were either less abundant or less stable to dilution when formed with GLP-26. SAXS analysis of the undiluted samples (Figure 4–B) showed that the latter was the likely explanation: Intensity at zero angle I(0), that should be roughly proportional to the concentration of capsid-sized objects, was larger with GLP-26 than without (1.86 cm⁻¹ compared to 1.54 cm⁻¹), suggesting a similar concentration of slightly larger objects with GLP-26. The resulting SAXS curves demonstrated a commonality among all assembly conditions: The initial oscillation occurred at q = 0.022 Å-1, indicating that most of the large objects formed were the size and shape of T = 4 capsids. In the presence of ρ = 3 for GLP-26 during assembly, the resulting SAXS curve (blue) closely resembled the curve of capsids formed under normal conditions (black curve), although the oscillations were more subdued. This confirms the mass photometry observations that with GLP-26 the capsid-like particles formed are more heterogeneous. These conclusions were further confirmed by EM analysis of samples at a concentration of 6 μM of subunits (Figure 4–C): In the presence of GLP-26, slightly larger capsids and open capsids were observed. Finally, as in our previous work, ¹⁴ all SAXS curves overlapped at large q values (Figure 1-B, pink area), indicating that the presence of GLP-26 molecules during assembly produced no significant change in the local organization of the capsid-like objects.

Figure 4: Cp149 capsid assembly in the presence of ρ = 3 for GLP-26.

(A) Mass photometry measurements of the mass distribution of objects formed without (left) or with (right) GLP-26 at modulator-to-dimer molar ratio ρ of 3. Samples assembled at 30 μM of subunits were diluted to 0.18 μM for measurements. The capsid-sized peak of Figure 5A, right, was used for mass calibration (see below Fig. 5 as well as methods and text for details). (B) Experimental SAXS curves of capsids formed without (ρ = 0, black curve) or with ρ = 3 for GLP-26 (blue curve). The result for ρ = 3 of D-CAM 6e/D-CAM-14 is also indicated (red curve) and the area marked in pink highlights the region where all SAXS curves are superimposed. (C) Micrographs of capsids assembled in the presence or absence of GLP-26 and observed by negative-staining electron microscopy. Left, assembly without modulator; right, assembly with 90 μM GLP-26. Scale bars are equal to 50 nm. (D) Cryo-EM 2D classification of particles. 30 μM of Cp149 subunits were assembled in the presence of GLP-26 and 200 mM ammonium acetate, then concentrated 11-fold. Box size is 50 nm.

To get a finer view of the effects of GLP-26 on the sizes and shapes of the final capsids formed, we used cryo-electron microscopy. For this method experimental constraints led us to concentrate the sample prior to deposition on grids (see methods). The results obtained for this condition are presented in Figure 4-D. The 2D classification analysis revealed that in the presence of ρ = 3 for GLP-26 (90 μM), the assembly resulted in the formation of three distinct sizes of objects. These included a minority population that appeared to be T = 3 capsids (1.8%), a majority of objects that was very similar to T = 4 capsids formed in the absence of modulators (81.3%), and a third population of larger objects with an ellipsoidal appearance, accounting for 17.5% of the total objects. The 2D averages do not allow for distinguishing between closed and open capsids but were consistent with most of the objects being either closed or missing only a few subunits. These observations were similar to a previous characterization of a compound classified as CAM-E, JNJ-632.¹⁴

Altogether, these results indicated that GLP-26 behaved as a CAM-E modulator. Indeed, even at high concentration and stoichiometry of GLP-26, most objects formed were morphologically similar to the T = 4 capsids of HBV, but with some moderate structural changes that are very apparent in a minority of objects.

- Assembly in the presence of 6e/D-CAM-14 of two populations of capsid-like objects, one a perfect T = 4 capsid

We next applied the same analysis to self-assembly of Cp149 dimers in the presence of ρ = 3 of 6e/D-CAM-14 (Figure 5). As before, we verified the shape of the objects formed by negative staining EM. Notably, the number of capsids observed on the grids was significantly reduced when CAM was added (after dilution to 6 μM of subunits, Figure 5-C). This reduction in the number of capsids on grids was reproducibly observed. This cannot be explained solely by a reduced number of capsids formed during assembly in the presence of 6e/D-CAM-14, as shown by our SAXS data of the undiluted sample, which are very similar to these without CAM (Figure 5-B). For instance, the I(0) value measured from the SAXS curves is 1.32 cm⁻¹ with 6e/D-CAM-14 compared to 1.54 cm⁻¹ without CAM. Thus, a sub-population of capsid-like objects formed in the presence of 6e/D-CAM-14 dissociates upon dilution to 6 μM of subunits before deposition and/or upon negative staining. We checked by mass photometry samples formed with or without 6e/D-CAM-14 and then diluted to 0.18 μM of dimeric subunits. Similarly to GLP-26, there were two peaks with one of capsid size, but in contrast to GLP-26, the peak of capsid-mass objects for 6e/D-CAM-14 was narrower than without CAM (Figure 5-A). This is consistent with the EM images where all capsids found were of the same size and without any apparent defects (Figure 5-C). Thus, we performed subsequent cryo-EM analyses of capsids assembled from 30 μM subunits in the presence of 90 μM 6e/D-CAM-14 on a higher-end microscope. Single-particle analysis showed that approximately 45% of the capsids displayed defects (Figure 5-D). They were removed from the final 3D reconstruction. The remaining particles were very homogeneous (Figure 5-E), and 3D reconstruction with icosahedral symmetry yielded a 3.3 Å resolution map (Figure 5-F). We directly fit an atomic model of the Cp183 capsid-like particle previously obtained by cryoEM.¹⁵ Thus the icosahedral capsids formed in the presence of high concentrations of 6e/D-CAM-14 are not inflated as was reported with other CAM-bound capsids.¹⁶ A careful examination of our map did not reveal any signs of density not accounted for by the protein model. Focused asymmetric refinement of fivefold and quasi-sixfold symmetry axes, including the CAM binding pockets, was also unsuccessful in locating 6e/D-CAM-14 in this reconstruction. A possible explanation is that the homogeneous T=4 capsids belong to an unliganded subpopulation, while the 6e/D-CAM-14 bound capsids were excluded from the reconstruction because they are heterogeneous and/or prone to dissociation.

Figure 5: Cp149 capsid assembly in the presence of ρ = 3 for 6e/D-CAM-14.

(A) Mass photometry measurements of the mass distribution of objects formed in the absence (left) or in the presence (right) of 6e/D-CAM-14 with modulator-to-subunit (-dimer) molar ratio ρ of 3. Samples were diluted to 0.18 μM of subunit during measurements. (B) Experimental SAXS curves of capsids formed with ρ = 0 (black curve) or with ρ = 3 for 6e/D-CAM-14 (red curve). -The blue curve corresponds to compound GLP-26 (see above Fig. 4). The area marked in pink highlights the region where all SAXS curves are superimposed. (C) Micrographs of capsids assembled in the presence or absence of 90 μM (ρ = 3) 6e/D-CAM-14 and observed by negative-staining electron microscopy. 30 μM of capsids were assembled in the absence of modulators (left) or in the presence of 6e/D-CAM-14 (right). Scale bars are equal to 50 nm. (D) Cryo-EM real-space slices after 3D classification (‘heterogeneous refinement’) of the capsid-sized particles present in the ice. The particles partition into a major class without apparent defects and classes with visible defects (indicated by black arrows). (E) 2D classification of particles. Box size is 50 nm. (F) The cryo-EM map of the Cp149 capsid at 3.3 Å from the major class of (D). The black scale bar is 10 nm.

- GLP-26 accelerates HBV capsid assembly kinetics while 6e/D-CAM-14 does not

To gain a better insight into modulators’ effects on HBV capsids, we performed time-resolved SAXS experiments to investigate their effects on the capsid assembly kinetics.

