NAD(P)H:quinone oxidoreductase type I (NQO1) is a target enzyme for triggered delivery of drugs at inflamed tissue and tumor sites, particularly those that challenge traditional therapies. Prodrugs, macromolecules, and molecular assemblies possessing trigger groups that can be cleaved by environmental stimuli are vehicles with the potential to yield active drug only at prescribed sites. Furthermore, quinone propionic acids (QPAs) covalently attached to prodrugs or liposome surfaces can be removed by application of a reductive trigger stimulus, such as that from NQO1; their rates of reductive activation should be tunable via QPA structure. We explored in detail the recombinant human NAD(P)H:quinone oxidoreductase type I (rhNQO1)-catalyzed NADH reduction of a family of substituted QPAs and obtained high precision kinetic parameters. It is found that small changes in QPA structure—in particular, single atom and function group substitutions on the quinone ring at R1—lead to significant impacts on the Michaelis constant (Km), maximum velocity (Vmax), catalytic constant (kcat), and catalytic efficiency (kcat/Km). Molecular docking simulations demonstrate that alterations in QPA structure result in large changes in QPA alignment and placement with respect to the flavin isoalloxazine ring in the active site of rhNQO1; a qualitative relationship exists between the kinetic parameters and the depth of QPA penetration into the rhNQO1 active site. From a quantitative perspective, a very good correlation is observed between log(kcat/Km) and the molecular-docking-derived distance between flavin hydride donor site and quinone hydride acceptor site in the QPAs, an observation that is in agreement with developing theories. The comprehensive kinetic and molecular modeling knowledge obtained for the interaction of recombinant human NQO1 with the quinone propionic acid analogues provides insight into the design and implementation of the QPA trigger groups for drug delivery applications.

Contents release from redox-responsive liposomes is anion specific. Liposomal contents release is initiated by contact of apposed liposome bilayers having in their outer leaflet 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), whose presence is due to redox-stimulated removal of a quinone propionic acid protecting group (Q) from Q-DOPE lipids. Contents release occurs upon the phase transition of DOPE from its lamellar liquid crystalline (Lα) to hexagonal-II inverted micelle (HII) phase. Contents release is slower in the presence of weakly hydrated chaotropic anions versus highly hydrated kosmotropic anions and is attributed to ion accumulation near the zwitterionic DOPE head groups, in turn altering head group hydration, as indicated by the Lα→HII phase transition temperature, TH, for DOPE. The results are significant, not only for mechanistic aspects of liposome contents release in DOPE-based systems, but also for drug delivery applications wherein exist at drug targeting sites variations in type and concentration of ions and neutral species.

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