The Impact of Inorganic Systems and Photoactive Metal Compounds on Cytochrome P450 Enzymes and Metabolism: From Induction to Inhibition.

While cytochrome P450 (CYP; P450) enzymes are commonly associated with the metabolism of organic xenobiotics and drugs or the biosynthesis of organic signaling molecules, they are also impacted by a variety of inorganic species. Metallic nanoparticles, clusters, ions, and complexes can alter CYP expression, modify enzyme interactions with reductase partners, and serve as direct inhibitors. This commonly overlooked topic is reviewed here, with an emphasis on understanding the structural and physiochemical basis for these interactions. Intriguingly, while both organometallic and coordination compounds can act as potent CYP inhibitors, there is little evidence for the metabolism of inorganic compounds by CYPs, suggesting a potential alternative approach to evading issues associated with rapid modification and elimination of medically useful compounds.

Triarylphosphine-Coordinated Bipyridyl Ru(II) Complexes Induce Mitochondrial Dysfunction.

While cancer cells rely heavily upon glycolysis to meet their energetic needs, reducing the importance of mitochondrial oxidative respiration processes, more recent studies have shown that their mitochondria still play an active role in the bioenergetics of metastases. This feature, in combination with the regulatory role of mitochondria in cell death, has made this organelle an attractive anticancer target. Here, we report the synthesis and biological characterization of triarylphosphine-containing bipyridyl ruthenium (Ru(II)) compounds and found distinct differences as a function of the substituents on the bipyridine and phosphine ligands. 4,4'-Dimethylbipyridyl-substituted compound 3 exhibited especially high depolarizing capabilities, and this depolarization was selective for the mitochondrial membrane and occurred within minutes of treatment in cancer cells. The Ru(II) complex 3 exhibited an 8-fold increase in depolarized mitochondrial membranes, as determined by flow cytometry, which compares favorably to the 2-fold increase observed by carbonyl cyanide chlorophenylhydrazone (CCCP), a proton ionophore that shuttles protons across membranes, depositing them into the mitochondrial matrix. Fluorination of the triphenylphosphine ligand provided a scaffold that maintained potency against a range of cancer cells but avoided inducing toxicity in zebrafish embryos at higher concentrations, displaying the potential of these Ru(II) compounds for anticancer applications. This study provides essential information regarding the role of ancillary ligands for the anticancer activity of Ru(II) coordination compounds that induce mitochondrial dysfunction.

A monoadduct generating Ru(ii) complex induces ribosome biogenesis stress and is a molecular mimic of phenanthriplatin.

Ruthenium complexes are often investigated as potential replacements for platinum-based chemotherapeutics in hopes of identifying systems with improved tolerability in vivo and reduced susceptibility to cellular resistance mechanisms. Inspired by phenanthriplatin, a non-traditional platinum agent that contains only one labile ligand, monofunctional ruthenium polypyridyl agents have been developed, but until now, few demonstrated promising anticancer activity. Here we introduce a potent new scaffold, based on [Ru(tpy)(dip)Cl]Cl (tpy = 2,2':6',2''-terpyridine and dip = 4,7-diphenyl-1,10-phenanthroline) in pursuit of effective Ru(ii)-based monofunctional agents. Notably, the extension of the terpyridine at the 4' position with an aromatic ring resulted in a molecule that was cytotoxic in several cancer cell lines with sub-micromolar IC50 values, induced ribosome biogenesis stress, and exhibited minimal zebrafish embryo toxicity. This study demonstrates the successful design of a Ru(ii) agent that mimics many of the biological effects and phenotypes seen with phenanthriplatin, despite numerous differences in both the ligands and metal center structure.

Synthesis and photobiological evaluation of Ru(II) complexes with expanded chelate polypyridyl ligands.

Photoreactive Ru(II) complexes capable of ejecting ligands have been used extensively for photocaging applications and for the creation of "photocisplatin" reagents. The incorporation of distortion into the structure of the coordination complex lowers the energy of dissociative excited states, increasing the yield of the photosubstitution reaction. While steric clash between ligands induced by adding substituents at the coordinating face of the ligand has been extensively utilized, a lesser known, more subtle approach is to distort the coordination sphere by altering the chelate ring size. Here a systematic study was performed to alter metal-ligand bond lengths, angles, and to cause intraligand distortion by introducing a "linker" atom or group between two pyridine rings. The synthesis, photochemistry, and photobiology of five Ru(II) complexes containing CH2, NH, O, and S-linked dipyridine ligands was investigated. All systems where stable in the dark, and three of the five were photochemically active in buffer. While a clear periodic trend was not observed, this study lays the foundation for the creation of photoactive systems utilizing an alternative type of distortion to facilitate photosubstitution reactions.

