Making the invisible visible: fast magic-angle-spinning NMR reveals the evasive hepatitis B virus capsid functional C-terminal domain

Experimentally determined protein structures often feature missing domains. One example is the C terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulated nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS is a game changer that allows to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain’s behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

Pharmacokinetics, safety and tolerability of single- and multiple-ascending doses of JNJ-64530440, a novel hepatitis B virus capsid assembly modulator, in healthy volunteers

Background Pharmacokinetics and safety of JNJ-64530440, a hepatitis B virus capsid assembly modulator producing normal empty capsids (CAM-N), in healthy volunteers were evaluated. Methods This Phase I study (NCT03439488) was a double-blind, randomised, placebo-controlled study. Adults ( n = 10/cohort, five Asian/five non-Asian), randomised 4:1, received single-ascending doses of oral JNJ-64530440 (first- and second-generation formulations) or placebo under fasted (50, 150, 300 and 900 mg) or fed (300, 750, 1,000, 2000 and 4000 mg) conditions. Multiple-ascending doses of 750 or 2000 mg once daily and 750 mg twice daily JNJ-64530440 (second-generation formulation) for 7 days were evaluated. Pharmacokinetic parameters were estimated from plasma concentrations. Safety was assessed throughout. Results Less than dose-proportional increases in maximum plasma concentrations (Cmax) and area under the plasma concentration–time curves (AUCs) were observed across the doses. Mean plasma half-lives ranged from 9.3 to 14.5 h. Cmax and AUC were ∼two fold higher under fed versus fasting conditions and slightly higher in Asians versus Caucasians. JNJ-64530440 doses ≥750 mg achieved plasma levels higher than protein-binding adjusted concentrations demonstrating in vitro antiviral activity. No serious adverse events (AEs), treatment discontinuations or dose-limiting toxicities were seen. AE frequency/severity did not increase with dose. Conclusions Single (up to 4000 mg) and multiple doses (up to 2000 mg for 7 days) of JNJ-64530440 were well tolerated in healthy volunteers. Multiple doses ≥750 mg/day achieved plasma concentrations expected to have antiviral activity that may lower hepatitis B surface antigen. No clinically relevant differences in tolerability or pharmacokinetic parameters were seen between Asians versus Caucasians.