Inhibition of Cyclic GMP-AMP Synthase Using a Novel Antimalarial Drug Derivative in Trex1-Deficient Mice.

OBJECTIVE: Type I interferon (IFN) is strongly implicated in the pathogenesis of systemic lupus erythematosus (SLE) as well as rare monogenic interferonopathies such as Aicardi-Goutières syndrome (AGS), a disease attributed to mutations in the DNA exonuclease TREX1. The DNA-activated type I IFN pathway cyclic GMP-AMP (cGAMP) synthase (cGAS) is linked to subsets of AGS and lupus. This study was undertaken to identify inhibitors of the DNA-cGAS interaction, and to test the lead candidate drug, X6, in a mouse model of AGS. METHODS: Trex1-/- mice were treated orally from birth with either X6 or hydroxychloroquine (HCQ) for 8 weeks. Expression of IFN-stimulated genes (ISGs) was quantified by quantitative polymerase chain reaction. Multiple reaction monitoring by ultra-performance liquid chromatography coupled with tandem mass spectrometry was used to quantify the production of cGAMP and X6 drug concentrations in the serum and heart tissue of Trex1-/- mice. RESULTS: On the basis of the efficacy-to-toxicity ratio established in vitro, drug X6 was selected as the lead candidate for treatment of Trex1-/- mice. X6 was significantly more effective than HCQ in attenuating ISG expression in mouse spleens (P < 0.01 for Isg15 and Isg20) and hearts (P < 0.05 for Isg15, Mx1, and Ifnb, and P < 0.01 for Cxcl10), and in reducing the production of cGAMP in mouse heart tissue (P < 0.05), thus demonstrating target engagement by the X6 compound. Of note, X6 was also more effective than HCQ in reducing ISG expression in vitro (P < 0.05 for IFI27 and MX1, and P < 0.01 for IFI44L and PKR) in human peripheral blood mononuclear cells from patients with SLE. CONCLUSION: This study demonstrates that X6 is superior to HCQ for the treatment of an experimental autoimmune myocarditis mediated in vivo by the cGAS/stimulator of IFN genes (cGAS/STING) pathway. The findings suggest that drug X6 could be developed as a novel treatment for AGS and/or lupus to inhibit activation of the cGAS/STING pathway.

Antimalarial Drugs as Immune Modulators: New Mechanisms for Old Drugs.

The best known of the naturally occurring antimalarial compounds are quinine, extracted from cinchona bark, and artemisinin (qinghao), extracted from Artemisia annua in China. These and other derivatives are now chemically synthesized and remain the mainstay of therapy to treat malaria. The beneficial effects of several of the antimalarial drugs (AMDs) on clinical features of autoimmune disorders were discovered by chance during World War II. In this review, we discuss the chemistry of AMDs and their mechanisms of action, emphasizing how they may impact multiple pathways of innate immunity. These pathways include Toll-like receptors and the recently described cGAS-STING pathway. Finally, we discuss the current and future impact of AMDs on systemic lupus erythematosus, rheumatoid arthritis, and devastating monogenic disorders (interferonopathies) characterized by expression of type I interferon in the brain.

DNA damage in dihydroartemisinin-resistant Molt-4 cells.

Artemisinin generates carbon-based free radicals when it reacts with iron, and induces molecular damage and apoptosis. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular free iron. Dihydroartemisinin (DHA), an analog of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A major concern is whether cancer cells could develop resistance to DHA, thus limiting its therapeutic efficacy. We have developed a DHA-resistant Molt-4 cell line (RTN) and found out that these cells exhibited resistance to DHA but no significant cross- resistance to artemisinin-tagged holotransferrin (ART-TF), a synthetic artemisinin compound. In the present study, we investigated DNA damage induced by DHA and ART-TF in both Molt-4 and RTN cells using the comet assay. RTN cells exhibited a significantly lower level of basal and X-ray-induced DNA damage compared to Molt-4 cells. Both DHA and ART-TF induced DNA damage in Molt-4 cells, whereas DNA damage was induced in RTN cells by ART-TF, and not DHA. The result of this study shows that by the cell selection method, it is possible to generate a Molt-4 cell line which is not sensitive to DHA, but sensitive to ART-TF, as measured by DNA damage.

pH-responsive artemisinin dimer in lipid nanoparticles are effective against human breast cancer in a xenograft model.

