Flavonol-Based Carbon Monoxide Delivery Molecule with Endoplasmic Reticulum, Mitochondria, And Lysosome Localization.

Light-triggered carbon monoxide (CO) delivery molecules are of significant current interest for evaluating the role of CO in biology and as potential therapeutics. Herein we report the first example of a metal free CO delivery molecule that can be tracked via confocal microscopy at low micromolar concentrations in cells prior to CO release. The NEt2-appended extended flavonol (4) localizes to the endoplasmic reticulum, mitochondria, and lysosomes. Subcellular localization of 4 results in CO-induced toxicity effects that are distinct as compared to a nonlocalized analog. Anti-inflammatory effects of 4, as measured by TNF-α suppression, occur at the nanomolar level in the absence of CO release, and are enhanced with visible-light-induced CO release. Overall, the highly trackable nature of 4 enables studies of the biological effects of both a localized flavonol and CO release at low micromolar to nanomolar concentrations.

Development of Triggerable, Trackable, and Targetable Carbon Monoxide Releasing Molecules.

Carbon monoxide (CO) is a gaseous signaling molecule produced in humans via the breakdown of heme in an O2-dependent reaction catalyzed by heme oxygenase enzymes. A long-lived species relative to other signaling molecules (e.g., NO, H2S), CO exerts its physiological effects via binding to low-valent transition metal centers in proteins and enzymes. Studies involving the administration of low doses of CO have shown its potential as a therapeutic agent to produce vasodilation, anti-inflammatory, antiapoptotic, and anticancer effects. In pursuit of developing tools to define better the role and therapeutic potential of CO, carbon monoxide releasing molecules (CORMs) were developed. To date, the vast majority of reported CORMs have been metal carbonyl complexes, with the most well-known being Ru2Cl4(CO)6 (CORM-2), Ru(CO)3Cl(glycinate) (CORM-3), and Mn(CO)4(S2CNMe(CH2CO2H)) (CORM-401). These complexes have been used to probe the effects of CO in hundreds of cell- and animal-based experiments. However, through recent investigations, it has become evident that these reagents exhibit complicated reactivity in biological environments. The interpretation of the effects produced by some of these complexes is obscured by protein binding, such that their formulation is not clear, and by CO leakage and potential redox activity. An additional weakness with regard to CORM-2 and CORM-3 is that these compounds cannot be tracked via fluorescence. Therefore, it is unclear where or when CO release occurs, which confounds the interpretation of experiments using these molecules. To address these weaknesses, our research team has pioneered the development of metal-free CORMs based on structurally tunable extended flavonol or quinolone scaffolds. In addition to being highly controlled, with CO release only occurring upon triggering with visible light (photoCORMs), these CO donors are trackable via fluorescence prior to CO release in cellular environments and can be targeted to specific cellular locations.In the Account, we highlight the development and application of a series of structurally related flavonol photoCORMs that (1) sense characteristics of cellular environments prior to CO release; (2) enable evaluation of the influence of cytosolic versus mitochondrial-localized CO release on cellular bioenergetics; (3) probe the cytotoxicity and anti-inflammatory effects of intracellular versus extracellular CO delivery; and (4) demonstrate that albumin delivery of a photoCORM enables potent anticancer and anti-inflammatory effects. A key advantage of using triggered CO release compounds in these investigations is the ability to examine the effects of the molecular delivery vehicle in the absence and presence of localized CO release, thus providing insight into the independent contributions of CO. Overall, flavonol-based CO delivery molecules offer opportunities for triggerable, trackable, and targetable CO delivery that are unprecedented in terms of previously reported CORMs and, thus, offer significant potential for applications in biological systems.

Extracellular vs Intracellular Delivery of CO: Does It Matter for a Stable, Diffusible Gasotransmitter?

Carbon monoxide (CO) is a gasotransmitter produced in humans. An essential unanswered question in the design of carbon monoxide releasing molecules (CORMs) is whether the delivery molecule should be localized extra- or intracellularly to produce desired biological effects. Herein we show that extracellular CO release is less toxic and is sufficient to produce an anti-inflammatory effect similar to that of intracellular CO release at nanomolar concentrations. This information is valuable for the design of CORMs.

