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INTRODUCTION: The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. METHODS: We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. RESULTS: SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. DISCUSSION: 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights: Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-contaning inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays.A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health.SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic.The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.
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Objectives: To address the knowledge gap in the practice of compassionate healthcare by elucidating patient perspectives on compassion, empathy, and sympathy. Methods: Semi-structured telephone interviews were conducted at two time points with patients undergoing head and neck cancer treatment. Questions explored participants' understanding of compassion, sympathy, and empathy, as they relate to each other and to healthcare. Interviewers manually recorded responses. Qualitative exploratory methods were used to analyze data; inductive line-by-line coding was conducted to develop primary codes. Themes emerged through categorization of codes. Results: Ninety-five interviews conducted with 63 participants across two time points revealed four major themes - Compassion-vs-Empathy-vs-Sympathy, Coping Methods, Showing Care, and Nature of Interaction - encompassing seven categories, with a total of 24 codes. Codes were consistent across time points, except for two new codes, "positivity" and "personalized" emerging during follow-up interviews. Conclusions: Patient narrative from this study supported the concept that compassion is multidimensional and enabled several dimensions to be identified, highlighting the importance of patient perspectives in improving the provision of compassionate healthcare. Findings should be considered in future training and practice.
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Objective: Identify the most salient elements of the head and neck cancer (HNC) care experience described by patients and caregivers in focus group interviews. Methods: Three focus groups of patients and caregivers were facilitated by research assistants and clinicians. Open-ended guiding questions captured/elicited aspects of care that were appreciated, warranted improvement, or enhanced communication and information. A four-step Delphi process derived consensus among focus group facilitators (n = 5) regarding salient discussion points from focus group conversations. Results: Seven salient themes were identified: (1) information provision, (2) burden related to symptoms and treatment side effects, (3) importance of social support, (4) quality of care at both hospital and provider levels, (5) caring for the person, not just treating cancer, (6) social and emotional impact of HNC, and (7) stigma and insufficient information regarding human papillomavirus-related HNC. Conclusion: Participants reported varying needs and support preferences, a desire for individualized communication, and to feel cared for as both a person and a patient. Findings illuminate the intricate details underlying high-quality, compassionate, person-centered HNC cancer care.
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There is an urgent need for new antibiotics to mitigate the existential threat posed by antibiotic resistance. Within the ketolide class, solithromycin has emerged as one of the most promising candidates for further development. Crystallographic studies of bacterial ribosomes and ribosomal subunits complexed with solithromycin have shed light on the nature of molecular interactions (π-stacking and H-bonding) between from the biaryl side-chain of the drug and key residues in the 50S ribosomal subunit. We have designed and synthesized a library of solithromycin analogs to study their structure-activity relationships (SAR) in tandem with new computational studies. The biological activity of each analog was evaluated in terms of ribosomal affinity (Kd determined by fluorescence polarization), as well as minimum inhibitory concentration assays (MICs). Density functional theory (DFT) studies of a simple binding site model identify key H-bonding interactions that modulate the potency of solithromycin analogs.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Macrolides/pharmacology , Staphylococcus aureus/drug effects , Triazoles/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Density Functional Theory , Dose-Response Relationship, Drug , Macrolides/chemical synthesis , Macrolides/chemistry , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistryABSTRACT
Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.
Subject(s)
Large-Conductance Calcium-Activated Potassium Channels , Potassium Channels, Calcium-Activated , Analgesics, Opioid , Calcium/metabolism , Humans , Loperamide/pharmacologyABSTRACT
Gaucher disease (GD) results from inherited mutations in the lysosomal enzyme ß-glucocerobrosidase (GCase). Currently available treatment options for Type 1 GD are not efficacious for treating neuronopathic Type 2 and 3 GD due to their inability to cross the blood-brain barrier. In an effort to identify small molecules which could be optimized for CNS penetration we identified tamoxifen from a high throughput phenotypic screen on Type 2 GD patient-derived fibroblasts which reversed the disease phenotype. Structure activity studies around this scaffold led to novel molecules that displayed improved potency, efficacy and reduced estrogenic/antiestrogenic activity compared to the original hits. Here we present the design, synthesis and structure activity relationships that led to the lead molecule Compound 31.