Artemisinin (ART), a well-known antimalaria drug, also exhibits anticancer activities. We previously reported a group of novel dimeric artemisinin piperazine conjugates (ADPs) possessing pH-dependent aqueous solubility and a proof-of-concept lipid nanoparticle formulation based on natural egg phosphatidylcholine (EPC). EPC may induce allergic reactions in individuals sensitive to egg products. Therefore, the goal of this report is to develop ADP-synthetic lipid particles suitable for in vivo evaluation. We found that ADP binds to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with greater than 90% efficiency and forms drug-lipid particles (d ∼ 80 nm). Cryo-electron microscopy of the ADP drug-lipid particles revealed unilamellar vesicle-like structures. Detailed characterization studies show insertion of the ADP lead compound, ADP109, into the DPPC membrane and the presence of an aqueous core. Over 50% of the ADP109 was released in 48 hours at pH4 compared with less than 20% at neutral. ADP109-lipid particles exhibited high potency against human breast cancer, but was tolerated well by nontumorigenic cells. In MDA-MB-231 mouse xenograft model, lipid-bound ADP109 particles were more effective than paclitaxel in controlling tumor growth. Cellular uptake studies showed endocytosis of the nanoparticles and release of core-trapped marker throughout the cytosol at 37°C. These results demonstrate, for the first time, the in vivo feasibility of lipid-bound ART dimer for cancer chemotherapy.

Cutting edge: Antimalarial drugs inhibit IFN-ß production through blockade of cyclic GMP-AMP synthase-DNA interaction.

Type I IFN is strongly implicated in the pathogenesis of systemic autoimmune diseases, such as lupus, and rare monogenic IFNopathies, including Aicardi-Goutières syndrome. Recently, a new DNA-activated pathway involving the enzyme cyclic GMP-AMP synthase (cGAS) was described and potentially linked to Aicardi-Goutières syndrome. To identify drugs that could potentially inhibit cGAS activity, we performed in silico screening of drug libraries. By computational analysis, we identified several antimalarial drugs (AMDs) that were predicted to interact with the cGAS/dsDNA complex. Our studies validated that several AMDs were effective inhibitors of IFN-ß production and that they functioned by inhibiting dsDNA stimulation of cGAS. Because AMDs have been widely used in human diseases and have an excellent safety profile, our findings suggest new therapeutic strategies for the treatment of severe debilitating diseases associated with type I IFNs due to cGAS activation.

Development of a dihydroartemisinin-resistant Molt-4 leukemia cell line.

Artemisinin generates cytotoxic free radicals when it reacts with iron. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular-free iron. We previously reported that dihydroartemisinin (DHA), an active metabolite of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A concern is whether cancer cells could develop resistance to DHA after repeated administration, thus limiting its therapeutic efficacy. In the present study, we developed a DHA-resistant Molt-4 cell line (RTN) by exposing Molt-4 cells to gradually increasing concentrations of DHA in vitro. The half-maximal inhibitory concentration (IC50) of DHA for RTN cells is 7.1-times higher than that of Molt-4 cells. RTN cells have a higher growth rate than Molt-4 cells. In addition, we investigated the toxicities of two more potent synthetic artemisinin compounds, artemisinin dimer-alcohol and artemisinin-tagged holotransferrin toward RTN cells; RTN cells showed no significant cross-resistance to these compounds.

Synthesis of artemisinin dimers using the Ugi reaction and their in vitro efficacy on breast cancer cells.

The Ugi four-component reaction was used to prepare a series of artemisinin monomers and dimers. We found that the endoperoxide group in artemisinin remains intact during the reaction. The new artemisinin dimers showed potent anti-cancer activity against two human breast cancer cell lines, MDA-MB-231 and BT-474. One of the Ugi artemisinin dimers showed an IC50 value of 12 nM when tested on BT474 cells, more than 600 times more potent than artesunate. Furthermore, the same Ugi artemisinin dimer showed a low toxicity when tested on MCF10A, a nontumorigenic cell line, resulting in a selectivity index of more than 8000.

pH-responsive artemisinin derivatives and lipid nanoparticle formulations inhibit growth of breast cancer cells in vitro and induce down-regulation of HER family members.

