New and better strategies to treat hepatitis B are urgently needed. Many people worldwide remain unvaccinated against the disease, leaving them vulnerable to infection and serious liver problems. Children are particularly at risk of developing long-term illness. Treatments exist to help manage the condition, but they can rarely cure it.

Hepatitis B virus is protected by a spiky shell called the capsid, made of HBc proteins. This structure is critical for survival, and therefore a promising therapeutic target. Current approaches rely on compounds disrupting the assembly or stability of this structure by binding onto the HBc protein. So far, most of these drug candidates target the same location at the base of the capsid’s spikes. Until now, other binding pockets on the capsid remained largely unexplored.

To investigate whether these sites could be potential drug targets, Khayenko et al. developed two types of molecules, peptides and geranyl dimers, that could theoretically attach to the capsid – the former at the tip of the spikes and the latter in their middle section. In vitro and in living cells, the compounds not only latched onto these sites, but caused HBc proteins to clump together, preventing the capsid from forming properly. A similar mode of action is observed with existing drug candidates that, however, all bind to the pocket at the base of the spikes.

These findings highlight alternative strategies for targeting hepatitis B. Future studies will need to determine how well these molecules work in clinical conditions, and whether they could complement or improve existing treatments.

The hepatitis B virus (HBV) infects the liver and can cause acute and chronic hepatitis. In childhood and infancy, the virus is particularly dangerous as the recovery rate among children is approximately 50%, while among infants infected through perinatal transmission, only 10% will naturally recover, the remainder will develop chronic infection (Thomas, 2019; Block et al., 2021). On the global scale, the most effective approach to address hepatitis B is through preventive treatment with vaccinations. However, the goals of achieving sufficient vaccination coverage and timely immunization have yet to be met (Thomas, 2019; Cox et al., 2020). Furthermore, vaccinations are ineffective for individuals who are already infected (Dienstag et al., 1982). With about 300 million chronic carriers and over 800,000 hepatitis-related yearly deaths, chronic hepatitis B is a global health problem (Block et al., 2021; World Health Organisation, 2022) that requires a solution.

Currently, there are two approved classes of medications for the treatment of chronic hepatitis B: nucleos(t)ide analogs (NAs) and interferon-α and its derivatives (IFN-α) (Hepatitis B Foundation, 2023; Jeng et al., 2023). NAs compete for binding with the natural nucleotide substrates, inhibiting the viral protein P in charge of the reverse transcription of the viral pre-genomic RNA (pgRNA) into HBV DNA (Menéndez-Arias et al., 2014) IFN-α serves as both an immunomodulator and immunostimulant, activating genes with diverse antiviral functions to target various steps of viral replication. Additionally, it indirectly suppresses HBV infection by modifying cell-mediated immunity (Liang et al., 2015).

Present treatments effectively suppress HBV replication, reduce liver inflammation, fibrosis, and the risk of cirrhosis and hepatocellular carcinoma (HCC), but IFN-α treatment is associated with significant adverse effects and NAs typically require long-term oral administration, often lifelong, as treatment discontinuation often results in viral rebound and disease recurrence in many patients. While current therapies manage the disease, a clinical cure is seldom achieved, and the risk of HCC, although reduced, remains (Jeng et al., 2023). Consequently, various classes of direct-acting antivirals and immunomodulatory therapies are currently under development, aiming to achieve a functional cure following a finite treatment duration (Cornberg et al., 2020).

New HBV antivirals capitalize on the enhanced understanding of the viral life cycle and can be categorized into several classes (Table 1): entry inhibitors that disrupt HBV entry into hepatocytes by blocking the binding to the sodium/taurocholate co-transporting polypeptide (NTCP) receptor (Yan et al., 2012). HBsAg inhibitors based on nucleic acid polymers that interfere with the production of HBV surface antigens, and viral gene repressors based on nucleases. Translation inhibitors based on small interfering RNAs or antisense oligonucleotides that silence HBV RNA, thereby decreasing the viral antigen production. Finally, the capsid assembly modulators (CAMs) target the hepatitis B core protein (HBc) that participates in multiple essential steps of the HBV life cycle (Jeng et al., 2023).

Among the direct acting antivirals that are in preclinical or clinical studies, a third are CAMs (Hepatitis B Foundation, 2023). Capsids are attractive targets due to the absence of human homologues for HBc and their involvement in crucial stages of the HBV life cycle, including nuclear entry, encapsulation of the pgRNA and polymerase, optional nuclear recycling to replenish the covalently closed circular DNA (cccDNA) pool, and eventual coating and secretion from infected cells (Jeng et al., 2023; Kim et al., 2021).