Subject(s)
Fibroblasts/metabolism , Gaucher Disease/pathology , Small Molecule Libraries/chemistry , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/drug effects , Gaucher Disease/metabolism , Glucosylceramidase/genetics , Glucosylceramidase/metabolism , Humans , Inhibitory Concentration 50 , Lysosomes/drug effects , Lysosomes/metabolism , Phenotype , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Tamoxifen/chemistry , Tamoxifen/metabolismABSTRACT
Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder. Neuronopathic manifestations in GD include neurodegeneration, which can be severe and rapidly progressive. GBA1 mutations are also the most frequent genetic risk factors for Parkinson's disease. Dysfunction of the autophagy-lysosomal pathway represents a key pathogenic event in GBA1-associated neurodegeneration. Using an induced pluripotent stem cell (iPSC) model of GD, we previously demonstrated that lysosomal alterations in GD neurons are linked to dysfunction of the transcription factor EB (TFEB). TFEB controls the coordinated expression of autophagy and lysosomal genes and is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). To further investigate the mechanism of autophagy-lysosomal pathway dysfunction in neuronopathic GD, we examined mTORC1 kinase activity in GD iPSC neuronal progenitors and differentiated neurons. We found that mTORC1 is hyperactive in GD cells as evidenced by increased phosphorylation of its downstream protein substrates. We also found that pharmacological inhibition of glucosylceramide synthase enzyme reversed mTORC1 hyperactivation, suggesting that increased mTORC1 activity is mediated by the abnormal accumulation of glycosphingolipids in the mutant cells. Treatment with the mTOR inhibitor Torin1 upregulated lysosomal biogenesis and enhanced autophagic clearance in GD neurons, confirming that lysosomal dysfunction is mediated by mTOR hyperactivation. Further analysis demonstrated that increased TFEB phosphorylation by mTORC1 results in decreased TFEB stability in GD cells. Our study uncovers a new mechanism contributing to autophagy-lysosomal pathway dysfunction in GD, and identifies the mTOR complex as a potential therapeutic target for treatment of GBA1-associated neurodegeneration.
Subject(s)
Gaucher Disease/pathology , Induced Pluripotent Stem Cells/pathology , Lysosomes/pathology , Mechanistic Target of Rapamycin Complex 1/metabolism , Neurons/metabolism , Autophagy/drug effects , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Biomarkers/metabolism , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Green Fluorescent Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Lipids/chemistry , Lysosomes/drug effects , Lysosomes/metabolism , Naphthyridines/pharmacology , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , Protein Stability/drug effects , Up-Regulation/drug effectsABSTRACT
Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3. These channels transduce light information into electrical signals carried by both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2), which results in attenuation or loss of color vision, daylight blindness, and reduced visual acuity. Classical techniques to measure CNG channel function utilize patch clamp electrophysiology measuring Na currents in the absence of divalent cations, yet intracellular Ca2+ regulates both light and dark adaptation in photoreceptors. We developed a fluorescence-based, high-throughput Ca2+ flux assay using yellow fluorescent protein (YFP) tagged CNGA3 channels expressed in HEK293 cells which allow monitoring for folding defects in mutant channels. The cell permeant cGMP analog, 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), was used to activate Ca2+ flux. The assay was validated using wild-type CNGA3 homomeric and heteromeric channels and ACHM2-associated homomeric mutant CNG channels, CNGA3-R427C, CNGA3-E590K, and CNGA3-L633P. Additionally, we examined two naturally occurring canine mutations causing day-blindness previously studied by patch clamp. We compared the CPT-cGMP K0.5 values of the channels with patch clamp values from previous studies. The assay provides a screen for modulation of gating and/or rescue of trafficking and/or misfolding defects in ACHM2-associated CNG channels. Importantly, the calcium flux assay is advantageous compared to patch clamp as it allows the ability to monitor CNG channel activity in the presence of calcium.
