Bisindole-based small molecules as transmembrane anion transporters and potential anticancer agents.

Few synthetic ion transporters have been reported incorporating indole as the core moiety. We have developed a novel bisindole-based transporter capable of efficient transmembrane anion antiport. This system induced cytotoxicity in MCF-7 breast cancer cells via chloride ion homeostasis disruption and the associated ROS generation, mitochondrial membrane depolarization, and lysosomal deacidification.

Tetrahydropalmatine from medicinal plants activates human glucokinase to regulate glucose homeostasis.

Many synthetic glucokinase activators (GKAs), modulating glucokinase (GK), an important therapeutic target in diabetes have failed to clear clinical trials. In this study, an in silico structural similarity search with differing scaffolds of reference GKAs have been used to identify derivatives from natural product databases. Ten molecules with good binding score and similar interactions to that in the co-crystallized GK as well good activation against recombinant human GK experimentally were identified. Tetrahydropalmatine, an alkaloid present in formulations and drugs from medicinal plants, has not been explored as an antidiabetic agent and no information regarding its mechanism of action or GK activation exists. Tetrahydropalmatine activates GK with EC50 value of 71.7 ± 17.9 µM while lowering the S0.5 (7.1 mM) and increasing Vmax (9.22 µM/min) as compared to control without activator (S0.5 = 10.37 mM; Vmax = 4.8 µM/min). Kinetic data (α and ß values) suggests it to act as mixed, nonessential type activator. Using microscale thermophoresis, Kd values of 3.8 µM suggests a good affinity for GK. In HepG2 cell line, the compound potentiated the uptake of glucose and maintained glucose homeostasis by increasing the expression of GK, glycogen synthase, and insulin receptor genes and lowering the expression of glucokinase regulatory protein (GKRP) and glucagon. Tetrahydropalmatine at low concentrations could elicit a good response by reducing expression of GKRP, increasing expression of GK while also activating it. Thus, it could be used alone or in combination as therapeutic drug as it could effectively modulate GK and alter glucose homeostasis.

A Benzohydrazide-Based Artificial Ion Channel that Modulates Chloride Ion Concentration in Cancer Cells and Induces Apoptosis by Disruption of Autophagy.

Fluctuations in the intracellular chloride ion concentration, mediated by synthetic ion transporters, have been known to induce cytotoxicity in cells by disrupting ionic homeostasis. However, the activity of these transporters in modulating autophagy remains largely unexplored. Here, we report a benzoylbenzohydrazide (1c) that self-assembles to form a supramolecular nanochannel lumen that allows selective and efficient transport of chloride ions across the cell membranes, disrupts ion homeostasis, and thus leads to the induction of apoptosis in cancer cells. It is important to note that the transporter was relatively nontoxic to cells of noncancerous origin. 1c was also shown to induce the deacidification of lysosomes, thereby disrupting autophagy in cancer cells. Taken together, these findings provide a rare example of an artificial ion channel that specifically targets cancer cells by induction of apoptosis via disruption of autophagy.

NAD(P)H:Quinone Acceptor Oxidoreductase 1 (NQO1) Activatable Salicylamide H+ /Cl- Transporters.

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a detoxifying enzyme overexpressed in tumors, plays a key role in protecting cancer cells against oxidative stress and thus has been considered an attractive candidate for activating prodrug(s). Herein, we report the first use of NQO1 for the selective activation of 'protransporter' systems in cancer cells leading to the induction of apoptosis. Salicylamides, easily synthesizable small molecules, have been effectively used for efficient H+ /Cl- symport across lipid membranes. The ion transport activity of salicylamides was efficiently abated by caging the OH group with NQO1 activatable quinones via either ether or ester linkage. The release of active transporters, following the reduction of quinone caged 'protransporters' by NQO1, was verified. Both the transporters and protransporters exhibited significant toxicity towards the MCF-7 breast cancer line, mediated via the induction of oxidative stress, mitochondrial membrane depolarization, and lysosomal deacidification. Induction of cell death via intrinsic apoptotic pathway was verified by monitoring PARP1 cleavage.

Human islet amyloid polypeptide (hIAPP) - a curse in type II diabetes mellitus: insights from structure and toxicity studies.