The capsid is composed of 120 units of HBc dimers, assembling into a T = 4 icosahedron. Within this structure, 60 asymmetric units are formed by four HBc monomers each, designated as A, B, C, and D, or AB dimers and CD dimers (Crowther et al., 1994). The ultrastructure formed by the HBc dimer reveals several binding pockets that can be exploited as potential targets for modulating protein activity (Figure 1).

Figure 1

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CAMs target the hydrophobic pocket at the HBc dimer–dimer interface, upon binding they strengthen the association energy between HBc-dimer subunits, thereby promoting capsid assembly, rather than inhibiting it (Venkatakrishnan et al., 2016; Stray et al., 2005). As a result, abnormal or empty capsids may form, sometimes accompanied by the aggregation of core proteins, consequently inhibiting HBV DNA replication. Additionally, CAMs can disrupt the disassembly of incoming virions and the intracellular recycling of capsids, thereby impeding the establishment and replenishment of cccDNA (Jeng et al., 2023; Kim et al., 2021; Zoulim et al., 2022; Schlicksup and Zlotnick, 2020).

Recently, a new potentially druggable site was discovered in the HBc dimer—a hydrophobic pocket formed at the base of the spike. This site was targeted by the detergent Triton X-100 (TX100), ultimately causing conformational alterations in the capsid structure (Lecoq et al., 2021; Makbul et al., 2021b). In addition to the spike base hydrophobic pocket, there is another less-explored interacting domain located on the spike tip of the HBc dimer. Previous studies have shown that peptides targeting the cleft on the spike tip reduced viral replication in a cell model, likely by interfering with viral assembly through modulation of the HBc interaction with the surface antigen (Böttcher et al., 1998). These two effector sites could serve as a foundation for the development of new types of HBc modulators and provide alternatives ways for controlling HBV infections.

In this study, we characterize and explore these alternative HBc binding pockets at the inner-dimer interface in the center and at the tips of capsid spikes (Lecoq et al., 2021; Makbul et al., 2021b; Makbul et al., 2021a), and unveil the HBc aggregating properties of a spike-binding dimeric peptide.

HBV capsid assembly modulation via the binding pockets on the HBc multimer ultrastructure represents a promising pharmacological strategy but until now only one site located on the HBc dimer–dimer interface was explored (Figure 1A; Kim et al., 2021; Zheng et al., 2023). We designed and synthesized bivalent binders specifically targeting two distinct regions of the HBV core protein (HBc)—the hydrophobic pocket at the dimer interface and the tips of the capsid spikes (Figure 1). These binders, designed to engage both sites with avidity enhanced affinity, were evaluated for their binding affinity and their effects on HBV capsids in both in vitro assays and living cell models.

Geranyl dimer targets the central hydrophobic pocket of HBc-dimers with micromolar affinity

Hydrophobic post-translational modifications, such as myristylation of the large hepatitis B virus surface protein (L-HBs), are essential for HBV infectivity and play a role in mediating viral assembly (Gripon et al., 1995). Additionally, farnesylation, another hydrophobic post-translational modification, is involved in the envelopment of hepatitis D virus (HDV) (Koh et al., 2015), which relies on the presence of HBV and its protein machinery for propagation. Recently, TX100, a nonionic surfactant sharing a similar hydrocarbon binding motif as the natural HBV post-translational modifications, was identified as a ligand of a distinct hydrophobic pocket in the center of HBc-dimers (Lecoq et al., 2021; Makbul et al., 2021b; Roseman et al., 2005).

Several of the pocket forming amino acids, such as K96 and 129-PPAY-132 (Rost et al., 2006) and the natural occurring point mutations HBcP5T, L60V, F97L, and P130T (; Le Pogam et al., 2000; Yuan and Shih, 2000; Yuan et al., 1999a; Ehata et al., 1992) are involved in the secretion of enveloped virions from the cell. These findings lead to the infectious HBV particles signal hypothesis where this hydrophobic pocket is involved in the regulation of the envelopment of nucleocapsids and thus could be an alternative druggable pocket to block virus envelopment (Roseman et al., 2005).

We reasoned that compounds mimicking the natural HBV/HDV compounds and sharing a hydrophobic motif similar to that of TX100 can prove to be potent binders of this key hydrophobic pocket. Therefore, we set out to test n-decyl-beta-d-maltopyranoside (DM) (1), geraniol (2), and its synthesized dimer (3), as the mimetics of myristic acid and farnesyl, respectively.

