Transition Metal Complexes and Photodynamic Therapy from a Tumor-Centered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433.

Transition metal complexes are of increasing interest as photosensitizers in photodynamic therapy (PDT) and, more recently, for photochemotherapy (PCT). In recent years, Ru(II) polypyridyl complexes have emerged as promising systems for both PDT and PCT. Their rich photochemical and photophysical properties derive from a variety of excited-state electronic configurations accessible with visible and near-infrared light, and these properties can be exploited for both energy- and electron-transfer processes that can yield highly potent oxygen-dependent and/or oxygen-independent photobiological activity. Selected examples highlight the use of rational design in coordination chemistry to control the lowest-energy triplet excited-state configurations for eliciting a particular type of photoreactivity for PDT and/or PCT effects. These principles are also discussed in the context of the development of TLD1433, the first Ru(II)-based photosensitizer for PDT to enter a human clinical trial. The design of TLD1433 arose from a tumor-centered approach, as part of a complete PDT package that includes the light component and the protocol for treating non-muscle invasive bladder cancer. Briefly, this review summarizes the challenges to bringing PDT into mainstream cancer therapy. It considers the chemical and photophysical solutions that transition metal complexes offer, and it puts into context the multidisciplinary effort needed to bring a new drug to clinical trial.

Enhanced Production and Anticancer Properties of Photoactivated Perylenequinones.

Hypocrellins and hypomycins are naturally occurring fungal perylenequinones with potential photodynamic activity against cancer and microbial diseases. This project pursued three lines of research. First, the production of perylenequinones was enhanced by investigating the effect of culture medium and light exposure on their biosynthesis. Solid-fermentation cultures on rice medium allowed for enhanced production of hypocrellins as compared to Cheerios or oatmeal medium. Alternatively, increased production of hypomycins, which are structurally related to the hypocrellins, was observed on oatmeal medium. In both cases, light exposure was an essential factor for the enhanced biosynthesis. In addition, this led to the discovery of two new perylenequinones, ent-shiraiachrome A (5) and hypomycin E (8), which were elucidated based on spectroscopic data. Finally, the photocytotoxic effects of both classes of compounds were evaluated against human skin melanoma, with EC50 values at nanomolar levels for hypocrellins and micromolar levels for hypomycins. In contrast, both classes of compounds showed reduced dark toxicity (EC50 values >100 µM), demonstrating promising phototherapeutic indices.

Neutral iridium(iii) complexes bearing BODIPY-substituted N-heterocyclic carbene (NHC) ligands: synthesis, photophysics, in vitro theranostic photodynamic therapy, and antimicrobial activity.

The synthesis, photophysics, and photobiological activities of a series of novel neutral heteroleptic cyclometalated iridium(iii) complexes incorporating boron dipyrromethene (BODIPY) substituted N-heterocyclic carbene (NHC) ligands (Ir1-Ir5) are reported. The effect of the substitution position of BODIPY on the NHC ligands, either on C4 of the phenyl ring (Ir1-Ir3) or C5 of the benzimidazole unit (Ir4 and Ir5), and its linker type (single or triple bond) on the photophysical properties was studied. Ir1-Ir5 exhibited BODIPY-localized intense 1IL (intraligand transition)/1MLCT (metal-to-ligand charge transfer) absorption at 530-543 nm and 1,3IL/1,3CT (charge transfer) emission at 582-610 nm. The nanosecond transient absorption results revealed that the lowest triplet excited states of these complexes were the BODIPY-localized 3π,π* states. Complexes Ir4 and Ir5 exhibited blue-shifted 1IL absorption and 1,3IL/1,3CT emission bands compared to the corresponding absorption and emission bands in complexes Ir1 and Ir3. However, replacing the methyl substituents on N3 of benzimidazole in complexes Ir1 and Ir4 with oligoether substituents in Ir3 and Ir5, respectively, did not impact the energies of the low-energy absorption and emission bands in the corresponding complexes. Water-soluble complexes Ir3 and Ir5 have been explored as photosensitizers for in vitro photodynamic therapy (PDT) effects toward human SKMEL28 melanoma cells. Ir3 showed no dark cytotoxicity (EC50 > 300 µM) but good photocytotoxic activity (9.66 ± 0.28 µM), whereas Ir5 exhibited a higher dark cytotoxicity (20.2 ± 1.26 µM) and excellent photocytotoxicity (0.15 ± 0.01 µM). The phototherapeutic indices with visible light (400-700 nm) activation were >31 for Ir3 and 135 for Ir5. Ir3 and Ir5 displayed 1O2 quantum yields of 38% and 22% in CH3CN, respectively, upon 450 nm excitation. Ir5 was more effective at generating reactive oxygen species (ROS) in vitro. Ir5 was also active against Staphylococcus aureus upon visible light activation, with a phototherapeutic index of >15 and EC50 value of 6.67 µM. These photobiological activities demonstrated that these neutral Ir(iii) complexes are promising in vitro PDT reagents, and substitution at C5 on the benzimidazole group of the NHC ligand was superior to C4 substitution on the phenyl ring.