Design of Cytochrome P450 1B1 Inhibitors via a Scaffold-Hopping Approach.

Cytochrome P450 1B1 (CYP1B1) is a potential drug target in cancer research that is overexpressed in several solid tumors but is present only at low levels in healthy tissues. Its expression is associated with resistance to common chemotherapeutics, while inhibitors restore efficacy to these drugs in model systems. The majority of CYP1B1 inhibitors are derived from a limited number of scaffolds, and few have achieved outstanding selectivity against other human CYPs, which could impede clinical development. This study explores a new chemical space for CYP1B1 inhibitors using a scaffold-hopping approach and establishes 2,4-diarylthiazoles as a promising framework for further development. From a small library, compound 15 emerged as the lead, with picomolar CYP1B1 inhibition, and over 19,000-fold selectivity against its relative, CYP1A1. To investigate the activity of 15, molecular dynamics, optical spectroscopy, point mutations, and traditional structure-activity relationships were employed and revealed key interactions important for the development of CYP1B1 inhibitors.

Ru(II) photocages enable precise control over enzyme activity with red light.

The cytochrome P450 family of enzymes (CYPs) are important targets for medicinal chemistry. Recently, CYP1B1 has emerged as a key player in chemotherapy resistance in the treatment of cancer. This enzyme is overexpressed in a variety of tumors, and is correlated with poor treatment outcomes; thus, it is desirable to develop CYP1B1 inhibitors to restore chemotherapy efficacy. However, possible off-target effects, such as inhibition of liver CYPs responsible for first pass metabolism, make selective inhibition a high priority to avoid possible drug-drug interactions and toxicity. Here we describe the creation of light-triggered CYP1B1 inhibitors as "prodrugs", and achieve >6000-fold improvement in potency upon activation with low energy (660 nm) light. These systems provide a selectivity index of 4,000-100,000 over other off-target CYPs. One key to the design was the development of coordinating CYP1B1 inhibitors, which suppress enzyme activity at pM concentrations in live cells. The metal binding group enforces inhibitor orientation in the active site by anchoring to the iron. The second essential component was the biologically compatible Ru(II) scaffold that cages the inhibitors before photochemical release. These Ru(II) photocages are anticipated to provide similar selectivity and control for any coordinating CYP inhibitors.

Fine-Feature Modifications to Strained Ruthenium Complexes Radically Alter Their Hypoxic Anticancer Activity†.

In an earlier study of π-expansive ruthenium complexes for photodynamic and photochemo-therapies, it was shown that a pair of structural isomers differing only in the connection point of a naphthalene residue exhibited vastly different biological activity. These isomers are further explored in this paper through the activity of their functionalized derivatives. In normoxia, the inactive 2-NIP isomer (5) can be made as photocytotoxic as the active 1-NIP isomer (1) by functionalizing with methyl or methoxy groups, while methoxy variants of the 1-NIP isomer became inactive. In all cases, the singlet oxygen sensitization quantum yield was below 1%. Hypoxic photocytotoxicity was attenuated, with only three of the series showing any activity, notwithstanding the photodissociative ligands. The results here are consistent with the earlier findings in that seemingly minor structural modifications on the non-strained ligand can dramatically modulate the normoxic and hypoxic activity of these strained compounds and that these changes appear to exert a greater influence on photocytotoxicity than singlet oxygen sensitization or rates of photosubstitution in cell-free conditions would suggest.

Biological Investigations of Ru(II) Complexes With Diverse ß-diketone Ligands.

The ß-diketone scaffold is a commonly used synthetic intermediate, and is a functional group found in natural products such as curcuminoids. This core structure can also act as a chelating ligand for a variety of metals. In order to assess the potential of this scaffold for medicinal inorganic chemistry, seven different κ2-O,O'-chelating ligands were used to construct Ru(II) complexes with polypyridyl co-ligands, and their biological activity was evaluated. The complexes demonstrated promising structure-dependent cytotoxicity. Three complexes maintained high activity in a tumor spheroid model, and all complexes demonstrated low in vivo toxicity in a zebrafish model. From this series, the best compound exhibited a ~ 30-fold window between cytotoxicity in a 3-D tumor spheroid model and potential in vivo toxicity. These results suggest that κ2-O,O'-ligands can be incorporated into Ru(II)-polypyridyl complexes to create favorable candidates for future drug development.

A Fluorometric CYP19A1 (Aromatase) Activity Assay in Live Cells.

