Biomolecular Interactions and Anticancer Mechanisms of Ru(II)-Arene Complexes of Cinnamaldehyde-Derived Thiosemicarbazone Ligands: Analysis Combining In Silico and In Vitro Approaches.

Our study focuses on synthesizing and exploring the potential of three N-(4) substituted thiosemicarbazones derived from cinnamic aldehyde, alongside their Ru(II)-(η6 -p-cymene)/(η6-benzene) complexes. The synthesized compounds were comprehensively characterized using a range of analytical techniques, including FT-IR, UV-visible spectroscopy, NMR (1H, 13C), and HRMS. We investigated their electronic and physicochemical properties via density functional theory (DFT). X-ray crystal structures validated structural differences identified by DFT. Molecular docking predicted promising bioactivities, supported by experimental observations. Notably, docking with EGFR suggested an inhibitory potential against this cancer-related protein. Spectroscopic titrations revealed significant DNA/BSA binding affinities, particularly with DNA intercalation and BSA hydrophobic interactions. RuPCAM displayed the strongest binding affinity with DNA (Kb = 6.23 × 107 M-1) and BSA (Kb = 9.75 × 105 M-1). Assessed the cytotoxicity of the complexes on cervical cancer cells (HeLa), and breast cancer cells (MCF-7 and MDA-MB 231), revealing remarkable potency. Additionally, selectivity was assessed by examining MCF-10a normal cell lines. The active complexes were found to trigger apoptosis, a vital cellular process crucial for evaluating their potential as anticancer agents utilizing staining assays and flow cytometry analysis. Intriguingly, complexation with Ru(II)-arene precursors significantly amplified the bioactivity of thiosemicarbazones, unveiling promising avenues toward the creation of powerful anticancer agents.

Targeting lysosomes by design: novel N-acridine thiosemicarbazones that enable direct detection of intracellular drug localization and overcome P-glycoprotein (Pgp)-mediated resistance.

Innovative N-acridine thiosemicarbazones (NATs) were designed along with their iron(iii), copper(ii), and zinc(ii) complexes. Lysosomal targeting was promoted by specifically incorporating the lysosomotropic Pgp substrate, acridine, into the thiosemicarbazone scaffold to maintain the tridentate N, N, S-donor system. The acridine moiety enables a significant advance in thiosemicarbazone design, since: (1) it enables tracking of the drugs by confocal microscopy using its inherent fluorescence; (2) it is lysosomotropic enabling lysosomal targeting; and (3) as acridine is a P-glycoprotein (Pgp) substrate, it facilitates lysosomal targeting, resulting in the drug overcoming Pgp-mediated resistance. These new N-acridine analogues are novel, and this is the first time that acridine has been specifically added to the thiosemicarbazone framework to achieve the three important properties above. These new agents displayed markedly greater anti-proliferative activity against resistant Pgp-expressing cells than very low Pgp-expressing cells. The anti-proliferative activity of NATs against multiple Pgp-positive cancer cell-types (colon, lung, and cervical carcinoma) was abrogated by the third generation Pgp inhibitor, Elacridar, and also Pgp siRNA that down-regulated Pgp. Confocal microscopy demonstrated that low Pgp in KB31 (-Pgp) cells resulted in acridine's proclivity for DNA intercalation promoting NAT nuclear-targeting. In contrast, high Pgp in KBV1 (+Pgp) cells led to NAT lysosomal sequestration, preventing its nuclear localisation. High Pgp expression in KBV1 (+Pgp) cells resulted in co-localization of NATs with the lysosomal marker, LysoTracker™, that was significantly (p < 0.001) greater than the positive control, the di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) Zn(ii) complex, [Zn(DpC)2]. Incorporation of acridine into the thiosemicarbazone scaffold led to Pgp-mediated transport into lysosomes to overcome Pgp-resistance.

