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1.
Proc Natl Acad Sci U S A ; 120(28): e2219543120, 2023 07 11.
Article in English | MEDLINE | ID: mdl-37406092

ABSTRACT

Although HIV-1 Gag is known to drive viral assembly and budding, the precise mechanisms by which the lipid composition of the plasma membrane is remodeled during assembly are incompletely understood. Here, we provide evidence that the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) interacts with HIV-1 Gag and through the hydrolysis of sphingomyelin creates ceramide that is necessary for proper formation of the viral envelope and viral maturation. Inhibition or depletion of nSMase2 resulted in the production of noninfectious HIV-1 virions with incomplete Gag lattices lacking condensed conical cores. Inhibition of nSMase2 in HIV-1-infected humanized mouse models with a potent and selective inhibitor of nSMase2 termed PDDC [phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl) pyrrolidin-3-yl)-carbamate] produced a linear reduction in levels of HIV-1 in plasma. If undetectable plasma levels of HIV-1 were achieved with PDDC treatment, viral rebound did not occur for up to 4 wk when PDDC was discontinued. In vivo and tissue culture results suggest that PDDC selectively kills cells with actively replicating HIV-1. Collectively, this work demonstrates that nSMase2 is a critical regulator of HIV-1 replication and suggests that nSMase2 could be an important therapeutic target with the potential to kill HIV-1-infected cells.


Subject(s)
HIV-1 , Sphingomyelin Phosphodiesterase , Mice , Animals , Sphingomyelin Phosphodiesterase/metabolism , HIV-1/metabolism , Sphingomyelins/metabolism , Cell Membrane/metabolism
2.
Proc Natl Acad Sci U S A ; 120(28): e2219475120, 2023 07 11.
Article in English | MEDLINE | ID: mdl-37406093

ABSTRACT

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.


Subject(s)
HIV-1 , Infectious Anemia Virus, Equine , Animals , Cats , Horses , Mice , HIV-1/physiology , Sphingomyelin Phosphodiesterase/metabolism , Virus Assembly , Lentivirus
3.
Mol Psychiatry ; 2024 Apr 13.
Article in English | MEDLINE | ID: mdl-38615102

ABSTRACT

We report a mechanism that underlies stress-induced cognitive inflexibility at the molecular level. In a mouse model under subacute cellular stress in which deficits in rule shifting tasks were elicited, the nuclear glyceraldehyde dehydrogenase (N-GAPDH) cascade was activated specifically in microglia in the prelimbic cortex. The cognitive deficits were normalized with a pharmacological intervention with a compound (the RR compound) that selectively blocked the initiation of N-GAPDH cascade without affecting glycolytic activity. The normalization was also observed with a microglia-specific genetic intervention targeting the N-GAPDH cascade. At the mechanistic levels, the microglial secretion of High-Mobility Group Box (HMGB), which is known to bind with and regulate the NMDA-type glutamate receptors, was elevated. Consequently, the hyperactivation of the prelimbic layer 5 excitatory neurons, a neural substrate for cognitive inflexibility, was also observed. The upregulation of the microglial HMGB signaling and neuronal hyperactivation were normalized by the pharmacological and microglia-specific genetic interventions. Taken together, we show a pivotal role of cortical microglia and microglia-neuron interaction in stress-induced cognitive inflexibility. We underscore the N-GAPDH cascade in microglia, which causally mediates stress-induced cognitive alteration.

