Insights into distinct signaling profiles of the µOR activated by diverse agonists.

Drugs targeting the µ-opioid receptor (µOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two µOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and ß-arrestin recruitment. Cryo-EM structures of µOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and ß-arrestins bind. These observations highlight how drugs engaging different parts of the µOR orthosteric pocket can lead to distinct signaling outcomes.

EVI1 upregulates PTGS1 (COX1) and decreases the action of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia cells.

Tyrosine kinase inhibitors (TKIs) are highly effective in treating chronic myelogenous leukemia (CML). However, primary and acquired drug resistance to TKIs have been reported. In this study, we used RNA sequencing followed by RQ-PCR to show that the proto-oncogene EVI1 targets the drug-metabolizing gene PTGS1 in CML. The PTGS1 promoter element had an EVI1 binding site, and CHIP assay confirmed its presence. Data from a publicly available CML microarray dataset and an independent set of CML samples showed a significant positive correlation between EVI1 and PTGS1 expression in CML. Downregulation of EVI1 in K562 cells and subsequent treatment with TKIs resulted in a lower IC50 in the control cells. Furthermore, combined inhibition of BCR-ABL with imatinib and PTGS1 with FR122047 (PTGS1 inhibitor) synergistically reduced the viability of imatinib-resistant K562 cells. We conclude that elevated EVI1 expression contributes to TKIs resistance and that combined inhibition of PTGS1 and BCR-ABL may represent a novel therapeutic approach.

AKT inhibition sensitizes EVI1 expressing colon cancer cells to irinotecan therapy by regulating the Akt/mTOR axis.

PURPOSE: Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor that has been attributed to chemotherapy resistance in different cancers. As yet, however, its role in colon cancer drug resistance is not completely understood. Here, we set out to investigate the functional and therapeutic relevance of EVI1 in colon cancer drug resistance. METHODS: The EVI1 gene was knocked down in colon cancer cells that were subsequently tested for susceptibility to irinotecan using in vitro assays and in vivo subcutaneous mouse colon cancer models. The effect of EVI1 knockdown on the AKT-mTOR signaling pathway was assessed using cell line models, immunohistochemistry and bioinformatics tools. The anti-proliferative activity of AKT inhibitor GSK690693 and its combination with irinotecan was tested in colon cancer cell line models (2D and 3D). Finally, the therapeutic efficacy of GSK690693 and its combination with irinotecan was evaluated in xenografted EVI1 expressing colon cancer mouse models. RESULTS: We found that EVI1 knockdown decreased cancer stem cell-like properties and improved irinotecan responses in both cell line and subcutaneous mouse models. In addition, we found that EVI1 downregulation resulted in inhibition of AKT/mTOR signaling and RICTOR expression. Knocking down RICTOR expression increased the cytotoxic effects of irinotecan in EVI1 downregulated colon cancer cells. Co-treatment with irinotecan and ATP-competitive AKT inhibitor GSK690693 significantly reduced colon cancer cell survival and tumor progression rates. CONCLUSION: Inhibition of the AKT signaling cascade by GSK690693 may serve as an alternative to improve the irinotecan response in EVI1-expressing colon cancer cells.

EVI1 promotes metastasis by downregulating TIMP2 in metastatic colon and breast cancer cells.

Ecotropic viral integration site-1 (EVI1) is an oncogenic zinc finger transcription factor whose expression is frequently upregulated in a variety of cancers, including both myeloid malignancies and solid tumors. Previously, our group has shown that EVI1 knockdown minimizes the metastatic potential of colon cancer cells compared to that of control cells. In this study, to identify the potential targets that regulate cancer metastasis, control and EVI1 knockdown colon cancer cells were subjected to microarray. Differential gene expression analysis revealed significant downregulation of tissue inhibitor of matrix metalloproteinase-2 (TIMP2) in EVI1 expressing cells. EVI1 knockdown increased TIMP2 protein expression levels and reduced wound healing and migration capacity in metastatic cells. Mechanistically, the TIMP2 promoter harbors potential binding sites for EVI1; EVI1 binds to TIMP2 promoter and represses its expression, as observed using ChIP and luciferase assay, respectively. TIMP2 is an important metastasis suppressor gene; however, its function is suppressed in many cancers through hypermethylation. Thus, demethylation could prove to be a potential alternative to reactivate TIMP2 functional activity. Immunoprecipitation analysis showed that DNA-methyltransferase 1 (DNMT1), which plays a vital role in maintaining the genome methylation pattern during DNA replication and repair, interacts with EVI1 to promote TIMP2 silencing. Treating cancer cells in vitro with a known demethylation agent, 5-aza-2'-deoxycytidine (Aza-D), restored the optimal TIMP2 expression without altering EVI1 binding efficiency and reduced relative wound healing potential of cancer cells. Animal studies showed that Aza-D treated cells injected through the intravenous route exhibited reduced liver and skin metastasis when compared to non-treated cells. Furthermore, Aza-D treatment in mice delayed the metastasis progression compared to the vehicle treated group. Thus, the present study provides an insight into the therapeutic applications of demethylating agents to reduce cancer metastasis in models with EVI1 overexpressing tumors.