Isosteric Replacement of Sulfur to Selenium in a Thiosemicarbazone: Promotion of Zn(II) Complex Dissociation and Transmetalation to Augment Anticancer Efficacy.

We implemented isosteric replacement of sulfur to selenium in a novel thiosemicarbazone (PPTP4c4mT) to create a selenosemicarbazone (PPTP4c4mSe) that demonstrates potentiated anticancer efficacy and selectivity. Their design specifically incorporated cyclohexyl and styryl moieties to sterically inhibit the approach of their Fe(III) complexes to the oxy-myoglobin heme plane. Importantly, in contrast to the Fe(III) complexes of the clinically trialed thiosemicarbazones Triapine, COTI-2, and DpC, the Fe(III) complexes of PPTP4c4mT and PPTP4c4mSe did not induce detrimental oxy-myoglobin oxidation. Furthermore, PPTP4c4mSe demonstrated more potent antiproliferative activity than the homologous thiosemicarbazone, PPTP4c4mT, with their selectivity being superior or similar, respectively, to the clinically trialed thiosemicarbazone, COTI-2. An advantageous property of the selenosemicarbazone Zn(II) complexes relative to their thiosemicarbazone analogues was their greater transmetalation to Cu(II) complexes in lysosomes. This latter effect probably promoted their antiproliferative activity. Both ligands down-regulated multiple key receptors that display inter-receptor cooperation that leads to aggressive and resistant breast cancer.

Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/ß-catenin signaling by stabilization of protein kinase Cα.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/ß-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased ß-catenin and downregulated glycogen synthase kinase-3ß (GSK-3ß) protein levels and its activation. However, ß-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3ß was significantly increased after NDRG1 overexpression, suggesting a GSK-3ß-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Cα (PKCα), with PKCα silencing preventing ß-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing ß-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in ß-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCα, NDRG1, and ß-catenin was identified, with the formation of a potential metabolon that promotes the latter ß-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in ß-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased ß-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCα and inhibit WNT signaling.

Differential transmetallation of complexes of the anti-cancer thiosemicarbazone, Dp4e4mT: effects on anti-proliferative efficacy, redox activity, oxy-myoglobin and oxy-hemoglobin oxidation.

The di-2-pyridylthiosemicarbazone (DpT) analogs demonstrate potent and selective anti-proliferative activity against human tumors. The current investigation reports the synthesis and chemical and biological characterization of the Fe(iii), Co(iii), Ni(ii), Cu(ii), Zn(ii), Ga(iii), and Pd(ii) complexes of the promising second generation DpT analog, di-2-pyridylketone-4-ethyl-4-methyl-3-thiosemicarbazone (Dp4e4mT). These studies demonstrate that the Dp4e4mT Co(iii), Ni(ii), and Pd(ii) complexes display distinct biological activity versus those with Cu(ii), Zn(ii), and Ga(iii) regarding anti-proliferative efficacy against cancer cells and a detrimental off-target effect involving oxidation of oxy-myoglobin (oxy-Mb) and oxy-hemoglobin (oxy-Hb). With regards to anti-proliferative activity, the Zn(ii) and Ga(iii) Dp4e4mT complexes demonstrate facile transmetallation with Cu(ii), resulting in efficacy against tumor cells that is strikingly similar to the Dp4e4mT Cu(ii) complex (IC50: 0.003-0.006 µM and 72 h). Relative to the Zn(ii) and Ga(iii) Dp4e4mT complexes, the Dp4e4mT Ni(ii) complex demonstrates kinetically slow transmetallation with Cu(ii) and intermediate anti-proliferative effects (IC50: 0.018-0.076 µM after 72 h). In contrast, the Co(iii) and Pd(ii) complexes demonstrate poor anti-proliferative activity (IC50: 0.262-1.570 µM after 72 h), probably due to a lack of transmetallation with Cu(ii). The poor efficacy of the Dp4e4mT Co(iii), Ni(ii), and Pd(ii) complexes to transmetallate with Fe(iii) markedly suppresses the oxidation of oxy-Mb and oxy-Hb. In contrast, the 2 : 1 Dp4e4mT: Cu(ii), Zn(ii), and Ga(iii) complexes demonstrate facile reactions with Fe(iii), leading to the redox active Dp4e4mT Fe(iii) complex and oxy-Mb and oxy-Hb oxidation. This study demonstrates the key role of differential transmetallation of Dp4e4mT complexes that has therapeutic ramifications for their use as anti-cancer agents.