Visible-Light-Activated Quinolone Carbon-Monoxide-Releasing Molecule: Prodrug and Albumin-Assisted Delivery Enables Anticancer and Potent Anti-Inflammatory Effects.

The delivery of controlled amounts of carbon monoxide (CO) to biological targets is of significant current interest. Very few CO-releasing compounds are currently known that can be rigorously controlled in terms of the location and amount of CO released. To address this deficiency, we report herein a new metal-free, visible-light-induced CO-releasing molecule (photoCORM) and its prodrug oxidized form, which offer new approaches to controlled, localized CO delivery. The new photoCORM, based on a 3-hydroxybenzo[ g]quinolone framework, releases 1 equiv of CO upon visible-light illumination under a variety of biologically relevant conditions. This nontoxic compound can be tracked prior to CO release using fluorescence microscopy and produces a nontoxic byproduct following CO release. An oxidized prodrug form of the photoCORM is reduced by cellular thiols, providing an approach toward activation in the reducing environment of cancer cells. Strong noncovalent affinity of the nonmetal photoCORM to albumin enables use of an albumin:photoCORM complex for targeted CO delivery to cancer cells. This approach produced cytotoxicity IC50 values among the lowest reported to date for CO delivery to cancer cells by a photoCORM. This albumin:photoCORM complex is also the first CO delivery system to produce significant anti-inflammatory effects when introduced at nanomolar photoCORM concentration.

Mitochondrial-Localized Versus Cytosolic Intracellular CO-Releasing Organic PhotoCORMs: Evaluation of CO Effects Using Bioenergetics.

While interactions between carbon monoxide (CO) and mitochondria have been previously studied, the methods used to deliver CO (gas or CO-releasing metal carbonyl compounds) lack subcellular targeting and/or controlled delivery. Thus, the effective concentration needed to produce changes in mitochondrial bioenergetics is yet to be fully defined. To evaluate the influence of mitochondrial-targeted versus intracellularly released CO on mitochondrial oxygen consumption rates, we developed and characterized flavonol-based CO donor compounds that differ at their site of release. These molecules are metal-free, visible light triggered CO donors (photoCORMs) that quantitatively release CO and are trackable in cells via confocal microscopy. Our studies indicate that at a concentration of 10 µM, the mitochondrial-localized and cytosolic CO-releasing compounds are similarly effective in terms of decreasing ATP production, maximal respiration, and the reserve capacity of A549 cells. This concentration is the lowest to impart changes in mitochondrial bioenergetics for any CO-releasing molecule (CORM) reported to date. The results reported herein demonstrate the feasibility of using a structurally tunable organic photoCORM framework for comparative intracellular studies of the biological effects of carbon monoxide.

Sense and Release: A Thiol-Responsive Flavonol-Based Photonically Driven Carbon Monoxide-Releasing Molecule That Operates via a Multiple-Input AND Logic Gate.

Molecular structures capable of intracellular information processing that couple responses from biomarker signals to the release of drug molecules represent intelligent delivery systems. Herein we report a chemophotonically driven, sense-of-logic carbon monoxide-releasing molecule (SL-photoCORM). This extended flavonol motif operates via an AND logic gate by first sensing the cellular environment via detection of thiols and then releasing CO when triggered with visible light and O2. Overall, this approach couples the detection of a cellular redox biomarker with the ability to release a small-molecule gasotransmitter known to trigger pathways involved in pro- and anti-inflammatory effects in a dose-dependent manner. Significantly, the fluorescence properties of the flavonol-based SL-photoCORM produce a series of chemophotonic input responses via two photochromatic switches (blue-to-green and green-to-colorless), leading to trackability and spatiotemporal control of CO release. Examination of the O2 requirements of the CO release step revealed that the SL-photoCORM is suitable for use under conditions of variable cellular levels of O2. These combined properties within a single-molecular framework advance the field of CO-releasing molecules by providing feedback on the diversity and complexity of the cellular environment prior to CO release.