Cinchonine and cinchonidine alleviate cisplatin-induced ototoxicity by regulating PI3K-AKT signaling.

AIM: Cinchonine (CN) and its isomer cinchonidine (CD), two of the common cinchona alkaloids, are wildly used as antimalarial drugs. However, the effects of CN and CD on the auditory system are unknown. METHODS: Molecular docking and molecular dynamics (MD) simulation were used for predicting effective drugs. The CCK-8 assay was conducted for assessing cell viability in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. MitoSox Red staining revealed reactive oxygen species (ROS) amounts. TMRM staining was used to assess the mitochondrial membrane potential (ΔΨm). Immunofluorescence staining of myosin 7a was used to examine hair cells (HCs) in cisplatin-treated neonatal mouse cochlear explants, while TUJ-1 immunostaining was used for the detection of spiral ganglion neurons (SGNs). Cleaved caspase-3 and TUNEL immunostaining were utilized for apoptosis assessment. Immunoblot was carried out to detect PI3K-AKT signaling effectors. RESULTS: Pretreatment with CN or CD significantly increased cell viability and reduced mitochondrial dysfunction and ROS accumulation in cisplatin-treated HEI-OC1 cells. Immunofluorescent staining of cochlear explants showed that CN and CD attenuated cisplatin-induced damage to SGNs and HCs. Immunoblot revealed that CN and CD downregulated the expression of cleaved caspase-3 and activated PI3K-AKT signaling in cisplatin-injured HEI-OC1 cells. CONCLUSION: CD and CN can reduce ototoxicity caused by cisplatin and might help treat cisplatin-associated hearing loss.

Synthesis, Anti-Oomycete and Anti-fungal Activities of Novel Cinchona Alkaloid Derivatives Containing Sulfonate Moiety.

Using cinchona alkaloid as the lead compound, twenty-four cinchona alkaloid sulfonate derivatives (1 a-l, 2 a-c, 3 a-c, 4 a-c, and 5 a-c) were designed and prepared by modifying their C9 position, and structurally confirmed by 1 H-NMR, 13 C-NMR, HR-MS and melting points. Moreover, the stereochemical configurations of compounds 1 f and 1 l were unambiguously confirmed by single-crystal X-ray diffraction. Furthermore, we determined the anti-oomycete and anti-fungal activities of these target compounds against Phytophthora capsici and Fusarium graminearum in vitro. The results showed that two compounds 4 b and 4 c exhibited prominent anti-oomycete activity, and the median effective concentration (EC50 ) values of 4 b and 4 c against P. capsici were 22.55 and 16.32 mg/L, respectively. This study suggested that when the C9 position of cinchona alkaloid sulfonate derivatives is in the S configuration and the 6'-position methoxy group is not present, the anti-oomycete activity is superior. In addition, five compounds 1 e, 1 f, 1 k, 3 c and 4 c displayed significant anti-fungal activity, with EC50 values of 43.64, 45.07, 80.18, 48.58 and 41.88 mg/L against F. graminearum, respectively. This result indicates that only when a specific substituent is introduced into the structural framework of the target compound, the corresponding compound exhibits significant inhibitory activity against fungi.

Attenuation of indoxyl sulfate-induced cell damage by cinchonidine-a Cinchona alkaloid-through the downregulation of p53 signaling pathway by promoting MDM2 cytoplasmic-nuclear shuttling in endothelial cells.

Renocardiac syndromes are a critical concern among patients with chronic kidney disease (CKD). High level of indoxyl sulfate (IS), a protein-bound uremic toxin, in plasma is known to promote the pathogenesis of cardiovascular diseases by impairing endothelial function. However, the therapeutic effects of the adsorbent of indole, a precursor of IS, on renocardiac syndromes is still debated. Therefore, novel therapeutic approaches should be developed to treat IS-associated endothelial dysfunction. In the present study, we have found that cinchonidine, a major Cinchona alkaloid, exhibited superior cell-protective effects among the 131 test compounds in IS-stimulated human umbilical vein endothelial cells (HUVECs). IS-induced cell death, cellular senescence, and impairment of tube formation in HUVECs were substantially reversed after treatment with cinchonidine. Despite the cinchonidine did not alter reactive oxygen species formation, cellular uptake of IS and OAT3 activity, RNA-Seq analysis showed that the cinchonidine treatment downregulated p53-modulated gene expression and substantially reversed IS-caused G0/G1 cell cycle arrest. Although the mRNA levels of p53 were not considerably downregulated by cinchonidine in IS-treated HUVECs, the treatment of cinchonidine promoted the degradation of p53 and the cytoplasmic-nuclear shuttling of MDM2. Cinchonidine exhibited cell-protective effects against the IS-induced cell death, cellular senescence, and impairment of vasculogenic activity in HUVECs through the downregulation of p53 signaling pathway. Collectively, cinchonidine may be a potential cell-protective agent to rescue IS-induced endothelial cell damage.