Thirty μM of Cp149 dimers were assembled with 200 mM of ammonium acetate at 37°C. Following TR-SAXS data analysis, we found the ${〈R_g〉}_w$ value plateaued to 176 Å, with an associated time scale of 237 ± 62 ms, and the average number of aggregation at equilibrium ${\langle N\rangle }_w^{\infty }$ value plateaued to 156, above the expected value of 120 dimers for T = 4 capsids, with an associated time scale of 699 ± 138 ms (Table 4).

Table 4:

Cp149 capsid assembly kinetics monitored by time-resolved SAXS in the presence of ρ = 3 for GLP-26 and 6e/D-CAM-14.

	ρ = 0	GLP-26 ρ = 3	6e ρ = 3	JNJ-632 ρ = 3
${〈R_g〉}_w$	176 ± 6 Å	180 ± 1Å	142 ± 0.3 Å	176 ± 0.5 Å
Associated timescale	237 ± 62 ms	116 ± 63 ms	131 ± 21 ms	100 ± 24 ms
${\langle N\rangle }_w^{\infty }$	156 ± 6	190 ± 11	126 ± 3	150 ± 4
Associated timescale	699 ± 138 ms	487 ± 178 ms	633 ± 77 ms	273 ± 46 ms
Subunit binding energies −Δg	9.0 kT	10.4 kT	9.5 kT	15.8 kT

Characteristic values and associated time scales of the mean radius of gyration ${〈{\mathrm{R}}_{\mathrm{g}}〉}_{\mathrm{w}}$ and of the mean aggregation number ${〈\mathrm{N}〉}_{\mathrm{w}}$ for capsid assembly from 30 μM subunits in the absence or in the presence of ρ = 3 of GLP-26, 6e/D-CAM-14 or JNJ-632 and 200 mM ammonium acetate. Timescales and limit values were obtained by fitting an exponential decay function to the curves of Figures SI-1, SI-2 and SI-3. Binding energies in thermal energy unit – k being the Boltzmann constant and T the temperature – were extracted by kinetic modeling of the TR-SAXS data as in our previous work.¹⁴

During the assembly of 30 μM of Cp149 dimers, with 200 mM of ammonium acetate and 90 μM of GLP-26 at 37°C, the formation of objects was observed, and an oscillation at q = 0.023 Å⁻¹ was detected. The value of $q$ was the same as that seen during the formation of T = 4 capsids in assemblies without modulators. The SAXS curves of the capsid formation kinetics under this condition are shown in Figure SI-1, and Figures SI-2 and SI-3 show the evolution of ${〈R_g〉}_w$ and ${〈N〉}_w$ during the assembly of capsids formed in this condition. With GLP-26 at ρ = 3 during assembly, three of these four characteristic figures are significantly different: The radius of gyration ${〈R_g〉}_w$ and average number of aggregation ${\langle N\rangle }_w^{\infty }$ plateaued to 180 Å and 190 dimers, respectively, showing that the objects formed are larger on average than regular T=4 capsids. The characteristic time scale for ${〈N〉}_w$, 487 ± 178 ms was shorter, and the time scale for ${〈R_g〉}_w$, 108 ± 66 ms, was also shorter. Thus, we concluded that this molecule accelerates the assembly kinetics.

Next, 30 μM of Cp149 dimers were assembled with 200 mM of ammonium acetate in the presence of 90 μM of 6e/D-CAM-14 at 37ºC. The SAXS curves of the capsid formation kinetics under this condition are shown in Figure SI-2. Objects had formed in the presence of 90 μM of 6e/D-CAM-14, and a first oscillation at q = 0.023 Å⁻¹ was observed. Figures S3.3 and S3.4 show the evolution of ${〈R_g〉}_w$ and ${〈N〉}_w$ during the assembly of capsids formed in this condition. In the presence of 6e/D-CAM-14 at ρ = 3, Table 1 shows that the time scale for ${〈R_g〉}_w$ was shorter (131 ± 21 ms), but the time scale for ${\langle N\rangle }_w^{\infty }$ was similar (633 ± 77 ms). Therefore, the overall capsid formation was not accelerated. The objects formed appeared slightly smaller. ${〈R_g〉}_w$ plateauing to 141 Å vs 176 Å without CAMs, but the number of subunits ${\langle N\rangle }_w^{\infty }$ plateaued at 126, close to the expected value for T=4 capsids.

Finally, kinetic modeling from our TR-SAXS data allowed us to determine the subunit binding energies during capsid assembly (Table 3). In the absence of modulators, the subunit binding energy was equal to 9 kT, where kT stands for thermal energy with k being the Boltzmann constant and T the temperature. In the presence of 6e/D-CAM-14, the subunit binding energy was equal to 9.5 kT, and to 10.4 kT in the presence of GLP-26 during assembly, indicating that the latter significantly enhances the attractive interaction between subunits.

To compare to another well-characterized CAM, we performed the same TR-SAXS analysis in the same conditions with the CAM-E JNJ-632 (Figure SI-3). Although the effects are slightly less pronouncedfor GLP-26, both JNJ-632 and GLP-26 behave as expected for an HBV Core assembly modulator, by both accelerating capsid assembly and increasing subunit binding energies. In sharp contrast, 6e/D-CAM-14 does neither of these.

Taken together, our TR-SAXS, EM, and mass photometry data suggest an unusual behavior of 6e/D-CAM-14: The compound did not appear to accelerate the self-assembly of Cp149, but seems to promote the formation of two types of capsid-like objects: one consisting of well-formed, stable capsids that are indistinguishable from HBV capsids even at near-atomic resolution but do not seem to have the CAM bound, and another of capsid-sized, but easily dissociated, objects that are presumably the capsid-inhibitor complexes.

Effect of 6e/D-CAM-14 on HBc distribution (HepAD38)

The effect of 6e/D-CAM-14 on the cellular localization of HBc proteins at different concentrations was determined by confocal microscopy in HepAD38 cells (Figure 6). At lower concentration, dimer 6e/D-CAM-14 had only a minor effect on the nucleocytoplasmic distribution of HBc in HepAD38 cells. However, by increasing the concentration of 6e/D-CAM-14, we observed a clear decrease in the levels of HBc inside the nucleus, an effect similar to that observed with GLP-26.

Figure 6:

Nucleo-cytoplasmic distribution of HBc in HepAD38. A, HepAD38 cells were seeded in a tetracycline-free medium with 1% DMSO. Cells were observed following incubation with various 6e/D-CAM-14 concentrations or 5 μM GLP-26. B, Fraction of capsids present in the nucleus relative to the total detected signal within cells, expressed as a percentage. (>300 cells per condition)

Effect of 6e/D-CAM-14 on capsid morphology

To observe changes in the hydrodynamic radius of pre-assembled HBV capsids upon incubation with GLP-26 and 6e/D-CAM-14, dynamic light scattering (DLS) experiments were conducted. Capsids made of the HBV Cp149 protein were first assembled overnight in 1 M NaCl. In the presence of a 1% DMSO control, the average detectable radius of particles was 24.6 nm (Table 5), similar to the expected T=4 capsid radius of ~18 nm. In the presence of 20 μM GLP-26, there was an apparent increase in detectable particle size, as the average radius was 53.0 nm (Table 5). This could be a result of aggregation of Cp149 protomers and/or assembled particles due to the CAM-E mechanism of GLP-26, which promotes capsid assembly. Meanwhile, administering 20 μM of 6e/D-CAM-14 significantly increased particle size, averaging 214 nm in particle radius upon treatment (Table 5). In both treatment conditions, slight inconsistencies remained among repeat DLS readouts, with select acquisitions reporting either low or high particle radii.

Table 5.

DLS measurements of preassembled HBV capsid radii upon CAM treatments.