4.
J Immunol ; 211(12): 1767-1782, 2023 12 15.
Article in English | MEDLINE | ID: mdl-37947442

ABSTRACT

Understanding the mechanisms underlying the acquisition and maintenance of effector function during T cell differentiation is important to unraveling how these processes can be dysregulated in the context of disease and manipulated for therapeutic intervention. In this study, we report the identification of a previously unappreciated regulator of murine T cell differentiation through the evaluation of a previously unreported activity of the kinase inhibitor, BioE-1197. Specifically, we demonstrate that liver kinase B1 (LKB1)-mediated activation of salt-inducible kinases epigenetically regulates cytokine recall potential in effector CD8+ and Th1 cells. Evaluation of this phenotype revealed that salt-inducible kinase-mediated phosphorylation-dependent stabilization of histone deacetylase 7 (HDAC7) occurred during late-stage effector differentiation. HDAC7 stabilization increased nuclear HDAC7 levels, which correlated with total and cytokine loci-specific reductions in the activating transcription mark histone 3 lysine 27 acetylation (H3K27Ac). Accordingly, HDAC7 stabilization diminished transcriptional induction of cytokine genes upon restimulation. Inhibition of this pathway during differentiation produced effector T cells epigenetically poised for enhanced cytokine recall. This work identifies a previously unrecognized target for enhancing effector T cell functionality.


Subject(s)
Cytokines , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases , Animals , Mice , Cell Differentiation , Cytokines/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism
5.
Prostate ; 84(11): 1086-1088, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38678435

ABSTRACT

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a biomarker and therapeutic target of high relevance in prostate cancer. Although upregulated PSMA expression is a well-documented feature of prostatic neoplasia in both humans and canids, to date humans are the only species known to express PSMA basally in the prostate. Thus, traditional laboratory animal species have limited utility for studying PSMA biology in the prostate or for predicting efficacy or toxicity of PSMA-targeted agents. METHODS: PSMA expression in human, macaque, and marmoset prostates was determined by immunohistochemistry, employing an antibody with validated cross-species reactivity in a PSMA-positive control tissue; kidney. RESULTS: We newly discover that the common marmoset endogenously expresses PSMA in non-diseased prostate, similar to humans, and thus may be a valuable preclinical model for researchers studying PSMA.


Subject(s)
Antigens, Surface , Callithrix , Glutamate Carboxypeptidase II , Prostate , Male , Animals , Glutamate Carboxypeptidase II/metabolism , Antigens, Surface/metabolism , Humans , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Immunohistochemistry
6.
Rapid Commun Mass Spectrom ; : e9772, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38867136

ABSTRACT

RATIONALE: Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to yield glutamate (Glu) and N-acetylaspartate (NAA). Inhibition of GCPII has been shown to remediate the neurotoxicity of excess Glu in a variety of cell and animal disease models. A robust high-throughput liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was needed to quantify GCPII enzymatic activity in a biochemical high-throughput screening assay. METHODS: A dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2 × 30 mm) columns. Each LC channel was run independently, and the cycle time was 2 min per channel. Overall throughput was 1 min per sample for the dual-channel integrated system. Multiply injected acquisition files were split during data review, and batch metadata were automatically paired with raw data during the review process. RESULTS: Two LC sorbents, BEH-Amide and Penta-HILIC, were tested to separate the NAAG cleavage product Glu from isobaric interference and ion suppressants in the bioassay matrix. Early elution of NAAG and NAA on BEH-Amide allowed interfering species to be diverted to waste. The limit of quantification was 0.1 pmol for Glu. The Z-factor of this assay averaged 0.85. Over 36 000 compounds were screened using this method. CONCLUSIONS: A fast gradient dual-stream LC/MS/MS method for Glu quantification in GCPII biochemical screening assay samples was developed and validated. HILIC separation chemistry offers robust performance and unique selectivity for targeted positive mode quantification of Glu, NAA, and NAAG.

7.
Int J Mol Sci ; 25(13)2024 Jun 23.
Article in English | MEDLINE | ID: mdl-39000003

ABSTRACT

Peripheral nerve injuries (PNIs) represent a significant clinical challenge, particularly in elderly populations where axonal remyelination and regeneration are impaired. Developing therapies to enhance these processes is crucial for improving PNI repair outcomes. Glutamate carboxypeptidase II (GCPII) is a neuropeptidase that plays a pivotal role in modulating glutamate signaling through its enzymatic cleavage of the abundant neuropeptide N-acetyl aspartyl glutamate (NAAG) to liberate glutamate. Within the PNS, GCPII is expressed in Schwann cells and activated macrophages, and its expression is amplified with aging. In this study, we explored the therapeutic potential of inhibiting GCPII activity following PNI. We report significant GCPII protein and activity upregulation following PNI, which was normalized by the potent and selective GCPII inhibitor 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). In vitro, 2-PMPA robustly enhanced myelination in dorsal root ganglion (DRG) explants. In vivo, using a sciatic nerve crush injury model in aged mice, 2-PMPA accelerated remyelination, as evidenced by increased myelin sheath thickness and higher numbers of remyelinated axons. These findings suggest that GCPII inhibition may be a promising therapeutic strategy to enhance remyelination and potentially improve functional recovery after PNI, which is especially relevant in elderly PNI patients where this process is compromised.