The isothermal calorimetric titration (ITC) of HBc capsids with DM (1) resolved micromolar affinity (K_D = 133 ± 38 µM) to all four hydrophobic pockets of HBc capsids’ asymmetric unit (N = 1.05 ± 0.1) (Figure 2B, Figure 2—figure supplement 1). ITC of geraniol with HBc showed a slightly enhanced micromolar affinity (K_D = 94 ± 8 µM) (Figure 2A and B, Supplementary file 1, Supplementary file 2 and Figure 2—figure supplement 1) and a stoichiometry of N = 1.01 ± 0.04, implying that all four hydrophobic pockets of the asymmetric unit are occupied simultaneously. To confirm geraniol’s binding to the capsids and resolve the molecular details of this interaction, we conducted cryo-EM of a mixture of HBc with excess of geraniol followed by single-particle analysis. This experiment resolved an additional density for geraniol in all four hydrophobic pockets within the asymmetric unit of HBc capsids (Figure 2D, Figure 2—figure supplement 2), confirming the thermodynamic binding data and further defining the underlying molecular interactions of the involved HBV residues P5, L60, K96, and F97.

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Encouraged by structural confirmation of geraniol binding to the central hydrophobic pocket, we designed and synthesized a dimeric version of geraniol potentially capable of simultaneous binding to the HBc dimer. We connected the two geranyl moieties with a polyethylene glycol (PEG) linker that could bridge the distance of 38 Å between the two opposing hydrophobic pockets (see Figure 2—figure supplement 3 for the design rationale). After synthesis, purification and mass spectrometric validation (Appendix 1) we determined the HBc capsid binding parameters of the geranyl dimer via ITC. The analysis suggested that the dimer engages with both HBc binding sites simultaneously, resulting, however, only in a moderately enhanced micromolar affinity of 63 ± 8 µM (Figure 2B).

Targeting the pocket of capsid spike tips with sub-micromolar affinity peptide dimers

Although geraniol and geranyl dimer displayed improved affinity to HBc and allowed structural insights on a binding pocket located at the center of HBc dimers, micromolar affinity is suboptimal for a functional compound. Therefore, we proceeded to explore another binding site located on the capsid spike tips formed by HBc dimers (Figure 1; Böttcher et al., 1998).

Earlier studies have shown that phage display-derived peptides were binding to the spike tips of recombinant HBc capsids. These peptides were also observed to disrupt the interaction between HBc and HBV’s surface protein, L-HBs (Wang et al., 1995). Recently, we have shown that these peptides MHRSLLGRMKGA (P1), GSLLGRMKGA (P2), and the core binding motif SLLGRM bind to wild-type (wt) and mutant HBc variants (P5T, L60V and F97L) with intermediate micromolar K_Ds of 26, 68, and 130 µM, respectively (Makbul et al., 2021a).

Here, we designed dimeric peptides with a PEG linker capable of bridging the distance of 50 Å between the capsid spikes, thus tailoring our binders for simultaneous binding of two HBc dimers (Figure 1B, Figure 2—figure supplement 3). The three distinct dimeric peptides, the minimal SLLGRM dimer (4), the P2 dimer (P2d) (5), and two P1 dimers (P1d) (6) and P1dC (7), were synthesized, purified, and validated using mass spectrometry (Appendix 1). Subsequently, their binding to the HBV capsid was evaluated through ITC (Figure 3A, Supplementary file 2, Figure 2—figure supplement 1, Figure 3—figure supplement 1). With a K_D value of 4.9 ± 0.7 µM, the SLLGRM-dimer (4) has the lowest affinity to HBc, followed by the P2-dimer (5) (K_D = 1.9 ± 0.4 µM). Finally, the P1-dimers (6) and (7) displayed sub-micromolar affinities of 312 nM and 420 nM. Thus, P1-, P2-, and SLLGRM-dimers show 83-, 36-, and 27-fold increased affinities compared to their monomeric counterparts.

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The significant increase in affinity of the P1-dimer over the monomer, by almost two orders of magnitude, may not be solely attributed to binding to two sites simultaneously. Once the P1-dimer binds, it can interact with up to four binding partners in its vicinity (Figure 3—figure supplement 2; Wynne et al., 1999). This may enable detachment and immediate reattachment to a nearby binding partner, further enhancing the local concentration and the overall binding strength of the P1 dimer.