Steric Blockade of Oxy-Myoglobin Oxidation by Thiosemicarbazones: Structure-Activity Relationships of the Novel PPP4pT Series.

The di-2-pyridylketone thiosemicarbazones demonstrated marked anticancer efficacy, prompting progression of DpC to clinical trials. However, DpC induced deleterious oxy-myoglobin oxidation, stifling development. To address this, novel substituted phenyl thiosemicarbazone (PPP4pT) analogues and their Fe(III), Cu(II), and Zn(II) complexes were prepared. The PPP4pT analogues demonstrated potent antiproliferative activity (IC50: 0.009-0.066 µM), with the 1:1 Cu:L complexes showing the greatest efficacy. Substitutions leading to decreased redox potential of the PPP4pT:Cu(II) complexes were associated with higher antiproliferative activity, while increasing potential correlated with increased redox activity. Surprisingly, there was no correlation between redox activity and antiproliferative efficacy. The PPP4pT:Fe(III) complexes attenuated oxy-myoglobin oxidation significantly more than the clinically trialed thiosemicarbazones, Triapine, COTI-2, and DpC, or earlier thiosemicarbazone series. Incorporation of phenyl- and styryl-substituents led to steric blockade, preventing approach of the PPP4pT:Fe(III) complexes to the heme plane and its oxidation. The 1:1 Cu(II):PPP4pT complexes were inert to transmetalation and did not induce oxy-myoglobin oxidation.

Synthesis of potent MDA-MB 231 breast cancer drug molecules from single step.

We have prepared novel potent breast cancer drug molecules from non-toxic and inexpensive method. Column chromatography is not necessary for purification of target molecules. The value of overall atom economy, environmental factor, environmental catalyst and product mass intensity gives additional merits for this synthetic method. Synthesized flexible dimeric imidazolium bromides showed less toxicity and gives excellent anticancer response against normal mammary epithelial cells. Novel dimeric pyridinium bromides showed excellent anticancer response against tested cancer cell lines. In cell cycle, novel flexible dimeric pyridinium bromides showed significant arrest in the G2/M phase by nearly three folds, when compared with control drug. We have studied the targeting epidermal growth factor receptor for all the synthesized flexible amino substituted and methyl substituted dimeric pyridinium bromides.

Ru(II)-p-Cymene Complexes of Furoylthiourea Ligands for Anticancer Applications against Breast Cancer Cells.

Half-sandwich Ru(II) complexes containing nitro-substituted furoylthiourea ligands, bearing the general formula [(η6-p-cymene)RuCl2(L)] (1-6) and [(η6-p-cymene)RuCl(L)(PPh3)]+ (7--12), have been synthesized and characterized. In contrast to the spectroscopic data which revealed monodentate coordination of the ligands to the Ru(II) ion via a "S" atom, single crystal X-ray structures revealed an unusual bidentate N, S coordination with the metal center forming a four-membered ring. Interaction studies by absorption, emission, and viscosity measurements revealed intercalation of the Ru(II) complexes with calf thymus (CT) DNA. The complexes showed good interactions with bovine serum albumin (BSA) as well. Further, their cytotoxicity was explored exclusively against breast cancer cells, namely, MCF-7, T47-D, and MDA-MB-231, wherein all of the complexes were found to display more pronounced activity than their ligand counterparts. Complexes 7-12 bearing triphenylphosphine displayed significant cytotoxicity, among which complex 12 showed IC50 values of 0.6 ± 0.9, 0.1 ± 0.8, and 0.1 ± 0.2 µM against MCF-7, T47-D, and MDA-MB-231 cell lines, respectively. The most active complexes were tested for their mode of cell death through staining assays, which confirmed apoptosis. The upregulation of apoptotic inducing and downregulation of apoptotic suppressing proteins as inferred from the western blot analysis also corroborated the apoptotic mode of cell death. The active complexes effectively generated reactive oxygen species (ROS) in MDA-MB-231 cells as analyzed from the 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining. Finally, in vivo studies of the highly active complexes (6 and 12) were performed on the mice model. Histological analyses revealed that treatment with these complexes at high doses of up to 8 mg/kg did not induce any visible damage to the tested organs.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Discovery of Novel Dimeric Pyridinium Bromide Analogues Inhibits Cancer Cell Growth by Activating Caspases and Downregulating Bcl-2 Protein.