Subject(s)
Glutamate Carboxypeptidase II , Peripheral Nerve Injuries , Remyelination , Animals , Mice , Peripheral Nerve Injuries/drug therapy , Peripheral Nerve Injuries/metabolism , Remyelination/drug effects , Glutamate Carboxypeptidase II/antagonists & inhibitors , Glutamate Carboxypeptidase II/metabolism , Myelin Sheath/metabolism , Myelin Sheath/drug effects , Aging/drug effects , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Mice, Inbred C57BL , Nerve Regeneration/drug effects , Sciatic Nerve/injuries , Sciatic Nerve/drug effects , Male , Axons/drug effects , Axons/metabolism
8.
Neurobiol Dis ; 177: 105987, 2023 02.
Article in English | MEDLINE | ID: mdl-36603748

ABSTRACT

Extracellular vesicles (EVs) have been proposed to regulate the deposition of Aß. Multiple publications have shown that APP, amyloid processing enzymes and Aß peptides are associated with EVs. However, very little Aß is associated with EVs compared with the total amount Aß present in human plasma, CSF, or supernatants from cultured neurons. The involvement of EVs has largely been inferred by pharmacological inhibition or whole body deletion of the sphingomyelin hydrolase neutral sphingomyelinase-2 (nSMase2) that is a key regulator for the biogenesis of at-least one population of EVs. Here we used a Cre-Lox system to selectively delete nSMase2 from pyramidal neurons in APP/PS1 mice (APP/PS1-SMPD3-Nex1) and found a âˆ¼ 70% reduction in Aß deposition at 6 months of age and âˆ¼ 35% reduction at 12 months of age in both cortex and hippocampus. Brain ceramides were increased in APP/PS1 compared with Wt mice, but were similar to Wt in APP/PS1-SMPD3-Nex1 mice suggesting that elevated brain ceramides in this model involves neuronally expressed nSMase2. Reduced levels of PSD95 and deficits of long-term potentiation in APP/PS1 mice were normalized in APP/PS1-SMPD3-Nex1 mice. In contrast, elevated levels of IL-1ß, IL-8 and TNFα in APP/PS1 mice were not normalized in APP/PS1-SMPD3-Nex1 mice compared with APP/PS1 mice. Mechanistic studies showed that the size of liquid ordered membrane microdomains was increased in APP/PS1 mice, as were the amounts of APP and BACE1 localized to these microdomains. Pharmacological inhibition of nSMase2 activity with PDDC reduced the size of the liquid ordered membrane microdomains, reduced the localization of APP with BACE1 and reduced the production of Aß1-40 and Aß1-42. Although inhibition of nSMase2 reduced the release and increased the size of EVs, very little Aß was associated with EVs in all conditions tested. We also found that nSMase2 directly protected neurons from the toxic effects of oligomerized Aß and preserved neural network connectivity despite considerable Aß deposition. These data demonstrate that nSMase2 plays a role in the production of Aß by stabilizing the interaction of APP with BACE1 in liquid ordered membrane microdomains, and directly protects neurons from the toxic effects of Aß. The effects of inhibiting nSMase2 on EV biogenesis may be independent from effects on Aß production and neuronal protection.