Photophysical Properties and Photobiological Activities of Ruthenium(II) Complexes Bearing π-Expansive Cyclometalating Ligands with Thienyl Groups.

A new family of cyclometalated ruthenium(II) complexes [Ru(N^N)2(C^N)]+ derived from the π-extended benzo[h]imidazo[4,5-f]quinolone ligand appended with thienyl groups (n = 1-4, compounds 1-4) was prepared and its members were characterized for their chemical, photophysical, and photobiological properties. The lipophilicities of 1-4, determined as octanol-water partition coefficients (log Po/w), were positive and increased with the number of thienyl units. The absorption and emission bands of the C^N compounds were red-shifted by up to 200 nm relative to the analogous Ru(II) diimine systems. All of the complexes exhibited dual emission with the intraligand fluorescence (1IL, C^N-based) shifting to lower energies with increasing n and the metal-to-ligand charge transfer phosphorescence (3MLCT, N^N-based) remaining unchanged. Compounds 1-3 exhibited excited state absorption (ESA) profiles consistent with lowest-lying 3MLCT states when probed by nanosecond transient absorption (TA) spectroscopy with 532 nm excitation and had contributions from 1IL(C^N) states with 355 nm excitation. These assignments were supported by the lifetimes observed (<10 ns for the 1IL states and around 20 ns for the 3MLCT states) as well as a noticeable ESA for 3 with 355 nm excitation that did not occur with 532 nm excitation. Compound 4 was the only member of the family with two 3MLCT emissive lifetimes (15, 110 ns), and the TA spectra collected with both 355 and 532 nm excitation was assigned to the 3IL state, which was corroborated by its 4-6 µs lifetime. The ESA for 4 had a rise time of approximately 10 ns and an initial decay of 110 ns, which suggests a possible 3MLCT-3IL excited state equilibrium that results in delayed emission from the 3MLCT state. Compound 4 was nontoxic toward human skin melanoma cells (SKMEL28) in the dark (EC50 = >300 µM); 1-3 were cytotoxic and yielded EC50 values between 1 and 20 µM. The photocytotoxicites with visible light ranged from 87 nM with a phototherapeutic index (PI) of 13 for 1 to approximately 1 µM (PI = >267) for 4. With red light, EC50 values varied from 270 nM (PI = 21) for 3 to 12 µM for 4 (PI = >25). The larger PIs for 4, especially with visible light, were attributed to the much lower dark cytotoxicity for this compound. Because the dark cytotoxicity contributes substantially to the observed photocytotoxicity for 1-3, it was not possible to assess whether the 3IL state of 4 led to a much more potent phototoxic mechanism in the absence of dark toxicity. There was no stark contrast in cellular uptake and accumulation by laser scanning confocal and differential interference contrast microscopy to explain the large differences in dark toxicities between 1-3 and 4. Nevertheless, the study highlights a new family of Ru(II) C^N complexes where π-conjugation beyond a certain point results in low dark cytotoxicity with high photocytotoxicity, opposing the notion that cyclometalated Ru(II) systems are too toxic to be phototherapeutic agents.