Cytotoxicity of cinchona alkaloid organocatalysts against MES-SA and MES-SA/Dx5 multidrug-resistant uterine sarcoma cell lines.

Since the first application of natural quinine as an anti-malarial drug, cinchona alkaloids and their derivatives have been exhaustively studied for their biological activity. In our work, we tested 13 cinchona alkaloid organocatalysts, synthesised from quinine. These derivatives were screened against MES-SA and Dx5 uterine sarcoma cell lines for in vitro anticancer activity and to investigate their potential to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR). Decorating quinine with hydrogen-bond donor units, such as thiourea and (thio)squaramide, resulted in decreased half-maximal growth inhibition values on both cell lines (1.3-21 µM) compared to quinine and other cinchona alcohols (47-111 µM). Further cytotoxicity studies conducted in the presence of the P-gp inhibitor tariquidar indicated that several analogues, especially cinchona amines and squaramides, but not thiosquaramide, were expelled from MDR cells by P-gp. Similarly to the established P-gp inhibitor quinine, 6 cinchona analogues were shown to inhibit calcein-AM efflux. Interestingly, quinine and didehydroquinine exhibited a marginally increased toxicity against the multidrug resistant Dx5 cells. Collateral sensitivity of the MDR cell line was more pronounced when the cinchona thiosquaramide was complexed with Cu(II) acetate. Based on the results, cinchona derivatives are good anticancer candidates for further drug development.

Treatment of Erythroid Precursor Cells from ß-Thalassemia Patients with Cinchona Alkaloids: Induction of Fetal Hemoglobin Production.

ß-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding ß-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for ß-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from ß-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine ß-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of ß-thalassemia.

Cinchona Alkaloid-Inspired Urea-Containing Autophagy Inhibitor Shows Single-Agent Anticancer Efficacy.

Autophagy is upregulated in response to metabolic stress, a hypoxic tumor microenvironment, and therapeutic stress in various cancers and mediates tumor progression and resistance to cancer therapy. Herein, we identified a cinchona alkaloid derivative containing urea (C1), which exhibited potential cytotoxicity and inhibited autophagy in hepatocellular carcinoma (HCC) cells. We showed that C1 not only induced apoptosis but also blocked autophagy in HCC cells, as indicated by the increased expression of LC3-II and p62, inhibition of autophagosome-lysosome fusion, and suppression of the Akt/mTOR/S6k pathway in the HCC cells. Finally, to improve its solubility and efficacy, we encapsulated C1 into PEGylated lipid-poly(lactic-co-glycolic acid) (PLGA) nanoscale drug carriers. Systemic administration of nanoscale C1 significantly suppressed primary tumor growth and prevented distant metastasis while maintaining a desirable safety profile. Our findings demonstrate that C1 combines autophagy modulation and apoptosis induction in a single molecule, making it a promising therapeutic option for HCC.

Cinchonine inhibits osteoclast differentiation by regulating TAK1 and AKT, and promotes osteogenesis.

Cinchonine (CN) has been known to exert antimalarial, antiplatelet, and antiobesity effects. It was also recently reported to inhibit transforming growth factor ß-activated kinase 1 (TAK1) and protein kinase B (AKT) through binding to tumor necrosis factor receptor-associated factor 6 (TRAF6). However, its role in bone metabolism remains largely unknown. Here, we showed that CN inhibits osteoclast differentiation with decreased expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key determinant of osteoclastogenesis. Immunoblot and quantitative real-time polymerase chain reaction analysis as well as the reporter assay revealed that CN inhibits nuclear factor-κB and activator protein-1 by regulating TAK1. CN also attenuated the activation of AKT, cyclic AMP response element-binding protein, and peroxisome proliferator-activated receptor-γ coactivator 1ß (PGC1ß), an essential regulator of mitochondrial biogenesis. Collectively, these results suggested that CN may inhibit TRAF6-mediated TAK1 and AKT activation, which leads to downregulation of NFATc1 and PGC1ß resulting in the suppression of osteoclast differentiation. Interestingly, CN not only inhibited the maturation and resorption function of differentiated osteoclasts but also promoted osteoblast differentiation. Furthermore, CN protected lipopolysaccharide- and ovariectomy-induced bone destruction in mouse models, suggesting its therapeutic potential for treating inflammation-induced bone diseases and postmenopausal osteoporosis.