Artemisinin (ART) dimers show potent anti-proliferative activities against breast cancer cells. To facilitate their clinical development, novel pH-responsive artemisinin dimers were synthesized for liposomal nanoparticle formulations. A new ART dimer was designed to become increasingly water-soluble as pH declines. The new artemisinin dimer piperazine derivatives (ADPs) remained tightly associated with liposomal nanoparticles (NPs) at neutral pH but were efficiently released at acidic pH's that are known to exist within solid tumors and organelles such as endosomes and lysosomes. ADPs incorporated into nanoparticles down regulated the anti-apoptotic protein, survivin, and cyclin D1 when incubated at low concentrations with breast cancer cell lines. We demonstrate for the first time, for any ART derivative, that ADP NPs can down regulate the oncogenic protein HER2, and its counterpart, HER3 in a HER2+ cell line. We also show that the wild type epidermal growth factor receptor (EGFR or HER1) declines in a triple negative breast cancer (TNBC) cell line in response to ADP NPs. The declines in these proteins are achieved at concentrations of NP109 at or below 1 µM. Furthermore, the new artemisinin derivatives showed improved cell-proliferation inhibition effects compared to known dimer derivatives.

Effects of transferrin conjugates of artemisinin and artemisinin dimer on breast cancer cell lines.

Transferrin (Tf) conjugates of monomeric artemisinin (ART) and artemisinin dimer were synthesized. The two conjugates, ART-Tf and dimer-Tf, retained the original protein structure, and formed stable aggregates in aqueous buffer. ART-Tf induced declines in proteins involved in apoptosis (survivin), cell cycling (cyclin D1), oncogenesis (c-myelocytomatosis oncogene product (c-MYC)), and dysregulated WNT signaling (beta-catenin) in both the human prostate (DU145) and breast (MCF7) cancer cell lines. Both ART-Tf and dimer-Tf induced down-regulation of survivin, c-MYC and mutated human epidermal growth factor receptor-2 (ERBB2 or HER2) in the BT474 breast cancer cell line. To our knowledge, this is the first demonstration that an ART derivative can cause a decline of ERBB2 in a human cancer cell line. Potential mechanisms for the observed effects are presented. Both transferrin conjugates strongly inhibited the growth of BT474 cells in the same concentration range that the conjugates caused declines in the levels of ERBB2, survivin, and c-MYC, while showing essentially no toxicity towards MCF10A normal breast cells.

Development of artemisinin compounds for cancer treatment.

Artemisinin contains an endoperoxide moiety that can react with iron to form cytotoxic free radicals. Cancer cells contain significantly more intracellular free iron than normal cells and it has been shown that artemisinin and its analogs selectively cause apoptosis in many cancer cell lines. In addition, artemisinin compounds have been shown to have anti-angiogenic, anti-inflammatory, anti-metastasis, and growth inhibition effects. These properties make artemisinin compounds attractive cancer chemotherapeutic drug candidates. However, simple artemisinin analogs are less potent than traditional cancer chemotherapeutic agents and have short plasma half-lives, and would require high dosage and frequent administration to be effective for cancer treatment. More potent and target-selective artemisinin-compounds are being developed. These include artemisinin dimers and trimers, artemisinin hybrid compounds, and tagging of artemisinin compounds to molecules that are involved in the intracellular iron-delivery mechanism. These compounds are promising potent anticancer compounds that produce significantly less side effect than traditional chemotherapeutic agents.

Effects of artemisinin dimers on rat breast cancer cells in vitro and in vivo.

Artemisinin has been shown to be an effective antimalarial and anticancer compound. Dimers of artemisinin have been synthesized and shown to be potent antimalarials compared with monomers. In the present study, we investigated the effect of two artemisinin dimers (dimer-alcohol and dimer-hydrazone) on rat mammary adenocarcinoma cells (MTLn3) in vitro and in vivo compared with that of the artemisinin monomer dihydroartemisinin (DHA). We found that the dimers are considerably more potent than DHA in killing MTLn3 cells in vitro and suppressing the growth of MTLn3 breast tumors in vivo.

Flavonoids from Artemisia annua L. as antioxidants and their potential synergism with artemisinin against malaria and cancer.

Artemisia annua is currently the only commercial source of the sesquiterpene lactone artemisinin.Since artemisinin was discovered as the active component of A. annua in early 1970s, hundreds of papers have focused on the anti-parasitic effects of artemisinin and its semi-synthetic analogs dihydroartemisinin, artemether, arteether, and artesunate. Artemisinin per se has not been used in mainstream clinical practice due to its poor bioavailability when compared to its analogs. In the past decade, the work with artemisinin-based compounds has expanded to their anti-cancer properties. Although artemisinin is a major bioactive component present in the traditional Chinese herbal preparations (tea), leaf flavonoids, also present in the tea, have shown a variety of biological activities and may synergize the effects of artemisinin against malaria and cancer. However, only a few studies have focused on the potential synergistic effects between flavonoids and artemisinin. The resurgent idea that multi-component drug therapy might be better than monotherapy is illustrated by the recent resolution of the World Health Organization to support artemisinin-based combination therapies (ACT), instead of the previously used monotherapy with artemisinins. In this critical review we will discuss the possibility that artemisinin and its semi-synthetic analogs might become more effective to treat parasitic diseases (such as malaria) and cancer if simultaneously delivered with flavonoids. The flavonoids present in A. annua leaves have been linked to suppression of CYP450 enzymes responsible for altering the absorption and metabolism of artemisinin in the body, but also have been linked to a beneficial immunomodulatory activity in subjects afflicted with parasitic and chronic diseases.

Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells.

Artemisinin is a plant-derived anti-malarial drug that has relatively low toxicity in humans and is activated by heme and/or intracellular iron leading to intracellular free radical formation. Interestingly, artemisinin has displayed anti-cancer activity, with artemisinin dimers being more potent than monomeric artemisinin. Intracellular iron uptake is regulated by the transferrin receptor (TfR), and the activity of artemisinin depends on the availability of iron. We examined the level of TfR in prostate cancer (PCa) tumor cells, synthesized two new artemisinin dimers, and evaluated the effect of dihydroartemisinin and artemisinin dimers, ON-2Py and 2Py, on proliferation and apoptosis in PCa cells. TfR was expressed in the majority of PCa bone and soft tissue metastases, all 24 LuCaP PCa xenografts, and PCa cell lines. After treatment with dihydroartemisinin, ON-2Py, or 2Py all PCa cell lines displayed dose-dependent decrease in cell number. 2Py was most effective in decreasing cell number. An increase in apoptotic events and growth arrest was observed in the C4-2 and LNCaP cell lines. Growth arrest was observed in PC-3 cells, but no significant change was observed in DU 145 cells. Treatment with 2Py resulted in a loss of the anti-apoptotic protein survivin in all four cell lines. 2Py treatment also decreased androgen receptor and prostate-specific antigen expression in C4-2 and LNCaP cells, with a concomitant loss of cell cycle regulatory proteins cyclin D1 and c-Myc. This study shows the potential use of artemisinin derivatives as therapeutic candidates for PCa and warrants the initiation of preclinical studies.

Artemisinin-transferrin conjugate retards growth of breast tumors in the rat.

BACKGROUND: Artemisinin is a compound isolated from the wormwood Artemisia annua L. It reacts with iron and forms cytotoxic free radicals. It is selectively more toxic to cancer than normal cells because cancer cells contain significantly more intracellular free iron. Previously, we found that covalently tagging artemisinin to transferrin enhanced the selectivity and toxicity of artemisinin toward cancer cells in vitro. In the present research, artemisinin-transferrin conjugate was tested in a rat breast cancer model. MATERIALS AND METHODS: Breast tumors were induced in rats by subcutaneous implantation of rat MTLn3 breast cancer cells. Once tumors were formed, daily intravenous injections of artemisinin-transferrin conjugate were administered. RESULTS: The conjugate significantly retarded the growth rate of breast tumors in the rat. No significant side effect was observed in the rats during treatment. CONCLUSION: Artemisinin-transferrin conjugate could be developed into a potent therapeutic agent for cancer in humans.

Transferrin receptor-dependent cytotoxicity of artemisinin-transferrin conjugates on prostate cancer cells and induction of apoptosis.

Artemisinin, a natural product isolated from Artemisia annua, contains an endoperoxide group that can be activated by intracellular iron to generate toxic radical species. Cancer cells over-express transferrin receptors (TfR) for iron uptake while most normal cells express nearly undetectable levels of TfR. We prepared a series of artemisinin-tagged transferrins (ART-Tf) where different numbers of artemisinin units are attached to the N-glycoside chains of transferrin (Tf). The Tf bearing approximately 16 artemisinins retains the functionality of both Tf and artemisinin. Reduction of TfRs by TfR siRNA transfection significantly impaired the ability of ART-Tf, but not dihydroartemisinin, to kill cells. We also demonstrate that the ART-Tf conjugate kills the prostate carcinoma cell line DU 145 by the mitochondrial pathway of apoptosis.

Synthesis and anti-cancer activity of covalent conjugates of artemisinin and a transferrin-receptor targeting peptide.

Artemisinin, a natural product isolated from Artemisia annua L., shows a unique anti-cancer activity by an iron dependent mechanism. Artemisinin was covalently conjugated to a transferrin-receptor targeting peptide, HAIYPRH that binds to a cavity on the surface of transferrin receptor. This enables artemisinin to be co-internalized with receptor-bound transferrin. The iron released from transferrin can activate artemisinin to generate toxic radical species to kill cells. The artemisinin-peptide conjugates showed potent anti-cancer activity against Molt-4 leukemia cells with a significantly improved cancer/normal cells selectivity.