	HBV Capsids + 1% DMSO	HBV Capsids + 20 μM GLP-26	HBV Capsids + 20 μM 6e/D-CAM-14
Radius (nM)	24.6 ± 3.4	53.0 ± 13.6	214 ± 61.3
PD (%)	32.3 ± 18.6	26.8 ± 9.8	Multimodal distribution

Results for overnight-assembled HBV capsids (7.5 μM Cp149 protein) that were subsequently incubated with 1% DMSO control (left), 20 μM GLP-26 (middle), or 20 μM 6e/D-CAM-14 (right) for 30 min. PD = polydispersity.

At the same time, there were differences in sample polydispersity between the GLP-26 and 6e/D-CAM-14 treatments. While a vast majority of acquisitions produced both radius and polydispersity values for GLP-26 conditions, 6e/D-CAM-14 treatment led to “multimodal” polydispersity output in most of the acquisitions. This multimodality could have resulted from a heterogeneous sample in which 6e/D-CAM-14 treatment produced distinct subpopulations irreconcilable by DLS alone.

Upon noticing differences in the resulting capsid radii between CAM treatments, we consulted negative-staining electron microscopy (EM) to visualize these readouts. We used the HBV C150 construct for negative staining EM of pre-assembled capsids treated with DMSO, GLP-26, or 6e/D-CAM-14. Images of pre-assembled capsids (40 μM HBV C150 protein) treated with 20 μM GLP-26 or 10 μM 6e/D-CAM-14 were consistent with micrographs of capsid assembly in the presence of the CAMs (Figures 4C and 5C). GLP-26 treatment resulted in a subset of capsids having altered curvatures, but images of 6e/D-CAM-14-treated capsids showed a decrease in the number of detectable particles and an increase in background noise overall (Figure 7). Given that pre-assembled cores with the hyper-stable C150 construct still disappeared after incubation with the D-CAM for 30 min, this strengthens the idea that 6e/D-CAM-14 does not act identically to GLP-26, for it can uniquely “dissolve” capsids.

Figure 7. TEM analysis of pre-assembled HBV capsids with various CAMs.

Capsids assembled overnight (40 μM HBV C150 protein) were incubated in the presence of (A) 1% DMSO control, (C) 20 μM GLP-26, or (E) 10 μM 6e/D-CAM-14 for 30 min. Negative stain images were taken at 57kX (left column) and 92kX (right column) magnifications. Scale bars for A, C, and E are 200 nm; scale bars for B, D, and F are 100 nm.

Molecular modeling – Discussion

As shown in Figure 2, we hypothesized that if the capsids were intact after the binding of one of the GLP moiety present on dimer 6e/D-CAM-14, it could either lead to the binding of two capsids together (Inter-capsid connection) (Figure 2C) or the formation of a “staple” within the same capsid (Intra-capsid connection) (Figure 2D).

HBV capsids consist of core proteins (Cp) that assemble first into dimers, yielding ultimately particles with T = 4 (120 dimers) and T = 3 (90 dimers) icosahedral symmetries; T = 4 capsids represent the predominant form in vivo.

To evaluate either hypothesis (inter or intra capsid linkage), we first generated the biological unit of a wild-type HBV core protein capsid (T = 4) from the reported cryo-EM structure (PDB ID:6HTX) of HBV core protein dimers, using the protein preparation wizard in Schrodinger Suite 2024–2. The capsid is formed with arrangement of four capsid protein conformations (A, B, C, D) in dimer form as shown in Figure 5. The binding mode of GLP-26 was then predicted using a previously reported approach. ¹⁷ The docked pose of GLP-26 between two dimers (AB-CD) was then transferred into the 2-fold symmetry of the capsid (T = 4). Because it has been established that most of CAMs bind at the interface between B and C molecules of neighboring AB and CD dimers and then between C and D molecules of two neighboring CD dimers, i.e., around the quasi-six-fold vertices at the two-fold symmetry axes, but not to the pentameric five-fold axes^{18, 19}, we incorporated GLP-26 in the two-fold symmetry. (Figure 8)

Figure 8. Dimer assembly of the Cp into icosahedral capsid shells.

A) an icosahedron with its defining 2-fold, 3-fold, and 5-fold symmetry axes. The arrangement of Cp dimers in T = 4 particles; B) A subunits (red) cluster around 5-fold axes, C (green) and D (cyan) subunits around 3-fold axes. The two B subunits (orange), two C subunits and two D subunits form 2-fold symmetry. C) The model on the right shows 2, 3 and 5-fold symmetry in a capsid (PDB:6HTX) with zoomed in 2-fold symmetry with docked GLP-26 (pink) between B and C, C and D molecules. The dotted black lines represent the distance in angstrom between the terminal carbon of the acetylene groups.

Hypothesis 1- Inter-capsid connection:

The crystal structure of WT HBV capsid showed that the surface is neither flat nor “smooth” and is instead covered with “spikes”. The distance between the binding site of a GLP-type of molecule and the top of these “spikes” can be estimated to be between 42 to 50 Å (Figure 9). Based on these basic measurements, it is estimated that the minimal distance between two binding sites on two different, completely formed, non-aberrant capsids is about 84 to 100 Å (Figure 9). Because the size of the linker in 6e/D-CAM-14 is about 18 to 20 Å, it is unlikely that 6e/D-CAM-14 can connect two intact capsids.

Figure 9. Hypothesis-1 −6e/D-CAM-14 binds between two intact capsids.

Left side: Two capsids at their minimum distance with the GLP-26 binding location. The zoomed in figure, with GLP-26 binding between A(red)-B(orange) and C(green)-D(cyan) dimers, shows that the estimated distance between the tip of the Cp spike and the alkyne group of GLP-26 is ~42–50 Å. For comparison, the lower figure shows the calculated length of the linker in 6e/D-CAM-14.

Hypothesis 2- Intra-capsid connection:

Since most of the known CAMs bind between B and C molecules first and then between C and D molecules of neighboring CD,^18,19 we hypothesized that, in the context of a connection within the same capsid (intra-), D-CAM-14 could bind in 3 different ways within the 2-fold symmetry (Figure 10). To study these three potential binding modes, GLP-26 molecules were initially kept tethered in their respective binding pockets and then linked in all three possible ways to form 6e/D-CAM-14 computationally. The three protein-D-CAM-14 complexes were then minimized and superimposed onto the protein-GLP-26 complex. By doing so, we were able to note that binding poses of the monomeric core of 6e/D-CAM-14, in all three hypothesized intra-capsid binding modes, remain similar to that of GLP-26, validating the possible staple effect of dimer 6e/D-CAM-14 (Figure 10).

Figure 10: Possible binding modes of 6e/D-CAM-14 in the 2-fold symmetry.

A) 6e/D-CAM-14 linking two BC interfaces; B) 6e/D-CAM-14 linking a BC interface and a neighboring CD interface; C) 6e/D-CAM-14 linking a BC interface and a CD interface from the opposite side.

Conclusions

As part of our ongoing HBV research program, we have designed a novel series of dimeric HBV CAMs (D-CAMs) susceptible of acting as an inter- or intra- capsid staple. Among them, D-CAM-14 exhibited better potency than its monomeric counterpart (GLP-26) against several markers of HBV replication, including HBV DNA, HBeAg and HBcAg. In addition, unlike GLP-26, D-CAM-14 leads to the formation of two populations of capsid-like objects and does not accelerate HBV capsid assembly kinetics. Moreover, DLS and EM experiments demonstrated that GLP-26 and D-CAM-14 had distinct impacts on capsid radii and the morphology of pre-assembled capsids. Based on their high potency and distinct effects on HBV capsid, when compared to existing CAM-E and CAM-A, D-CAMs represent a novel and intriguing class of HBV CAM. Although molecular modeling studies suggest the possibility of intra-capsid connections, further mechanistic studies are currently being conducted to understand how these dimers bind to the HBV capsids.