Subject(s)
Alzheimer Disease , Mice , Humans , Animals , Amyloid Precursor Protein Secretases , Mice, Transgenic , Aspartic Acid Endopeptidases , Amyloid beta-Peptides , Neurons , Amyloid beta-Protein Precursor/genetics , Presenilin-1 , Disease Models, Animal , Sphingomyelin Phosphodiesterase/genetics
9.
Mol Psychiatry ; 27(10): 4252-4263, 2022 10.
Article in English | MEDLINE | ID: mdl-35732693

ABSTRACT

Glutamate carboxypeptidase-II (GCPII) expression in brain is increased by inflammation, e.g. by COVID19 infection, where it reduces NAAG stimulation of metabotropic glutamate receptor type 3 (mGluR3). GCPII-mGluR3 signaling is increasingly linked to higher cognition, as genetic alterations that weaken mGluR3 or increase GCPII signaling are associated with impaired cognition in humans. Recent evidence from macaque dorsolateral prefrontal cortex (dlPFC) shows that mGluR3 are expressed on dendritic spines, where they regulate cAMP-PKA opening of potassium (K+) channels to enhance neuronal firing during working memory. However, little is known about GCPII expression and function in the primate dlPFC, despite its relevance to inflammatory disorders. The present study used multiple label immunofluorescence and immunoelectron microscopy to localize GCPII in aging macaque dlPFC, and examined the effects of GCPII inhibition on dlPFC neuronal physiology and working memory function. GCPII was observed in astrocytes as expected, but also on neurons, including extensive expression in dendritic spines. Recordings in dlPFC from aged monkeys performing a working memory task found that iontophoresis of the GCPII inhibitors 2-MPPA or 2-PMPA markedly increased working memory-related neuronal firing and spatial tuning, enhancing neural representations. These beneficial effects were reversed by an mGluR2/3 antagonist, or by a cAMP-PKA activator, consistent with mGluR3 inhibition of cAMP-PKA-K+ channel signaling. Systemic administration of the brain penetrant inhibitor, 2-MPPA, significantly improved working memory performance without apparent side effects, with largest effects in the oldest monkeys. Taken together, these data endorse GCPII inhibition as a potential strategy for treating cognitive disorders associated with aging and/or neuroinflammation.


Subject(s)
COVID-19 , Dorsolateral Prefrontal Cortex , Humans , Animals , Haplorhini , Macaca , Cognition , Glutamates
10.
Neurobiol Dis ; 169: 105734, 2022 07.
Article in English | MEDLINE | ID: mdl-35462006

ABSTRACT

People living with HIV (PLH) have significantly higher rates of cognitive impairment (CI) and major depressive disorder (MDD) versus the general population. The enzyme neutral sphingomyelinase 2 (nSMase2) is involved in the biogenesis of ceramide and extracellular vesicles (EVs), both of which are dysregulated in PLH, CI, and MDD. Here we evaluated EcoHIV-infected mice for behavioral abnormalities relevant to depression and cognition deficits, and assessed the behavioral and biochemical effects of nSMase2 inhibition. Mice were infected with EcoHIV and daily treatment with either vehicle or the nSMase2 inhibitor (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC) began 3 weeks post-infection. After 2 weeks of treatment, mice were subjected to behavior tests. EcoHIV-infected mice exhibited behavioral abnormalities relevant to MDD and CI that were reversed by PDDC treatment. EcoHIV infection significantly increased cortical brain nSMase2 activity, resulting in trend changes in sphingomyelin and ceramide levels that were normalized by PDDC treatment. EcoHIV-infected mice also exhibited increased levels of brain-derived EVs and altered microRNA cargo, including miR-183-5p, miR-200c-3p, miR-200b-3p, and miR-429-3p, known to be associated with MDD and CI; all were normalized by PDDC. In conclusion, inhibition of nSMase2 represents a possible new therapeutic strategy for the treatment of HIV-associated CI and MDD.