Subject(s)
Calcium/metabolism , Color Vision Defects/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Retinal Cone Photoreceptor Cells/metabolism , HEK293 Cells , Humans , Patch-Clamp Techniques , Protein Transport , Retina/metabolismABSTRACT
After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can thus serve as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We optimized an HCS assay based on this readout, which showed highly robust performance in a 96-well format. Using this assay we screened a library of 1200 FDA-approved compounds and found that ketoconazole, the compound that most dramatically affected DLK localization in our primary screen, dose-dependently inhibited DLK palmitoylation in follow-up biochemical assays. Moreover, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to trophic deprivation, a well known model of DLK-dependent pro-degenerative signaling. Our HCS platform is thus capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.
Subject(s)
High-Throughput Screening Assays/methods , Ketoconazole/pharmacology , Lipoylation , MAP Kinase Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational , Combinatorial Chemistry Techniques , Cytochrome P-450 CYP3A Inhibitors/pharmacology , HEK293 Cells , Humans , MAP Kinase Kinase Kinases/metabolism , Signal TransductionABSTRACT
Tay-Sachs disease is an inherited lysosomal storage disease resulting from mutations in the lysosomal enzyme, ß-hexosaminidase A, and leads to excessive accumulation of GM2 ganglioside. Tay-Sachs patients with the infantile form do not live beyond 2-4 years of age due to rapid, progressive neurodegeneration. Enzyme replacement therapy is not a therapeutic option due to its inability to cross the blood-brain barrier. As an alternative, small molecules identified from high-throughput screening could provide leads suitable for chemical optimization to target the central nervous system. We developed a new high-throughput phenotypic assay utilizing infantile Tay-Sachs patient cells based on disrupted lysosomal calcium signaling as a monitor of diseased phenotype. The assay was validated in a pilot screen on a collection of Food and Drug Administration-approved drugs to identify compounds that could reverse or attenuate the disease. Pyrimethamine, a known pharmacological chaperone of ß-hexosaminidase A, was identified from the primary screen. The mechanism of action of pyrimethamine in reversing the defective lysosomal phenotype was by improving autophagy. This new high-throughput screening assay in patient cells will enable the screening of larger chemical compound collections. Importantly, this approach could lead to identification of new molecular targets previously unknown to impact the disease and accelerate the discovery of new treatments for Tay-Sachs disease.
Subject(s)
High-Throughput Screening Assays/methods , Lysosomes/physiology , Small Molecule Libraries/analysis , Tay-Sachs Disease/physiopathology , Autophagy , Calcium Signaling/drug effects , Cell Line , Drug Evaluation, Preclinical , Humans , Lysosomes/metabolism , Pilot Projects , Proof of Concept Study , Small Molecule Libraries/therapeutic use , Tay-Sachs Disease/drug therapy , Tay-Sachs Disease/metabolismABSTRACT
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Subject(s)
Cyclin-Dependent Kinase 9/metabolism , Animals , Cell Line, Tumor , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/genetics , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Methylation , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
PURPOSE OF REVIEW: Late dysphagia has a profound impact on quality of life in patients treated with chemoradiation therapy for advanced head and neck cancer and the number of survivors is growing. Traditional treatment methods are not uniformly successful in reducing the swallow impairment in these patients. RECENT FINDINGS: Manifestations of late dysphagia are complex and do not follow a uniform trajectory. Patient's experience of eating and engagement in treatment is critical given the complex, variable nature of late dysphagia. Novel swallow treatment methods target respiratory function including coordination and strength as well as patient adherence. SUMMARY: Head and neck cancer survivors deserve routine and fastidious surveillance beyond the 5-year survival point with respect to swallow function if the enormous biopsychosocial burden of late dysphagia is to be addressed.
Subject(s)
Chemoradiotherapy/adverse effects , Deglutition Disorders/therapy , Deglutition/drug effects , Deglutition/radiation effects , Head and Neck Neoplasms/therapy , Deglutition Disorders/diagnosis , Deglutition Disorders/etiology , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/radiotherapy , Humans , Time FactorsABSTRACT
PURPOSE OF REVIEW: The head and neck cancer (HNC) survivorship experience is unique among cancer populations. This review seeks to explore the HNC survivor experience associated with altered upper aerodigestive tract (UADT) function using principles of survivorship. RECENT FINDINGS: HNC survivors experience complex physical, functional, and psychosocial challenges related to UADT dysfunction. Interventions need to address all of these dimensions being mindful of the survivor experience. Studies reveal related unmet needs of both HNC survivors and their family members. An expose of the HNC survivorship experience is timely since 2016/2017 contains the release of landmark position papers and guidelines in the field. These address the unique challenges faced by the HNC population, their management, and the triad psychosocial, functional, and physical survivor burden. Such developments will drive future care. SUMMARY: The head and neck survivorship experience is characterized by complex changes with broad impact when examined in an experiential framework. HNC survivor care demands clinical excellence from multiple disciplines that are positioned to enact recent HNC guidelines and adopt survivorship principles. Future research in this population that explores experiential aspects of altered UADT function would be valuable in informing clinical practice. A biopsychosocial framework is presented for conceptualizing the HNC survivorship experience.