Notably, while performing the ITC titrations we have noticed fast fluctuations of the heat signature baseline across the tested ligands (Figure 3, Figure 3—figure supplement 1), at least for the P1 dimer this phenomenon may be attributed to aggregation. To rule out any non-specific interactions caused by the PEG linker or handle for both the geranyl dimers as well as the P1/2 dimers, a scrambled dimeric peptide was used as a negative control. This scrambled peptide showed no detectable binding (Figure 3C), thereby confirming that the observed binding is specific to the designed peptide sequence and not influenced by the linker or other structural components.

To further substantiate and quantify a possible dimer-induced HBc aggregation, we next performed a turbidity assay (Zhao et al., 2016). We found that P1 dimer induces turbidity of a HBc solution already at 1:10 equivalents of HBc, whereas the P2 dimer was slightly less potent and the SLLGRM dimer did not induce turbidity at the same conditions and further required significantly higher concentrations ratio relative to HBc (Figure 3—figure supplement 3). To shed light on the seemingly sequence-specific aggregation properties of the different dimers, we analyzed the binding of 240-point mutated P1 peptide variants in array format (Figure 3D, Supplementary file 3). The analysis recapitulated our earlier resolved sequence requirement for HBc binding and substantiated that the minimal sequence SLLGRM is the major mediator of HBc binding. Importantly, it further indicates that the additional N-terminal residues in P1 sequence are neither conserved nor critically required for binding despite their importance in inducing HBc aggregation.

P1dC aggregates HBc in living HEK293 cells

The sub-micromolar affinity of the P1-dimer, along with its ability to induce capsid aggregation in vitro, prompted us to evaluate its effect on HBV core protein in living cells. To adapt the peptide for the intracellular delivery, we synthesized a C-terminally cysteinated version of P1-dimer, P1dC (7) (Figure 3A), and its scrambled counterpart scrP1dC scr(7), as well as a thiol-reactive polyarginine-based cell penetrating peptide (CPP), containing a cysteine, with a 5-thio-2-nitrobenzoic acid (TNB)-modified thiol (Figure 4A, Appendix 1). At the core of this intracellular delivery method is the in situ conjugation of the cargo molecule to a molar excess of a CPP via a disulfide bond, and the application of this reaction mix on living cells. The excess of the CPP over the active compound enable the reaction of CPP-thiols with the cellular surface, facilitating the penetration of the cargo-CPP conjugate. In turn, the disulfide bond between CPP and the cargo is reduced in the cytosol, separating the cargo from the CPP, allowing unhindered activity of the cargo molecule within the cell (Figure 4B; Schneider et al., 2021a; Schneider et al., 2021b).

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To verify that the P1dC performs similarly to P1-dimer,, we performed another ITC assay to determine the affinity of the compound to HBc. The ITC confirmed that P1dC has an affinity of 420 ± 38 nM, comparable to P1-dimer, while the scrambled peptide did not display binding to HBc (Figure 3, Figure 3—figure supplement 1, Supplementary file 2). Thereafter we transfected mammalian cells (HEK293) with a plasmid coding for HBc. The cells expressed the protein for 2 days and were then treated for 1 hour with the thiol-activated cell penetrating peptide and P1dC or the respective negative control peptide scrP1dC (Figure 4B). Afterward, the cells were immediately washed and fixed and HBc was visualized with anti-HBc antibody and a secondary DyLight650 conjugated antibody. Transfected but otherwise untreated cells showed a homogeneous distribution of recombinant HBc molecules in the nucleus and to a lesser extent in the cytoplasm (Figure 4C). Yet, upon administration of 10 µM of P1dC (7), we observed aggregates of HBc (in the form of large bright spots) within the cells (Figure 4C, Figure 4—figure supplement 1). At a concentration of 10 µM, the scrambled dimer scrP1dC did not induce aggregation and the distribution of HBc remained largely homogenous.

Our live cell experiments have corroborated our in vitro findings, providing us a visual proof of P1dC-mediated HBc aggregation in a living cell. Thus, the peptide dimer causes an aggregation that resembles the HAP induced aggregation of the core protein and, like CAMs, can be expected to have the potential to disrupt the HBV life cycle.