Experimental section

Chemistry

Reagents and solvents were purchased from commercial sources and were used as received without further purification unless otherwise noted. Unless otherwise stated, intermediates and final compounds were obtained from readily available commercial suppliers or synthesized by standard methods known to individuals skilled in the art of chemical synthesis. Intermediates and final compounds were purified by either flash chromatography or preparative TLC. Flash chromatography was performed using Teledyne ISCO combi-flash chromatography instrument model R_f-200. The silica gel cartridges were purchased from Agela Technologies Silica (CS) irregular 40–60 μm 60Å. Preparative TLC plates were purchased from Analtech, GF silica gel plates. ¹H, ¹³C, and ¹⁹F NMR spectra were taken on a Bruker AscendTM 400 MHz spectrometer at rt and reported in ppm downfield using residual solvent lines as an internal standard for ¹H and ¹³C NMR. No standard was used for ¹⁹F NMR spectra. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), m (multiplet). Mass spectra were determined on a Micromass Platform LC spectrometer using electrospray ionization. The purity of the final compounds evaluated is ≥ 95%, as determined by ¹H and ¹³C NMR and UPLC.

General procedure for the synthesis of diazido alkyl intermediates (2a-e). ²⁰

To a solution of compound 1a-e (2.5 g, 10.25 mmol, 1.0 eq) in DMF/H₂O (54 mL/ 6 mL) was added NaN₃ (1.3 g, 20.49 mmol, 2.0 eq). The resulting mixture was stirred at 90°C overnight and was quenched with cold H₂O (50 mL). The water layer was then extracted with EtOAc (2 × 100 mL). The organic layers were finally combined, washed with water, brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash chromatography (Hexane /Ethyl acetate 10:1 to 1:4) to afford product 2a-e (1.4 g, 82%).

2a: 1,6-diazidohexane. (1.41 g, 82%). ¹H NMR (400 MHz, CDCl₃) δ3.27 (d, J = 8.0 Hz, 4H),1.63–1.58 (m, 4H), 1.44–1.39 (m, 4H). 2b: 1,8-diazidooctane. (651 mg, 82%). ¹H NMR (400 MHz, CDCl₃) δ 3.26 (t, J = 8.0 Hz, 4H), 1.63–1.56 (m, 4H), 1.39–1.34 (m, 8H). 2c: 1,10-diazidodecane. (1.68 g, 90%). ¹H NMR (400 MHz, CDCl₃) δ 3.26 (t, J = 8.0 Hz, 4H), 1.63–1.56 (m, 4H), 1.38–1.30 (m, 12H). 2d: 1,12-diazidododecane. (993 mg, 86%). ¹H NMR (400 MHz, CDCl₃) δ 3.24 (t, J = 8.0 Hz, 4H), 1.62–1.55 (m, 4H), 1.38–1.28 (m, 16H). 2e: 1,14-diazidotetradecane. (559 mg, 71%). ¹H NMR (400 MHz, CDCl₃) δ 3.25 (t, J = 8.0 Hz, 4H), 1.63–1.56 (m, 4H), 1.38–1.27 (m, 20H).

General procedure for the synthesis of diazido alkyl intermediates (5a-b).

To a solution of compound 3a-b (2.5 g, 10.25 mmol, 1.0 eq) in CHCl₃ (54 mL) was added Et₃N (2.5 g, 10.25 mmol, 1.0 eq) at 0°C, then TsCl (1.3 g, 20.49 mmol, 2.0 eq) was added portion wise. The resulting mixture was stirred at r.t overnight and was quenched with cold saturated NaHCO₃ (50 mL). The water layer was then extracted with EtOAc (2 × 100 mL). The organic layers were finally combined, washed with water, brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to obtain crude 4a-b. To the above crude mixture of 4a-b in DMF was added NaN₃ (1.3 g, 20.49 mmol, 2.0 eq). The resulting mixture was stirred at 90–100°C overnight and was quenched with cold H₂O (50 mL). The water layer was then extracted with EtOAc (2 × 100 mL). The organic layers were finally combined, washed with water, brine, and dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash chromatography (Hexane /Ethyl acetate 10:1 to 1:4) to afford product 5a-b (1.4 g, 80–85%).

5a: 1,16-diazidohexadecane. (80 mg, 61%). ¹H NMR (400 MHz, CDCl₃) δ 3.20–3.14 (m, 4H), 1.56–1.49 (m, 4H), 1.29–1.19 (m, 24H). ¹³C NMR (101 MHz, CDCl₃) δ 51.5, 29.7, 29.6, 29.5, 29.4, 29.2, 28.8, 26.7. 5b: 1,18-diazidooctadecane. (1.4 g, 82%). ¹H NMR (400 MHz, CDCl₃) δ 3.27–3.24 (m, 4H), 1.62–1.57 (m, 4H), 1.38–1.26 (m, 28H). ¹³C NMR (101 MHz, CDCl₃) δ 51.5, 29.7, 29.7, 29.6, 29.6, 29.5, 29.2, 28.9, 26.7.

General procedure for the synthesis of D-CAMs (6a-g).

To a mixture of compound 2a-e or 5a-b (100 mg, 0.27 mmol, 1.0 eq) and GLP-26 (22.5 mg, 0.13 mmol, 0.5 eq) in H₂O/CH₃CN (3 mL/5 mL) was added CuSO₄.5H₂O (24 mg) and Na ascorbate (48 mg) under Ar atmosphere. The resulting mixture was stirred at 90°C for 8 h. The mixture was diluted with 40 mL of cold H₂O and extracted with ethyl acetate (3 × 50 mL). The organic layers were combined, washed with water, brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash chromatography (DCM/MeOH, from 100:1 to 5:1) to afford dimers 6a-g in 40–60% yield.

4,4’-(2,2’-(((Hexane-1,6-diylbis(1H-1,2,3-triazole-1,4-diyl))bis(methylene))bis(azanediyl))bis(2-oxoacetyl))bis(N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide) (6a).

¹H NMR (400 MHz, DMSO-d₆) δ 10.42 (s, 2H), 9.18 (t, J = 5.6 Hz, 2H), 7.99 (s, 2H), 7.90–7.84 (m, 2H), 7.46–7.38 (m, 4H), 4.44 (d, J = 5.6 Hz, 4H), 4.31 (t, J = 7.0 Hz, 4H), 3.58 (s, 6H), 2.18 (s, 6H), 2.34 (s, 6H). 1.77 (t, J = 6.6 Hz, 4H), 1.23 (s, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 188.1, 167.7, 160.5, 150.6, 148.2, 147.2, 144.7, 141.1, 136.3, 127.2, 123.6,122.8, 117.8, 116.6, 116.4, 109.1, 49.6, 34.3, 32.3, 30.0, 25.6, 11.8. ¹⁹F NMR (377 MHz, DMSO-d₆) δ −137.1 to −137.2 (m, 1 F), −144.2 to −144.4 (m, 1 F). HRMS (ESI) m/z calcd for C₄₄H₄₇F₄N₁₂O₆ [M+H]⁺: 915.3678; found 915.3663.

4,4’-(2,2’-(((Octane-1,8-diylbis(1H-1,2,3-triazole-1,4-diyl))bis(methylene))bis(azanediyl))bis(2-oxoacetyl))bis(N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide) (6b).

¹H NMR (400 MHz, CDCl₃) δ 8.87 (s, 2H), 8.09 (s, 2H), 7.75–7.70 (m, 2H), 7.61 (s, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.11–7.04 (m, 2H), 4.45 (s, 4H), 4.27 (t, J = 6.2 Hz, 4H), 3.57 (s, 6H), 2.25 (s, 6H), 2.15 (s, 6H), 8.09 (t, J = 6.6 Hz, 4H), 1.23–1.19 (m, 8H). ¹³C NMR (101 MHz, CDCl₃) δ 186.9, 166.0, 160.4, 151.2, 148.7, 148.1, 145.7, 141.8, 134.6, 126.4, 123.5, 122.6, 117.2, 115.8, 109.7, 53.4, 50.2, 34.7, 32.2, 29.9, 28.2, 25.8, 11.9. ¹⁹F NMR (377 MHz, CDCl₃) δ −135.9 to −136.0 (m, 1 F), −142.5 to −142.6 (m, 1 F). HRMS (ESI) m/z calcd for C₄₆H₅₁F₄N₁₂O₆ [M+H] ⁺: 943.3991; found 943.3978.