Oxidative Metabolism as a Modulator of Kratom's Biological Actions.

The leaves of Mitragyna speciosa (kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitro and in vivo pharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom's major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.

Evaluation of Kratom Opioid Derivatives as Potential Treatment Option for Alcohol Use Disorder.

Background and Purpose: Mitragyna speciosa extract and kratom alkaloids decrease alcohol consumption in mice at least in part through actions at the δ-opioid receptor (δOR). However, the most potent opioidergic kratom alkaloid, 7-hydroxymitragynine, exhibits rewarding properties and hyperlocomotion presumably due to preferred affinity for the mu opioid receptor (µOR). We hypothesized that opioidergic kratom alkaloids like paynantheine and speciogynine with reduced µOR potency could provide a starting point for developing opioids with an improved therapeutic window to treat alcohol use disorder. Experimental Approach: We characterized paynantheine, speciociliatine, and four novel kratom-derived analogs for their ability to bind and activate δOR, µOR, and κOR. Select opioids were assessed in behavioral assays in male C57BL/6N WT and δOR knockout mice. Key Results: Paynantheine (10 mg∙kg-1, i.p.) produced aversion in a limited conditioned place preference (CPP) paradigm but did not produce CPP with additional conditioning sessions. Paynantheine did not produce robust antinociception but did block morphine-induced antinociception and hyperlocomotion. Yet, at 10 and 30 mg∙kg-1 doses (i.p.), paynantheine did not counteract morphine CPP. 7-hydroxypaynantheine and 7-hydroxyspeciogynine displayed potency at δOR but limited µOR potency relative to 7-hydroxymitragynine in vitro, and dose-dependently decreased voluntary alcohol consumption in WT but not δOR in KO mice. 7-hydroxyspeciogynine has a maximally tolerated dose of at least 10 mg∙kg-1 (s.c.) at which it did not produce significant CPP neither alter general locomotion nor induce noticeable seizures. Conclusion and Implications: Derivatizing kratom alkaloids with the goal of enhancing δOR potency and reducing off-target effects could provide a pathway to develop novel lead compounds to treat alcohol use disorder with an improved therapeutic window.

A Novel Mitragynine Analog with Low-Efficacy Mu Opioid Receptor Agonism Displays Antinociception with Attenuated Adverse Effects.

Mitragynine and 7-hydroxymitragynine (7OH) are the major alkaloids mediating the biological actions of the psychoactive plant kratom. To investigate the structure-activity relationships of mitragynine/7OH templates, we diversified the aromatic ring of the indole at the C9, C10, and C12 positions and investigated their G-protein and arrestin signaling mediated by mu opioid receptors (MOR). Three synthesized lead C9 analogs replacing the 9-OCH3 group with phenyl (4), methyl (5), or 3'-furanyl [6 (SC13)] substituents demonstrated partial agonism with a lower efficacy than DAMGO or morphine in heterologous G-protein assays and synaptic physiology. In assays limiting MOR reserve, the G-protein efficacy of all three was comparable to buprenorphine. 6 (SC13) showed MOR-dependent analgesia with potency similar to morphine without respiratory depression, hyperlocomotion, constipation, or place conditioning in mice. These results suggest the possibility of activating MOR minimally (G-protein Emax ≈ 10%) in cell lines while yet attaining maximal antinociception in vivo with reduced opioid liabilities.

Kratom Alkaloids as Probes for Opioid Receptor Function: Pharmacological Characterization of Minor Indole and Oxindole Alkaloids from Kratom.