Subject(s)
Cancer Survivors/psychology , Head and Neck Neoplasms/psychology , Quality of Life , Deglutition Disorders/psychology , Drinking , Eating/psychology , Head and Neck Neoplasms/rehabilitation , Head and Neck Neoplasms/surgery , Health Status , Humans , Laryngectomy/psychologyABSTRACT
BACKGROUND: Hypoglossal nerve palsies are infrequent complications of head and neck radiotherapy. Treatments focus on maintaining function and prevention of abnormal airway-related swallowing events. METHODS: A patient with longstanding cranial neuropathies, including bilateral hypoglossal involvement, secondary to chemoradiotherapy for nasopharyngeal carcinoma, experienced repeated episodes of life-threatening complications. Initially, 2 courses of 2 weekly 24-hour intravenous methylprednisolone (IVMP) infusions were administered 2 years apart. We report the results of a third course comprising 5 weekly cycles. RESULTS: Patient-reported outcomes revealed significant improvement in swallowing function, speech, and psychosocial status. Airway invasion during swallowing and pharyngeal retention were assessed videofluoroscopically and evaluated using the Penetration-Aspiration Scale (PAS) and a residue rating scale, respectively. PAS ratings after infusions 2 and 5, improved dramatically from baseline and were maintained at 1-year follow-up. CONCLUSION: High doses of IVMP may improve radiation-induced neuropathies. Further testing in similar patients is needed to prove reproducibility. © 2016 Wiley Periodicals, Inc. Head Neck 39: E23-E28, 2017.
Subject(s)
Carcinoma/therapy , Chemoradiotherapy/adverse effects , Hypoglossal Nerve Diseases/drug therapy , Methylprednisolone/administration & dosage , Nasopharyngeal Neoplasms/therapy , Radiation Injuries/drug therapy , Radiotherapy, Intensity-Modulated/adverse effects , Adult , Carcinoma/diagnosis , Chemoradiotherapy/methods , Deglutition/drug effects , Deglutition Disorders/drug therapy , Deglutition Disorders/etiology , Dose-Response Relationship, Drug , Follow-Up Studies , Humans , Hypoglossal Nerve Diseases/etiology , Infusions, Intravenous , Male , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/diagnosis , Radiation Injuries/diagnosis , Radiotherapy Dosage , Radiotherapy, Intensity-Modulated/methods , Retreatment , Speech Intelligibility/drug effects , Treatment OutcomeABSTRACT
Investigation of a novel amino-aza-benzimidazolone structural class of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 2 (mGluR2) identified [2.2.2]-bicyclic amine 12 as an intriguing lead structure due to its promising physicochemical properties and lipophilic ligand efficiency (LLE). Further optimization led to chiral amide 18, which exhibited strong in vitro activity and attractive pharmacokinetic (PK) properties. Hypothesis-driven target design identified compound 21 as a potent, highly selective, orally bioavailable mGluR2 PAM, which addressed a CYP time-dependent inhibition (TDI) liability of 18, while maintaining excellent drug-like properties with robust in vivo activity in a clinically validated model of antipsychotic potential.
ABSTRACT
Over half of all antibiotics target the bacterial ribosome-nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a 16-component reaction (i.e., azide 2 and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).