The human islet amyloid polypeptide (hIAPP) or amylin, a neuroendocrine peptide hormone, is known to misfold and form amyloidogenic aggregates that have been observed in the pancreas of 90% subjects with Type 2 Diabetes Mellitus (T2DM). Under normal physiological conditions, hIAPP is co-stored and co-secreted with insulin; however, under chronic hyperglycemic conditions associated with T2DM, the overexpression of hIAPP occurs that has been associated with the formation of amyloid deposits; as well as the death and dysfunction of pancreatic ß-islets in T2DM. Hitherto, various biophysical and structural studies have shown that during this process of aggregation, the peptide conformation changes from random structure to helix, then to ß-sheet, subsequently to cross ß-sheets, which finally form left-handed helical aggregates. The intermediates, formed during this process, have been shown to induce higher cytotoxicity in the ß-cells by inducing cell membrane disruption, endoplasmic reticulum stress, mitochondrial dysfunction, oxidative stress, islet inflammation, and DNA damage. As a result, several research groups have attempted to target both hIAPP aggregation phenomenon and the destabilization of preformed fibrils as a therapeutic intervention for T2DM management. In this review, we have summarized structural aspects of various forms of hIAPP viz. monomer, oligomers, proto-filaments, and fibrils of hIAPP. Subsequently, cellular toxicity caused by toxic conformations of hIAPP has been elaborated upon. Finally, the need for performing structural and toxicity studies in vivo to fill in the gap between the structural and cellular aspects has been discussed.

Myricetin protects pancreatic ß-cells from human islet amyloid polypeptide (hIAPP) induced cytotoxicity and restores islet function.

The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)-a hormone that is co-secreted with insulin from pancreatic ß-cells-into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of ß-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like Syzygium cumini). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.

Azadirachtin inhibits amyloid formation, disaggregates pre-formed fibrils and protects pancreatic ß-cells from human islet amyloid polypeptide/amylin-induced cytotoxicity.

The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with ß-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)-a secondary metabolite isolated from traditional medicinal plant Neem (Azadirachta indica)-was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic ß-cells. Furthermore, AZD supplementation in pancreatic ß-cells (INS-1E) resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.

A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels.

Cancer cells use elevated glutathione (GSH) levels as an inner line of defense to evade apoptosis and develop drug resistance. In this study, we describe a novel 2,4-nitrobenzenesulfonyl (DNS) protected 2-hydroxyisophthalamide system that exploits GSH for its activation into free 2-hydroxyisophthalamide forming supramolecular M+ /Cl- channels. Better permeation of the DNS protected compound into MCF-7 cells compared to the free 2-hydroxyisophthalamide and GSH-activatable ion transport resulted in higher cytotoxicity, which was associated with increased oxidative stress that further reduced the intracellular GSH levels and altered mitochondrial membrane permeability leading to the induction of the intrinsic apoptosis pathway. The GSH-activatable transport-mediated cell death was further validated in rat insulinoma cells (INS-1E); wherein the intracellular GSH levels showed a direct correlation to the resulting cytotoxicity. Lastly, the active compound was found to restrict the growth and proliferation of 3D spheroids of MCF-7 cells with efficiency similar to that of the anticancer drug doxorubicin.

Metabolic signatures suggest o-phosphocholine to UDP-N-acetylglucosamine ratio as a potential biomarker for high-glucose and/or palmitate exposure in pancreatic ß-cells.

INTRODUCTION: Chronic exposure to high-glucose and free fatty acids (FFA) alone/or in combination; and the resulting gluco-, lipo- and glucolipo-toxic conditions, respectively, have been known to induce dysfunction and apoptosis of ß-cells in Diabetes. The molecular mechanisms and the development of biomarkers that can be used to predict similarities and differences behind these conditions would help in easier and earlier diagnosis of Diabetes. OBJECTIVES: This study aims to use metabolomics to gain insight into the mechanisms by which ß-cells respond to excess-nutrient stress and identify associated biomarkers. METHODS: INS-1E cells were cultured in high-glucose, palmitate alone/or in combination for 24 h to mimic gluco-, lipo- and glucolipo-toxic conditions, respectively. Biochemical and cellular experiments were performed to confirm the establishment of these conditions. To gain molecular insights, abundant metabolites were identified and quantified using 1H-NMR. RESULTS: No loss of cellular viability was observed in high-glucose while exposure to FFA alone/in combination with high-glucose was associated with increased ROS levels, membrane damage, lipid accumulation, and DNA double-strand breaks. Forty-nine abundant metabolites were identified and quantified using 1H-NMR. Chemometric pair-wise analysis in glucotoxic and lipotoxic conditions, when compared with glucolipotoxic conditions, revealed partial overlap in the dysregulated metabolites; however, the dysregulation was more significant under glucolipotoxic conditions. CONCLUSION: The current study compared gluco-, lipo- and glucolipotoxic conditions in parallel and elucidated differences in metabolic pathways that play major roles in Diabetes. o-phosphocholine and UDP-N-acetylglucosamine were identified as common dysregulated metabolites and their ratio was proposed as a potential biomarker for these conditions.

