Efficacies and ADME properties of redox active methylene blue and phenoxazine analogues for use in new antimalarial triple drug combinations with amino-artemisinins.

Efforts to develop new artemisinin triple combination therapies effective against artemisinin-tolerant strains of Plasmodium falciparum based on rational combinations comprising artemisone or other amino-artemisinins, a redox active drug and a third drug with a different mode of action have now been extended to evaluation of three potential redox partners. These are the diethyl analogue AD01 of methylene blue (MB), the benzo [α]phenoxazine PhX6, and the thiosemicarbazone DpNEt. IC50 values in vitro against CQ-sensitive and resistant P. falciparum strains ranged from 11.9 nM for AD01-41.8 nM for PhX6. PhX6 possessed the most favourable pharmacokinetic (PK) profile: intrinsic clearance rate CLint was 21.47 ± 1.76 mL/min/kg, bioavailability was 60% and half-life was 7.96 h. AD01 presented weaker, but manageable pharmacokinetic properties with a rapid CLint of 74.41 ± 6.68 mL/min/kg leading to a half-life of 2.51 ± 0.07 h and bioavailability of 15%. DpNEt exhibited a half-life of 1.12 h and bioavailability of 8%, data which discourage its further examination, despite a low CLint of 10.20 mL/min/kg and a high Cmax of 6.32 µM. Efficacies of AD01 and PhX6 were enhanced synergistically when each was paired with artemisone against asexual blood stages of P. falciparum NF54 in vitro. The favourable pharmacokinetics of PhX6 indicate this is the best partner among the compounds examined thus far for artemisone. Future work will focus on extending the drug combination studies to artemiside in vitro, and conducting efficacy studies in vivo for artemisone with each of PhX6 and the related benzo[α]phenoxazine SSJ-183.

Intracellular Accumulation of Novel and Clinically Used TB Drugs Potentiates Intracellular Synergy.

The therapeutic repertoire for tuberculosis (TB) remains limited despite the existence of many TB drugs that are highly active in in vitro models and possess clinical utility. Underlying the lack of efficacy in vivo is the inability of TB drugs to penetrate microenvironments inhabited by the causative agent, Mycobacterium tuberculosis, including host alveolar macrophages. Here, we determined the ability of the phenoxazine PhX1 previously shown to be active against M. tuberculosis in vitro to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. We also investigated the extent of permeation into uninfected and M. tuberculosis-infected human macrophage-like Tamm-Horsfall protein 1 (THP-1) cells directly and by comparing to results obtained in vitro in synergy assays. Our data indicate that PhX1 (4,750 ± 127.2 ng/ml) penetrates more effectively into THP-1 cells than do the clinically used anti-TB agents, rifampin (3,050 ± 62.9 ng/ml), moxifloxacin (3,374 ± 48.7 ng/ml), bedaquiline (4,410 ± 190.9 ng/ml), and linezolid (770 ± 14.1 ng/ml). Compound efficacy in infected cells correlated with intracellular accumulation, reinforcing the perceived importance of intracellular penetration as a key drug property. Moreover, we detected synergies deriving from redox-stimulatory combinations of PhX1 or clofazimine with the novel prenylated amino-artemisinin WHN296. Finally, we used compound synergies to elucidate the relationship between compound intracellular accumulation and efficacy, with PhX1/WHN296 synergy levels shown to predict drug efficacy. Collectively, our data support the utility of the applied assays in identifying in vitro active compounds with the potential for clinical development. IMPORTANCE This study addresses the development of novel therapeutic compounds for the eventual treatment of drug-resistant tuberculosis. Tuberculosis continues to progress, with cases of Mycobacterium tuberculosis (M. tuberculosis) resistance to first-line medications increasing. We assess new combinations of drugs with both oxidant and redox properties coupled with a third partner drug, with the focus here being on the potentiation of M. tuberculosis-active combinations of compounds in the intracellular macrophage environment. Thus, we determined the ability of the phenoxazine PhX1, previously shown to be active against M. tuberculosis in vitro, to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. In addition, the extent of permeation into human macrophage-like THP-1 cells and H37Rv-infected THP-1 cells was measured via mass spectrometry and compared to in vitro two-dimensional synergy and subsequent intracellular efficacy. Collectively, our data indicate that development of new drugs will be facilitated using the methods described herein.