4,4’-(2,2’-(((Decane-1,10-diylbis(1H-1,2,3-triazole-1,4-diyl)) bis(methylene)) bis(azanediyl)) bis (2-oxoacetyl))bis(N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide) (6c).

¹H NMR (400 MHz, CDCl₃) δ 8.66 (s, 2H), 7.99 (s, 2H), 7.75–7.66 (m, 4H), 7.31–7.30 (m, 2H), 7.11–7.05 (m, 2H), 4.51 (s, 4H), 4.30 (s, 4H), 3.59 (s, 6H), 2.27 (s, 6H), 2.18 (s, 6H), 1.83 (s, 4H), 1.24–1.19 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 186.7, 165.6, 160.3, 151.2, 148.8, 148.2, 145.7, 141.7, 134.6, 126.4, 123.4, 117.3, 115.7, 109.6, 50.6, 34.6, 32.2, 29.9, 28.7, 28.5, 26.0, 12.0. ¹⁹F NMR (377 MHz, CDCl₃) δ −135.8 to −135.9 (m, 1 F), −142.5 to −142.6 (m, 1 F). HRMS (ESI) m/z calcd for C₄₈H₅₅F₄N₁₂O₆ [M+H] ⁺: 971.4304; found 971.4301.

4,4’-(2,2’-(((Dodecane-1,12-diylbis(1H-1,2,3-triazole-1,4-diyl)) bis (methylene)) bis(azanediyl)) bis(2-oxoacetyl))bis(N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide) (6d).

¹H NMR (400 MHz, DMSO-d₆) δ 10.41 (s, 2H), 9.19 (t, J = 8.0 Hz, 2H), 7.98 (s, 2H), 7.90–7.84 (m, 2H), 7.44–7.38 (m, 4H), 4.44 (d, J = 4.0 Hz, 4H), 4.32 (t, J = 8.0 Hz, 4H), 3.36 (s, 6H), 2.35 (s, 6H), 2.15 (s, 6H). 1.77 (t, J = 6.0 Hz, 4H), 1.18 (s, 16H). ¹³C NMR (101 MHz, DMSO-d₆) δ 188.1, 167.7, 160.5, 150.8, 148.2, 147.1, 144.7, 144.1, 141.1, 136.4, 127.2, 123.5, 122.7, 117.9, 116.4, 109.0, 49.7, 34.3, 32.3, 30.3, 29.4, 29.3, 28.8, 26.3, 11.8. ¹⁹F NMR (377 MHz, CDCl₃) δ −137.2 to −137.3 (m, 1 F), −144.3 to −144.4 (m, 1 F). HRMS (ESI) m/z calcd for C₅₀H₅₉F₄N₁₂O₆ [M+H] ⁺: 999.4617; found 999.4622.

4,4’-(2,2’-(((Tetradecane-1,14-diylbis(1H-1,2,3-triazole-1,4-diyl))bis(methylene)) bis(azanediyl)) bis(2-oxoacetyl))bis(N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide) (6e).

¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 2H), 7.76–7.71 (m, 2H), 7.57 (s, 2H), 7.54 (t, J = 5.9 Hz, 2H), 7.24–7.22 (m, 2H), 7.22–7.09 (m, 2H), 4.56 (d, J = 5.9 Hz, 4H), 4.32 (t, J = 7.2 Hz, 4H), 3.67 (s, 6H), 2.34 (s, 6H), 2.29 (s, 6H), 1.87 (t, J = 7.2 Hz, 4H), 1.29–1.22 (m, 20H).¹³C NMR (101 MHz, DMSO-d₆) δ 186.8, 165.5, 160.3, 151.3, 148.8, 148.2, 145.8, 143.8, 141.8, 134.6, 134.5, 126.4, 123.5, 122.4, 117.5, 117.1, 115.8, 109.8, 50.5, 34.8, 32.3, 30.2, 29.3, 29.2, 28.9, 26.4, 12.0. ¹⁹F NMR (377 MHz, DMSO-d₆) δ −137.1 to −137.3 (m, 1 F), −144.3 to −144.4 (m, 1 F). HRMS (ESI) m/z calcd for C₅₂H₆₃F₄N₁₂O₆ [M+H] ⁺: 1027.4930; found 1027.4932.

4,4’-(2,2’-(((Hexadecane-1,16-diylbis(1H-1,2,3-triazole-1,4-diyl))bis(methylene)) bis(azanediyl)) bis(2-oxoacetyl))bis(N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide) (6f).

¹H NMR (400 MHz, DMSO-d₆) δ 10.40 (s, 2H), 9.18 (t, J = 5.7 Hz, 2H), 7.97 (s, 2H), 7.88–7.83 (m, 2H), 7.43–7.39 (m, 4H), 4.42 (d, J = 5.7 Hz, 2H), 4.32 (t, J = 6.9 Hz, 4H), 3.57 (s, 6H), 2.34 (s, 6H), 2.13 (s, 6H), 1.76 (t, J = 6.9 Hz, 4H), 1.19–1.16 (m, 26H). ¹³C NMR (101 MHz, DMSO-d₆) δ 187.6, 167.2, 160.1, 150.2, 147.7, 146.7, 144.3, 143.6, 140.7, 135.9, 126.7, 123.1, 122.2, 117.4, 116.3, 115.9, 108.7, 108.4, 49.2, 33.8, 31.8, 29.8, 29.0, 28.9, 28.8, 28.4, 25.8, 11.8. ¹⁹F NMR (377 MHz, DMSO-d₆) δ −137.1 to −137.3 (m, 1 F), −144.3 to −144.4 (m, 1 F). HRMS (ESI) m/z calcd for C₅₄H₆₇F₄N₁₂O₆ [M+H] ⁺: 1055.5243; found 1055.5245.

4,4’-(2,2’-(((Octadecane-1,18-diylbis(1H-1,2,3-triazole-1,4-diyl))bis(methylene)) bis(azanediyl)) bis(2-oxoacetyl))bis(N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide) (6g).

¹H NMR (400 MHz, DMSO-d₆) δ 10.40 (s, 2H), 9.18 (t, J = 5.7 Hz, 2H), 7.97 (s, 2H), 7.89–7.83 (m, 2H), 7.44–7.37 (m, 4H), 4.43 (d, J = 5.7 Hz, 4H), 4.32 (t, J = 7.0 Hz, 4H), 3.57 (s, 6H), 2.35 (s, 6H), 2.14 (s, 6H), 1.76 (t, J = 7.0 Hz, 4H), 1.24–1.20 (m, 28H). ¹³C NMR (101 MHz, DMSO-d₆) δ 188.1, 167.7, 160.5, 150.6, 148.2, 147.2, 144.7, 144.1, 141.1, 136.4, 127.2, 123.5, 122.7, 117.9, 116.7, 116.4, 109.0, 49.7, 34.2, 32.3, 30.3, 29.5, 29.4, 29.3, 28.8, 26.3, 11.8. ¹⁹F NMR (377 MHz, DMSO-d₆) δ −137.2 to −137.3 (m, 1 F), −144.3 to −144.4 (m, 1 F). HRMS (ESI) m/z calcd for C₅₆H₇₁F₄N₁₂O₆ [M+H] ⁺: 1083.5556; found 1083.5555.

4-(2-(((1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoacetyl)-N-(3,4-difluorophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (7).

