Gold(I) ion and the phosphine ligand are necessary for the anti-Toxoplasma gondii activity of auranofin.

Auranofin, an FDA-approved drug for rheumatoid arthritis, has emerged as a promising antiparasitic medication in recent years. The gold(I) ion in auranofin is postulated to be responsible for its antiparasitic activity. Notably, aurothiomalate and aurothioglucose also contain gold(I), and, like auranofin, they were previously used to treat rheumatoid arthritis. Whether they have antiparasitic activity remains to be elucidated. Herein, we demonstrated that auranofin and similar derivatives, but not aurothiomalate and aurothioglucose, inhibited the growth of Toxoplasma gondii in vitro. We found that auranofin affected the T. gondii biological cycle (lytic cycle) by inhibiting T. gondii's invasion and triggering its egress from the host cell. However, auranofin could not prevent parasite replication once T. gondii resided within the host. Auranofin treatment induced apoptosis in T. gondii parasites, as demonstrated by its reduced size and elevated phosphatidylserine externalization (PS). Notably, the gold from auranofin enters the cytoplasm of T. gondii, as demonstrated by scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).IMPORTANCEToxoplasmosis, caused by Toxoplasma gondii, is a devastating disease affecting the brain and the eyes, frequently affecting immunocompromised individuals. Approximately 60 million people in the United States are already infected with T. gondii, representing a population at-risk of developing toxoplasmosis. Recent advances in treating cancer, autoimmune diseases, and organ transplants have contributed to this at-risk population's exponential growth. Paradoxically, treatments for toxoplasmosis have remained the same for more than 60 years, relying on medications well-known for their bone marrow toxicity and allergic reactions. Discovering new therapies is a priority, and repurposing FDA-approved drugs is an alternative approach to speed up drug discovery. Herein, we report the effect of auranofin, an FDA-approved drug, on the biological cycle of T. gondii and how both the phosphine ligand and the gold molecule determine the anti-parasitic activity of auranofin and other gold compounds. Our studies would contribute to the pipeline of candidate anti-T. gondii agents.

Next Generation Gold Drugs and Probes: Chemistry and Biomedical Applications.

The gold drugs, gold sodium thiomalate (Myocrisin), aurothioglucose (Solganal), and the orally administered auranofin (Ridaura), are utilized in modern medicine for the treatment of inflammatory arthritis including rheumatoid and juvenile arthritis; however, new gold agents have been slow to enter the clinic. Repurposing of auranofin in different disease indications such as cancer, parasitic, and microbial infections in the clinic has provided impetus for the development of new gold complexes for biomedical applications based on unique mechanistic insights differentiated from auranofin. Various chemical methods for the preparation of physiologically stable gold complexes and associated mechanisms have been explored in biomedicine such as therapeutics or chemical probes. In this Review, we discuss the chemistry of next generation gold drugs, which encompasses oxidation states, geometry, ligands, coordination, and organometallic compounds for infectious diseases, cancer, inflammation, and as tools for chemical biology via gold-protein interactions. We will focus on the development of gold agents in biomedicine within the past decade. The Review provides readers with an accessible overview of the utility, development, and mechanism of action of gold-based small molecules to establish context and basis for the thriving resurgence of gold in medicine.

Aurothiomalate-Based Drugs as Potentially Novel Agents Against Leishmania major: A Mini Review.

PURPOSE: Leishmaniasis is a major public health problem worldwide in many parts of the world. Current anti-leishmanial drugs have only limited clinical efficacy. Aurothiomalate derivatives are useful for treating rheumatoid arthritis, but have emerged as a promising therapeutic candidate for leishmaniasis. This paper gives a review of the literature about the usefulness of aurothiomalate derivatives against leishmaniasis. METHODS: In this study, we reviewed the proposed mechanisms of action of aurothiomalate and related compounds on the metabolism of L. major and collected data by searching relevant articles. RESULTS: Aurothiomalate-based drugs could be effective against leishmaniasis through two direct and indirect mechanisms: first, cytotoxic effects on parasites via thiomalate's false substrate role in the citric acid cycle against malate; and second, immunosuppressive and anti-inflammatory effects of aurothiomalate derivatives with prostaglandin production inhibitory effects. CONCLUSIONS: The current study documented that aurothiomalate-based drugs could be effective against leishmaniasis through two direct and indirect mechanisms of action. Gold thiomalate as a promising hit should be evaluated against L. major in vitro and in vivo conditions in the future.