Anti-Mycobacterial Peroxides: A New Class of Agents for Development Against Tuberculosis.

BACKGROUND: With few exceptions, existing tuberculosis drugs were developed many years ago and resistance profiles have emerged. This has created a need for new drugs with discrete modes of action. There is evidence that tuberculosis (like other bacteria) is susceptible to oxidative pressure and this has yet to be properly utilised as a therapeutic approach in a manner similar to that which has proven highly successful in malaria therapy. OBJECTIVE: To develop an alternative approach to the incorporation of bacterial siderophores that results in the creation of antitubercular peroxidic leads for subsequent development as novel agents against tuberculosis. METHODS: Eight novel peroxides were prepared and the antitubercular activity (H37Rv) was compared to existing artemisinin derivatives in vitro. The potential for toxicity was evaluated against the L6 rat skeletal myoblast and HeLa cervical cancer lines in vitro. RESULTS: The addition of a pyrimidinyl residue to an artemisinin or, preferably, a tetraoxane peroxidic structure results in antitubercular activity in vitro. The same effect is not observed in the absence of the pyrimidine or with other heteroaromatic substituents. CONCLUSION: The incorporation of a pyrimidinyl residue adjacent to the peroxidic function in an organic peroxide results in anti-tubercular activity in an otherwise inactive peroxidic compound. This will be a useful approach for creating oxidative drugs to target tuberculosis.

In Vitro Efficacies, ADME, and Pharmacokinetic Properties of Phenoxazine Derivatives Active against Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a leading infectious killer globally, demanding the urgent development of faster-acting drugs with novel mechanisms of action. Riminophenazines such as clofazimine are clinically efficacious against both drug-susceptible and drug-resistant strains of M. tuberculosis We determined the in vitro anti-M. tuberculosis activities, absorption, distribution, metabolism, and excretion properties, and in vivo mouse pharmacokinetics of a series of structurally related phenoxazines. One of these, PhX1, displayed promising drug-like properties and potent in vitro efficacy, supporting its further investigation in an M. tuberculosis-infected animal model.

Development of pyridyl thiosemicarbazones as highly potent agents for the treatment of malaria after oral administration.

OBJECTIVES: Drug resistance exists to all current and investigational antimalarial drug classes. Consequently, we have set out to develop chemically and mechanistically discrete antimalarials. Here we report on the development of thiosemicarbazone (TSC) antimalarials, with TSC3 as the most advanced lead. METHODS: Thiosemicarbazones were generated through simple condensation reactions of thiosemicarbazides and ketones. TSC3 was selected and tested for in vitro antimalarial activities against MDR Plasmodium falciparum lines using the [3H]hypoxanthine growth assay, in vitro cytotoxicity against mammalian cell lines using the alamarBlue fluorescence cell viability assay, in vivo potency in the mouse-Plasmodium berghei model and blood exposure in mice measured by LC-MS for pharmacokinetic analysis. RESULTS: TSC3 showed potent in vitro activity against atovaquone-, dihydroartemisinin-, chloroquine- and mefloquine-resistant P. falciparum lines (EC50 <15 nM). The selectivity index (EC50 cells/EC50Pf W2 line) of TSC3 was >500 in two of three mammalian cell lines. In P. berghei-infected mice, TSC3 showed potent activity in the Peters 4 day suppression test (ED50 1.2 mg/kg/day) and was as potent as artesunate and chloroquine in the curative modified Thompson test. A single oral dose of TSC3 at 16 mg/kg in healthy mice achieved a mean maximum blood concentration of 1883 ng/mL at 1 h after dosing and an elimination half-life of 48.7 h in groups of five mice. CONCLUSIONS: TSC3 shows promise as a persistent, potent and orally effective antimalarial. This, coupled with the extremely low cost of synthesis, suggests that the further development of antimalarial thiosemicarbazones is clearly warranted.

The Evaluation of Metal Co-ordinating Bis-Thiosemicarbazones as Potential Anti-malarial Agents.