Recombinant human islet amyloid polypeptide forms shorter fibrils and mediates ß-cell apoptosis via generation of oxidative stress.

Protein misfolding and aggregation play an important role in many human diseases including Alzheimer's, Parkinson's and type 2 diabetes mellitus (T2DM). The human islet amyloid polypeptide (hIAPP) forms amyloid plaques in the pancreas of T2DM subjects (>95%) that are involved in deteriorating islet function and in mediating ß-cell apoptosis. However, the detailed mechanism of action, structure and nature of toxic hIAPP species responsible for this effect remains elusive to date mainly due to the high cost associated with the chemical synthesis of pure peptide required for these studies. In the present work, we attempted to obtain structural and mechanistic insights into the hIAPP aggregation process using recombinant hIAPP (rhIAPP) isolated from Escherichia coli Results from biophysical and structural studies indicate that the rhIAPP self-assembled into highly pure, ß-sheet-rich amyloid fibrils with uniform morphology. rhIAPP-mediated apoptosis in INS-1E cells was associated with increased oxidative stress and changes in mitochondrial membrane potential. The transcript levels of apoptotic genes - Caspase-3 and Bax were found to be up-regulated, while the levels of the anti-apoptotic gene - Bcl2 were down-regulated in rhIAPP-treated cells. Additionally, the expression levels of genes involved in combating oxidative stress namely Catalase, SOD1 and GPx were down-regulated. rhIAPP exposure also affected glucose-stimulated insulin secretion from isolated pancreatic islets. The aggregation of rhIAPP also occurred significantly faster when compared with that of the chemically synthesized peptide. We also show that the rhIAPP fibrils were shorter and more cytotoxic. In summary, our study is one among the few to provide comprehensive evaluation of structural, biophysical and cytotoxic properties of rhIAPP.

BRCA1 promotes the ubiquitination of PCNA and recruitment of translesion polymerases in response to replication blockade.

Breast cancer gene 1 (BRCA1) deficient cells not only are hypersensitive to double-strand breaks but also are hypersensitive to UV irradiation and other agents that cause replication blockade; however, the molecular mechanisms behind these latter sensitivities are largely unknown. Here, we report that BRCA1 promotes cell survival by directly regulating the DNA damage tolerance pathway in response to agents that create cross-links in DNA. We show that BRCA1 not only promotes efficient mono- and polyubiquitination of proliferating cell nuclear antigen (PCNA) by regulating the recruitment of replication protein A, Rad18, and helicase-like transcription factor to chromatin but also directly recruits translesion polymerases, such as Polymerase eta and Rev1, to the lesions through protein-protein interactions. Our data suggest that BRCA1 plays a critical role in promoting translesion DNA synthesis as well as DNA template switching.

REV1 and polymerase ζ facilitate homologous recombination repair.

REV1 and DNA Polymerase ζ (REV3 and REV7) play important roles in translesion DNA synthesis (TLS) in which DNA replication bypasses blocking lesions. REV1 and Polζ have also been implicated in promoting repair of DNA double-stranded breaks (DSBs). However, the mechanism by which these two TLS polymerases increase tolerance to DSBs is poorly understood. Here we demonstrate that full-length human REV1, REV3 and REV7 interact in vivo (as determined by co-immunoprecipitation studies) and together, promote homologous recombination repair. Cells lacking REV3 were hypersensitive to agents that cause DSBs including the PARP inhibitor, olaparib. REV1, REV3 or REV7-depleted cells displayed increased chromosomal aberrations, residual DSBs and sites of HR repair following exposure to ionizing radiation. Notably, cells depleted of DNA polymerase η (Polη) or the E3 ubiquitin ligase RAD18 were proficient in DSB repair following exposure to IR indicating that Polη-dependent lesion bypass or RAD18-dependent monoubiquitination of PCNA are not necessary to promote REV1 and Polζ-dependent DNA repair. Thus, the REV1/Polζ complex maintains genomic stability by directly participating in DSB repair in addition to the canonical TLS pathway.

Dual Functional Microcapsule based on Monodisperse Short PEG Amphiphile for Drug Encapsulation and Protein Affinity Controlled Release.

