A frontier area in the development of activatable (turn-on) fluorescence-based probes is that concerned with rapid and selective stimulus triggering of probe activation so as to allow for biomarker identification and cellular imaging. The work here is concerned with a cloaked fluorophore composed of a reporter whose fluorescence is efficiently quenched by it being bound to an activatable trigger group through a novel self-immolative linker. Highly selective and rapid activation of the trigger group is achieved by chemical and enzymatic means that result in activated trigger group detachment from the self-immolative linker, with the latter subsequently cleaved from the reporter autonomously, thereby unmasking intense, red-shifted fluorescence emission. To achieve this success, we used a trimethyl-locked quinone propionic acid trigger group and an N-methyl-p-aminobenzyl alcohol self-immolative linker attached to the reporter. Delineated here are the synthesis and characterization of this cloaked fluorophore and the evaluation of its triggered turning on in the presence of an up-regulated enzyme in human cancer cells, NAD(P)H:quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2).

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.

Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage‐derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.g., TLR7 agonists) have proven too toxic to administer systemically. Based on the expression of folate receptor β solely on activated myeloid cells, we have created a folate‐targeted TLR7 agonist (FA‐TLR7‐54) that selectively accumulates in profibrotic macrophages and suppresses fibrosis‐inducing cytokine production. We demonstrate that FA‐TLR7‐54 reprograms M2‐like fibrosis‐inducing macrophages into fibrosis‐suppressing macrophages, resulting in dramatic declines in profibrotic cytokine release, hydroxyproline biosynthesis, and collagen deposition, with concomitant increases in alveolar airspaces. Although nontargeted TLR7‐54 is lethal at fibrosis‐suppressing doses, FA‐TLR7‐54 halts fibrosis without evidence of toxicity. Taken together, FA‐TLR7‐54 is shown to constitute a novel and potent approach for treating fibrosis without causing dose‐limiting systemic toxicities.

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Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome. To develop a more effective therapy, we have exploited the fact that collagen-producing myofibroblasts express a membrane-spanning protein, fibroblast activation protein (FAP), that exhibits limited if any expression on other cell types. Because collagen-producing myofibroblasts are only found in fibrotic tissues, solid tumors, and healing wounds, FAP constitutes an excellent marker for targeted delivery of drugs to tissues undergoing pathologic fibrosis. We demonstrate here that a low–molecular weight FAP ligand can be used to deliver imaging and therapeutic agents selectively to FAP-expressing cells. Because induction of collagen synthesis is associated with phosphatidylinositol 3-kinase (PI3K) activation, we designed a FAP-targeted PI3K inhibitor that selectively targets FAP-expressing human IPF lung fibroblasts and potently inhibited collagen synthesis. Moreover, we showed that administration of the inhibitor in a mouse model of IPF inhibited PI3K activation in fibrotic lungs, suppressed production of hydroxyproline (major building block of collagen), reduced collagen deposition, and increased mouse survival. Collectively, these studies suggest that a FAP-targeted PI3K inhibitor might be promising for treating IPF.