Inhibition of estrogen synthesis is an integral component of the frontline pharmacologic therapy for the treatment of estrogen receptor positive cancers. However, there is currently no direct, high-throughput-ready assay for aromatase (also known as CYP19A1) that can be performed in live cells. Herein we present a cell-based assay that allows for multiplexed assessment of enzyme activity, protein half-life, cell viability, and identification of inhibitors with slow off-rates.

Organoplatinum(II) Complexes Self-Assemble and Recognize AT-Rich Duplex DNA Sequences.

The specific recognition of AT-rich DNA sequences opens up the door to promising diagnostic and/or therapeutic strategies against gene-related diseases. Here, we demonstrate that amphiphilic PtII complexes of the type [Pt(dmba)(N∧N)]NO3 (dmba = N,N-dimethylbenzylamine-κN, κC; N∧N = dpq (3), dppz (4), and dppn (5)) recognize AT-rich oligonucleotides over other types of DNA, RNA, and model proteins. The crystal structure of 4 shows the presence of significant π-stacking interactions and a distorted coordination sphere of the d8 PtII atom. Complex 5, containing the largest π-conjugated ligand, forms supramolecular assemblies at high concentrations under aqueous environment. However, its aggregation can be promoted in the presence of DNA at concentrations as low as 10 µM in a process that "turns on" its excimer emission around 600 nm. Viscometry, gel electrophoresis, and theoretical calculations demonstrate that 5 binds to minor groove when self-assembled, while the monomers of 3 and 4 intercalate into the DNA. The complexes also inhibit cancer cell growth with low-micromolar IC50 values in 2D tissue culture and suppress tumor growth in 3D tumor spheroids with a multicellular resistance (MCR) index comparable to that of cisplatin.

Avobenzone incorporation in a diverse range of Ru(II) scaffolds produces potent potential antineoplastic agents.

Four structurally distinct classes of polypyridyl ruthenium complexes containing avobenzone exhibited low micromolar and submicromolar potencies in cancer cells, and were up to 273-fold more active than the parent ligand. Visible light irradiation enhanced the cytotoxicity of some complexes, making them promising candidates for combined chemo-photodynamic therapy.

Photochemical and Photobiological Properties of Pyridyl-pyrazol(in)e-Based Ruthenium(II) Complexes with Sub-micromolar Cytotoxicity for Phototherapy.

The discovery of new light-triggered prodrugs based on ruthenium (II) complexes is a promising approach for photoactivated chemotherapy (PACT). The light-mediated activation of "strained" Ru(II) polypyridyl complexes resulted in ligand release and produced a ligand-deficient metal center capable of forming covalent adducts with biomolecules such as DNA. Based on the strategy of exploiting structural distortion to activate photochemistry, biologically active small molecules were coordinated to a Ru(II) scaffold to create light-triggered dual-action agents. Thirteen new Ru(II) complexes with pyridyl-pyrazol(in)e ligands were synthesized, and their photochemical reactivity and anticancer properties were investigated. Isomeric bidentate ligands were investigated, where "regular" ligands (where the coordinated nitrogens in the heterocycles are linked by C-C atoms) were compared to "inverse" isomers (where the coordinated nitrogens in the heterocycles are linked by C-N atoms). Coordination of the regular 3-(pyrid-2-yl)-pyrazol(in)es to a Ru(II) bis-dimethylphenanthroline scaffold yielded photoresponsive compounds with promising photochemical and biological properties, in contrast to the inverse 1-(pyrid-2-yl)-pyrazolines. The introduction of a phenyl ring to the 1N-pyrazoline cycle increased the distortion in complexes and improved ligand release upon light irradiation (470 nm) up to 5-fold in aqueous media. Compounds 1-8, containing pyridyl-pyrazol(in)e ligands, were at least 20-80-fold more potent than the parent pyridyl-pyrazol(in)es, and exhibited biological activity in the dark, with half-maximal inhibitory concentration (IC50) values ranging from 0.2 to 7.6 µM in the HL60 cell line, with complete growth inhibition upon light irradiation. The diversification of coligands and introduction of a carboxylic acid into the Ru(II) complex resulted in compounds 9-12, with up to 146-fold improved phototoxicity indices compared with complexes 1-8.

Bis-tridentate N-Heterocyclic Carbene Ru(II) Complexes are Promising New Agents for Photodynamic Therapy.