BACKGROUND: The emergence of resistance to the artemisinins which are the current mainstays for antimalarial chemotheraphy has created an environment where the development of new drugs acting in a mechanistally discrete manner is a priority. OBJECTIVE: The goal of this work was to synthesize ane evaluate bis-thiosemicarbazones as potential antimalarial agents. METHODS: Fifteen compounds were generated using two condensation protocols and evaluated in vitro against the NF54 (CQ sensitive) strain of Plasmodium falciparum. A preliminary assessment of the potential for human toxicity was conducted in vitro against the MRC5 human lung fibroblast line. RESULTS: The activity of the bis-thiosemicarbazones was highly dependent on the nature of the arene at the core of the structure. The inclusion of a non-coordinating benzene core resulted in inactive compounds, while the inclusion of a pyridyl core resulted in compounds of moderate or potent antimalarial activity (4 compounds showing IC50 < 250 nM). CONCLUSION: Bis-thiosemicarbazones containing a central pyridyl core display potent antimalarial activity in vitro. Sequestration and activation of ferric iron appears to play a significant role in this activity. Ongoing studies are aimed at further development of this series as potential antimalarials.

Cytotoxic activity of expanded coordination bis-thiosemicarbazones and copper complexes thereof.

A series of bis-thiosemicarbazone agents with coordinating groups capable of multiple metal coordination modes has been generated and evaluated for potential cytotoxic effects against melanoma (MelRm) and breast adenocarcinoma (MCF-7) cell lines. The bis-thiosemicarbazones in this study generally demonstrated superior cytotoxic activity against MelRm than MCF-7 in the absence of metal ion supplementation, but in most cases could not be considered superior to the reference thiosemicarbazone Dp44mT. The key structural features for the cytotoxic activity were the central metal binding atom on the aromatic core, the thiocarbonyl residue and the nature of substitution on the N4-terminus in terms of size and lipophilicity. The cytotoxicity of bis-thiosemicarbazone ligands improved significantly with Cu(II) supplementation, particularly against MCF-7 cells. The mechanism of cytotoxicity of bis-thiosemicarbazones was proposed to be dependent on the combined effect of metal mobilisation and ROS generation which is so called a "double-punch effect".

Investigation of the cytotoxic implications of metal chelators against melanoma and approaches to improve the cytotoxicity profiles of metal coordinating agents.

The cytotoxic activity of thiosemicarbazones (TSC) and thiocarbohydrazones was investigated against the MelRm melanoma cell line. In general, the melanoma line was susceptible to metal coordinating agents, the most useful of which incorporated the dipyridyl ketone hydrazone sub-structure. The impact of copper supplementation on the cytotoxic activity towards the melanoma line (MelRm) of metal coordinating agents when acting as ionophores is less predictable than the general improvement that has been seen in other cancer cells such as breast adenocarcinoma (MCF-7). The bimetallic nature of thiocarbohydrazone complexes with resultant loss of lipophilicity is a limiting factor in usage against MelRm. The cytotoxic activity of TSC against MelRm when used as copper ionophores could be markedly improved through combination with a partner drug capable of disrupting cellular defences to oxidative stress. In the absence of copper supplementation, both TSC and thiocarbohydrazones could be used to initiate cell cycle arrest and this could be employed to improve cytotoxicity profiles of other metallodrugs such as cisplatin.

Design, synthesis, and In vitro antituberculosis activity of 2(5H)-Furanone derivatives.

A series of 2(5H)-furanone-based compounds were synthesized from commercially available mucohalic acids. From the first-generation compounds, three showed inhibitory activity (10 µg/mL) of at least 35% against Mycobacterium smegmatis mc(2) 155 growth (Bioscreen C system). In screening the active first-generation compounds for growth inhibition against Mycobacterium tuberculosis H37Rv, the most active compound was identified with a minimum inhibitory concentration (MIC99 ) of 8.07 µg/mL (15.8 µM) using BACTEC 460 system. No cross-resistance was observed with some current first-line anti-TB drugs, since it similarly inhibited the growth of multidrug resistant (MDR) clinical isolates. The compound showed a good selectivity for mycobacteria since it did not inhibit the growth of selected Gram-positive and Gram-negative bacteria. It also showed synergistic activity with rifampicin (RIF) and additive activity with isoniazid (INH) and ethambutol (EMB). Additional time-kill studies showed that the compound is bacteriostatic to mycobacteria, but cytotoxic to the Chinese Hamster Ovarian (CHO) cell line. From a second generation library, two compounds showed improved anti-TB activity against M. tuberculosis H37Rv and decreased CHO cell cytotoxicity. The compounds exhibited MIC values of 2.62 µg/mL (5.6 µM) and 3.07 µg/mL (5.6 µM) respectively. The improved cytotoxicity against CHO cell line of the two compounds ranged from IC50 = 38.24 µg/mL to IC50 = 45.58 µg/mL when compared to the most active first-generation compound (IC50 = 1.82 µg/mL). The two second generation leads with selectivity indices (SI) of 14.64 and 14.85 respectively, warrant further development as anti-TB drug candidates. © 2016 IUBMB Life, 68(8):612-620, 2016.