Subject(s)
Depressive Disorder, Major , Extracellular Vesicles , HIV Infections , MicroRNAs , Animals , Ceramides , HIV Infections/complications , HIV Infections/drug therapy , Humans , Mice , MicroRNAs/genetics , MicroRNAs/pharmacology , Sphingomyelin Phosphodiesterase/genetics
11.
Eur J Immunol ; 51(9): 2317-2329, 2021 09.
Article in English | MEDLINE | ID: mdl-34272885

ABSTRACT

Endothelial cells are key regulators of transendothelial migration and their secretion of chemokines and expression of adhesion molecules facilitates lymphocyte entry into tissues. Previously, we demonstrated that Tregs can reduce transendothelial migration of T cells into tumors by decreasing endothelial CXCL10 secretion, but the mechanism by which this occurs is still not known. In this study, we aimed to define how Tregs decrease transendothelial migration into tumors. mRNA sequencing of intestinal tumor endothelial cells from Treg depleted mice identified neutral sphingomyelinase 2 (nSMase2) as a gene downregulated in the presence of Tregs. nSMase2 is expressed in human umbilical vein endothelial cells (HUVECs) and was decreased after coculture with Tregs. Furthermore, blocking of nSMase2 activity in vitro decreased VCAM1, CX3CL1, and CXCL10 expression in HUVECs, mirroring the same decrease found in Treg cocultures. In the APCmin/+ mouse model of intestinal cancer, nSMase2 is lower in tumor endothelial cells than in unaffected small intestine and chronic treatment with a nSMase2 inhibitor suppressed the increased migration that is otherwise seen in the absence of Tregs. We conclude that nSMase2 is an important mediator in endothelial cells supporting transendothelial migration, which may be targeted by Tregs to reduce T-cell migration into tumors.


Subject(s)
Chemokine CXCL10/metabolism , Colonic Neoplasms/pathology , Lymphocytes, Tumor-Infiltrating/immunology , Sphingomyelin Phosphodiesterase/metabolism , T-Lymphocytes, Regulatory/immunology , Transendothelial and Transepithelial Migration/physiology , Animals , Cell Adhesion Molecules/biosynthesis , Cell Line , Chemokine CX3CL1/biosynthesis , Chemokine CXCL10/biosynthesis , Colonic Neoplasms/immunology , Down-Regulation , Female , Human Umbilical Vein Endothelial Cells , Humans , Male , Mice , Mice, Transgenic , T-Lymphocyte Subsets/immunology , Versicans/biosynthesis
12.
Bioorg Med Chem Lett ; 50: 128321, 2021 10 15.
Article in English | MEDLINE | ID: mdl-34400301

ABSTRACT

Two distinct diazo precursors, imidazotetrazine and nitrous amide, were explored as promoieties in designing prodrugs of 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist. As a model for an imidazotetrazine-based prodrug, we synthesized (S)-2-acetamido-6-(8-carbamoyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazin-3(4H)-yl)-5-oxohexanoic acid (4) containing the entire scaffold of temozolomide, a precursor of the DNA-methylating agent clinically approved for the treatment of glioblastoma multiforme. For a nitrous amide-based prodrug, we synthesized 2-acetamido-6-(((benzyloxy)carbonyl)(nitroso)amino)-5-oxohexanoic acid (5) containing a N-nitrosocarbamate group, which can be converted to a diazo moiety via a mechanism similar to that of streptozotocin, a clinically approved diazomethane-releasing drug containing an N-nitrosourea group. Preliminary characterization confirmed formation of N-acetyl DON (6), also known as duazomycin A, from compound 4 in a pH-dependent manner while compound 5 did not exhibit sufficient stability to allow further characterization. Taken together, our model studies suggest that further improvements are needed to translate this prodrug approach into glutamine antagonist-based therapy.


Subject(s)
Diazooxonorleucine/analogs & derivatives , Diazooxonorleucine/pharmacology , Glutamine/antagonists & inhibitors , Prodrugs/chemistry , Prodrugs/pharmacology , Diazooxonorleucine/chemistry , Drug Design , Drug Stability , Molecular Structure
13.
Proc Natl Acad Sci U S A ; 115(51): E12024-E12033, 2018 12 18.
Article in English | MEDLINE | ID: mdl-30514812

ABSTRACT

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.