Multilevel Approach for the Treatment of Giardiasis by Targeting Arginine Deiminase.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

The Role of NF-κВ in the Realization of Functions of Various Types of Regeneration-Competent Cells of the Nervous Tissue in Ethanol-Induced Neurodegeneration.

The role of NF-κВ in the realization of the growth potential of neural progenitor cells from the subventricular area of cerebral hemispheres and secretion of neurotrophins by glial elements was studied under conditions of in vitro and in vivo modeled ethanol-induced neurodegeneration. It was found that this transcription factor does not participate in the regulation of mitotic activity of neural stem cells and neuronal-committed progenitors under optimal conditions and under the influence of ethanol in vitro. At the same time, NF-κВ suppresses differentiation/maturation of neural progenitor cells. Long-term peroral administration of ethanol to mice was accompanied by the inhibitory influence of NF-κВ on proliferation of progenitor cells. Blockade of NF-κВ in neural stem cells and committed neuronal precursors in animals with neurodegeneration induced cell cycle progression in these elements. The involvement of NF-κВ in the secretory function of astrocytes and oligodendrogliocytes was established. Inactivation of the nuclear transcription factor reduced the production of neurotrophins, in particular, in the case of ethanol exposure. At the same time, no changes in the function of microglia were noted.

Involvement of Signaling Cascades in Granulocytopoiesis Regulation under Conditions of Cytostatic Treatment.

Suppression of the production of granulocytic CSF under the effect of 5-fluorouracyl is related to disorders in the NF-κB-, cAMP-dependent signaling pathways and MAPK cascade. These secondary messengers are involved in the regulation of functional activity of nonadherent myelokaryocytes starting from day 10 of the experiment (initial period of the hemopoietic granulocytic stem regeneration after antimetabolite challenge). Granulocytic CSF does not play essential role in the formation of colony-stimulating activity of cells of the adherent and nonadherent fractions of the bone marrow. Only cAMP-dependent pathway is involved in the regulation of the realization of the granulocytic precursor growth potential in response to the challenge.

Role of Signaling Molecules in the Regulation of Granulocytopoiesis during Stress-Inducing Stimulation.

The role of signaling molecules in synthesis of humoral regulators of granulocytopoiesis by the hematopoietic microenvironmental cells during stress was analyzed using specific inhibitors. The major role in stimulation of the synthesis of granulocytic CSF during stressful stimulation is played by PI3K/Akt signaling cascade. Nuclear transcription factor NF-κB plays an auxiliary role in the regulation of functional activity of the bone marrow mononuclears. However, this factor affects the synthesis of granulocytic CSF by CD4+ cells of the bone marrow in response to stressful stimulation. Different degree and specific character of involvement of the signaling proteins in the regulation of the production of humoral factors determining colony-stimulating activity are explained by changes in functional state of monocyte-derived macrophages in different periods of stress response.

Targeting oncogenic protein kinase Cι for treatment of mutant KRAS LADC.

Lung cancer is the leading cause of cancer death in the US with ∼124,000 new cases annually, and a 5 y survival rate of ∼16%. Mutant KRAS-driven lung adenocarcinoma (KRAS LADC) is a particularly prevalent and deadly form of lung cancer. Protein kinase Cι (PKCι) is an oncogenic effector of KRAS that activates multiple signaling pathways that stimulate transformed growth and invasion, and maintain a KRAS LADC tumor-initiating cell (TIC) phenotype. PKCι inhibitors used alone and in strategic combination show promise as new therapeutic approaches to treatment of KRAS LADC. These novel drug combinations may improve clinical management of KRAS LADC.

A small molecule inhibitor of atypical protein kinase C signaling inhibits pancreatic cancer cell transformed growth and invasion.