Increased generation of intracellular reactive oxygen species initiates selective cytotoxicity against the MCF-7 cell line resultant from redox active combination therapy using copper-thiosemicarbazone complexes.

The combination of cytotoxic copper-thiosemicarbazone complexes with phenoxazines results in an up to 50-fold enhancement in the cytotoxic potential of the thiosemicarbazone against the MCF-7 human breast adenocarcinoma cell line over the effect attributable to drug additivity-allowing minimization of the more toxic copper-thiosemicarbazone component of the therapy. The combination of a benzophenoxazine with all classes of copper complex examined in this study proved more effective than combinations of the copper complexes with related isoelectronic azines. The combination approach results in rapid elevation of intracellular reactive oxygen levels followed by apoptotic cell death. Normal fibroblasts representative of non-cancerous cells (MRC-5) did not display a similar elevation of reactive oxygen levels when exposed to similar drug levels. The minimization of the copper-thiosemicarbazone component of the therapy results in an enhanced safety profile against normal fibroblasts.

Improved cytotoxicity of pyridyl-substituted thiosemicarbazones against MCF-7 when used as metal ionophores.

Zinc is the second most abundant transition metal in the human body, between 3 and 10% of human genes encoding for zinc binding proteins. We have investigated the interplay of reactive oxygen species and zinc homeostasis on the cytotoxicity of the thiosemicarbazone chelators against the MCF-7 cell line. The cytotoxicity of thiosemicarbazone chelators against MCF-7 can be improved through supplementation of ionic zinc provided the zinc ion is at a level exceeding the thiosemicarbazone concentration. Elimination of the entire cell population can be accomplished with this regime, unlike the plateau of cytotoxicity observed on thiosemicarbazone monotherapy. The cytotoxic effects of copper complexes of the thiosemicarbazone are not enhanced by zinc supplementation, displacement of copper from the complex being disfavoured. Treatment of MCF-7 with uncomplexed thiosemicarbazone initiates post G1 blockade alongside the induction of apoptosis, cell death being abrogated through subsequent supplementation with zinc ion after drug removal. This would implicate a metal depletion mechanism in the cytotoxic effect of the un-coordinated thiosemicarbazone. The metal complexes of the species, however, fail to initiate similar G1 blockade and apparently exert their cytotoxic effect through generation of reactive oxygen species, suggesting that multiple mechanisms of cytotoxicity can be associated with the thiosemicarbazones dependant on the level of metal ion association.

Selective induction of oxidative stress in cancer cells via synergistic combinations of agents targeting redox homeostasis.

Cancer cell resistance to chemotherapy is still a heavy burden that impairs the response of many cancer patients to conventional chemotherapy. Using drug combinations is one therapeutic approach to overcome the developing resistance to any one drug. Oxidative stress is now a generally regarded hallmark of cancer that can be one approach to selectively target cancer cells while sparing normal cells. With the aim of increasing oxidative stress in cancer cells to a lethal set point, we have generated and combined several series of redox active compounds that act at different points of the cellular oxidative cascade. The premise of such combinations is to deplete of endogenous antioxidant defence proteins (e.g., Glutathione) while concomitantly increasing the generation of ROS via metal redox recycling and Fenton chemistry which eventually leads to the disruption of cellular redox homeostasis and induction of cell death. Through this approach, we have identified highly synergistic combinations of two distinctive classes of compounds (Azines and Copper(II) complexes of 2-pyridyl ketone thiosemicarbazones) which are capable of eliminating cancer cells without concomitant increase in toxicity toward normal cells. In one of our most potent combinations, a combination index (CI) value of 0.056 was observed, representing a 17 fold enhancement in activity beyond additive effects. Such new combination regimen of redox active compounds can be one step closer to potentially safer low dose chemotherapy.