Cryo-EM confirms peptide-induced HBc aggregation

To affirm the capsid-aggregation property of our peptide dimers, we incubated solubilized purified capsid-like particles (CLPs, spherical capsid-like HBc multimers purified from Escherichia coli) with an excess of SLLGRM-dimer or P1dC, applied them on carbon grids, and imaged them using cryo-EM. The effect of peptide dimers on CLPs was already seen on the microscale cryo-EM images (Figure 5—figure supplements 1 and 2), with P1dC inducing large protein aggregates with multimicron diameter. The less potent SLLGRM-dimer also induced visible aggregation, although with smaller aggregate size, while geraniol-treated samples showed minimal aggregation, and the smallest observed aggregate sizes. In the nanoscale, we observed clumped CLPs (Figure 5—figure supplement 3) and resolved the binding of both peptide dimers to the spike tips (Figure 5, Figure 5—figure supplement 3). The densities corresponding to bound peptide-dimers in both EM-reconstructions have volumes, which can accommodate a peptide chain of approximately six amino acid residues (Figure 5, Figure 5—figure supplement 3).

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The asymmetric unit of HBc capsids (T = 4) is a tetramer consisting of an A/B- and C/D-dimer, which have slightly different 3D structures. Interestingly, the SLLGRM-dimer binds the A/B-dimer as well as the C/D-dimer, in contrast to the monomeric SLLGRM, which binds only to the tip of the C/D-dimer (Makbul et al., 2021a; Figure 5), in line with the multivalent binding and the higher affinity we measured with ITC. A possible mode of action of the peptide-dimer-induced aggregation is shown in Figure 1B.

The live cell experiments showed the formation of HBc aggregates upon incubation with P1dC. Yet, in live cells HBc may exist as a monomer, a dimer, a multimer, or a whole capsid; therefore, the observed aggregates were not necessarily formed by whole capsids. The cryo-EM experiment, however, provides confirmation that the peptide dimers have the capability to interact with complete CLPs, an important feature, which implies that the dimers have the potential to affect intact capsids upon cell infection.

In this study, we focused on the HBV core protein, a protein essential for HBV proliferation and virulence. We explored the druggability of two alternative, non-HAP, binding pockets on the HBc ultrastructure and developed synthetic dimers that target these pockets with sub-micromolar affinity, resulting in the aggregation of HBc.

DM and geraniol were selected as the water-soluble mimetics of the natural post-translational modifications of HBV/HDV components. We have demonstrated their ability to interact with the vital central hydrophobic pocket of HBV and showed a binding affinity improvement upon dimerization of the geraniol ligand. Higher affinity ligands may be developed into even more potent binders of the hydrophobic pocket using the outlined linker design, potentially exerting a pharmacological effect on HBV (Briday et al., 2022).

Earlier works demonstrated that point mutations within the HBc can significantly affect HBV infectivity, particularly through disruptions in HBc interactions (Yuan et al., 1999b; Bruss, 2007; Ponsel and Bruss, 2003; Koschel et al., 1999). These mutations at the base of the spike and the groove between spikes highlight the importance of these regions in viral replication. Using our structural knowledge of the capsid, particularly the distances between the spikes, we designed peptide dimers with the ability to simultaneously bind to neighboring spikes on the same capsid or attach to two distinct capsids (Figure 3—figure supplement 2). Our in vitro assays demonstrated that these peptide dimers display a robust affinity ranging from low micromolar to sub-micromolar levels (Figure 3 and Supplementary file 2). Specifically, the peptide dimer (P1dC) with sub-micromolar affinity (K_D = 420 ± 40 nM) is a promising candidate for a lead molecule for new a new class of CAMs. The peptide dimer, but not its scrambled dimeric counterpart, induced HBc aggregation in live mammalian cells expressing HBc. An effect resembling the aggregation was observed after a treatment with the classical CAM HAP (Wu et al., 2013).

An intriguing possibility would be that the spike and hydrophobic pocket interact or influence each other when binding different ligands. Molecular dynamics simulations reveal notable flexibility in HBV capsids, suggesting that structural asymmetry might impact ligand binding. Structural analysis of the HBV capsid shows that P2 peptide binding to the capsid spikes increases flexibility, while exerting a minimal impact on the underlying hydrophobic pocket. However, TX100 binding within the hydrophobic pocket influences the spike tips by flipping Phe97, whereas geraniols bound within the same pocket do not induce this change, indicating that this interaction is ligand-specific. Nevertheless, simultaneous application of spike binders and hydrophobic pocket ligands that modify spike conformation may offer valuable structural and functional insights into HBV capsids.

