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The metabolic characteristics of tumors present considerable hurdles to immune cell function and cancer immunotherapy. Using a glutamine antagonist, we metabolically dismantled the immunosuppressive microenvironment of tumors. We demonstrate that glutamine blockade in tumor-bearing mice suppresses oxidative and glycolytic metabolism of cancer cells, leading to decreased hypoxia, acidosis, and nutrient depletion. By contrast, effector T cells responded to glutamine antagonism by markedly up-regulating oxidative metabolism and adopting a long-lived, highly activated phenotype. These divergent changes in cellular metabolism and programming form the basis for potent antitumor responses. Glutamine antagonism therefore exposes a previously undefined difference in metabolic plasticity between cancer cells and effector T cells that can be exploited as a “metabolic checkpoint” for tumor immunotherapy.

As one of the most successful human pathogens, Mycobacterium tuberculosis ( Mtb ) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomic analysis of lungs from JHU083-treated Mtb -infected mice reveals citrulline accumulation, suggesting elevated nitric oxide (NO) synthesis, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. JHU083-treated macrophages also produce more NO potentiating their antibacterial activity. When tested in an immunocompromised mouse model of Mtb infection, JHU083 loses its therapeutic efficacy suggesting the drug’s host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis. Parveen et al., using immunological, and metabolomics-based assays, demonstrate the importance of host glutamine metabolism in tuberculosis pathogenesis and show host-directed activity against tuberculosis.

Glucosinolates (GS) are metabolized to isothiocyanates that may enhance human healthspan by protecting against a variety of chronic diseases. Moringa oleifera , the drumstick tree, produces unique GS but little is known about GS variation within M . oleifera , and even less in the 12 other Moringa species, some of which are very rare. We assess leaf, seed, stem, and leaf gland exudate GS content of 12 of the 13 known Moringa species. We describe 2 previously unidentified GS as major components of 6 species, reporting on the presence of simple alkyl GS in 4 species, which are dominant in M . longituba . We document potent chemoprotective potential in 11 of 12 species, and measure the cytoprotective activity of 6 purified GS in several cell lines. Some of the unique GS rank with the most powerful known inducers of the phase 2 cytoprotective response. Although extracts of most species induced a robust phase 2 cytoprotective response in cultured cells, one was very low ( M . longituba ), and by far the highest was M . arborea , a very rare and poorly known species. Our results underscore the importance of Moringa as a chemoprotective resource and the need to survey and conserve its interspecific diversity.

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Amplification of c-MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates glutamine metabolism across many types of cancer. We modified the naturally occurring glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) by adding 2 promoeities to increase its lipophilicity and brain penetration creating the prodrug isopropyl 6-diazo-5-oxo-2-(((phenyl (pivaloyloxy) methoxy) - carbonyl) amino) hexanoate, termed JHU395. This prodrug was shown to have a 10-fold improved CSF-to-plasma ratio and brain-to-plasma ratio relative to DON. We hypothesized that JHU395 would have superior cell penetration compared with DON and would effectively and more potently kill MYC-expressing medulloblastoma. JHU395 treatment caused decreased growth and increased apoptosis in multiple human high-MYC medulloblastoma cell lines at lower concentrations than DON. Parenteral administration of JHU395 in Nu/Nu mice led to the accumulation of micromolar concentrations of DON in brain. Treatment of mice bearing orthotopic xenografts of human MYC-amplified medulloblastoma with JHU395 increased median survival from 26 to 45 days compared with vehicle control mice (p < 0.001 by log-rank test). These data provide preclinical justification for the ongoing development and testing of brain-targeted DON prodrugs for use in medulloblastoma.

There are a number of clinically effective treatments for stress-associated psychiatric diseases, including major depressive disorder (MDD). Nonetheless, many patients exhibit resistance to first-line interventions calling for novel interventions based on pathological mechanisms. Accumulating evidence implicates altered glutamate signaling in MDD pathophysiology, suggesting that modulation of glutamate signaling cascades may offer novel therapeutic potential. Here we report that JHU-083, our recently developed prodrug of the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON) ameliorates social avoidance and anhedonia-like behaviors in mice subjected to chronic social defeat stress (CSDS). JHU-083 normalized CSDS-induced increases in glutaminase activity specifically in microglia-enriched CD11b+ cells isolated from the prefrontal cortex and hippocampus. JHU-083 treatment also reverses the CSDS-induced inflammatory activation of CD11b+ cells. These results support the importance of altered glutamate signaling in the behavioral abnormalities observed in the CSDS model, and identify glutaminase in microglia-enriched CD11b+ cells as a pharmacotherapeutic target implicated in the pathophysiology of stress-associated psychiatric conditions such as MDD.

Cytotoxic neuronal swelling and glutamate excitotoxicity are two hallmarks of ischemic stroke. However, the underlying molecular mechanisms are not well understood. Here, it is reported that SWELL1, the essential subunit of the volume‐regulated anion channel (VRAC), plays a dual role in ischemic injury by promoting neuronal swelling and glutamate excitotoxicity. SWELL1 expression is upregulated in neurons and astrocytes after experimental stroke in mice. The neuronal SWELL1 channel is activated by intracellular hypertonicity, leading to Cl− influx‐dependent cytotoxic neuronal swelling and subsequent cell death. Additionally, the SWELL1 channel in astrocytes mediates pathological glutamate release, indicated by increases in neuronal slow inward current frequency and tonic NMDAR current. Pharmacologically, targeting VRAC with a new inhibitor, an FDA‐approved drug Dicumarol, attenuated cytotoxic neuronal swelling and cell death, reduced astrocytic glutamate release, and provided significant neuroprotection in mice when administered either before or after ischemia. Therefore, these findings uncover the pleiotropic effects of the SWELL1 channel in neurons and astrocytes in the pathogenesis of ischemic stroke and provide proof of concept for therapeutically targeting it in this disease. This study reveals the critical involvement of the SWELL1 channel in ischemic brain injury pathogenesis, highlighting its role in cytotoxic neuronal swelling and astrocytic glutamate excitotoxicity. Notably, pharmacological inhibition of SWELL1 by the FDA‐approved drug Dicumarol effectively blocks these deleterious pathways, underscoring SWELL1 as a promising therapeutic target for ischemic stroke.