A short monodisperse poly(ethylene glycol) (PEG) and a neutral organic rotamer conjugate TEG-BTA-2 amphiphile was designed for the construction of a stimuli-responsive switchable self-assembled structure for drug encapsulation by noncovalent interaction and targeted controlled delivery. A short PEG, tetraethylene glycol (TEG) was covalently attached with a neutral organic rotamer benzothiazole dye (BTA-2) affording the neutral TEG-BTA-2 (<500 D). The TEG-BTA-2 is self-assembled into a microsphere in an aqueous medium, but remarkably undergoes morphology change switching to a rice-like microcapsule for curcumin encapsulation. Curcumin-loaded microcapsules were stable in an aqueous solution, however, were noticed disintegrating upon the addition of BSA protein. This is possibly due to an interaction with BSA protein leading to a protein affinity-controlled curcumin release in a neutral PBS buffer. Moreover, cell internalization of the neutral amphiphile TEG-BTA-2 into A549 cells was observed by fluorescence microscopy, providing an opportunity for application as a molecular vehicle for targeted drug delivery and monitoring.

Identification of potential serum biomarkers associated with HbA1c levels in Indian type 2 diabetic subjects using NMR-based metabolomics.

BACKGROUND: The prevalence of type 2 diabetes mellitus (T2DM), a progressive metabolic disorder characterized by chronic hyperglycemia and the development of insulin resistance, has increased globally, with worrying statistics coming from children, adolescents, and young adults from developing countries like India. Here, we investigated unique circulating metabolic signatures associated with prediabetes and T2DM in an Indian cohort using NMR-based metabolomics. MATERIALS AND METHODS: The study subjects included healthy volunteers (N = 101), prediabetic subjects (N = 75), and T2DM patients (N = 108). Serum metabolic profiling was performed using 1H NMR spectroscopy and major perturbed metabolites were identified by multivariate analysis and receiver operating characteristic (ROC) modules. RESULTS: Of the 36 aqueous abundant metabolites, 24 showed a statistically significant difference between healthy volunteers, prediabetics, and established T2DM subjects. On performing multivariate ROC curve analysis with 5 commonly dysregulated metabolites (namely, glucose, pyroglutamate, o-phosphocholine, serine, and methionine) in prediabetes and T2DM, AUC values obtained were 0.96 (95 % confidence interval (CI) = 0.93, 0.98) for T2DM; and 0.88 (95 % CI = 0.81, 0.93) for prediabetic subjects, respectively. CONCLUSION: We propose that the identified metabolite panel can be used in the future as a biomarker for clinical diagnosis, patient surveillance, and for predicting individuals at risk for developing diabetes.

Correction: Molybdenum-maltolate as a molybdopterin mimic for bioinspired oxidation reaction.

Correction for 'Molybdenum-maltolate as a molybdopterin mimic for bioinspired oxidation reaction' by Swapnil S. Pawar et al., Dalton Trans., 2024, 53, 5770-5774, https://doi.org/10.1039/D3DT04296K.

Molybdenum-maltolate as a molybdopterin mimic for bioinspired oxidation reaction.

A novel cis-dioxomolybdenum(VI)-maltolate [MoO2(Mal)2] (1) is prepared as a stable molybdopterin model for the biomimetic catalysis of the oxidation of hypoxanthine in acetonitrile-water at room temperature. Compound 1 efficiently catalyzes the oxidation reaction of toluene, diphenylmethane, and styrene. Cyto- and oral-toxicity studies suggest its tremendous potential for application as a molybdenum supplement.

The roles of DNA polymerase ζ and the Y family DNA polymerases in promoting or preventing genome instability.

Cancer cells display numerous abnormal characteristics which are initiated and maintained by elevated mutation rates and genome instability. Chromosomal DNA is continuously surveyed for the presence of damage or blocked replication forks by the DNA Damage Response (DDR) network. The DDR is complex and includes activation of cell cycle checkpoints, DNA repair, gene transcription, and induction of apoptosis. Duplicating a damaged genome is associated with elevated risks to fork collapse and genome instability. Therefore, the DNA damage tolerance (DDT) pathway is also employed to enhance survival and involves the recruitment of translesion DNA synthesis (TLS) polymerases to sites of replication fork blockade or single stranded DNA gaps left after the completion of replication in order to restore DNA to its double stranded form before mitosis. TLS polymerases are specialized for inserting nucleotides opposite DNA adducts, abasic sites, or DNA crosslinks. By definition, the DDT pathway is not involved in the actual repair of damaged DNA, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and lessening the chance for genome instability. However this may be associated with increased mutagenesis. In this review, we will describe the specialized functions of Y family polymerases (Rev1, Polη, Polι and Polκ) and DNA polymerase ζ in lesion bypass, mutagenesis, and prevention of genome instability, the latter due to newly appreciated roles in DNA repair. The recently described role of the Fanconi anemia pathway in regulating Rev1 and Polζ-dependent TLS is also discussed in terms of their involvement in TLS, interstrand crosslink repair, and homologous recombination.