Pancreatic cancer is highly resistant to current chemotherapies. Identification of the critical signaling pathways that mediate pancreatic cancer transformed growth is necessary for the development of more effective therapeutic treatments. Recently, we demonstrated that protein kinase C iota (PKCι) and zeta (PKCζ) promote pancreatic cancer transformed growth and invasion, by activating Rac1→ERK and STAT3 signaling pathways, respectively. However, a key question is whether PKCι and PKCζ play redundant (or non-redundant) roles in pancreatic cancer cell transformed growth. Here we describe the novel observations that 1) PKCι and PKCζ are non-redundant in the context of the transformed growth of pancreatic cancer cells; 2) a gold-containing small molecule known to disrupt the PKCι/Par6 interaction, aurothiomalate, also disrupts PKCζ/Par6 interaction; 3) aurothiomalate inhibits downstream signaling of both PKCι and PKCζ, and blocks transformed growth of pancreatic cancer cells in vitro; and 4) aurothiomalate inhibits pancreatic cancer tumor growth and metastasis in vivo. Taken together, these data provide convincing evidence that an inhibitor of atypical PKC signaling inhibits two key oncogenic signaling pathways, driven non-redundantly by PKCι and PKCζ, to significantly reduce tumor growth and metastasis. Our results demonstrate that inhibition of atypical PKC signaling is a promising therapeutic strategy to treat pancreatic cancer.

Reactions of model proteins with aurothiomalate, a clinically established gold(I) drug: The comparison with auranofin.

Aurothiomalate (AuTm) is an old, clinically established, antiarthritic gold drug that is currently being reconsidered as a candidate drug for cancer treatment and for other therapeutic indications within a more general drug repositioning program. As the biological effects of gold drugs seem to be mediated, mainly, by their interactions with protein targets we have analyzed here, in detail, the metalation patterns produced by aurothiomalate in a few model proteins. In particular, the reactions of aurothiomalate with the small proteins ribonuclease A, cytochrome c and lysozyme were explored through ESI MS (electrospray ionization mass spectrometry) analysis. Notably, characteristic and rather constant features emerged in the protein metalation patterns induced by AuTm that are markedly distinct from those caused by auranofin; a non-covalent interaction mode is invoked for AuTm binding to the mentioned proteins. The affinity constants of AuTm toward the three mentioned proteins were also initially assessed. The implications of the present findings are discussed.

Role of NF-κB/IKK-dependent signaling in functional stimulation of mesenchymal progenitor cells by alkaloid songorine.

We studied the role of NF-κB/IKK-mediated signaling in the stimulation of growth potential of mesenchymal progenitor cells by alkaloid songorine in vitro. Specific NF-κB inhibitor oridonin abolished activation of proliferation and differentiation of progenitor cells. Aurothiomalate, a selective blocker of IKK-2, also suppressed mitotic activity of fibroblast precursors, but had no effect on the rate of the differentiation.

Aurothiomalate inhibits the expression of mPGES-1 in primary human chondrocytes.

OBJECTIVES: Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal enzyme in the production of prostaglandin E2 (PGE2) and its expression is upregulated during inflammation. mPGES-1 is considered as a potential drug target for the treatment of arthritis to reduce adverse effects related to the current non-steroidal anti-inflammatory drugs (NSAIDs). Our aim was to study the expression of mPGES-1 in primary human chondrocytes and whether the expression is affected by clinically used antirheumatic drugs. METHOD: Primary human chondrocytes were isolated from cartilage samples obtained from patients undergoing total knee replacement surgery. Expression of mPGES-1 was studied by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis. PGE2 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: mPGES-1 expression in primary human chondrocytes was enhanced when the cells were exposed to interleukin-1ß (IL-1ß) and mPGES-1 protein levels continued to increase up to the 96-h follow-up. Aurothiomalate inhibited mPGES-1 expression and PGE2 production in a dose-dependent manner, as did the anti-inflammatory steroid dexamethasone. Other disease-modifying antirheumatic drugs (DMARDs) studied (sulfasalazine, methotrexate, and hydroxychloroquine) did not alter mPGES-1 expression. CONCLUSIONS: The results introduce aurothiomalate as the first, and so far the only, DMARD found to be able to inhibit mPGES-1 expression. The effect is likely involved in the mechanisms of action of this gold-containing DMARD in rheumatic diseases. The results are implicated in the regulatory mechanisms of mPGES-1 expression, which are under intensive research.