Subject(s)
Diazooxonorleucine/antagonists & inhibitors , Diazooxonorleucine/therapeutic use , Glutamine/antagonists & inhibitors , Magnetic Resonance Imaging/methods , Malaria, Cerebral/drug therapy , Malaria, Cerebral/pathology , Malaria, Falciparum/drug therapy , Malaria, Falciparum/pathology , Adult , Animals , Antimalarials/therapeutic use , Biomarkers , Blood-Brain Barrier/pathology , Brain/parasitology , Brain/pathology , Brain Edema/diagnostic imaging , Brain Edema/pathology , Child , Diazooxonorleucine/administration & dosage , Disease Models, Animal , Disease Progression , Female , Humans , Malaria, Cerebral/diagnostic imaging , Malaria, Cerebral/parasitology , Malaria, Falciparum/diagnostic imaging , Mice , Mice, Inbred C57BL , Plasmodium falciparum/pathogenicity
15.
Neurochem Res ; 45(6): 1256-1267, 2020 Jun.
Article in English | MEDLINE | ID: mdl-31749072

ABSTRACT

In tribute to our friend and colleague Michael Robinson, we review his involvement in the identification, characterization and localization of the metallopeptidase glutamate carboxypeptidase II (GCPII), originally called NAALADase. While Mike was characterizing NAALADase in the brain, the protein was independently identified by other laboratories in human prostate where it was termed prostate specific membrane antigen (PSMA) and in the intestines where it was named Folate Hydrolase 1 (FOLH1). It was almost a decade to establish that NAALADase, PSMA, and FOLH1 are encoded by the same gene. The enzyme has emerged as a therapeutic target outside of the brain, with the most notable progress made in the treatment of prostate cancer and inflammatory bowel disease (IBD). PSMA-PET imaging with high affinity ligands is proving useful for the clinical diagnosis and staging of prostate cancer. A molecular radiotherapy based on similar ligands is in trials for metastatic castration-resistant prostate cancer. New PSMA inhibitor prodrugs that preferentially block kidney and salivary gland versus prostate tumor enzyme may improve the clinical safety of this radiotherapy. The wide clinical use of PSMA-PET imaging in prostate cancer has coincidentally led to clinical documentation of GCPII upregulation in a wide variety of tumors and inflammatory diseases, likely associated with angiogenesis. In IBD, expression of the FOLH1 gene that codes for GCPII is strongly upregulated, as is the enzymatic activity in diseased patient biopsies. In animal models of IBD, GCPII inhibitors show substantial efficacy, suggesting potential theranostic use of GCPII ligands for IBD.


Subject(s)
Antineoplastic Agents/therapeutic use , Brain/drug effects , Brain/metabolism , Glutamate Carboxypeptidase II/antagonists & inhibitors , Glutamate Carboxypeptidase II/metabolism , Prodrugs/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/metabolism , Humans , Male , Prodrugs/chemistry , Prodrugs/pharmacology , Prostate/drug effects , Prostate/metabolism
16.
Biomacromolecules ; 21(9): 3909-3922, 2020 09 14.
Article in English | MEDLINE | ID: mdl-32786523

ABSTRACT

Mitochondria mediate critical cellular processes, including proliferation, apoptosis, and immune responses; as such, their dysfunction is pathogenic in many neurodegenerative disorders and cancers. In glioblastoma, targeted delivery of mitochondria-focused anticancer therapies has failed to translate into clinical success due to the nonspecific cellular localization, heterogeneity of receptor expression across patients, poor transport across biological barriers to reach the brain, tumor, and mitochondria, and systemic side effects. Strategies that can overcome brain and solid tumor barriers and selectively target mitochondria within specific cell types may lead to improvements in glioblastoma treatment. Developments in dendrimer-mediated nanomedicines have shown promise targeting tumor-associated macrophages (TAMs) in glioblastoma, following systemic administration. Here, we present a novel dendrimer conjugated to the translocator protein (18 kDa) (TSPO) ligand 5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-ylacetamide (DPA). We developed a clickable DPA for conjugation on the dendrimer surface and demonstrated in vitro that the dendrimer-DPA conjugate (D-DPA) significantly increases dendrimer colocalization with mitochondria. Compared to free TSPO ligand PK11195, D-DPA stimulates greater antitumor immune signaling. In vivo, we show that D-DPA targets mitochondria specifically within TAMs following systemic administration. Our results demonstrate that dendrimers can achieve TAM-specific targeting in glioblastoma and can be further modified to target specific intracellular compartments for organelle-specific drug delivery.