Anticancer properties of artemisinin derivatives and their targeted delivery by transferrin conjugation.

Artemisinin and its derivatives are well known antimalaria drugs and particularly useful for the treatment of infection of Plasmodium falciparum malaria parasites resistant to traditional antimalarials. Artemisinin has an endoperoxide bridge that is activated by intraparasitic heme-iron to form free radicals, which kill malaria parasites by alkylating biomolecules. In recent years, there are many reports of anticancer activities of artemisinins both in vitro and in vivo. Artemisinins have inhibitory effects on cancer cell growth, including many drug- and radiation-resistant cancer cell lines. The cytotoxic effect of artemisinin is specific to cancer cells because most cancer cells express a high concentration of transferrin receptors on cell surface and have higher iron ion influx than normal cells via transferrin mechanism. In addition, some artemisinin analogs have been shown to have antiangiogenesis activity. Artemisinin tagged to transferrin via carbohydrate chain has also been shown to have high potency and specificity against cancer cells. The conjugation enables targeted delivery of artemisinin into cancer cells. In this review, we discuss the anticancer activities and mechanisms of action of artemisinins and the transferrin-conjugate.

Electron capture in spin-trap capped peptides. An experimental example of ergodic dissociation in peptide cation-radicals.

Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4'-carboxypyrid-2'-yl)-4-carboxamide group (pepy), e.g., pepy-AEQLLQEEQLLQEL-NH(2), pepy-AQEFGEQGQKALKQL-NH(2), and pepy-AQEGSEQAQKFFKQL-NH(2). Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b(11-14) + 1)(+)* fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1' positions in the pepy moiety had topical gas-phase basicities (GB = 923 kJ mol(-1)) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE = 5.43-5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by >160 kJ mol(-1), which exceeded the hydrogen atom affinity of the backbone amide groups (21-41 kJ mol(-1)). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD.

Targeted treatment of cancer with artemisinin and artemisinin-tagged iron-carrying compounds.

Artemisinin is a chemical compound that reacts with iron to form free radicals which can kill cells. Cancer cells require and uptake a large amount of iron to proliferate. They are more susceptible to the cytotoxic effect of artemisinin than normal cells. Cancer cells express a large concentration of cell surface transferrin receptors that facilitate uptake of the plasma iron-carrying protein transferrin via endocytosis. By covalently tagging artemisinin to transferrin, artemisinin could be selectively picked up and concentrated by cancer cells. Futhermore, both artemisinin and iron would be transported into the cell in one package. Once an artemisinin-tagged transferrin molecule is endocytosed, iron is released and reacts with artemisinin moieties tagged to transferrin. Formation of free radicals kills the cancer cell. The authors have found that artemisinin-tagged transferrin is highly selective and potent in killing cancer cells. Thus, artemisinin and artemisinin-tagged iron-carrying compounds could be developed into powerful anticancer drugs.

Effects of artemisinin-tagged holotransferrin on cancer cells.

Artemisinin reacts with iron to form free radicals that kill cells. Since cancer cells uptake relatively large amount of iron than normal cells, they are more susceptible to the toxic effect of artemisinin. In previous research, we have shown that artemisinin is more toxic to cancer cells than to normal cells. In the present research, we covalently attached artemisinin to the iron-carrying plasma glycoprotein transferrin. Transferrin is transported into cells via receptor-mediated endocytosis and cancer cells express significantly more transferrin receptors on their cell surface and endocytose more transferrin than normal cells. Thus, we hypothesize that by tagging artemisinin to transferrin, both iron and artemisinin would be transported into cancer cells in one package. Once inside a cell, iron is released and can readily react with artemisinin close by tagged to the transferrin. This would enhance the toxicity and selectivity of artemisinin towards cancer cells. In this paper, we describe a method to synthesize such a compound in which transferrin was conjugated with an analog of artemisinin artelinic acid via the N-glycoside chains on the C-domain. The resulting conjugate ('tagged-compound') was characterized by MALDI-MS, UV/Vis spectroscopy, chemiluminescence, and HPLC. We then tested the compound on a human leukemia cell line (Molt-4) and normal human lymphocytes. We found that holotransferrin-tagged artemisinin, when compared with artemisinin, was very potent and selective in killing cancer cells. Thus, this 'tagged-compound' could potentially be developed into an effective chemotherapeutic agent for cancer treatment.