Role of NF-κB-dependent signaling and p38 MAPK signaling pathway in the control of hemopoiesis during cytostatic administration.

The study examines the role of signal pathways in triggering the compensatory reactions in the blood system in response to the cytostatic administration. In vitro experiments elucidated the involvement of protein kinase p38 in the synthesis of granulocytic CSF by cells of hemopoietic microenvironment. The important role of the transcriptional factor NF-κB and protein kinase p38 in limitation of the processes of maturation of the hemopoietic precursors was demonstrated.

The aPKCι blocking agent ATM negatively regulates EMT and invasion of hepatocellular carcinoma.

Epithelial-to-mesenchymal transition (EMT) has an important role in invasion and metastasis of hepatocellular carcinoma (HCC). To explore the regulatory mechanism of atypical protein kinase C ι (aPKCι) signaling pathways to HCC development, and find an agent for targeted therapy for HCC, immortalized murine hepatocytes were employed to establish an EMT cell model of HCC, MMH-RT cells. Our study showed that EMT took place in MMH-R cells under the effect of transforming growth factor-ß1 (TGF-ß1) overexpressing aPKCι. Furthermore, we showed that the aPKCι blocking agent aurothiomalate (ATM) inhibited EMT and decreased invasion of hepatocytes. Moreover, ATM selectively inhibited proliferation of mesenchymal cells and HepG2 cells and induced apoptosis. However, ATM increased proliferation of epithelial cells and had little effect on apoptosis and invasion of epithelial cells. In conclusion, our result suggested that aPKCι could be an important bio-marker of tumor EMT, and used as an indicator of invasion and malignancy. ATM might be a promising agent for targeted treatment of HCC.

Effects of aurothiomalate treatment on canine osteosarcoma in a murine xenograft model.

Osteosarcoma is a highly fatal cancer, with most patients ultimately succumbing to metastatic disease. The purpose of this study was to evaluate the effects of the antirheumatoid drug aurothiomalate on canine and human osteosarcoma cells and on canine osteosarcoma growth and metastasis in a mouse xenograft model. We hypothesized that aurothiomalate would decrease osteosarcoma cell survival, tumor cellular proliferation, tumor growth, and metastasis. After performing clonogenic assays, aurothiomalate or a placebo was administered to 54 mice inoculated with canine osteosarcoma. Survival, tumor growth, embolization, metastasis, histopathology, cell proliferation marker Ki67, and apoptosis marker caspase-3 were compared between groups. Statistical analysis was carried out using the Kaplan-Meier method with the log-rank test and one-way analysis of variance with the Tukey's test or Dunn's method. Aurothiomalate caused dose-dependent inhibition of osteosarcoma cell survival (P<0.001) and decreased tumor growth (P<0.001). Pulmonary macrometastasis and Ki67 labeling were reduced with low-dose aurothiomalate (P=0.033 and 0.005, respectively), and tumor emboli and pulmonary micrometastases were decreased with high-dose aurothiomalate (P=0.010 and 0.011, respectively). There was no difference in survival, tumor development, ulceration, mitotic indices, tumor necrosis, nonpulmonary metastases, and caspase-3 labeling. Aurothiomalate treatment inhibited osteosarcoma cell survival and reduced tumor cell proliferation, growth, embolization, and pulmonary metastasis. Given aurothiomalate's established utility in canine and human medicine, our results suggest that this compound may hold promise as an adjunctive therapy for osteosarcoma. Further translational research is warranted to better characterize the dose response of canine and human osteosarcoma to aurothiomalate.

Conditional pharmacology/toxicology V: ambivalent effects of thiocyanate upon the development and the inhibition of experimental arthritis in rats by aurothiomalate (Myocrysin®) and metallic silver.