Predictive Strength of Photophysical Measurements for in Vitro Photobiological Activity in a Series of Ru(II) Polypyridyl Complexes Derived from π-Extended Ligands.

This study investigates the correlation between photocytotoxicity and the prolonged excited-state lifetimes exhibited by certain Ru(II) polypyridyl photosensitizers comprised of π-expansive ligands. The eight metal complexes selected for this study differ markedly in their triplet state configurations and lifetimes. Human melanoma SKMEL28 and human leukemia HL60 cells were used as in vitro models to test photocytotoxicity induced by the compounds when activated by either broadband visible or monochromatic red light. The photocytotoxicities of the metal complexes investigated varied over 2 orders of magnitude and were positively correlated with their excited-state lifetimes. The complexes with the longest excited-state lifetimes, contributed by low-lying 3IL states, were the most phototoxic toward cancer cells under all conditions.

Photophysical and Photobiological Properties of Dinuclear Iridium(III) Bis-tridentate Complexes.

A series of cationic dinuclear iridium(III) complexes (Ir1-Ir5) bearing terpyridine-capped fluorenyl bridging ligands and different polypyridyl or cyclometalating terminal tridentate ligands were synthesized, characterized, and evaluated for their photophysical and photobiological activities. The influence of the bridging and terminal ligands on the photophysical properties of the complexes was investigated by UV-vis absorption, emission, and transient absorption spectroscopy and simulated by TDDFT calculations. All of the complexes displayed strong bridging-ligand localized visible 1π,π* absorption and red- or near-infrared phosphorescence as well as broad triplet excited-state absorption across both visible and NIR wavelengths. These triplet states were assigned as predominantly 3π,π* for Ir1 (τ = 3.1 µs) and Ir4 (τ = 48 µs) and 3CT (charge transfer) for Ir2, Ir3, and Ir5 (τ = 1.7-2.7 µs). Complexes Ir1-Ir5 acted as in vitro photodynamic therapy (PDT) agents toward human SK-MEL-28 melanoma cells when activated with visible light, with submicromolar photocytotoxicity and phototherapeutic indices ranging from 20 to almost 300. The in vitro PDT effects with visible light did not correlate with singlet oxygen (1O2) quantum yields or DNA photocleaving capacity probed under cell-free conditions. All of the Ir(III) complexes phosphoresced brightly when associated with compromised cells (with or without light treatment) and exhibited photoactivated cellular uptake, highlighting the theranostic potential of this new class of Ir(III) complex photosensitizers.

Cyclometalated Ruthenium(II) Complexes Derived from α-Oligothiophenes as Highly Selective Cytotoxic or Photocytotoxic Agents.

The photophysical and photobiological properties of a new class of cyclometalated ruthenium(II) compounds incorporating π-extended benzo[ h]imidazo[4,5- f]quinoline (IBQ) cyclometalating ligands (C^N) bearing thienyl rings ( n = 1-4, compounds 1-4) were investigated. Their octanol-water partition coefficients (log Po/w) were positive and increased with n. Their absorption and emission energies were red-shifted substantially compared to the analogous Ru(II) diimine (N^N) complexes. They displayed C^N-based intraligand (IL) fluorescence and triplet excited-state absorption that shifted to longer wavelengths with increasing n and N^N-based metal-to-ligand charge transfer (MLCT) phosphorescence that was independent of n. Their photoluminescence lifetimes (τem) ranged from 4-10 ns for 1IL states and 12-18 ns for 3MLCT states. Transient absorption lifetimes (τTA) were 5-8 µs with 355 nm excitation, ascribed to 3IL states that became inaccessible for 1-3 with 532 nm excitation (1-3, τTA = 16-17 ns); the 3IL of 4 only was accessible by lower energy excitation, τTA = 3.8 µs. Complex 4 was nontoxic (EC50 > 300 µM) to SK-MEL-28 melanoma cells and CCD1064-Sk normal skin fibroblasts in the dark, while 3 was selectively cytotoxic to melanoma (EC50= 5.1 µM) only. Compounds 1 and 2 were selective for melanoma cells in the dark, with submicromolar potencies (EC50 = 350-500 nM) and selectivity factors (SFs) around 50. The photocytotoxicities of compounds 1-4 toward melanoma cells were similar, but only compounds 3 and 4 displayed significant phototherapeutic indices (PIs; 3, 43; 4, >1100). The larger cytotoxicities for compounds 1 and 2 were attributed to increased cellular uptake and nuclear accumulation, and possibly related to the DNA-aggregating properties of all four compounds as demonstrated by cell-free gel mobility-shift assays. Together, these results demonstrate a new class of thiophene-containing Ru(II) cyclometalated compounds that contain both highly selective chemotherapeutic agents and extremely potent photocytotoxic agents.

