Synthetic prodrug design enables biocatalytic activation in mice to elicit tumor growth suppression

Considering the intrinsic toxicities of transition metals, their incorporation into drug therapies must operate at minimal amounts while ensuring adequate catalytic activity within complex biological systems. As a way to address this issue, this study investigates the design of synthetic prodrugs that are not only tuned to be harmless, but can be robustly transformed in vivo to reach therapeutically relevant levels. To accomplish this, retrosynthetic prodrug design highlights the potential of naphthylcombretastatin-based prodrugs, which form highly active cytostatic agents via sequential ring-closing metathesis and aromatization. Structural adjustments will also be done to improve aspects related to catalytic reactivity, intrinsic bioactivity, and hydrolytic stability. The developed prodrug therapy is found to possess excellent anticancer activities in cell-based assays. Furthermore, in vivo activation by intravenously administered glycosylated artificial metalloenzymes can also induce significant reduction of implanted tumor growth in mice.

Designer Compartment for Artificial Metalloenzymes through Controlled Liquid-Liquid Phase Separation

Artificial metalloenzymes with different protein scaffolds, cofactors and functions have been prepared to expand the natural enzymatic repertoire with abiotic reactions. However, due to the sensitivity of metal centers toward various biomolecules, especially glutathione, low activity and turnover of artificial metalloenzymes in vivo are systematic problems not fully solved. Apart from straightforward routes such as the use of neutralizing agents, metal cofactors modification and directed evolution, one may notice that nature can create isolated microenvironments for diverse biological processes within cells. Following this way, here we report the in vivo assembly of artificial metalloenzymes based on HaloTag-SNAPTag fusion protein. These metalloenzymes have metal cofactors bound on protein interfaces, and can trigger liquid-liquid phase separation to form liquid condensates inside Escherichia coli. These condensates serve as membraneless, isolated compartments for artificial metalloenzymes to efficiently perform intracellular catalysis, mediating abiotic unmasking, coupling and polymerization reactions. The cellular compartmentalization also enables spatial control of reactions, either facilitating a cascade reaction within the confined spaces, or concurrent reactions with spatial separation. Such engineered Escherichia coli can work as whole-cell catalyst with confined metal species, colonizing at mice intestine to effect in vivo abiotic transformations with a lower chance of heavy metal poisoning. These results represent a systematic strategy to stabilize and potentiate artificial metalloenzyme in vivo, with potential applications in fields such as non-natural metabolism, fermentation and drug delivery.

Controlling the Optical and Catalytic Properties of Artificial Metalloenzyme Photocatalysts Using Chemogenetic Engineering

Visible light photocatalysis enables a broad range of organic transformations that proceed via single electron or energy transfer. Metal polypyridyl complexes are among the most commonly employed visible light photocatalysts. The photophysical properties of these complexes have been extensively studied and can be tuned by modifying the substituents on the pyridine ligands. On the other hand, ligand modifications that enable substrate binding to control reaction selectivity remain rare. Given the exquisite control that enzymes exert over electron and energy transfer processes in nature, we envisioned that artificial metalloenzymes (ArMs) created by incorporating Ru(II) polypyridyl complexes into a suitable protein scaffold could provide a means to control photocatalyst properties. This study describes approaches to create covalent and non-covalent ArMs from a variety of Ru(II) polypyridyl cofactors and a prolyl oligopeptidase scaffold. A panel of ArMs with enhanced photophysical properties were engineered, and the nature of the scaffold/cofactor interactions in these systems was investigated. These ArMs provided higher yields and rates than Ru(Bpy)32+ for the reductive cyclization of dienones and the [2+2] photocycloaddition between C-cinnamoyl imidazole and 4-methoxystyrene, suggesting that protein scaffolds could provide a means to improve the efficiency of visible light photocatalysts.

Artificial metalloenzymes in a nutshell: the quartet for efficient catalysis

Artificial metalloenzymes combine the inherent reactivity of transition metal catalysis with the sophisticated reaction control of natural enzymes. By providing new opportunities in bioorthogonal chemistry and biocatalysis, artificial metalloenzymes have the potential to overcome certain limitations in both drug discovery and green chemistry or related research fields. Ongoing advances in organometallic catalysis, directed evolution, and bioinformatics are enabling the design of increasingly powerful systems that outperform conventional catalysis in a growing number of cases. Therefore, this review article collects challenges and opportunities in designing artificial metalloenzymes described in recent review articles. This will provide an equitable insight for those new to and interested in the field.