This article discusses the bizarre and contrary effects of thiocyanate, the major detoxication product of hydrogen cyanide inhaled from tobacco smoke or liberated from cyanogenic foods, e.g. cassava. Thiocyanate both (1) promotes inflammatory disease in rats and (2) facilitates the anti-inflammatory action of historic metal therapies based on gold (Au) or silver (Ag) in three models of chronic polyarthritis in rats. Low doses of nanoparticulate metallic silver (NMS) preparations, i.e. zerovalent silver (Ag°) administered orally, suppressed the mycobacterial ('adjuvant')-induced arthritis (MIA) in rats. Similar doses of cationic silver, Ag(I), administered orally as silver oxide or soluble silver salts were inactive. By contrast, NMS only inhibited the development of the collagen-induced arthritis (CIA) and pristane-induced arthritis (PIA) in rats when thiocyanate was also co-administered in drinking water. These (a) arthritis-selective and (b) thiocyanate-inducible effects of Ag° were also observed in some previous, and now extended, studies with the classic anti-arthritic drug, sodium aurothiomalate (ATM, Myocrisin(®)) and its silver analogue (STM), administered subcutaneously to rats developing the same three forms of polyarthritis. In the absence of either Ag° or ATM, thiocyanate considerably increased the severity of the MIA, CIA and PIA, i.e. acting as a pro-pathogen. Hitherto, thiocyanate was considered relatively harmless. This may not be true in rats/people with immuno-inflammatory stress and concomitant leukocyte activation. Collectively, these findings show how the drug action of a xenobiotic might be determined by the nature (and severity) of the experimental inflammation, as an example of conditional pharmacology. They also suggest that an incipient toxicity, even of normobiotics such as thiocyanate, might likewise be modulated beneficially by well-chosen xenobiotics (drugs, nutritional supplements, etc.), i.e. conditional toxicology (Powanda 1995). Thus, both the disease and the environment may determine (1) the therapeutic action and/or (2) adverse effect(s) of xenobiotics--and even some normobiotics.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

Effect of NF-κB inhibitor aurothiomalate on local inflammation in experimental Th1- and Th2-type immune response.

We compared the effect of NF-κB inhibitor aurothiomalate and voltaren on local inflammation in different types of immune response. Both substances reduced edema caused by sheep erythrocytes (Th1-type immune response) and local immediate-type hypersensitivity response induced with ovalbumin (Th2-dependent response). The anti-inflammatory effects of aurothiomalate were similar to those of voltaren during Th1-type immune response.

Aurothiomalate as preventive and chain-breaking antioxidant in radical degradation of high-molar-mass hyaluronan.

The potential anti- or pro-oxidative effects of a disease-modifying antirheumatic drug, aurothiomalate, to protect high-molar-mass hyaluronan against radical degradation were investigated along with L-glutathione - tested in similar functions. Hyaluronan degradation was induced by the oxidative system Cu(II) plus ascorbate known as the Weissberger's oxidative system. The time- and dose-dependent changes of the dynamic viscosity of the hyaluronan solutions were studied by the method of rotational viscometry. Additionally, the antioxidative activity of aurothiomalate expressed as a radical-scavenging capacity based on a decolorization 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay was inspected. At the higher concentrations tested, L-glutathione showed excellent scavenging of (.) OH and peroxyl-type radicals, however, at the lowest concentration applied, its pro-oxidative effect was revealed. The effects of aurothiomalate on hyaluronan degradation were similar to that of L-glutathione, however, at the lowest concentration tested, no significant pro-oxidant effect was observed.

Study of anti-inflammatory action of aurothiomalate, an inhibitor of NF-κB.

We compared the effects of NF-κB inhibitor aurothiomalate and voltaren on NO production by mouse macrophages in vitro, their ability to cause local edema at the site of injection, and their effect on carrageenan-induced inflammation. High concentrations of aurothiomalate reduced NO production, while in low concentrations both aurothiomalate and voltaren stimulated this process. When injected into mouse footpad, aurothiomalate in a dose >1 mM and voltaren in a dose >1.6 µM induce paw edema. Both compounds suppressed carrageenan-induced inflammation, but the efficacy of aurothiomalate 2-fold exceeded that of voltaren.