To a mixture of NaN₃ (100 mg, 0.27 mmol, 1.0 eq) and GLP-26 (22.5 mg, 0.13 mmol, 0.5 eq) in H₂O/CH₃CN (3 mL/5 mL) was added CuSO₄.5H₂O (24 mg) and Na ascorbate (48 mg) under an argon atmosphere. The resulting mixture was stirred at 90°C for 8 h. The mixture was diluted with 40 mL of cold H₂O and extracted with ethyl acetate (3 × 50 mL). The organic layers were combined, washed with water, brine, and dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash chromatography (DCM/MeOH, from 100:1 to 5:1) to afford product 7 (62 mg, 51%). ¹H NMR (400 MHz, MeOH-d₄) δ 7.82–7.77 (m, 2H), 7.36–7.34 (m, 1H), 7.29–7.22 (m, 1H), 4.64 (s, 2H), 3.65 (s, 3H), 2.41 (s, 3H), 2.29 (s, 3H). ¹³C NMR (101 MHz, MeOH-d₄) δ 187.2, 168.1, 161.2, 151.0, 148.6, 148.0, 145.5, 142.2, 141.9, 135.2, 126.8, 123.5, 116.9, 116.6, 160.0, 109.3, 33.5, 31.1,10.5. ¹⁹F NMR (377 MHz, MeOH-d₄) δ −138.9 to −139.0 (m, 1 F), −145.5 to −145.6 (m, 1 F). HRMS (ESI): m/z [M+1] ⁺ calcd for C₁₉H₁₉F₂N₆O₃: 417.1487, found 417.1475.

1-azidododecane (9). ²¹

To a solution of 1-bromododecane 8 (2.5 g, 10.25 mmol, 1.0 eq) in DMF/H₂O (54 mL/ 6 mL) was added NaN₃ (1.3 g, 20.49 mmol, 2.0 eq). The resulting mixture was stirred at 90°C overnight and was quenched with cold H₂O (50 mL). The water layer was then extracted with EtOAc (2 × 100 mL). The organic layers were finally combined, washed with water, brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash chromatography (Hexane /Ethyl acetate 10:1 to 1:4) to afford product 9 (1.4 g, 82%). ¹H NMR (400 MHz, DMSO-d₆) δ 3.33 (d, J = 6.8 Hz, 4H), 1.58–1.51 (m, 4H), 1.39–1.33 (m, 4H).

N-(3,4-difluorophenyl)-4-(2-(((1-dodecyl-1H-1,2,3-triazol-4-yl) methyl) amino)-2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (10).

To a mixture of compound 9 (100 mg, 0.27 mmol, 1.0 eq) and GLP-26 (22.5 mg, 0.13 mmol, 0.5 eq) in H₂O/CH₃CN (3 mL/5 mL) was added CuSO₄.5H₂O (24 mg) and Na ascorbate (48 mg) under Ar atmosphere. The resulting mixture was stirred at 90°C for 8 h. The mixture was diluted with 40 mL of cold H₂O and extracted with ethyl acetate (3 × 50 mL). The organic layers were combined, washed with water, brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash chromatography (DCM/MeOH, from 100:1 to 5:1) to afford product 10 (62 mg, 51%). ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 7.78–7.72 (m, 2H), 7.58 (s, 1H), 7.31–7.28 (m, 1H), 7.14–7.08 (m, 1H), 4.50 (d, J = 5.9 Hz, 2H), 4.30 (t, J = 7.3 Hz, 2H), 3.64 (s, 3H), 2.31 (s, 3H), 2.25 (s, 3H), 1.86 (t, J = 7.0 Hz, 2H), 1.30–1.24 (m, 18H), 0.89–0.86 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 186.8, 165.3, 160.3, 151.3, 148.8, 148.2, 145.8, 143.7, 141.8, 134.5, 126.3, 123.5, 122.4, 117.5, 117.2, 115.6, 109.7, 50.5, 34.8, 32.3, 31.9, 30.3, 29.5, 29.3, 29.0, 26.5, 22.7, 14.1, 12.1.¹⁹F NMR (377 MHz, DMSO-d₆) δ −135.7 to −135.8 (m, 1 F), −142.4 to −142.5 (m, 1 F). HRMS (ESI) m/z calcd for C₃₁H₄₃F₂N₆O₃ [M+H] ⁺: 585.3365; found 585.3366.

Anti-HBV activity in HepAd38

The HBV 7-day assay was performed in HepAD38 wild type (HepAD38) cells as previously described. ²² Briefly, HepAD38 cells were seeded onto 96-well plates and incubated for two days at 37°C in a humidified 5% CO₂ atmosphere. On day two, medium was removed and cells were washed with 1X phosphate buffer saline (PBS). Forty mM stock solutions of the compounds were prepared for the assay. The desired aliquot of the solution was diluted in medium without tetracycline and added in duplicate at various concentrations to the wells. On day seven, total DNA was extracted using DNeasy 96 Tissue kit (Qiagen), and HBV DNA was amplified by RT-PCR. ²³ Antiviral activity was measured by determining the average threshold cycle for the HBV amplification of the compounds (alone or in combination), which was subtracted from the average cycle of the untreated-tetracycline control (ΔCT). Drugs were first tested individually for effective concentration, which inhibited 50% and 90% of HBV DNA replication (EC₅₀ and EC₉₀) using CalcuSyn software program (Biosoft, Cambridge, UK).

Cytotoxicity assays.

Cytotoxicity was determined by using the CellTiter 96 non-radioactive cell proliferation colorimetric assay (Promega) in human peripheral blood mononuclear (PBM) cells and in human T lymphoblast (CEM), African green monkey kidney (Vero), and human hepatocellular carcinoma (HepG2) cells. Toxicity levels were measured as the concentration of test compound that inhibited cell growth by 50% (CC₅₀) as determined by the CalcuSyn software program (Biosoft, Cambridge, UK).

Anti-HBV activity in HBV-infected HepG2-NTCP

HepG2-NTCP cells were engineered from HepG2 cells by lentivectors expressing NTCP (gene coding the sodium taurocholate co-transporting peptide) and handled as previously described. ²⁴ They are cultivated in DMEM medium (Gibco), supplemented with FBS (10%), glutamine, sodium pyruvate, non-essential amino acids, and antibiotics, at 37°C in a humidified 5% CO₂ atmosphere. These cells can be infected with HBV produced by HepAD38 cells;²² infection is usually made at a multiplicity of infection of 100 to 500 virus-genome-equivalent/cell in 80% confluent HepG2-NTCP cells. One day prior infection, the medium is supplemented with 2% DMSO to stop cell division and allow experiments to be conducted over the next two weeks of culture.²⁴ Treatment with drugs can be done either before the virus is inoculated to cells or after infection. Intracellular and extracellular HBV DNA were extracted with silica column (from Qiagen or Machereay Nagel, following provider’s instructions) and were quantified by qPCR using specific HBV primers (forward primer: 5’-ACCGAATGTTGCCCAAGGTC-3’; reverse primer: 5’-TATGCCTCAAGGTCGGTCGT-3’). HBeAg was quantified by ELISA or CLIA (Autobio). HBc protein was detected in Western blotting experiments following SDS-PAGE and transfer onto a nitrocellulose membrane using a rabbit polyclonal antibody (a generous gift from Pr. Adam Zlotnick, Indiana University).

Biophysical and structural characterization Sample preparation

Expression of Cp149 Capsids.

Hepatitis B virus capsids were expressed in E. coli as previously described. In summary, pRSF-T7-HBc149 opt plasmid-transfected E. Coli BL21*Codon Plus cells were cultivated at 37°C overnight on an LB-agar plate supplemented with 50 mg.ml⁻¹ of kanamycin and 34 mg.ml⁻¹ of chloramphenicol. After that, a single colony was introduced into 2 X 10 mL of LB medium and cultivated overnights at 37°C. Subsequently, it was diluted into 2 × 500 mL of LB medium with the same antibiotic concentration and incubated for 5 h at 37°C. When the OD₆₀₀ reached 0.8, the induction was done by adding 1 mM IPTG, and the bacterial cells were then grown at 25°C overnight.

Purification of Cp149 Capsids.