Subject(s)
Dendrimers , Glioblastoma , Cell Line, Tumor , Dendrimers/pharmacology , Dendrimers/therapeutic use , Glioblastoma/drug therapy , Humans , Ligands , Mitochondria , Receptors, GABA/therapeutic use , Tumor-Associated Macrophages
17.
Bioorg Med Chem ; 28(20): 115698, 2020 10 15.
Article in English | MEDLINE | ID: mdl-33069080

ABSTRACT

A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a mercaptoethyl (SCH2CH2) linker were synthesized in an effort to further expand the structural diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl linker between the two thiadiazole rings was found to potently inhibit GLS with an IC50 value of 50 nM. Interestingly, the corresponding derivative with an n-propyl (CH2CH2CH2) linker showed substantially lower inhibitory potency (IC50 = 2.3 µM) while the derivative with a dimethylsulfide (CH2SCH2) linker showed no inhibitory activity at concentrations up to 100 µM, underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and tested as GLS inhibitors to further explore SAR within this scaffold including derivatives possessing a pyridazine as a replacement for one of the two thiadiazole moiety.


Subject(s)
Benzene Derivatives/pharmacology , Enzyme Inhibitors/pharmacology , Glutaminase/antagonists & inhibitors , Kidney/enzymology , Sulfhydryl Compounds/pharmacology , Allosteric Site/drug effects , Benzene Derivatives/chemical synthesis , Benzene Derivatives/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Glutaminase/metabolism , Humans , Molecular Structure , Solubility , Structure-Activity Relationship , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/chemistry
18.
Addict Biol ; 25(4): e12791, 2020 07.
Article in English | MEDLINE | ID: mdl-31192517

ABSTRACT

The guidance cue receptor DCC controls mesocortical dopamine development in adolescence. Repeated exposure to an amphetamine regimen of 4 mg/kg during early adolescence induces, in male mice, downregulation of DCC expression in dopamine neurons by recruiting the Dcc microRNA repressor, microRNA-218 (miR-218). This adolescent amphetamine regimen also disrupts mesocortical dopamine connectivity and behavioral control in adulthood. Whether low doses of amphetamine in adolescence induce similar molecular and developmental effects needs to be established. Here, we quantified plasma amphetamine concentrations in early adolescent mice following a 4 or 0.5 mg/kg dose and found peak levels corresponding to those seen in humans following recreational and therapeutic settings, respectively. In contrast to the high doses, the low amphetamine regimen does not alter Dcc mRNA or miR-218 expression; instead, it upregulates DCC protein levels. Furthermore, high, but not low, drug doses downregulate the expression of the DCC receptor ligand, Netrin-1, in the nucleus accumbens and prefrontal cortex. Exposure to the low-dose regimen did not alter the expanse of mesocortical dopamine axons or their number/density of presynaptic sites in adulthood. Strikingly, adolescent exposure to the low-dose drug regimen does not impair behavioral inhibition in adulthood; instead, it induces an overall increase in performance in a go/no-go task. These results show that developmental consequences of exposure to therapeutic- versus abused-like doses of amphetamine in adolescence have dissimilar molecular signatures and opposite behavioral effects. These findings have important clinical relevance since amphetamines are widely used for therapeutic purposes in youth.


Subject(s)
Amphetamine/pharmacology , Central Nervous System Stimulants/pharmacology , DCC Receptor/drug effects , Dopaminergic Neurons/drug effects , MicroRNAs/drug effects , Amphetamine/administration & dosage , Amphetamine-Related Disorders , Animals , Behavior, Animal/drug effects , Central Nervous System Stimulants/administration & dosage , DCC Receptor/genetics , DCC Receptor/metabolism , Dose-Response Relationship, Drug , Inhibition, Psychological , Male , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Netrin-1/drug effects , Netrin-1/metabolism , Neural Pathways , Nucleus Accumbens/drug effects , Nucleus Accumbens/metabolism , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism
19.
Proteomics ; 19(21-22): e1800451, 2019 11.
Article in English | MEDLINE | ID: mdl-31231915

