Plant-Produced Viral Nanoparticles as a Functionalized Catalytic Support for Metabolic Engineering.

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed.

A minimal PBPK model to accelerate preclinical development of drugs against tuberculosis.

Introduction: Understanding drug exposure at disease target sites is pivotal to profiling new drug candidates in terms of tolerability and efficacy. Such quantification is particularly tedious for anti-tuberculosis (TB) compounds as the heterogeneous pulmonary microenvironment due to the infection may alter lung permeability and affect drug disposition. Murine models have been a longstanding support in TB research so far and are here used as human surrogates to unveil the distribution of several anti-TB compounds at the site-of-action via a novel and centralized PBPK design framework. Methods: As an intermediate approach between data-driven pharmacokinetic (PK) models and whole-body physiologically based (PB) PK models, we propose a parsimonious framework for PK investigation (minimal PBPK approach) that retains key physiological processes involved in TB disease, while reducing computational costs and prior knowledge requirements. By lumping together pulmonary TB-unessential organs, our minimal PBPK model counts 9 equations compared to the 36 of published full models, accelerating the simulation more than 3-folds in Matlab 2022b. Results: The model has been successfully tested and validated against 11 anti-TB compounds-rifampicin, rifapentine, pyrazinamide, ethambutol, isoniazid, moxifloxacin, delamanid, pretomanid, bedaquiline, OPC-167832, GSK2556286 - showing robust predictability power in recapitulating PK dynamics in mice. Structural inspections on the proposed design have ensured global identifiability and listed free fraction in plasma and blood-to-plasma ratio as top sensitive parameters for PK metrics. The platform-oriented implementation allows fast comparison of the compounds in terms of exposure and target attainment. Discrepancies in plasma and lung levels for the latest BPaMZ and HPMZ regimens have been analyzed in terms of their impact on preclinical experiment design and on PK/PD indices. Conclusion: The framework we developed requires limited drug- and species-specific information to reconstruct accurate PK dynamics, delivering a unified viewpoint on anti-TB drug distribution at the site-of-action and a flexible fit-for-purpose tool to accelerate model-informed drug design pipelines and facilitate translation into the clinic.