Dissecting the Interrelationship between COVID-19 and Diabetes Mellitus.

COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to enormous morbidity and mortality worldwide. After gaining entry into the human host, the virus initially infects the upper and lower respiratory tract, subsequently invading multiple organs, including the pancreas. While on one hand, diabetes mellitus (DM) is a significant risk factor for severe COVID-19 infection and associated death, recent reports have shown the onset of DM in COVID-19-recovered patients. SARS-CoV-2 infiltrates the pancreatic islets and activates stress response and inflammatory signaling pathways, impairs glucose metabolism, and consequently leads to their death. Indeed, the pancreatic autopsy samples of COVID-19 patients reveal the presence of SARS-CoV-2 particles in ß-cells. The current review describes how the virus enters the host cells and activates an immunological response. Further, it takes a closer look into the interrelationship between COVID-19 and DM with the aim to provide mechanistic insights into the process by which SARS-CoV-2 infects the pancreas and mediates dysfunction and death of endocrine islets. The effects of known anti-diabetic interventions for COVID-19 management are also discussed. The application of mesenchymal stem cells (MSCs) as a future therapy for pancreatic ß-cells damage to reverse COVID-19-induced DM is also emphasized.

Mapping metabolic perturbations induced by glutathione activatable synthetic ion channels in human breast cancer cells.

Ion channels and transporters play key roles in various biological processes, including cell proliferation and programmed cell death. Recently, we reported that 2,4-dinitrobenzene-sulfonyl-protected N1,N3-dihexy-2-hydroxyisophthalamide (1) forms ion channels upon activation by glutathione (GSH) and results in the induction of apoptosis by depleting the intracellular GSH reservoir in cancer cells. However, the detailed molecular events leading to the induction of apoptosis by these synthetic transport systems in cancer cells still need to be uncovered. Along these lines, we investigated the alterations in cellular metabolites and the associated metabolic pathways by performing untargeted global metabolic profiling of breast cancer cells - MCF-7 - using 1H NMR-based metabolomics. The evaluation of spectral profiles from MCF-7 cells exposed to 1 and their comparison with those corresponding to untreated (control) cells identified 14 significantly perturbed signature metabolites. These metabolites belonged mostly to antioxidant defence, energy metabolism, amino acid biosynthesis, and lipid metabolism pathways and included GSH, o-phosphocholine, malate, and aspartate, to name a few. These results would help us gain deeper insights into the molecular mechanism underlying 1-mediated cytotoxicity of MCF-7 cells and eventually help identify potential novel therapeutic targets for more effective cancer management.

DNA polymerase ζ is a major determinant of resistance to platinum-based chemotherapeutic agents.

Oxaliplatin, satraplatin, and picoplatin are cisplatin analogs that interact with DNA forming intrastrand and interstrand DNA cross-links (ICLs). Replicative bypass of cisplatin DNA adducts requires the cooperative actions of at least three translesion DNA synthesis (TLS) polymerases: Polη, REV1, and Polζ. Because oxaliplatin, satraplatin, and picoplatin contain bulkier chemical groups attached to the platinum core compared with cisplatin, we hypothesized that these chemical additions may impede replicative bypass by TLS polymerases and reduce tolerance to platinum-containing adducts. We examined multiple responses of cancer cells to oxaliplatin, satraplatin, or picoplatin treatment under conditions where expression of a TLS polymerase was limited. Our studies revealed that, although Polη contributes to the tolerance of cisplatin adducts, it plays a lesser role in promoting replication through oxaliplatin, satraplatin, and picoplatin adducts. REV1 and Polζ were necessary for tolerance to all four platinum analogs and prevention of hyperactivation of the DNA damage response after treatment. In addition, REV1 and Polζ were important for the resolution of DNA double-stranded breaks created during replication-associated repair of platinum-containing ICLs. Consistent with ICLs being the predominant cytotoxic lesion, depletion of REV1 or Polζ rendered two different model cell systems extremely sensitive to all four drugs, whereas Polη depletion had little effect. Together, our data suggest that REV1 and Polζ are critical for promoting resistance to all four clinically relevant platinum-based drugs by promoting both translesion DNA synthesis and DNA repair.