ABSTRACT

The targeting of glutamine metabolism specifically via pharmacological inhibition of glutaminase 1 (GLS1) has been translated into clinical trials as a novel therapy for several cancers. The results, though encouraging, show room for improvement in terms of tumor reduction. In this study, the glutaminase II pathway is found to be upregulated for glutamate production upon GLS1 inhibition in pancreatic tumors. Moreover, genetic suppression of glutamine transaminase K (GTK), a key enzyme of the glutaminase II pathway, leads to the complete inhibition of pancreatic tumorigenesis in vivo unveiling GTK as a new metabolic target for cancer therapy. These results suggest that current trials using GLS1 inhibition as a therapeutic approach targeting glutamine metabolism in cancer should take into account the upregulation of other metabolic pathways that can lead to glutamate production; one such pathway is the glutaminase II pathway via GTK.


Subject(s)
Enzyme Inhibitors/pharmacology , Glutaminase/genetics , Lyases/genetics , Pancreatic Neoplasms/drug therapy , Transaminases/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glutamic Acid/metabolism , Glutaminase/antagonists & inhibitors , Glutamine/genetics , Glutamine/metabolism , Humans , Lyases/antagonists & inhibitors , Metabolic Networks and Pathways/drug effects , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Transaminases/antagonists & inhibitors
20.
Mol Pharm ; 16(10): 4292-4301, 2019 10 07.
Article in English | MEDLINE | ID: mdl-31503493

ABSTRACT

2-(Phosphonomethyl)-pentanedioic acid (2-PMPA) is a potent (IC50 = 300 pM) and selective inhibitor of glutamate carboxypeptidase II (GCPII) with efficacy in multiple neurological and psychiatric disease preclinical models and more recently in models of inflammatory bowel disease (IBD) and cancer. 2-PMPA (1), however, has not been clinically developed due to its poor oral bioavailability (<1%) imparted by its four acidic functionalities (c Log P = -1.14). In an attempt to improve the oral bioavailability of 2-PMPA, we explored a prodrug approach using (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL), an FDA-approved promoiety, and systematically masked two (2), three (3), or all four (4) of its acidic groups. The prodrugs were evaluated for in vitro stability and in vivo pharmacokinetics in mice and dog. Prodrugs 2, 3, and 4 were found to be moderately stable at pH 7.4 in phosphate-buffered saline (57, 63, and 54% remaining at 1 h, respectively), but rapidly hydrolyzed in plasma and liver microsomes, across species. In vivo, in a single time-point screening study in mice, 10 mg/kg 2-PMPA equivalent doses of 2, 3, and 4 delivered significantly higher 2-PMPA plasma concentrations (3.65 ± 0.37, 3.56 ± 0.46, and 17.3 ± 5.03 nmol/mL, respectively) versus 2-PMPA (0.25 ± 0.02 nmol/mL). Given that prodrug 4 delivered the highest 2-PMPA levels, we next evaluated it in an extended time-course pharmacokinetic study in mice. 4 demonstrated an 80-fold enhancement in exposure versus oral 2-PMPA (AUC0-t: 52.1 ± 5.9 versus 0.65 ± 0.13 h*nmol/mL) with a calculated absolute oral bioavailability of 50%. In mouse brain, 4 showed similar exposures to that achieved with the IV route (1.2 ± 0.2 versus 1.6 ± 0.2 h*nmol/g). Further, in dogs, relative to orally administered 2-PMPA, 4 delivered a 44-fold enhanced 2-PMPA plasma exposure (AUC0-t for 4: 62.6 h*nmol/mL versus AUC0-t for 2-PMPA: 1.44 h*nmol/mL). These results suggest that ODOL promoieties can serve as a promising strategy for enhancing the oral bioavailability of multiply charged compounds, such as 2-PMPA, and enable its clinical translation.


Subject(s)
Microsomes, Liver/metabolism , Organophosphorus Compounds/metabolism , Prodrugs/metabolism , Administration, Oral , Animals , Biological Availability , Dogs , Male , Mice , Organophosphorus Compounds/administration & dosage , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacokinetics , Prodrugs/administration & dosage , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Tissue Distribution
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