To obtain the Cp149 capsids, the bacterial cells were subjected to centrifugation, and the pellets were resuspended in lysis buffer containing 300 mM NaCl, 2 mM DTT, 50 mM Tris-HCl pH 7.5, 1 g.L⁻¹ of lysozyme, 0.5 % Triton-X-100, and a protease inhibitor cocktail. The cells were mixed on ice for 1 h and then treated with 4 μl of commercial Pierce nuclease at room temperature for 45 min. Before centrifugation, the cells were lysed by sonication. After centrifugation, the supernatant was placed on a 10 % to 60 % sucrose gradient and centrifuged for three hr at 4°C using a SW-32 Ti rotor (Beckman, France). After centrifugation, a 16% SDS-PAGE gel was used to identify the fractions containing Cp149 capsids. Then, ammonium sulfate was added gradually until 40% saturation was obtained to precipitate the capsids. The solution was incubated at room temperature for an hr and on ice for two hr before centrifugation. To get rid of the insoluble pellet, the pellet was resuspended in a purification buffer containing 5% sucrose, 1 MM DTT, 50 mM Tris-HCl, pH 7.5 and centrifuged. The purified Cp149 capsids were stored at 4°C.

Purification of Cp149 Subunits.

Cp149 subunits were purified using previously described methods. After a dialysis at 4°C overnight in the disassembly buffer containing 1 mM DTT, and 50 mM CHES pH 9, the Cp149 capsids were dissociated by adding solid urea up to 3 M. Then, the subunits were purified by size exclusion chromatography (SEC), using a Superdex S200 increase GL pre-equilibrated with the disassembly buffer. The subunits obtained from the SEC were then dialyzed in the assembly buffer, which contained 500 mM NaCl, 1 mM DTT, and 50 mM Tris-HCl at pH 7.5. The reassembled capsids were purified using size exclusion chromatography, to remove inactive subunits. A final dissociation-purification step was performed, and the purified assembly-competent subunits were stored at −80°C.

Expression of HBV C150 protein.

An HBV C150 expression plasmid was generated as previously described,² encoding the HBV capsid protein assembly domain (amino acids 1–149) with an attached C-terminal cysteine.³ Plasmid was transformed into BL21 DE3 RIL E. coli cells. Protein expression and purification were performed as detailed prior, with several moderations.^{2, 4} At 37°C, cultures were grown to an OD₆₀₀ of ~0.8 and induced for 3 h with 1 mM IPTG. Cells were then pelleted at 4,000 RCF for 10 min.

Purification of HBV C150 protein.

Cell pellets were resuspended in 50 mM Tris (pH 7.5), 1 mM EDTA, and 10 mM β-mercaptoethanol (BME). 150 μg/mL lysozyme and 1 mM PMSF were then added, and suspension was incubated on ice for 30 min with swirling every 5 min. 0.2 mg/mL DNase I and 10 mM MgCl₂ were added, followed by incubation on ice for 30 min with swirling every 5 min. Cells were lysed by sonication on ice for 6 min (15 s on, 15 s off). Polyethyleneimine (PEI) was added to a 0.15% (w/v) concentration to precipitate DNA, and lysate was centrifuged at 17,000 RCF for 1 hr at 4°C. After isolating supernatant, ammonium sulfate was added to 40% (v/v) saturation. Solution was rocked for 1 h at 4°C and centrifuged at 17,000 RCF for 1 h at 4°C. Pellet was resuspended in Buffer A (100 mM Tris [pH 7.5], 100 mM NaCl, 5 mM BME) to an A₂₈₀ of ~6–10 mg/mL. Suspension was centrifuged at 17,000 RCF for 20 min at 4°C. Supernatant was isolated and filtered through a cheese cloth and 0.2 μM syringe filter tip. Sample was loaded onto a Buffer A-equilibrated HiLoad 26/60 Superdex 200 pg column (GE Healthcare) and eluted at 2 mL/min. Protein-containing fractions were pooled per chromatogram readout and concentrated to ~2–4 mg/mL. At 4°C, sample was twice dialyzed into Buffer N (50 mM sodium bicarbonate [pH 9.6], 10 mM BME), first for 3 hr and then overnight. Solid urea was added to 3 M to dissociate intact capsids, and solution was kept on ice for 1 h with swirling every 10 min. Sample was filtered through a 0.2 μM syringe filter tip, loaded onto a Buffer N-equilibrated HiLoad 26/60 Superdex 200 pg column, and eluted at 2 mL/min. Protein-containing fractions were pooled per chromatogram readout, concentrated, and stored at −80°C.

Protein concentrations (A₂₈₀) were determined spectrophotometrically (ε: 29.45 M⁻¹cm⁻¹; MW: 16.78 kDa). SDS-PAGE was conducted after each centrifugation and chromatography step to confirm presence of the HBV C150 protein in the intended fractions.

Mass photometry measurements

Mass photometry studies were performed using a TwoMP system from Refeyn. Mass calibrations were done by generating standard curves using BSA (66 kDa), immunoglobulins (150 kDa & 300 kDa), and Cp149 capsids assembled from Cp149 subunits in the presence of 200 mM ammonium acetate and 6e/D-CAM-14 (4042 kDa). The latter choice was made as all data point to the capsid-mass peak in this sample being made of complete T = 4 capsids. The masses for the capsid peaks indicated on Figures 5 and 6 are given under this assumption. Before measurements, all samples were diluted to a dimer concentration of 0.72 μM (6 nM of capsids). Then, all samples were run by setting focus with 15 μL of the appropriate buffer and then adding 5 μL of the sample for data collection. The final concentration during measurement was thus equal to 0.18 μM of dimers. Data were collected using AcquireMP software and analyzed using DiscoverMP software.

Time-resolved and static Small-Angle X-ray Scattering

Before any measurements, Cp149 subunits were incubated for 1 hr with 3 M of solid urea and then desalted in a buffer containing 1 mM DTT and 50 mM Tris-HCl pH 7.5 for 2 hr.

Time-resolved small-angle X-ray scattering experiments were conducted at the ID02 beamline of the ESRF synchrotron facility, with the sample-to-detector distance set to 2 m, momentum transfer q ranging from 3.42 × 10⁻² to 3.83 nm⁻¹ were obtained. Subunits, the assembly buffer composed of 200 mM AmAc, and the molecules at a modulators-to-subunit ratio equal to 3 were mixed into a 1 mm quartz capillary at 37°C to initiate assembly using a stopped-flow mixer (BioLogic SFM-400). The beam exposure time was set to 5 ms.

Static Small-angle X-ray scattering measurements were performed at the SWING beamline of the SOLEIL synchrotron facility. Subunits were manually mixed with 500 mM AmAc in the presence of modulators to prepare the samples for static small-angle X-ray scattering measurements. The samples were then incubated and stored at 4°C for two to three days. During measurements, the sample-to-detector distance was set to 2.5 m and the samples were injected using an autosampler into a quartz capillary cell with a 1.5 mm diameter. A heat bath was used to keep the temperature at 37°C during measurements.

The software available on Foxtrot at SWING and SAXSUtilities2 at ID02 was used to radially average two-dimensional images into one-dimensional scattering profiles. The contribution of buffer solutions was subtracted and intensities were converted into absolute units. AUTORG and PRIMUS from the ATSAS suite were used to determine the forward scattering intensity $I_0=I\left(q\to 0\right)$ and the radius of gyration $R_{\text{g}}$ – or the mean (weight-averaged) radius of gyration ${⟨R_{\text{g}}⟩}_{\text{w}}$ for mixtures – with the condition $q{⟨R_{\text{g}}⟩}_{\text{w}}<1.3$ defining the Guinier region. $I_0$ was used to estimate the mean aggregation number ${〈N〉}_{\text{w}}$ corresponding to the weight-averaged number of subunits per object:

$${⟨N⟩}_{\text{w}}\left(t\right)=\frac{c^{\text{S}}}{c}\frac{I_0\left(t\right)}{I_0^{\text{S}}}$$ (4)

where $I_0^{\mathrm{S}}$ stands for the forward scattering intensity of subunits measured separately at a molar concentration $c^{\text{S}}$, while $c$ is the initial molar concentration of subunits in an assembly experiment.