Repurposing of quinoline alkaloids identifies their ability to enhance doxorubicin-induced sub-G0/G1 phase cell cycle arrest and apoptosis in cervical and hepatocellular carcinoma cells.

The ability of quinoline alkaloids (cinchonine, cinchonidine, quinine, and quinidine) to sensitize different human cancer cell lines to doxorubicin (DOX)-induced cell death was evaluated. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the alkaloids ability to enhance DOX-induced apoptosis was explored using Western blotting analysis. Also, flow cytometry was applied to analyze cell fractions in the different cell cycle phases. All alkaloids showed a significant enhancement of DOX-induced cell death in HeLa and HepG2 cell lines. The chemosensitizing activity of the quinoline alkaloids was attributed to the induction of apoptosis as indicated by splitting of caspase-3 and its substrate poly (ADP-ribose) polymerase (PARP). In addition, there was an increase in the cell fractions in sub-G0/G1 phase in case of DOX combination with the alkaloids. This study proves the ability of the quinoline alkaloids to enhance DOX-induced apoptotic cell death in human cervical and hepatocellular carcinoma cells.

Click Inspired Synthesis of Novel Cinchonidine Glycoconjugates as Promising Plasmepsin Inhibitors.

Among all the malaria parasites, P. falciparum is the most predominant species which has developed drug resistance against most of the commercial anti-malarial drugs. Thus, finding a new molecule for the inhibition of enzymes of P. falciparum is the pharmacological challenge in present era. Herein, ten novel molecules have been designed with an amalgamation of cinchonidine, carbohydrate moiety and triazole ring by utilizing copper-catalyzed click reaction of cinchonidine-derived azide and clickable glycosyl alkynes. The molecular docking of developed molecules showed promising results for plasmepsin inhibition in the form of effective binding with target proteins.

Image-Based Morphological Profiling Identifies a Lysosomotropic, Iron-Sequestering Autophagy Inhibitor.

Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe2+ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.

Hybrids of Cinchona Alkaloids and Bile Acids as Antiparasitic Agents Against Trypanosoma cruzi.

The current chemotherapy of Chagas disease needs to be urgently improved. With this aim, a series of 16 hybrids of Cinchona alkaloids and bile acids were prepared by functionalization at position C-2 of the quinoline nucleus by a radical attack of a norcholane substituent via a Barton-Zard decarboxylation reaction. The antitrypanosomal activity of the hybrids was tested on different stages and strains of T. cruzi. In particular, eight out of 16 hybrids presented an IC50 ≤1 µg/mL against trypomastigotes of the CL Brener strain and/or a selectivity index higher than 10. These promising hybrids yielded similar results when tested on trypomastigotes from the RA strain of T. cruzi (discrete typing unit-DTU-VI). Surprisingly, trypomastigotes of the Y strain (DTU II) were more resistant to benznidazole and to most of the hybrids than those of the CL Brener and RA strains. However, the peracetylated and non-acetylated forms of the cinchonine/chenodeoxycholic bile acid conjugate 4f and 5f were the most trypanocidal hybrids against Y strain trypomastigotes, with IC50 values of 0.5 and 0.65 µg/mL, respectively. More importantly, promising results were observed in invasion assays using the Y strain, where hybrids 5f and 4f induced a significant reduction in intracellular amastigotes and on the release of trypomastigotes from infected cells.

Effects of cinchonine, a Cinchona bark alkaloid, on spontaneous and induced rat ileum contractions.

AIM: Quinine, a frequently used anti-malaria alkaloid isolated from the Cinchona bark, possesses numerous toxic properties, the majority of which arrive from a dysfunction of the gastrointestinal tract. Similarly, cinchonine, another alkaloid from the Cinchona bark, displays a great potential for treating malaria (especially the resistant forms). METHODS: In this work, we aimed to evaluate the effects of cinchonine on spontaneous and induced Wistar rat ileum contractions in order to uncover potential side effects that might arise after its application. RESULTS: Cinchonine produced a concentration-dependent spasmolytic activity, which was found to be reversible (i.e. disappeared after tissue wash-up), with an IC50 value of 273 µM. Furthermore, the mechanism of action of cinchonine at IC50 elucidated through experiments with acetylcholine and Ca2+-induced ileum contractions. The applied IC50 concentration of cinchonine statistically significantly prevented the occurrence of contractions after the application of specific agonist. The obtained results are in a range with the effects seen with standard receptor antagonists, i.e. atropine and verapamil. CONCLUSIONS: The obtained results showed that cinchonine inhibited both types of induced contractions, suggesting a Ca2+-channels mediated modus operandi (Fig. 4, Ref. 19).

