Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer's disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure-activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.

• MeSH
• Alzheimerova nemoc farmakoterapie   patologie   MeSH
• exozómy metabolismus   MeSH
• inhibitory enzymů chemie   metabolismus   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• mozek metabolismus   MeSH
• myši transgenní MeSH
• myši MeSH
• pyridaziny chemie   metabolismus   terapeutické užití   MeSH
• sfingomyelinfosfodiesterasa antagonisté a inhibitory   metabolismus   farmakologie   MeSH
• tělesná hmotnost účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory enzymů MeSH
• pyridaziny MeSH
• sfingomyelinfosfodiesterasa MeSH
• SMPD3 protein, human MeSH Prohlížeč

BACKGROUND AND PURPOSE: Extracellular vesicles (EVs) are constitutively shed from cells and released by various stimuli. Their protein and RNA cargo are modified by the stimulus, and in disease conditions can carry pathological cargo involved in disease progression. Neutral sphingomyelinase 2 (nSMase2) is a major regulator in at least one of several independent routes of EV biogenesis, and its inhibition is a promising new therapeutic approach for neurological disorders. Unfortunately, known inhibitors exhibit μM potency, poor physicochemical properties, and/or limited brain penetration. Here, we sought to identify a drug-like inhibitor of nSMase2. EXPERIMENTAL APPROACH: We conducted a human nSMase2 high throughput screen (>365,000 compounds). Selected hits were optimized focusing on potency, selectivity, metabolic stability, pharmacokinetics, and ability to inhibit EV release in vitro and in vivo. KEY RESULTS: We identified phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC), a potent (pIC50 = 6.57) and selective non-competitive inhibitor of nSMase2. PDDC was metabolically stable, with excellent oral bioavailability (%F = 88) and brain penetration (AUCbrain /AUCplasma = 0.60). PDDC dose-dependently (pEC50 = 5.5) inhibited release of astrocyte-derived extracellular vesicles (ADEV). In an in vivo inflammatory brain injury model, PDDC robustly inhibited ADEV release and the associated peripheral immunological response. A closely related inactive PDDC analogue was ineffective. CONCLUSION AND IMPLICATIONS: PDDC is a structurally novel, potent, orally available, and brain penetrant inhibitor of nSMase2. PDDC inhibited release of ADEVs in tissue culture and in vivo. PDDC is actively being tested in animal models of neurological disease and, along with closely related analogues, is being considered for clinical translation.

• MeSH
• astrocyty chemie   metabolismus   MeSH
• extracelulární vezikuly účinky léků   metabolismus   MeSH
• jaterní mikrozomy chemie   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární struktura MeSH
• mozek účinky léků   metabolismus   MeSH
• myši MeSH
• potkani Sprague-Dawley MeSH
• rychlé screeningové testy MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

During this process, we found that we need add the following sentence in the manuscript within the Acknowledgement section.

• Publikační typ
• časopisecké články MeSH
• tisková chyba MeSH

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.

• MeSH
• antigeny CD11b * MeSH
• chování zvířat účinky léků   MeSH
• deprese etiologie   prevence a kontrola   MeSH
• diazooxonorleucin * farmakologie   MeSH
• glutaminasa účinky léků   MeSH
• hipokampus účinky léků   imunologie   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• prefrontální mozková kůra účinky léků   imunologie   metabolismus   MeSH
• prekurzory léčiv * farmakologie   MeSH
• psychický stres komplikace   MeSH
• signální transdukce MeSH
• zánět farmakoterapie   imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antigeny CD11b * MeSH
• diazooxonorleucin * MeSH
• glutaminasa MeSH
• Itgam protein, mouse MeSH Prohlížeč
• prekurzory léčiv * MeSH

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.

• Klíčová slova
• Crystal structure, Glutamate carboxypeptidase II, Metallopeptidase, Prostate-specific membrane antigen,
• MeSH
• buněčné linie MeSH
• Drosophila genetika   MeSH
• enzymatické testy MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   chemie   metabolismus   MeSH
• inhibitory proteas chemická syntéza   chemie   metabolismus   MeSH
• karbamáty chemická syntéza   chemie   metabolismus   MeSH
• katalytická doména MeSH
• kvantová teorie MeSH
• lidé MeSH
• močovina analogy a deriváty   chemická syntéza   chemie   metabolismus   MeSH
• molekulární modely MeSH
• stereoizomerie MeSH
• vazba proteinů MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• glutamátkarboxypeptidasa II MeSH
• inhibitory proteas MeSH
• karbamáty MeSH
• močovina MeSH
• ZJ43 MeSH Prohlížeč

[11C]-PK11195 (PK11195) has been widely used with positron emission tomography (PET) to assess levels of the translocator protein 18 kDa (TSPO) as a marker of neuroinflammation. Recent ligands, such as [11C]-PBR28 and [11C]-DPA713, have improved signal-to-noise ratio and specificity for TSPO over PK11195. However, these second generation radiotracers exhibit binding differences due to a single polymorphism (rs6971) that leads to three genotypes: C/C, C/T and T/T associated with high, mixed and low binding affinities, respectively. Here we report that [3H]-DPA-713 in the presence of cholesterol or PK11195 has an accelerated dissociation rate from TSPO in platelets isolated from individuals with the T/T genotype. This allosteric interaction was not observed in platelets isolated from individuals with the C/C or C/T genotype. The results provide a molecular rationale for low binding affinity of T/T TSPO and further support the exclusion of these subjects from PET imaging studies using second generation TSPO ligands.