Dry leaves of kratom (mitragyna speciosa) are anecdotally consumed as pain relievers and antidotes against opioid withdrawal and alcohol use disorders. There are at least 54 alkaloids in kratom; however, investigations to date have focused around mitragynine, 7-hydroxy mitragynine (7OH), and mitragynine pseudoindoxyl (MP). Herein, we probe a few minor indole and oxindole based alkaloids, reporting the receptor affinity, G-protein activity, and ßarrestin-2 signaling of corynantheidine, corynoxine, corynoxine B, mitraciliatine, and isopaynantheine at mouse and human opioid receptors. We identify corynantheidine as a mu opioid receptor (MOR) partial agonist, whereas its oxindole derivative corynoxine was an MOR full agonist. Similarly, another alkaloid mitraciliatine was found to be an MOR partial agonist, while isopaynantheine was a KOR agonist which showed reduced ßarrestin-2 recruitment. Corynantheidine, corynoxine, and mitraciliatine showed MOR dependent antinociception in mice, but mitraciliatine and corynoxine displayed attenuated respiratory depression and hyperlocomotion compared to the prototypic MOR agonist morphine in vivo when administered supraspinally. Isopaynantheine on the other hand was identified as the first kratom derived KOR agonist in vivo. While these minor alkaloids are unlikely to play the majority role in the biological actions of kratom, they represent excellent starting points for further diversification as well as distinct efficacy and signaling profiles with which to probe opioid actions in vivo.

Kratom Alkaloids, Natural and Semi-Synthetic, Show Less Physical Dependence and Ameliorate Opioid Withdrawal.

Chronic administration of opioids produces physical dependence and opioid-induced hyperalgesia. Users claim the Thai traditional tea "kratom" and component alkaloid mitragynine ameliorate opioid withdrawal without increased sensitivity to pain. Testing these claims, we assessed the combined kratom alkaloid extract (KAE) and two individual alkaloids, mitragynine (MG) and the analog mitragynine pseudoindoxyl (MP), evaluating their ability to produce physical dependence and induce hyperalgesia after chronic administration, and as treatments for withdrawal in morphine-dependent subjects. C57BL/6J mice (n = 10/drug) were administered repeated saline, or graded, escalating doses of morphine (intraperitoneal; i.p.), kratom alkaloid extract (orally, p.o.), mitragynine (p.o.), or MP (subcutaneously, s.c.) for 5 days. Mice treated chronically with morphine, KAE, or mitragynine demonstrated significant drug-induced hyperalgesia by day 5 in a 48 °C warm-water tail-withdrawal test. Mice were then administered naloxone (10 mg/kg, s.c.) and tested for opioid withdrawal signs. Kratom alkaloid extract and the two individual alkaloids demonstrated significantly fewer naloxone-precipitated withdrawal signs than morphine-treated mice. Additional C57BL/6J mice made physically dependent on morphine were then used to test the therapeutic potential of combined KAE, mitragynine, or MP given twice daily over the next 3 days at either a fixed dose or in graded, tapering descending doses. When administered naloxone, mice treated with KAE, mitragynine, or MP under either regimen demonstrated significantly fewer signs of precipitated withdrawal than control mice that continued to receive morphine. In conclusion, while retaining some liabilities, kratom, mitragynine, and mitragynine pseudoindoxyl produced significantly less physical dependence and ameliorated precipitated withdrawal in morphine-dependent animals, suggesting some clinical value.

Natural Products for the Treatment of Pain: Chemistry and Pharmacology of Salvinorin A, Mitragynine, and Collybolide.

Pain remains a very pervasive problem throughout medicine. Classical pain management is achieved through the use of opiates belonging to the mu opioid receptor (MOR) class, which have significant side effects that hinder their utility. Pharmacologists have been trying to develop opioids devoid of side effects since the isolation of morphine from papaver somniferum, more commonly known as opium by Sertürner in 1804. The natural products salvinorin A, mitragynine, and collybolide represent three nonmorphinan natural product-based targets, which are potent selective agonists of opioid receptors, and emerging next-generation analgesics. In this work, we review the phytochemistry and medicinal chemistry efforts on these templates and their effects on affinity, selectivity, analgesic actions, and a myriad of other opioid-receptor-related behavioral effects.

Predicted Mode of Binding to and Allosteric Modulation of the µ-Opioid Receptor by Kratom's Alkaloids with Reported Antinociception In Vivo.