Flexible dimeric substituted pyridinium bromides with primary and tertiary amines are prepared by conventional and solvent-free methods. The formation of compounds 2 and 4 is much easier than that of compounds 1 and 3 because of the benzyl carbon which is more electropositive than the primary alkyl carbon. The newly synthesized dimeric pyridinium compounds are optimized using DFT and B3LYP 6-31 g(d,p). The in vitro antiproliferative activity is studied in lung (A549) and breast cancer cell lines (MDA-MB 231). Among the four compounds, 1,1'-(1,3-phenylene bis(methylene)bis 2-aminopyridinium bromide 4 showed potent anticancer activity when compared to the standard drug 5-fluorouracil. 1,1'-(1,3-Phenylene bis(methylene)bis 2-aminopyridinium bromide 4 is not toxic to normal cell lines 3T3-L1 and MRC-5 cell lines. Also, 1,1'-(1,3-phenylene bis(methylene)bis 2-aminopyridinium bromide 4-induced apoptosis in cancer cell lines is examined using AO/EB and Hoechst staining, which is further supported by cell cycle analysis. Western blot analysis showed that 1,1'-(1,3-phenylene bis(methylene)bis 2-aminopyridinium bromide 4 induces apoptosis through the extrinsic apoptotic pathway by upregulating caspase 3 and caspase 9. This compound also downregulates intrinsic apoptotic proteins, including Bcl-2, Bcl-x, and Bad. From the present study results, it is confirmed that 1,1'-(1,3-phenylene bis(methylene)bis 2-aminopyridinium bromide 4 has potent anticancer activity when compared to other compounds.

Hinged Bipodal Furoylthiourea-Based Ru(II)-Arene Complexes: Effect of (ortho, meta, or para)-Substitution on Coordination and Anticancer Activity.

We set out to design and synthesize bipodal ligands with the phenyl group as the spacer and varied the substitution on the spacer between ortho (L1), meta (L2), and para (L3). The respective ligands and complexes containing either p-cymene (PL1-PL3) or benzene (BL1-BL3) as the arene unit were synthesized and characterized successfully. The influence of the ligands due to substitution change on their coordination behavior was quite minimal; however, the differences were seen in the anticancer activity of the complexes. DFT studies revealed the structural variations between the three different substitutions, which was further confirmed by single-crystal X-ray diffraction studies. The anticancer activity of the complexes could be correlated with their rate of hydrolysis and their lipophilicity index as determined by UV-visible spectroscopy. The cell death mechanism of the active complexes was deduced to be apoptotic via staining assays, flow cytometry, and Western blot analysis.

Multi-target activity of copper complexes: Antibacterial, DNA binding, and molecular docking with SARS-CoV-2 receptor.