π-Expansive Heteroleptic Ruthenium(II) Complexes as Reverse Saturable Absorbers and Photosensitizers for Photodynamic Therapy.

Five heteroleptic tris-diimine ruthenium(II) complexes [RuL(N^N)2](PF6)2 (where L is 3,8-di(benzothiazolylfluorenyl)-1,10-phenanthroline and N^N is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), 1,4,8,9-tetraazatriphenylene (tatp) (3), dipyrido[3,2-a:2',3'-c]phenazine (dppz) (4), or benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn) (5), respectively) were synthesized. The influence of π-conjugation of the ancillary ligands (N^N) on the photophysical properties of the complexes was investigated by spectroscopic methods and simulated by density functional theory (DFT) and time-dependent DFT. Their ground-state absorption spectra were characterized by intense absorption bands below 350 nm (ligand L localized 1π,π* transitions) and a featureless band centered at ∼410 nm (intraligand charge transfer (1ILCT)/1π,π* transitions with minor contribution from metal-to-ligand charge transfer (1MLCT) transition). For complexes 4 and 5 with dppz and dppn ligands, respectively, broad but very weak absorption (ε < 800 M-1 cm-1) was present from 600 to 850 nm, likely emanating from the spin-forbidden transitions to the triplet excited states. All five complexes showed red-orange phosphorescence at room temperature in CH2Cl2 solution with decreased lifetimes and emission quantum yields, as the π-conjugation of the ancillary ligands increased. Transient absorption (TA) profiles were probed in acetonitrile solutions at room temperature for all of the complexes. Except for complex 5 (which showed dppn-localized 3π,π* absorption with a long lifetime of 41.2 µs), complexes 1-4 displayed similar TA spectral features but with much shorter triplet lifetimes (1-2 µs). Reverse saturable absorption (RSA) was demonstrated for the complexes at 532 nm using 4.1 ns laser pulses, and the strength of RSA decreased in the order: 2 ≥ 1 ≈ 5 > 3 > 4. Complex 5 is particularly attractive as a broadband reverse saturable absorber due to its wide optical window (430-850 nm) and long-lived triplet lifetime in addition to its strong RSA at 532 nm. Complexes 1-5 were also probed as photosensitizing agents for in vitro photodynamic therapy (PDT). Most of them showed a PDT effect, and 5 emerged as the most potent complex with red light (EC50 = 10 µM) and was highly photoselective for melanoma cells (selectivity factor, SF = 13). Complexes 1-5 were readily taken up by cells and tracked by their intracellular luminescence before and after a light treatment. Diagnostic intracellular luminescence increased with increased π-conjugation of the ancillary N^N ligands despite diminishing cell-free phosphorescence in that order. All of the complexes penetrated the nucleus and caused DNA condensation in cell-free conditions in a concentration-dependent manner, which was not influenced by the identity of N^N ligands. Although the mechanism for photobiological activity was not established, complexes 1-5 were shown to exhibit potential as theranostic agents. Together the RSA and PDT studies indicate that developing new agents with long intrinsic triplet lifetimes, high yields for triplet formation, and broad ground-state absorption to near-infrared (NIR) in tandem is a viable approach to identifying promising agents for these applications.