Structure-activity relationship study of sesquiterpene lactones and their semi-synthetic amino derivatives as potential antitrypanosomal products.

Sesquiterpene lactones (STLs) are natural products that have potent antitrypanosomal activity in vitro and, in the case of cynaropicrin, also reduce parasitemia in the murine model of trypanosomiasis. To explore their structure-antitrypanosomal activity relationships, a set of 34 natural and semi-synthetic STLs and amino-STLs was tested in vitro against T. b. rhodesiense (which causes East African sleeping sickness) and mammalian cancer cells (rat bone myoblast L6 cells). It was found that the α-methylene-γ-lactone moiety is necessary for both antitrypanosomal effects and cytotoxicity. Antitrypanosomal selectivity is facilitated by 2-(hydroxymethyl)acrylate or 3,4-dihydroxy-2-methylenebutylate side chains, and by the presence of cyclopentenone rings. Semi-synthetic STL amines with morpholino and dimethylamino groups showed improved in vitro activity over the native STLs. The dimethylamino derivative of cynaropicrin was prepared and tested orally in the T. b. rhodesiense acute mouse model, where it showed reduced toxicity over cynaropicrin, but also lost antitrypanosomal activity.

ATP and luciferase assays to determine the rate of drug action in in vitro cultures of Plasmodium falciparum.

BACKGROUND: Knowledge of the rate of action of compounds against cultured malaria parasites is required to determine the optimal time-points for drug mode of action studies, as well as to predict likely in vivo parasite clearance rates in order to select optimal hit compounds for further development. In this study, changes in parasite ATP levels and transgenic luciferase reporter activity were explored as means to detect drug-induced stress in cultured parasites. METHODS: In vitro cultures of Plasmodium falciparum 3D7 wild-type or firefly luciferase-expressing parasites were incubated with a panel of six anti-malarial compounds for 10 hours and parasite ATP levels or luciferase activity determined at two-hour intervals using luminescence-based reagents. For comparative purposes, parasite morphology changes were evaluated by light microscopy, as well as the extent to which parasites recover after 48 hours from a six-hour drug treatment using a parasite lactate dehydrogenase assay. RESULTS: Changes in parasite ATP levels displayed three phenotypes: mild or no change (chloroquine, DFMO); 2-4 fold increase (mefloquine, artemisinin); severe depletion (ritonavir, gramicidin). The respective phenotypes and the rate at which they manifested correlated closely with the extent to which parasites recovered from a six-hour drug treatment (with the exception of chloroquine) and the appearance and severity of morphological changes observed by light microscopy. Luciferase activity decreased profoundly in parasites treated with mefloquine, artemisinin and ritonavir (34-67% decrease in 2 hours), while chloroquine and DFMO produced only mild changes over 10 hours. Gramicidin yielded intermediate decreases in luciferase activity. CONCLUSIONS: ATP levels and luciferase activity respond rapidly to incubation with anti-malarial drugs and provide quantitative read-outs to detect the appearance and magnitude of drug-induced stress in cultured parasites. The correlation between the observed changes and irreversible parasite toxicity is not yet sufficiently clear to predict clinical clearance rates, but may be useful for ranking compounds against each other and standard drugs vis-à-vis rate of action and for determining early time-points for drug mode of action studies.

Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer.

BACKGROUND: The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. RESULTS: A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile. CONCLUSIONS: The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.

Metathesis reactions for the synthesis of ring-fused carbazoles.

[reactions: see text] The metathesis reaction is used as a key step for the synthesis of the indolo[2,3-a]carbazole core of rebeccamycin 13 and the sulfur analog of furostifoline 21. Using the same methodology for the attempted synthesis of furostifoline, we unexpectedly formed tert-butyl-2a-methyl-1,2,2a,10c-tetrahydro-6H-cyclobuta[c]furo[3,2-a]carbazole-6-carboxylate 26 from the unstable diene, tert-butyl 2-(2-isopropenyl-3-furyl)-3-vinyl-1H-indole-1-carboxylate 25, presumably via a spontaneous pi8 electrocyclization reaction.