Negative staining electron microscopy

Capsids deposited on grids were assembled at 1 mg/mL (30 μM) and diluted extemporaneously to a concentration of 0.2 mg/mL. After ionizing the Formvar/Carbon film S162–3 300 mesh copper grids, 5 μl of the capsid-containing solution was deposited on the grid for 5 min. The excess solution was then removed using filter paper, and a drop of a previously filtered 1% ammonium molybdate solution was deposited for 45 seconds on the grid. The excess contrast agent was removed using filter paper. Then, a JEOL JEM-2010 microscope was used to observe the grids at 2 μm defocus and a magnification of ×50,000. Images were recorded with a GATAN Ultrascan 4K CCD camera and processed using the ImageJ analysis software.

To assess the effect of compounds on pre-assembled capsids, assembled capsids (40 μM HBV C150) with CAMs (20 μM GLP-26 or 10 μM 6e/D-CAM-14) were incubated for 30 min at room temperature in 100 mM HEPES (pH 7.5) and 1% DMSO. Carbon Film 400-Mesh Copper grids (Electron Microscopy Services) were glow discharged (15 mA, 15 s, atmospheric conditions). 4 μL per reaction were added to grids, incubated for 5 min, and blotted off. 3 μL of uranyl acetate solution were then used to stain grids for 5 min and blotted off. Stained grids were left to dry for 30 min at room temperature. Grids were imaged using an LVEM 25E Electron Microscope (Delong Instruments).

Cryo-Electron Microscopy

Assembly was triggered manually by mixing 30 μM (1 mg/ml) of Cp149 dimers in a buffer composed of 1 mM DTT and 20 mM Tris HCl at pH 7.5 in the presence or absence of modulators (90 μM – i.e., ρ = 3 for GLP-26 and 6e/D-CAM-14) and 200 mM ammonium acetate. The latter salt concentration was found to be necessary for capsid viability during cryofixation. Assembled capsids were then incubated at 37°C for 1 h and concentrated ten times using an Amicon tube with a 100 kDa cut-off, to facilitate a faster collection of an abundance of single particles during image processing. An FEI Vitrobot was used to freeze the grids. For the assembly in the presence of GLP-26, 4 μL of solution was deposited onto glow-discharged Quantifoil holey*carbon grids (R2/2). The grids were blotted with filter paper for 2 sec at room temperature with a blot force of 5, before being automatically submerged in liquid nitrogen-cooled ethane. The grids were then stored in liquid nitrogen until use. The frozen samples were transferred into a Gatan 626 cryoholder, and imaging was carried out at −180 °C on a JEOL JEM-2010 microscope equipped with a 200-kV field emission gun. The samples were imaged with a magnification of ×50,000 using a minimal dose system. Images were recorded with a Gatan Ultrascan 4K CCD camera at 2 μm of nominal defocus. For the assembly in the presence of 6e/D-CAM-14, 4 μL of solution was deposited onto glow-discharged Quantifoil holey*carbon grids (R 1.2/1.3). The grids were blotted with filter paper for 4.5 seconds at 4°C with a blot force of 2 before being automatically submerged in liquid nitrogen-cooled ethane. Imaging of the frozen samples was performed on a Glacios microscope equipped with a 200-kV field-emission gun. The samples were imaged with a magnification of ×150,000 using a minimal dose system (total dose 45 e⁻/ Ų). Two thousand eighteen images were recorded with a Falcon 3EC camera in linear mode with defoci in the range 0.6–3 μm.

Single Particle Analysis

Particle picking, 2D classification, and 3D reconstructions were carried out using standard CryoSPARC software protocols (Punjani et al., 2017). After motion correction and CTF estimation, particles were manually selected and extracted, and a 2D classification was performed. 2D classes were used to select particles from the 2018 micrographs, and after particle inspection, a second round of 2D classification was performed, selecting particles from good classes. Ab initio 3D volume generation was followed by three successive rounds of heterogeneous refinement. Homogeneous refinement with icosahedral symmetry of 55% of T = 4 capsid-sized particles in the best class yielded a 3.7 Å resolution reconstruction. CTF refinement improved the reconstruction to 3.3 Å resolution

Dynamic Light Scattering (DLS)

Aliquots of HBV Cp149 protein were incubated at room temperature overnight in an equal volume of assembly buffer (100 mM HEPES [pH 7.5], 1 M NaCl) in darkened conditions. Additional assembly buffer was filtered using a 0.02 μM syringe filter tip. Reactions of assembled capsids (7.5 μM HBV Cp149) and CAMs (20 μM GLP-26, 20 μM 6e/D-CAM-14) in filtered buffer and 1% DMSO were incubated for 30 min at room temperature. Reactions were plated in a 384-Well Sensoplate^™ Glass Bottom Plate (Greiner Bio-One). A DynaPro^™ Plate Reader III (Wyatt Technology) was used to generate DLS readings, and data were obtained using the DYNAMICS^™ analysis software v7.8.0.26 (Wyatt Technology). Each condition was done in repeated wells with multiple acquisitions per well. Based on sample heterogeneity, scanning times varied as automatically determined by the plate reader.

Confocal microscopy in HepAD38 cells

The compounds are prepared at a 100X concentration in DMSO and the treatment media contained 1% of the compound by volume in DMEM (supplemented with FBS and Pen/Strep) to achieve the displayed concentrations. HepAD38 cells expressing HBV were incubated with the compounds 24 hr after seeding. The experiment was stopped at 72 h by a 4% PFA fixation. Control GLP-26 was used at 5 μM. The images were acquired using the software Las X by Leica, on a Leica SP8 laser scanning confocal microscope. The cells were illuminated by a white light laser (405 nm, i.e., DAPI) and an argon laser (488 nm wavelength, i.e., HBc) passing through a 63X apochromatic oil objective. Patchworks of 4×4 pictures corresponding to 660 × 660 μm fields of view were used for bioinformatic analyses with Fiji. The areas of HBc contained inside nuclei were measured and normalized by the nuclei areas. Final values correspond to the proportion of nuclei overlapping with the labelled HBc: we call it here Nuclear HBc (%). More than 300 cells per condition were analysed.

Supplementary Material

The Supporting Information is available free of charge at XXX

General supplements containing Figure SI-1 to SI-3, NMR spectra, HRMS and UPLC traces for compounds 6a-g, 7 and 10

GLP-26 bound to the full HBV capsid (PDB)

Molecular string file for all the final compounds (CSV)

Abbreviations

CAM

capsid assembly modulator

CAM-A

CAM-aberrant

CAM-E

CAM-empty

cccDNA

covalently closed circular DNA

D-CAMs

dimeric capsid assembly modulators

DCM

dichloromethane

DLS

dynamic light scattering

DMEM

Dulbecco’s modified Eagle’s medium

DMF

dimethylformamide

DTT

dithiothreitol

EC₅₀

half-maximal effective concentration

EtOAc

ethyl acetate

FBS

fetal bovine serum

HBcAg

hepatitis B core antigen

HBeAg

hepatitis B envelope antigen

HBV

hepatitis B virus

hr

hour(s)

IC₅₀

half-maximal inhibitory concentration

IPTG

isopropyl β-D-1-thiogalactopyranoside

MeOH

methanol

μM

micromolar

nM

nanomolar

NaN₃

sodium azide

NTCP

sodium taurocholate cotransporting polypeptide

pgRNA

pregenomic RNA

PMSF

phenylmethylsulfonyl fluoride

rt

room temperature

RT-PCR

reverse transcription-polymerase chain reaction

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TLC

thin-layer chromatography

TsCl

p-toluenesulfonyl chloride