Ruthenium(II) complexes developed for photodynamic therapy (PDT) are almost exclusively tris-bidentate systems with C2 or D3 symmetry. This is due to the fact that this structural framework commonly produces long-lived excited states, which, in turn, allow for the generation of large amounts of singlet oxygen (1O2) and other reactive oxygen species. Complexes containing tridentate ligands would be advantageous for biological applications as they are generally achiral (D2d or C2v symmetry), which eliminates the possibility of multiple isomers which could exhibit potentially different interactions with chiral biological entities. However, Ru(II) complexes containing tridentate ligands are rarely studied as candidates for photobiological applications, such as PDT, since they almost exclusively exhibit low quantum yields and very short excited-state lifetimes and, thus, are not capable of generating sufficient 1O2 or engaging in electron transfer reactions. Here, we report a proof-of-concept approach to make bis-tridentate Ru(II) complexes useful for PDT applications by altering their photophysical properties through the inclusion of N-heterocyclic carbene (NHC) ligands. Three NHC and two terpyridine ligands were studied to evaluate the effects of structural and photophysical modulations of bis-substituted Ru(II) complexes. The NHC complexes were found to have superior excited-state lifetimes, 1O2 production, and photocytotoxicity. To the best of our knowledge, these complexes are the most potent light-activated bis-tridentate complexes reported.

Strained, Photoejecting Ru(II) Complexes that are Cytotoxic Under Hypoxic Conditions.

A series of strained Ru(II) complexes were studied for potential anticancer activity in hypoxic tissues. The complexes were constructed with methylated ligands that were photolabile and an imidizo[4,5-f][1,10]phenanthroline ligand that contained an appended aromatic group to potentially allow for contributions of ligand-centered excited states. A systematic variation of the size and energy of the aromatic group was performed using systems containing 1-4 fused rings, and the photochemical and photobiological behaviors of all complexes were assessed. The structure and nature of the aromatic group had a subtle impact on photochemistry, altering environmental sensitivity, and had a significant impact on cellular cytotoxicity and photobiology. Up to 5-fold differences in cytotoxicity were observed in the absence of light activation; this rose to 50-fold differences upon exposure to 453 nm light. Most significantly, one complex retained activity under conditions with 1% O2 , which is used to induce hypoxic changes. This system exhibited a photocytotoxicity index (PI) of 15, which is in marked contrast to most other Ru(II) complexes, including those designed for O2 -independent mechanisms of action.

Molecular Determinants of Substrate Affinity and Enzyme Activity of a Cytochrome P450BM3 Variant.

Cytochrome P450BM3 catalyzes the hydroxylation and/or epoxidation of fatty acids, fatty amides, and alcohols. Protein engineering has produced P450BM3 variants capable of accepting drug molecules normally metabolized by human P450 enzymes. The enhanced substrate promiscuity has been attributed to the greater flexibility of the lid of the substrate channel. However, it is not well understood how structurally different and highly polar drug molecules can stably bind in the active site nor how the activity and coupling efficiency of the enzyme may be affected by the lack of enzyme-substrate complementarity. To address these important aspects of non-native small molecule binding, this study investigated the binding of drug molecules with different size, charge, polar surface area, and human P450 affinity on the promiscuous R47L/F87V/L188Q/E267V/F81I pentuple mutant of P450BM3. Binding free energy data and energy decomposition analysis showed that pentuple mutant P450BM3 stably binds (i.e., negative ΔGb°) a broad range of substrate and inhibitor types because dispersion interactions with active site residues overcome unfavorable repulsive and electrostatic effects. Molecular dynamics simulations revealed that 1) acidic substrates tend to disrupt the heme propionate A-K69 salt bridge, which may reduce heme oxidizing ability, and 2) the lack of complementarity leads to high substrate mobility and water density in the active site, which may lead to uncoupling. These factors must be considered in future developments of P450BM3 as a biocatalyst in the large-scale production of drug metabolites.

Structure-activity relationships of anticancer ruthenium(II) complexes with substituted hydroxyquinolines.

8-Hydroxyquinolines (HQ), including clioquinol, possess cytotoxic properties and are widely used as ligands for metal-based anticancer drug research. The number and identity of substituents on the HQ can have a profound effect on activity for a variety of inorganic compounds. Ruthenium complexes of HQ exhibit radically improved potencies, and operate by a new, currently unknown, mechanism of action. To define structure-activity relationships (SAR), a family of 22 Ru(II) coordination complexes containing mono-, di- and tri-substituted hydroxyquinoline ligands were synthesized and their biological activity evaluated. The complexes exhibited promising cytotoxic activity against a cancer cell line, and the SAR data revealed the 2- and 7-positions as key sites for the incorporation of halogens to improve potency. The Ru(II) complexes potently inhibited translation, as demonstrated by an in-cell translation assay. The effects were seen at 2-15-fold higher concentrations than those required to observe cytotoxicity, suggesting that prevention of protein synthesis may be a primary, but not the exclusive mechanism for the observed cytotoxic activity.