An intriguing possibility would be that the spike and the hydrophobic pocket could interact or exert an effect on each other upon binding various ligands. Molecular dynamics simulations revealed significant flexibility of the HBV capsids and suggested that structural asymmetry may affect ligand binding (Pavlova et al., 2018; Perilla et al., 2016; Pavlova et al., 2022). HBV capsid structural analysis showed that P2 peptide binding to the capsid spikes increases flexibility (Makbul et al., 2021a) while exerting a minimal impact on the hydrophobic pocket beneath. However, TX100 binding to the hydrophobic pocket affected the spike tips by flipping Phe97 (Makbul et al., 2021b) in the pocket, but the geraniols resolved within the hydrophobic pocket did not flip Phe97, thus suggesting that this cross-talk is ligand-specific. Nevertheless, a simultaneous application of spike binders and hydrophobic pocket ligands able to affect the spike conformation may provide valuable structural and functional insights into HBV capsids.

While our results are highly encouraging, application in complex organisms may require alternative delivery methods, investigation of HBV proliferation in infection models, and further study of immunogenicity and stability. Future studies should thoroughly assess the cytotoxic potential of peptide-induced HBc aggregation to determine any adverse effects at the cellular level, which will be crucial for evaluating the therapeutic potential of these compounds. Long-term cytotoxicity studies on cellular viability are essential to optimize these binders for clinical applications. Although our study targets two ligand-binding pockets on the capsid surface, a direct effect on HBV infectivity remains to be demonstrated. Prior mutational data, however, suggest that even minor perturbations, such as ligand binding, could mimic deleterious mutations and impair viral function. This study’s insights into the unexplored pharmacological potential of these binding pockets and the compounds targeting them may lead to the development of new agents that impact viral capsids. Unlike classical CAMs, the peptide dimers exhibit a different mechanism of action and may act synergistically with CAMs or other antivirals. Further biological investigations will clarify the antiviral potential, applicability, and potency of compounds targeting the non-HAP binding pockets.

Unless otherwise noted, all resins and reagents were purchased from IRIS biotechnologies or Carl Roth and used without further purification. All solvents were HPLC grade. All water-sensitive reactions were performed in anhydrous solvents under positive pressure of argon.

Experimental Cryo-EM data acquisition and image processing data has been deposited in PDB and EMDB under accession codes: HBc CLPs + Geraniol (2), PDB 8PWO, EMD-17996; HBc CLP+ SLLGRM dimer, PDB 8PX6, EMD-18001; HBc CLP + P1dC, PDB: 8PX3, EMD-18000.

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   ID 8PX6. Hepatitis B core protein with bound SLLGRM-dimer.

   https://www.rcsb.org/structure/8PX6
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   ID EMD-18000. Hepatitis B core protein with bound P1dC.

   https://www.ebi.ac.uk/emdb/EMD-18000

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Author details

1. Vladimir Khayenko

   1. Rudolf Virchow Center, Center for Integrative and Translational Bioimaging; University of Würzburg, Würzburg, Germany
   2. Biocenter, University of Würzburg, Würzburg, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Cihan Makbul

   Competing interests

   No competing interests declared

2. Cihan Makbul

   1. Rudolf Virchow Center, Center for Integrative and Translational Bioimaging; University of Würzburg, Würzburg, Germany
   2. Biocenter, University of Würzburg, Würzburg, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Vladimir Khayenko

   Competing interests

   No competing interests declared

3. Clemens Schulte

   1. Rudolf Virchow Center, Center for Integrative and Translational Bioimaging; University of Würzburg, Würzburg, Germany
   2. Biocenter, University of Würzburg, Würzburg, Germany

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Naomi Hemmelmann

   1. Rudolf Virchow Center, Center for Integrative and Translational Bioimaging; University of Würzburg, Würzburg, Germany
   2. Biocenter, University of Würzburg, Würzburg, Germany

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Sonja Kachler

   1. Rudolf Virchow Center, Center for Integrative and Translational Bioimaging; University of Würzburg, Würzburg, Germany
   2. Biocenter, University of Würzburg, Würzburg, Germany

   Contribution

   Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Bettina Böttcher

   1. Rudolf Virchow Center, Center for Integrative and Translational Bioimaging; University of Würzburg, Würzburg, Germany
   2. Biocenter, University of Würzburg, Würzburg, Germany

   Contribution

   Data curation, Software, Supervision, Visualization, Project administration, Writing – review and editing

   For correspondence

   bettina.boettcher@uni-wuerzburg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7962-4849

7. Hans Michael Maric

   1. Rudolf Virchow Center, Center for Integrative and Translational Bioimaging; University of Würzburg, Würzburg, Germany
   2. Biocenter, University of Würzburg, Würzburg, Germany

   Contribution

   Conceptualization, Supervision, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   Hans.Maric@virchow.uni-wuerzburg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2719-4752

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