In vitro and in vivo cancer cell apoptosis triggered by competitive binding of Cinchona alkaloids to the RING domain of TRAF6.

TRAF6 is highly expressed in many tumors and plays an important role in the immune system. The aim of this study is to confirm anti-tumor activities of all naturally occurring Cinchona alkaloids that have been screened using computational docking program, and to validate the accuracy and specificity of the RING domain of TRAF6 as a potential anti-tumor target, and to explore their effect on the immune system. Results reported herein would demonstrate that Cinchona alkaloids could induce apoptosis in HeLa cells, inhibit the ubiquitination and phosphorylation of both AKT and TAK1, and up-regulate the ratio of Bax/Bcl-2. In addition, these compounds could induce apoptosis in vivo, and increase the secretion of TNF-α, IFN-γ, and IgG, while not significantly impacting the ratio of CD4+T/CD8+T. These investigations suggest that the RING domain of TRAF6 could serve as a de novo biological target for therapeutic treatment in cancers.

A Cinchona Alkaloid Antibiotic That Appears To Target ATP Synthase in Streptococcus pneumoniae.

Optochin, a cinchona alkaloid derivative discovered over 100 years ago, possesses highly selective antibacterial activity toward Streptococcus pneumoniae. Pneumococcal disease remains the leading source of bacterial pneumonia and meningitis worldwide. The structure-activity relationships of optochin were examined through modification to both the quinoline and quinuclidine subunits, which led to the identification of analogue 48 with substantially improved activity. Resistance and molecular modeling studies indicate that 48 likely binds to the c-ring of ATP synthase near the conserved glutamate 52 ion-binding site, while mechanistic studies demonstrated that 48 causes cytoplasmic acidification. Initial pharmacokinetic and drug metabolism analyses of optochin and 48 revealed limitations of these quinine analogues, which were rapidly cleared, resulting in poor in vivo exposure through hydroxylation pendants to the quinuclidine and O-dealkylation of the quinoline. Collectively, the results provide a foundation to advance 48 and highlight ATP synthase as a promising target for antibiotic development.

Cytotoxic and trypanocidal activities of cinchona alkaloid derivatives.

A series of 27 cinchona alkaloid derivatives (1f-w, 2a-e and 3a-d) were investigated for their cytotoxic and trypanocidal activities using seven different cancer cell lines (KB, HeLa, MCF-7, A-549, Hep-G2, U-87 and HL-60), two normal cell lines (HDF and CHO) and bloodstream forms of Trypanosoma brucei brucei, respectively. Four compounds (1u, 1w, 2e and 3d) were identified with promising cytotoxic activity with 50% growth inhibition (GI50 ) values below 10 µM. Two (2e and 3d) of the four compounds also exhibited potent anti-trypanosomal activity with GI50 values of 0.3-0.4 µM. All four active compounds represented derivatives modified at their C-9 hydroxy group. With respect to anti-proliferative activity and selectivity, 2e (epi-N-quinidyl-N'-bis(3,5-trifluoromethyl)phenylthiourea) proved to be the most promising derivative for both cancer cells and bloodstream forms of T. b. brucei. The cytotoxic activity of compounds 1u, 1w, 2e and 3d was attributed to their ability to induce apoptosis in cancer cells. The results demonstrate the potential of cinchona alkaloid derivatives as novel anti-cancer and anti-trypanosome drug candidates.

Cinchonine induces apoptosis of HeLa and A549 cells through targeting TRAF6.