A series of pendant-armed mixed-ligand copper(II) complexes of the type [CuL1-3(diimine)] (1-6) have been synthesized by the reaction of pendant-armed ligands N,N-bis(2-(((E)-2-hydroxy-5-methylbenzylidene)amino)ethyl)benzamide (H2L1), N,N-bis(2-(((E)-2-hydroxy-5-methylbenzylidene)amino)ethyl)-4-nitrobenzamide (H2L2) and N,N-bis(2-(((E)-2-hydroxy-5-methylbenzylidene)amino)ethyl)-3,5-dinitrobenzamide (H2L3) with diimine = 2,2'-bipyridyl (bpy) or 1,10-phenanthroline (phen) in the presence of copper(II) chloride and analyzed using various spectroscopic methods. All the spectroscopic results support that the complexes adopt a pentagonal-bipyramidal shape around the copper ion. Gram-positive and Gram-negative bacteria were used to test all the complexes for antibacterial activity and all the complexes had greater potency against gram-negative pathogens. DNA-binding experiments of complexes with calf thymus DNA revealed a major-groove binding pattern, further supported by molecular docking studies. Complexes have significantly interacted with SARS-CoV-2 receptor via π-π, π-σ, π-alkyl, π-anion, π-cation, alkyl, hydrogen bond, van der Waals, and electrostatic interactions. The estimated binding energy and inhibition constant of these complexes are higher than standard drugs, chloroquine, and molnupiravir.

Designing Tailored Thiosemicarbazones with Bespoke Properties: The Styrene Moiety Imparts Potent Activity, Inhibits Heme Center Oxidation, and Results in a Novel "Stealth Zinc(II) Complex".

A novel, potent, and selective antitumor agent, namely (E)-3-phenyl-1-(2-pyridinyl)-2-propen-1-one 4,4-dimethyl-3-thiosemicarbazone (PPP44mT), and its analogues were synthesized and characterized and displayed strikingly distinctive properties. This activity was mediated by the inclusion of a styrene moiety, which through steric and electrochemical mechanisms prevented deleterious oxy-myoglobin or oxy-hemoglobin oxidation relative to other potent thiosemicarbazones, i.e., di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Structure-activity relationship analysis demonstrated specific tuning of PPP44mT electrochemistry further inhibited oxy-myoglobin or oxy-hemoglobin oxidation. Both PPP44mT and its Cu(II) complexes showed conspicuous almost immediate cytotoxicity against SK-N-MC tumor cells (within 3 h). In contrast, [Zn(PPP44mT)2] demonstrated a pronounced delay in activity, taking 48 h before marked antiproliferative efficacy was apparent. As such, [Zn(PPP44mT)2] was designated as a "stealth Zn(II) complex" that overcomes the near immediate cytotoxicity of PPP44mT or its copper complexes. Upon examination of the suppression of oncogenic signaling, [Zn(PPP44mT)2] was superior at inhibiting cyclin D1 expression compared to DpC or Dp44mT.

Efficient Antibacterial Dimeric Nitro Imidazolium Type of Ionic Liquids from a Simple Synthetic Approach.

Synthesis of dimeric nitro-substituted imidazolium salts under the conventional/solvent-free method is reported. The solvent-free method is more important than the conventional one because of its shorter reaction time, higher yield from easily available starting material, environmental safety, and so forth. Counter anion exchange is carried out using inorganic salt, which is dissolved in deionized water at room temperature. In antibacterial studies, dimeric nitro-substituted imidazolium cations with bromide counter anions showed excellent inhibition against E. coli and P. aeruginosa bacteria. These experimental results were further supported by molecular docking studies. All the compounds (3-6) (a-d) showed excellent antibacterial activity than the standard drugs (gentamycin, nalidixic acid, oflaxacin, ciproflaxacin, and amikacin). Molecular docking studies showed strong hydrogen bonding, polar and hydrophobic interactions between the dimeric imidazolium salts, and Escherichia coli/Pseudomonas aeruginosa/Proteus vulgaris/Staphylococcus aureus receptors.