Pain management devoid of serious opioid adverse effects is still far from reach despite vigorous research and development efforts. Alternatives to classical opioids have been sought for years, and mounting reports of individuals finding pain relief with kratom have recently intensified research on this natural product. Although the composition of kratom is complex, the pharmacological characterization of its most abundant alkaloids has drawn attention to three molecules in particular, owing to their demonstrated antinociceptive activity and limited side effects in vivo. These three molecules are mitragynine (MG), its oxidized active metabolite, 7-hydroxymitragynine (7OH), and the indole-to-spiropseudoindoxy rearrangement product of MG known as mitragynine pseudoindoxyl (MP). Although these three alkaloids have been shown to preferentially activate the G protein signaling pathway by binding and allosterically modulating the µ-opioid receptor (MOP), a molecular level understanding of this process is lacking and yet important for the design of improved therapeutics. The molecular dynamics study and experimental validation reported here provide an atomic level description of how MG, 7OH, and MP bind and allosterically modulate the MOP, which can eventually guide structure-based drug design of improved therapeutics.

Diabetes self-management amid COVID-19 pandemic.

BACKGROUND AND AIMS: COVID-19 pandemic has challenged the physician-centered approach of diabetes care in India that is primarily based on routine clinic visits. We aim to review the various aspects of patient-centered care via diabetes self-management education based on available literature. METHODS: This is a narrative review using Pubmed, EMBASE and Google Scholar search till March 29, 2020. Search terms were "COVID-19", "diabetes self-care", "diabetes self-management education", "DSME", "diabetes self-management in India", "diabetes self-care in India" and "DSME in India". RESULTS: We have discussed an educational plan on diabetes self-management that can be adopted for people with diabetes mellitus in our country amid the ongoing pandemic. We have also identified the barriers to diabetes self-management in the current scenario and suggested possible solutions to overcome those. CONCLUSIONS: We have reemphasized the need for a simultaneous patient-centered approach in routine diabetes care that has to be coordinated by a multidisciplinary team amid the ongoing COVID-19 pandemic.

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells.

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric 'bias' into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

About a third of all drugs work by targeting a group of proteins known as G-protein coupled receptors, or GPCRs for short. These receptors are found on the surface of cells and transmit messages across the cell's outer barrier. When a signaling molecule, like a hormone, is released in the body, it binds to a GPCR and changes the receptor's shape. The change in structure affects how the GPCR interacts and binds to other proteins on the inside of the cell, triggering a series of reactions that alter the cell's activity. Scientists have previously seen that a GPCR can trigger different responses depending on which signaling molecule is binding on the surface of the cell. However, the mechanism for this is unknown. One hypothesis is that different signaling molecules change the GPCR's preference for binding to different proteins on the inside of the cell. The challenge has been to observe this happening without interfering with the process. Stoeber et al. have now tested this idea by attaching fluorescent tags to proteins that bind to activated GPCRs directly and without binding other signaling proteins. This meant these proteins could be tracked under a microscope as they made their way to bind to the GPCRs. Stoeber et al. focused on one particular GPCR, known as the opioid receptor, and tested the binding of two different opioid signaling molecules, etorphine and Dynorphin A. The experiments revealed that the different opioids did affect which of the engineered proteins would preferentially bind to the opioid receptor. This was followed by a similar experiment, where the engineered proteins were replaced with another protein called GRK2, which binds to the opioid receptor under normal conditions in the cell. This showed that GRK2 binds much more strongly to the opioid receptor when Dynorphin A is added compared to adding etorphine. These findings show that GPCRs can not only communicate that a signaling molecule is binding but can respond differently to convey what molecule it is more specifically. This could be important in developing drugs, particularly to specifically trigger the desired response and reduce side effects. Stoeber et al. suggest that an important next step for research is to understand how the GPCRs preferentially bind to different proteins.

PTBP2 exon 10 inclusion is associated with the progression of CML and it is BCR-ABL1 dependent.

Altered or aberrant expression of several splicing factors leads to the progression of different cancers. Though there are several ongoing studies underscoring the role of the splicing regulator polypyrimidine tract binding protein 2 (PTBP2) in neuronal cells, we unveil the role of PTBP2 in chronic myeloid leukemia (CML). Different RNA binding proteins (RBP's) earlier reported in chronic myeloid leukemia blast crisis (CML-BC) cases (n = 28) from Radich Oncomine leukemia dataset, were compared. We observed increased expression of MSI2 followed by PTBP2 in BC cases and increased PTBP2 expression in relapsed cases (n = 10) from the same dataset compared to other RBPs. We also observed increased PTBP2 exon 10 inclusion in KCL22, a granulocytic lineage CML cell line when compared to other CML cell lines of different lineages. As PTBP2 protein expression is associated with PTBP2 exon 10 inclusion, we observed in cell lines and in a set of progressed cases (n = 4) that increased BCR-ABL1 expression potentiates PTBP2 exon 10 inclusion and thus confers the existence of a functional protein. Inhibition of BCR-ABL1 with imatinib not only blocks the inclusion of exon 10 but also deregulates PTBP2 expression in CML cells. Knockdown of PTBP2 in KCL22 cells leads to reduced cell proliferation, increased G2/M cell cycle arrest and increased apoptosis. Taken together our study portrays PTBP2 as a new possible target for CML and progressive inclusion/exclusion of PTBP2 exon 10 might play an important role in CML progression.