For (M, [g]) a conformal manifold of signature (p, q) and dimension at least three, the conformal holonomy group Hol(M, [g]) ⊂ O(p + 1, q + 1) is an invariant induced by the canonical Cartan geometry of (M, [g]). We give a description of all possible connected conformal holonomy groups which act transitively on the Möbius sphere S p,q, the homogeneous model space for conformal structures of signature (p, q). The main part of this description is a list of all such groups which also act irreducibly on ℝp+1,q+1. For the rest, we show that they must be compact and act decomposably on ℝp+1,q+1, in particular, by known facts about conformal holonomy the conformal class [g] must contain a metric which is either Einstein (if p = 0 or q = 0) or locally isometric to a so-called special Einstein product.

Background: Extracellular vesicles (EVs) can carry pathological cargo, contributing to disease progression. The enzyme neutral sphingomyelinase 2 (nSMase2) plays a critical role in EV biogenesis, making it a promising therapeutic target. Our lab previously identified a potent and selective inhibitor of nSMase2, named DPTIP (IC50 = 30 nM). Although promising, DPTIP exhibits poor pharmacokinetics (PKs) with a low oral bioavailability (%F < 5), and a short half-life (t1/2 ≤ 0.5 h). To address these limitations, we previously developed DPTIP prodrugs by masking its phenolic hydroxyl group, demonstrating improved plasma exposure in mice. Recognizing that species-specific metabolic differences can influence prodrug PK, we expanded our studies to evaluate selected prodrugs in both mice and dogs. Methods: The scaleup of selected prodrugs was completed and two additional valine- ester based prodrugs were synthesized. Mice were dosed prodrugs via peroral route (10 mg/kg equivalent). For dog studies DPTIP was dosed via intravenous (1 mg/kg) or peroral route (2 mg/kg) and prodrugs were given peroral at a dose 2 mg/kg DPTIP equivalent. Plasma samples were collected at predetermined points and analyzed using developed LC/MS-MS methods. Results: In mice, several of the tested prodrugs showed similar or improved plasma exposures compared to DPTIP. However, in dog studies, the double valine ester prodrug 9, showed significant improvement with an almost two-fold increase in DPTIP plasma exposure (AUC0–t = 1352 vs. 701 pmol·h/mL), enhancing oral bioavailability from 8.9% to 17.3%. Conclusions: These findings identify prodrug 9 as a promising candidate for further evaluation and underscore the critical role of species-specific differences in prodrug PKs.

There is an unmet need for therapeutics with a novel mechanism to address Q9 symptoms associated with conditions where aberrant glutamatergic neurotransmission is presumed pathogenic. One enzyme of potential relevance is glutamate carboxypeptidase II (GCPII), a brain metallopeptidase with significantly upregulated activity in nervous tissues following neurodegeneration or injury. Current inhibitors are too polar and charged leading to minimal brain penetration necessitating high systemic doses or direct brain injection. Our efforts are focused on identifying new inhibitor scaffolds with favorable brain penetration. Herein, we used a newly developed dual-stream liquid chromatography mass spectrometry (LC/MS/MS) substrate cleavage assay to screen two small molecule libraries. The two top confirmed hits were cefsulodin (IC = 2 ± 0.1 μM) and amaranth (IC = 0.3 ± 0.01 μM). The interactions of Amaranth and cefsulodin with GCPII were characterized with mode of inhibition (MOI) studies, nano differential scanning fluorimetry (DSF) thermal shift assay, and binding site was modeled with in-silico docking. As cefsulodin is an antibiotic used clinically to treat bacterial meningitis, we tested the compound's brain pharmacokinetics (PK) in mice using a sensitive LC/MS method we developed. Moreover, following confirmation and characterization of cefsulodin and amaranth as viable hits an SAR investigation was conducted with analogs of both compounds. A first derivative analysis of the DSF data revealed a shift in melting temperature of Δ 0.76 °C (±0.04) for amaranth at 25 μM and 80.41 °C (±0.05) for cefsulodin at 250 μM, suggesting both compounds are acting as stabilizers for the enzyme. Increasing concentrations of cefsulodin increased the Km of N-acetyl-aspartyl-glutamate (NAAG) as a substrate with no change in Vmax, suggesting active site competitive inhibition. In contrast, increasing concentrations of amaranth led to reductions in Vmax while the Km remained constant, suggesting a non-competitive MOI. Results from in-silico docking studies complemented this MOI data, suggesting cefsulodin likely binds in the active site while amaranth likely binds in an allosteric site. Our PK study demonstrated that administration of cefsulodin (100 mg/kg IP) led to a Cmax of 4 μM in the brain, exceeding its GCPII IC value. Our new screening approaches identified novel inhibitors of GCPII that could serve as molecular templates for further structural optimization.

Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC0-t, i.n./AUC0-t, i.p. = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC0-t, i.n./AUC0-t, i.p. = 67), cortex (AUC0-t, i.n./AUC0-t, i.p. = 46) and cerebellum (AUC0-t, i.n./AUC0-t, i.p. = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity ex-vivo confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support intranasal delivery of 2-PMPA to deliver therapeutic concentrations in the brain and may facilitate its clinical development.