A spectroscopic study of substituted anthranilic acids as sensitive environmental probes for detecting cancer cells.

Small-molecule fluorescent reporters of disease states are highly sought after, yet they remain elusive. Anthranilic acids are extremely sensitive environmental probes, and hold promise as general but selective agents for cancer-cell detection if they can be equipped with the appropriate targeting groups. The optical properties of a small library of N-isopropyl invariant anthranilic acids were investigated in methanol and chloroform. Points of variation included: fluoro, trifluoromethyl, or cyano substitution on the aromatic ring, and derivitization of the parent carboxylic acid as esters or secondary carboxamides. Phenylboronic acid conjugation at the carboxylic acid alongside un-, mono-, and dimethylated 2-amino groups was also explored. The boron-containing anthranilic acids were also evaluated as sensitive fluorescent probes for cancer cells using laser scanning confocal microscopy. In general, the compounds produced blue fluorescence that was strongly influenced by substitution and environment. 4-Trifluoromethyl and 4-cyano esters proved to be the most sensitive environmental probes with quantum yields as large as 100% in chloroform, and enhancements of up to 30-fold on going from methanol to chloroform. Stokes shifts ranged from 63 to 120nm, generally increasing with ortho-substitution and environmental polarity. It was demonstrated that phenylboronic acid conjugation was an attractive method for cancer cell detection via boronate ester formation with overexpressed glycoproteins (with no interference from normal, healthy cells), presumably due to favorable boron-sialic acid interactions.

Influence of Protonation State on the Excited State Dynamics of a Photobiologically Active Ru(II) Dyad.

The influence of ligand protonation on the photophysics of a ruthenium (Ru) dyad bearing the 2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]-phenanthroline (ippy) ligand was investigated by time-resolved transient absorption spectroscopy. It was found that changes in the protonation state of the imidazole group led to changes in the electronic configuration of the lowest lying excited state. Formation of the fully deprotonated imidazole anion resulted in excited state signatures that were consistent with a low-lying intraligand (IL) triplet state. This assignment was supported by time-dependent density functional theory (TDDFT) calculations. IL triplet states have been suggested to be potent mediators of photodynamic effects. Thus, these results are of interest in the design of Ru metal complexes as photosensitizers (PSs) for photodynamic therapy (PDT).

Eight-membered ring-containing jadomycins: implications for non-enzymatic natural products biosynthesis.

Jadomycin Oct (1) was isolated from Streptomyces venezuelae ISP5230 and characterized as a structurally unique eight-membered l-ornithine ring-containing jadomycin. The structure was elucidated through the semisynthetic derivatization of starting material via chemoselective acylation of the l-ornithine α-amino group using activated succinimidyl esters. Incorporation of 5-aminovaleric acid led to jadomycin AVA, a second eight-membered ring-containing jadomycin. These natural products illustrate the structural diversity permissible from a non-enzymatic step within a biosynthetic pathway and exemplifies the potential for discovery of novel scaffolds.

Isolation and Synthetic Diversification of Jadomycin 4-Amino-l-phenylalanine.

Streptomyces venezuelae ISP5230 was grown in the presence of phenylalanine analogues to observe whether they could be incorporated into novel jadomycin structures. It was found that the bacteria successfully produced jadomycins incorporating 4-aminophenylalanine enantiomers. Upon isolation and characterization of jadomycin 4-amino-l-phenylalanine (1), it was synthetically derivatized, using activated succinimidyl esters, to yield a small jadomycin amide library. These are the first examples of oxazolone-ring-containing jadomycins that have incorporated an amino functionality subsequently used for derivatization.

In vitro multiwavelength PDT with 3IL states: teaching old molecules new tricks.