RhIII -Catalyzed Direct C8-Arylation of Quinoline N-Oxides using Diazonaphthalen-2(1H)-ones: A Practical Approach towards 8-aza BINOL.

An efficient RhIII -catalyzed redox-neutral method for the direct C8-arylation of quinoline N-oxides using diazonaphthalen-2(1H)-one as coupling partner has been demonstrated. The developed method is simple, scalable and straightforward with a wide range of substrate scope. The applicative potential was extended with a late-stage functionalization and straightforward synthesis of 8-azaBINOL derivative. A plausible reaction pathway was proposed after carrying out preliminary control studies.

Ecotropic viral integration site 1 promotes metastasis independent of epithelial mesenchymal transition in colon cancer cells.

The most indecipherable component of solid cancer is the development of metastasis which accounts for more than 90% of cancer-related mortalities. A developmental program termed epithelial-mesenchymal transition (EMT) has also been shown to play a critical role in promoting metastasis in epithelium-derived solid tumors. By analyzing publicly available microarray datasets, we observed that ecotropic viral integration site 1 (EVI1) correlates negatively with SLUG, a master regulator of EMT. This correlation was found to be relevant as we demonstrated that EVI1 binds to SLUG promoter element directly through the distal set of zinc fingers and downregulates its expression. Many studies have shown that the primary role of SLUG during EMT and EMT-like processes is the regulation of cell motility in most of the cancer cells. Knockdown of EVI1 in metastatic colon cancer cell and subsequent passage through matrigel not only increased the invading capacity but also induced an EMT-like morphological feature of the cells, such as spindle-shaped appearance and led to a significant reduction in the expression of the epithelial marker, E-CADHERIN and increase in the expression of the mesenchymal marker, N-CADHERIN. The cells, when injected into immunocompromised mice, failed to show any metastatic foci in distant organs however the ones with EVI1, metastasized in the intraperitoneal layer and also showed multiple micro metastatic foci in the lungs and spleen. These findings suggest that in colon cancer EVI1 is dispensable for epithelial-mesenchymal transition, however, is required for metastasis.

A Representative Synthetic Route for C5 Angucycline Glycosides: Studies Directed toward the Total Synthesis of Mayamycin.

This study discloses an efficient synthetic route for the regiospecific construction of a C5 glycoside angucycline representative of mayamycin. The key steps are intramolecular aldol condensation and Hauser annulation, and the key precursor for the aldol reaction is accessible through utilization of α-lithiation of a vinyl ether.

Multipotent adult progenitor cells improve the hematopoietic function in myelodysplasia.

BACKGROUND AIMS: Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders affecting the normal hematopoietic differentiation process and leading to abnormal maturation and differentiation of all blood cell lineages. Treatment options are limited, and there is an unmet medical need for effective therapies for patients with severe cytopenias. METHODS: We demonstrate that multipotent adult progenitor cells (MAPC) improve the function of hematopoietic progenitors derived from human MDS bone marrow (BM) by significantly increasing the frequency of primitive progenitors as well as the number of myeloid colonies. RESULTS: This effect was more pronounced in a non-contact culture, indicating the importance of soluble factors produced by the MAPC cells. Moreover, the cells did not stimulate the growth of the abnormal MDS clone, as shown by fluorescent in situ hybridization analysis on BM cells from patients with a known genetic abnormality. We also demonstrate that MAPC cells can provide stromal support for patient-derived hematopoietic cells. When MAPC cells were intravenously injected into a mouse model of MDS, they migrated to the site of injury and increased the hematopoietic function in diseased mice. DISCUSSION: The preclinical studies undertaken here indicate an initial proof of concept for the use of MAPC cell therapy in patients with MDS-related severe and symptomatic cytopenias and should pave the way for further investigation in clinical trials.