The thiosemicarbazone, DpC, broadly synergizes with multiple anti-cancer therapeutics and demonstrates temperature- and energy-dependent uptake by tumor cells.

BACKGROUND: The di-2-pyridylketone thiosemicarbazones, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), demonstrate potent and selective anti-tumor activity. In fact, DpC entered Phase I clinical trials for advanced and resistant tumors. METHODS: This investigation examined the activity of these thiosemicarbazones in five tumor cell-types compared to nine clinically used chemotherapeutics and also in combination with these drugs. RESULTS: Dp44mT and especially DpC demonstrated potent anti-proliferative activity that was significantly greater than a range of standard anti-cancer therapeutics. As most anti-cancer drugs are given in combination, further studies were performed to examine the synergistic activity of DpC or Dp44mT with these chemotherapeutics. Combination experiments revealed broad synergy between Dp44mT or DpC upon addition of these drugs, with a sequential protocol of treating first with standard chemotherapies followed by incubation with the thiosemicarbazones being optimal. However, combining DpC and Dp44mT resulted in a pronounced antagonistic drug interaction. To dissect the mechanism of this latter effect, custom-prepared 14C-DpC was implemented and examined for its uptake by cells. The avid uptake of 14C-DpC by tumor cells observed at 37 °C was suppressed at 4 °C and by the metabolic inhibitor, sodium fluoride, suggesting a temperature- and energy-dependent mechanism. Furthermore, competition studies using an excess of unlabeled Dp44mT or DpC inhibited 14C-DpC or 14C-Dp44mT uptake, respectively, suggesting these ligands utilize the same carrier/receptor, antagonizing the internalization of each other. CONCLUSIONS AND GENERAL SIGNIFICANCE: These studies demonstrate the potent and broad anti-proliferative activity of Dp44mT and particularly DpC, and are important for establishing optimized combinations with standard chemotherapies.

Calcium channels and iron metabolism: A redox catastrophe in Parkinson's disease and an innovative path to novel therapies?

Autonomously spiking dopaminergic neurons of the substantia nigra pars compacta (SNpc) are exquisitely specialized and suffer toxic iron-loading in Parkinson's disease (PD). However, the molecular mechanism involved remains unclear and critical to decipher for designing new PD therapeutics. The long-lasting (L-type) CaV1.3 voltage-gated calcium channel is expressed at high levels amongst nigral neurons of the SNpc, and due to its role in calcium and iron influx, could play a role in the pathogenesis of PD. Neuronal iron uptake via this route could be unregulated under the pathological setting of PD and potentiate cellular stress due to its redox activity. This Commentary will focus on the role of the CaV1.3 channels in calcium and iron uptake in the context of pharmacological targeting. Prospectively, the audacious use of artificial intelligence to design innovative CaV1.3 channel inhibitors could lead to breakthrough pharmaceuticals that attenuate calcium and iron entry to ameliorate PD pathology.

Innovative therapies for neuroblastoma: The surprisingly potent role of iron chelation in up-regulating metastasis and tumor suppressors and down-regulating the key oncogene, N-myc.

Iron is an indispensable requirement for essential biological processes in cancer cells. Due to the greater proliferation of neoplastic cells, their demand for iron is considerably higher relative to normal cells, making them highly susceptible to iron depletion. Understanding this sensitive relationship led to research exploring the effect of iron chelation therapy for cancer treatment. The classical iron-binding ligand, desferrioxamine (DFO), has demonstrated effective anti-proliferative activity against many cancer-types, particularly neuroblastoma tumors, and has the surprising activity of down-regulating the potent oncogene, N-myc, which is a major oncogenic driver in neuroblastoma. Even more significant is the ability of DFO to simultaneously up-regulate the potent metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), which plays a plethora of roles in suppressing a variety of oncogenic signaling pathways. However, DFO suffers the disadvantage of demonstrating poor membrane permeability and short plasma half-life, requiring administration by prolonged subcutaneous or intravenous infusions. Considering this, the specifically designed di-2-pyridylketone thiosemicarbazone (DpT) series of metal-binding ligands was developed in our laboratory. The lead agent from the first generation DpT series, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), showed exceptional anti-cancer properties compared to DFO. However, it exhibited cardiotoxicity in mouse models at higher dosages. Therefore, a second generation of agents was developed with the lead compound being di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) that progressed to Phase I clinical trials. Importantly, DpC showed better anti-proliferative activity than Dp44mT and no cardiotoxicity, demonstrating effective anti-cancer activity against neuroblastoma tumors in vivo.