The purpose of the present investigation was to ascertain whether (3)IL excited states with microsecond lifetimes are universally potent for photodynamic applications, and if these long-lived states are superior to their (3)MLCT counterparts as in vitro PDT agents. A family of blue-green absorbing, Ru(II)-based transition metal complexes derived from the π-expansive dppn ligand was prepared and characterized according to its photodynamic activity against HL-60 cells, and toward DNA in cell-free media. Complexes in this series that are characterized by low-energy and long-lived (3)IL excited states photocleaved DNA with blue, green, red, and near-IR light. This panchromatic photodynamic effect translated to in vitro multiwavelength photodynamic therapy (PDT) with red-light cytotoxicities as low as 1.5 µM (EC50) for the parent complex and 400 nM for its more lipophilic counterpart. This potency is similar to that achieved with Ru(II)-based dyads containing long-lived (3)IL excitons located on appended pyrenyl units, and appears to be a general property of sufficiently long-lived excited states. Moreover, the red PDT observed for certain members of this family was almost 5 times more potent than Photofrin with therapeutic indices 30 times greater. Related Ru(II) complexes having lowest-lying (3)MLCT states of much shorter duration (≤1 µs) did not yield DNA photodamage or in vitro PDT with red or near-IR light, nor did the corresponding Os(II) complex with a submicrosecond (3)IL excited state lifetime. Therefore, metal complexes that utilize highly photosensitizing (3)IL excited states, with suitably long lifetimes (≫ 1 µs), are well-poised to elicit PDT at wavelengths even where their molar extinction coefficients are very low (<100 M(-1) cm(-1)). Herein we demonstrate that such unexpected reactivity gives rise to very effective PDT in the typical therapeutic window (600-850 nm).

Ru(II) dyads derived from 2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]phenanthroline: versatile photosensitizers for photodynamic applications.

Combining the best attributes of organic photosensitizers with those of coordination complexes is an elegant way to achieve prolonged excited state lifetimes in Ru(II) dyads. Not only do their reduced radiative and nonradiative rates provide ample time for photosensitization of reactive oxygen species at low oxygen tension but they also harness the unique properties of (3)IL states that can act as discrete units or in concert with (3)MLCT states. The imidazo[4,5-f][1,10]phenanthroline framework provides a convenient tether for linking π-expansive ligands such as pyrene to a Ru(II) scaffold, and the stabilizing coligands can fine-tune the chemical and biological properties of these bichromophoric systems. The resulting dyads described in this study exhibited nanomolar light cytotoxicities against cancer cells with photocytotoxicity indices exceeding 400 for some coligands employed. This potency extended to bacteria, where concentrations as low as 10 nM destroyed 75% of a bacterial population. Notably, these dyads remained extremely active against biofilm with light photocytotoxicities against these more resistant bacterial populations in the 10-100 nM regime. The results from this study demonstrate the versatility of these highly potent photosensitizers in destroying both cancer and bacterial cells and expand the scope of compounds that utilize low-lying (3)IL states for photobiological applications.

Exploitation of long-lived 3IL excited states for metal-organic photodynamic therapy: verification in a metastatic melanoma model.

Members of a family of Ru(II)-appended pyrenylethynylene dyads were synthesized, characterized according to their photophysical and photobiological properties, and evaluated for their collective potential as photosensitizers for metal-organic photodynamic therapy. The dyads in this series possess lowest-lying (3)IL-based excited states with lifetimes that can be tuned from 22 to 270 µs in fluid solution and from 44 to 3440 µs in glass at 77 K. To our knowledge, these excited-state lifetimes are the longest reported for Ru(II)-based dyads containing only one organic chromophore and lacking terminal diimine groups. These excited states proved to be extremely sensitive to trace amounts of oxygen, owing to their long lifetimes and very low radiative rates. Herein, we demonstrate that (3)IL states of this nature are potent photodynamic agents, exhibiting the largest photocytotoxicity indices reported to date with nanomolar light cytotoxicities at very short drug-to-light intervals. Importantly, these new agents are robust enough to maintain submicromolar PDT in pigmented metastatic melanoma cells, where the presence of melanin in combination with low oxygen tension is known to compromise PDT. This activity underscores the potential of metal-organic PDT as an alternate treatment strategy for challenging environments such as malignant melanoma.