Subject(s)
Alkynes/chemistry , Anti-Bacterial Agents/chemical synthesis , Azides/chemistry , Macrolides/chemical synthesis , Ribosomes/chemistry , Triazoles/chemical synthesis , Alkynes/pharmacology , Anti-Bacterial Agents/pharmacology , Azides/pharmacology , Click Chemistry , Cycloaddition Reaction , Humans , Macrolides/pharmacology , Models, Molecular , Ribosomes/metabolism , Thermodynamics , Triazoles/pharmacologyABSTRACT
Optimization of a benzimidazolone template for potency and physical properties revealed 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as a key template on which to develop a new series of mGlu2 positive allosteric modulators (PAMs). Systematic investigation of aryl-SAR led to the identification of compound 27 as a potent and highly selective mGlu2 PAM with sufficient pharmacokinetics to advance to preclinical models of psychosis. Gratifyingly, compound 27 showed full efficacy in the PCP- and MK-801-induced hyperlocomotion assay in rats at CSF concentrations consistent with mGlu2 PAM potency.
Subject(s)
Imidazoles/chemistry , Pyridines/chemistry , Pyridones/chemistry , Receptors, Metabotropic Glutamate/chemistry , Allosteric Regulation , Animals , Disease Models, Animal , Drug Evaluation, Preclinical , Imidazoles/blood , Imidazoles/pharmacology , Imidazoles/therapeutic use , Locomotion/drug effects , Protein Binding , Psychotic Disorders/drug therapy , Psychotic Disorders/pathology , Pyridines/pharmacology , Pyridines/therapeutic use , Pyridones/blood , Pyridones/pharmacology , Rats , Receptors, Metabotropic Glutamate/metabolism , Structure-Activity RelationshipABSTRACT
Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. ß-adenergic receptors (ßAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous ßAR in primary neonatal rat cardiac fibroblasts (NRCF). Catecholamine stimulation of the cells induced a negative DMR deflection resulting in a concentration-dependent pharmacological response that was competitively blocked by ßAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Pharmacological profiling of subtype-selective ßAR agonists and antagonists revealed a dominant role of ß2AR in mediating the DMR responses, consistent with the relative expression levels of ß2AR and ß1AR in NRCF. Additionally, ßAR-mediated cAMP generation was assessed via a fluorescence biosensor, revealing similar kinetics between DMR responses and cAMP generation. As such, ßAR-dependent DMR responses were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, we assessed G protein-independent ßAR signaling through epidermal growth factor receptor (EGFR). While inhibition of EGFR reduced the DMR response to ßAR stimulation, our results demonstrate that G protein-dependent signaling produces a majority of the biological response to ßAR stimulation in NRCF. Altogether, measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous ßAR signaling via both G protein-dependent and -independent pathways.
ABSTRACT
The glutamatergic hypofunction hypothesis of schizophrenia has led to the development of novel therapeutic strategies modulating NMDA receptor function. One of these strategies targets the activation of the metabotropic glutamate receptor 5 (mGlu5 receptor) using positive allosteric modulators (PAMs). Our goal was to evaluate the potential for repeated administration of the mGlu5 receptor PAM, CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide) (30 mg/kg) to induce tolerance to the anti-psychotic like effect using the amphetamine-induced hyperlocomotion rat model, and to produce receptor desensitization in mGlu5 receptor-enriched brain regions. CDPPB dose dependently reduced the locomotor response to amphetamine when administered acutely, and the same effect was observed following 7-day pre-treatment regime. In addition, 7-day dosing of CDPPB did not affect mGlu5 receptor density in the striatum, nor did it change mGlu5 receptor PAM-induced phosphorylation of NMDA, GluN1 and GluN2b, receptor subunits in striatum compared to the levels measured acutely. In contrast, in the frontal cortex, repeated administration of CDPPB decreased mGlu5 receptor density and resulted in a loss of its ability to increase GluN1 and GluN2b levels. Consistent with a reduction of cortical mGlu5 receptor density and phosphorylation, CDPPB (30 mg/kg) significantly affected sleep architecture as determined by cortical EEG at day one however by the seventh day of dosing all sleep changes were absent. Together these results suggest that the development of tolerance induced by the repeated treatment with the mGlu5 receptor PAM, CDPPB, may depend not only on the system being measured (sleep architecture vs psychostimulant induced hyperactivity), but also on the brain region involved with frontal cortex being a more susceptible region to receptor desensitization and internalization than striatum.