BACKGROUND: Cancer cells are known to over-express TRAF6 that is critical for both AKT and TAK1 activations. The Really Interesting New Gene (RING) domain of TRAF6 is believed to be responsible for the E3 ligase activity, ZINC fingers of TRAF6 provide critical support for the activity of the RING domain which is critical for both AKT and TAK1 activations. METHODS: We employed computational docking program to identify small molecules that could effectively and competitively bind with the RING domain of TRAF6, which is believed to be responsible for its E3 ligase activity. MTT assay and flow cytometry were employed to analyze apoptosis of cancer cells. Signaling pathways were detected using immunoprecipitation and western blotting, and immunofluorescence was pursued to assess the nature of binding of cinchonine to TRAF6. We also performed animal experiments to test effect of cinchonine in vivo. RESULTS: Cinchonine, a naturally occurring Cinchona alkaloid identified from the docking study, could bind to TRAF6 in HeLa and A549 cells and induce apoptosis of these cancer cells. We found that AKT ubiquitination and phosphorylation as well as phosphorylation of TAK1 were decreased. These activities would lead to subsequent suppression anti-apoptotic protein Bcl-2, while elevating pro-apoptotic protein Bax. Immunofluorescence staining unambiguously demonstrated the binding of cinchonine specifically at the RING domain of TRAF6 in cells, thereby validating the computational modeling. Animal experiments showed that cinchonine could suppress tumor growth in mice without showing significant acute toxicity. CONCLUSION: These investigations suggest that through competitive binding with the RING domain of TRAF6, cinchonine could induce apoptosis via inhibiting AKT and TAK1 signaling pathways.

Discovery of Novel Cinchona-Alkaloid-Inspired Oxazatwistane Autophagy Inhibitors.

The cinchona alkaloids are a privileged class of natural products and are endowed with diverse bioactivities. However, for compounds with the closely-related oxazatricyclo[4.4.0.0]decane ("oxazatwistane") scaffold, which are accessible from cinchonidine and quinidine by means of ring distortion and modification, biological activity has not been identified. We report the synthesis of an oxazatwistane compound collection through employing state-of-the-art C-H functionalization, and metal-catalyzed cross-coupling reactions as key late diversity-generating steps. Exploration of oxazatwistane bioactivity in phenotypic assays monitoring different cellular processes revealed a novel class of autophagy inhibitors termed oxautins, which, in contrast to the guiding natural products, selectively inhibit autophagy by inhibiting both autophagosome biogenesis and autophagosome maturation.

Shining new light on ancient drugs: preparation and subcellular localisation of novel fluorescent analogues of Cinchona alkaloids in intraerythrocytic Plasmodium falciparum.

Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, suitable for cellular imaging have been synthesised by attaching the small extrinsic fluorophore, NBD. Interactions of these derivatives with ferriprotoporphyrin IX were evaluated to verify that insights generated by live-cell imaging were relevant to the parent molecules. These analogues are shown by confocal and super-resolution microscopy to accumulate selectively in Plasmodium falciparum. Localisation to the region corresponding to the digestive vacuole supports the putative primary role of these alkaloids as haemozoin inhibitors. Quantitative analysis revealed minimal accumulation within the nucleus, rejecting the disruption of DNA replication as a possible mode of action. While extensive localisation to phospholipid structures and associated organelles was observed, the analogues did not show evidence of association with neutral lipid bodies.

Structure-activity relationship of hybrids of Cinchona alkaloids and bile acids with in vitro antiplasmodial and antitrypanosomal activities.

In this work, a series of hybrid compounds were tested as antiparasitic substances. These hybrids were prepared from bile acids and a series of antiparasitic Cinchona alkaloids by the formation of a covalent C-C bond via a decarboxylative Barton-Zard reaction between the two entities. The bile acids showed only weak antiparasitic properties, but all the hybrids exhibited high in vitro activities (IC50: 0.48-5.39 µM) against Trypanosoma brucei. These hybrids were more active than their respective parent alkaloids (up to a 135 fold increase in activity), and displayed good selectivity indices. Aditionally, all these compounds inhibited the in vitro growth of a chloroquine-sensitive strain of Plasmodium falciparum (3D7: IC50: 36.1 nM to 8.72 µM), and the most active hybrids had IC50s comparable to that of artemisinin (IC50: 36 nM). Some structure-activity relationships among the group of 48 hybrids are discussed. The increase in antiparasitic activity may be explained by an improvement in bioavailability, since the more lipophilic derivatives showed the lowest IC50s.

Antiproliferative Activity of Polyether Antibiotic--Cinchona Alkaloid Conjugates Obtained via Click Chemistry.

A series of eight new conjugates of salinomycin or monensin and Cinchona alkaloids were obtained by the Cu(I)-catalysed 1,3-dipolar Huisgen cycloaddition (click chemistry) of respective N-propargyl amides of salinomycin or monensin with four different Cinchona alkaloid derived azides. In vitro antiproliferative activity of these conjugates evaluated against three cancer cell lines (LoVo, LoVo/DX, HepG2) showed that four of the compounds exhibited high antiproliferative activity (IC50 below 3.00 µm) and appeared to be less toxic and more selective against normal cells than two standard anticancer drugs.