Photodynamic Inactivation of Human Coronaviruses.

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.

Copper-mediated nuclease activity of jadomycin B.

The natural product jadomycin B, isolated from Streptomyces venezeulae ISP5230, has been found to cleave DNA in the presence of Cu(II) ions without the requirement for an external reducing agent. The efficiency of DNA cleavage was probed using supercoiled plasmid DNA in buffered solution as a model environment. EC50 and t(½) values for cleavage were 1.7 µM and 0.75 h, respectively, and varied ± 5% with the particular batch of plasmid and jadomycin employed. While UV-vis spectroscopy indicates that the cleavage event does not involve direct binding of jadomycin B to DNA, a stoichiometric Cu(II) preference for optimum cleavage suggests a weak binding interaction between jadomycin B and Cu(II) in the presence of DNA. The Cu(II)-mediated cleavage is greatly enhanced by UV light, which implicates the jadomycin B radical cation and Cu(I) as potential intermediates in DNA cleavage. Evidence in favor of this hypothesis was derived from a mechanistic assay which showed reduced cleavage as a function of added catalase and EDTA, scavengers of H2O2 and Cu(II), respectively. Thus, jadomycin B may serve as a source of electrons for Cu(II) reduction, producing Cu(I) which reacts with H2O2 to form hydroxyl radicals that cause DNA strand scission. In addition, scavengers of hydroxyl radicals and superoxide also display inhibitory effects, underscoring the ability of jadomycin B to produce a powerful arsenal of deleterious oxygen species when copper is present.

Jadomycins derived from the assimilation and incorporation of norvaline and norleucine.

Streptomyces venezuelae ISP5230 is recognized for the production of chloramphenicol and the jadomycin family of natural products. The jadomycins are angucycline natural products containing a unique oxazolone ring incorporating an amino acid present in the minimal culture media. Substitution of different amino acids results in products of varying biological activity. Analysis of cultures of S. venezuelae ISP5230 incubated with l- and d-norvaline and l- and d-norleucine indicated that only the d-configured amino acids were incorporated into the natural products. Subsequently, jadomycin DNV and jadomycin DNL were isolated and characterized (titers 4 and 9 mg L(-1), respectively). The compounds were evaluated in the National Cancer Institute cell line cancer growth inhibition and cytotoxicity screens, for antimicrobial activity against selected Gram-positive and Gram-negative bacteria, and as DNA-cleavage agents in vitro.

Photobiological activity of Ru(II) dyads based on (pyren-1-yl)ethynyl derivatives of 1,10-phenanthroline.

Several mononuclear Ru(II) dyads possessing 1,10-phenanthroline-appended pyrenylethynylene ligands were synthesized, characterized, and evaluated for their potential in photobiological applications such as photodynamic therapy (PDT). These complexes interact with DNA via intercalation and photocleave DNA in vitro at submicromolar concentrations when irradiated with visible light (lambda(irr) > or = 400 nm). Such properties are remarkably sensitive to the position of the ethynylpyrenyl substituent on the 1,10-phenanthroline ring, with 3-substitution showing the strongest binding under all conditions and causing the most deleterious DNA damage. Both dyads photocleave DNA under hypoxic conditions, and this photoactivity translates well to cytotoxicity and photocytotoxicity models using human leukemia cells, where the 5- and 3-substituted dyads show photocytotoxicity at 5-10 microM and 10-20 microM, respectively, with minimal, or essentially no, dark toxicity at these concentrations. This lack of dark cytotoxicity at concentrations where significant photoactivity is observed emphasizes that agents with strong intercalating units, previously thought to be too toxic for phototherapeutic applications, should not be excluded from the arsenal of potential photochemotherapeutic agents under investigation.