Bacterial Cytological Profiling Reveals the Mechanism of Action of Anticancer Metal Complexes.

Target identification and mechanistic studies of cytotoxic agents are challenging processes that are both time-consuming and costly. Here we describe an approach to mechanism of action studies for potential anticancer compounds by utilizing the simple prokaryotic system, E. coli, and we demonstrate its utility with the characterization of a ruthenium polypyridyl complex [Ru(bpy)2dmbpy2+]. Expression of the photoconvertible fluorescent protein Dendra2 facilitated both high throughput studies and single-cell imaging. This allowed for simultaneous ratiometric analysis of inhibition of protein production and phenotypic investigations. The profile of protein production, filament size and population, and nucleoid morphology revealed important differences between inorganic agents that damage DNA vs more selective inhibitors of transcription and translation. Trace metal analysis demonstrated that DNA is the preferred nucleic acid target of the ruthenium complex, but further studies in human cancer cells revealed altered cell signaling pathways compared to the commonly administrated anticancer agent cisplatin. This study demonstrates E. coli can be used to rapidly distinguish between compounds with disparate mechanisms of action and also for more subtle distinctions within in studies in mammalian cells.

High resolution measurement of membrane receptor endocytosis.

We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Upon exposure to 405 nm light, Dendra2 is photoconverted from green to red emission. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Conversion of Dendra2 works as a pulse chase experiment through monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. This method is not impacted by the insertion of new protein into the plasma membrane as newly synthesized protein only exhibits green emission. We used this approach to determine the half-life of ENaC on the plasma membrane illustrating the high temporal resolution capability of this technique compared to current methods.

Photochemical Properties and Structure-Activity Relationships of RuII Complexes with Pyridylbenzazole Ligands as Promising Anticancer Agents.

Ruthenium complexes capable of light-triggered cytotoxicity are appealing potential prodrugs for photodynamic therapy (PDT) and photoactivated chemotherapy (PACT). Two groups of Ru(II) polypyridyl complexes with 2-(2-pyridyl)-benzazole ligands were synthesized and investigated for their photochemical properties and anticancer activity to compare strained and unstrained systems that are likely to have different biological mechanisms of action. The structure-activity relationship was focused on the benzazole core bioisosterism and replacement of coligands in Ru(II) complexes. Strained compounds rapidly ejected the 2-(2-pyridyl)-benzazole ligand after light irradiation, and possessed strong toxicity in the HL-60 cell line both under dark and light conditions. In contrast, unstrained Ru(II) complexes were non-toxic in the absence of light, induced cytotoxicity at nanomolar concentrations after light irradiation, and are capable of light-induced DNA damage. The 90-220-fold difference in light and dark IC50 values provides a large potential therapeutic window to allow for selective targeting of cells by exposure to light.

Photochemical and Photobiological Activity of Ru(II) Homoleptic and Heteroleptic Complexes Containing Methylated Bipyridyl-type Ligands.

Light-activated compounds are powerful tools and potential agents for medical applications, as biological effects can be controlled in space and time. Ruthenium polypyridyl complexes can induce cytotoxic effects through multiple mechanisms, including acting as photosensitizers for singlet oxygen (1O2) production, generating other reactive oxygen species (ROS), releasing biologically active ligands, and creating reactive intermediates that form covalent bonds to biological molecules. A structure-activity relationship (SAR) study was performed on a series of Ru(II) complexes containing isomeric tetramethyl-substituted bipyridyl-type ligands. Three of the ligand systems studied contained strain-inducing methyl groups and created photolabile metal complexes, which can form covalent bonds to biomolecules upon light activation, while the fourth was unstrained and resulted in photostable complexes, which can generate 1O2. The compounds studied included both bis-heteroleptic complexes containing two bipyridine ligands and a third, substituted ligand and tris-homoleptic complexes containing only the substituted ligand. The photophysics, electrochemistry, photochemistry, and photobiology were assessed. Strained heteroleptic complexes were found to be more photoactive and cytotoxic then tris-homoleptic complexes, and bipyridine ligands were superior to bipyrimidine. However, the homoleptic complexes exhibited an enhanced ability to inhibit protein production in live cells. Specific methylation patterns were associated with improved activation with red light, and photolabile complexes were generally more potent cytotoxic agents than the photostable 1O2-generating compounds.