The Role of Extracellular Proteases in Tumor Progression and the Development of Innovative Metal Ion Chelators that Inhibit their Activity.

The crucial role of extracellular proteases in cancer progression is well-known, especially in relation to the promotion of cell invasion through extracellular matrix remodeling. This also occurs by the ability of extracellular proteases to induce the shedding of transmembrane proteins at the plasma membrane surface or within extracellular vesicles. This process results in the regulation of key signaling pathways by the modulation of kinases, e.g., the epidermal growth factor receptor (EGFR). Considering their regulatory roles in cancer, therapeutics targeting various extracellular proteases have been discovered. These include the metal-binding agents di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which increase c-MET degradation by multiple mechanisms. Both the direct and indirect inhibition of protease expression and activity can be achieved through metal ion depletion. Considering direct mechanisms, chelators can bind zinc(II) that plays a catalytic role in enzyme activity. In terms of indirect mechanisms, Dp44mT and DpC potently suppress the expression of the kallikrein-related peptidase-a prostate-specific antigen-in prostate cancer cells. The mechanism of this activity involves promotion of the degradation of the androgen receptor. Additional suppressive mechanisms of Dp44mT and DpC on matrix metalloproteases (MMPs) relate to their ability to up-regulate the metastasis suppressors N-myc downstream regulated gene-1 (NDRG1) and NDRG2, which down-regulate MMPs that are crucial for cancer cell invasion.

The potential of the novel NAD+ supplementing agent, SNH6, as a therapeutic strategy for the treatment of Friedreich's ataxia.

Friedreich's ataxia (FA) is due to deficiency of the mitochondrial protein, frataxin, which results in multiple pathologies including a deadly, hypertrophic cardiomyopathy. Frataxin loss leads to deleterious accumulations of redox-active, mitochondrial iron, and suppressed mitochondrial bioenergetics. Hence, there is an urgent need to develop innovative pharmaceuticals. Herein, the activity of the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazone (SNH6), was assessed in vivo using the well-characterized muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse model of FA. The design of SNH6 incorporated a dual-mechanism mediating: (1) NAD+-supplementation to restore cardiac bioenergetics; and (2) iron chelation to remove toxic mitochondrial iron. In these studies, MCK wild-type (WT) and KO mice were treated for 4-weeks from the asymptomatic age of 4.5-weeks to 8.5-weeks of age, where the mouse displays an overt cardiomyopathy. SNH6-treatment significantly elevated NAD+ and markedly increased NAD+ consumption in WT and KO hearts. In SNH6-treated KO mice, nuclear Sirt1 activity was also significantly increased together with the NAD+-metabolic product, nicotinamide (NAM). Therefore, NAD+-supplementation by SNH6 aided mitochondrial function and cardiac bioenergetics. SNH6 also chelated iron in cultured cardiac cells and also removed iron-loading in vivo from the MCK KO heart. Despite its dual beneficial properties of supplementing NAD+ and chelating iron, SNH6 did not mitigate cardiomyopathy development in the MCK KO mouse. Collectively, SNH6 is an innovative therapeutic with marked pharmacological efficacy, which successfully enhanced cardiac NAD+ and nuclear Sirt1 activity and reduced cardiac iron-loading in MCK KO mice. No other pharmaceutical yet designed exhibits both these effective pharmacological properties.