• Klíčová slova
• Allosteric modulation, Residence time, Translocator protein 18 KDa (TSPO),
• MeSH
• genotyp MeSH
• lidé MeSH
• neurozobrazování metody   MeSH
• pozitronová emisní tomografie metody   MeSH
• radiofarmaka chemie   MeSH
• receptory GABA analýza   chemie   genetika   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• radiofarmaka MeSH
• receptory GABA MeSH
• TSPO protein, human MeSH Prohlížeč

2-(Phosphonomethyl)pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase-II (GCPII) with efficacy in multiple neurological and psychiatric disease models, but its clinical utility is hampered by low brain penetration due to the inclusion of multiple acidic functionalities. We recently reported an improvement in the brain-to-plasma ratio of 2-PMPA after intranasal (IN) dosing in both rodents and primates. Herein, we describe the synthesis of several 2-PMPA prodrugs with further improved brain delivery of 2-PMPA after IN administration by masking of the γ-carboxylate. When compared to IN 2-PMPA in rats at 1 h post dose, γ-(4-acetoxybenzyl)-2-PMPA (compound 1) resulted in significantly higher 2-PMPA delivery to both plasma (4.1-fold) and brain (11-fold). Subsequent time-dependent evaluation of 1 also showed high brain as well as plasma 2-PMPA exposures with brain-to-plasma ratios of 2.2, 0.48, and 0.26 for olfactory bulb, cortex, and cerebellum, respectively, as well as an improved sciatic nerve to plasma ratio of 0.84. In contrast, IV administration of compound 1 resulted in similar plasma exposure of 2-PMPA versus the IN route (AUCIV: 76 ± 9 h·nmol/mL versus AUCIN: 99 ± 24 h·nmol/mL); but significantly lower nerve and brain tissue exposures with tissue-to-plasma ratios of 0.21, 0.03, 0.04, and 0.04 in nerve, olfactory bulb, cortex, and cerebellum, respectively. In primates, IN administration of 1 more than doubled 2-PMPA concentrations in the cerebrospinal fluid relative to previously reported levels following IN 2-PMPA. The results of these experiments provide a promising strategy for testing GCPII inhibition in neurological and psychiatric disorders.

• Klíčová slova
• 2-PMPA, glutamate carboxypeptidase II, intranasal, neurological disease, pharmacokinetics, prodrugs,
• MeSH
• aplikace intranazální MeSH
• estery analýza   chemie   farmakologie   MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   MeSH
• hematoencefalická bariéra účinky léků   MeSH
• intravenózní podání MeSH
• krysa rodu Rattus MeSH
• Macaca mulatta MeSH
• mozek účinky léků   MeSH
• mozkomíšní mok účinky léků   MeSH
• neuroprotektivní látky analýza   chemie   farmakologie   MeSH
• organofosforové sloučeniny analýza   chemie   farmakologie   MeSH
• potkani Wistar MeSH
• prekurzory léčiv analýza   chemie   farmakologie   MeSH
• tkáňová distribuce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• 2-(phosphonomethyl)pentanedioic acid MeSH Prohlížeč
• estery MeSH
• glutamátkarboxypeptidasa II MeSH
• neuroprotektivní látky MeSH
• organofosforové sloučeniny MeSH
• prekurzory léčiv MeSH

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

• Klíčová slova
• Angiogenesis, Extracellular matrix, GCPII, Laminin, PSMA, Peptides,
• MeSH
• angiogenní proteiny metabolismus   MeSH
• antigeny CD29 metabolismus   MeSH
• antigeny povrchové genetika   metabolismus   MeSH
• buněčná adheze MeSH
• dipeptidy metabolismus   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• endoteliální buňky cytologie   metabolismus   MeSH
• fyziologická neovaskularizace MeSH
• glutamátkarboxypeptidasa II genetika   metabolismus   MeSH
• hydrolýza MeSH
• laminin metabolismus   MeSH
• lidé MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• peptidové fragmenty metabolismus   MeSH
• proteolýza MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• angiogenní proteiny MeSH
• antigeny CD29 MeSH
• antigeny povrchové MeSH
• dipeptidy MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• isospaglumic acid MeSH Prohlížeč
• laminin MeSH
• mutantní proteiny MeSH
• peptidové fragmenty MeSH

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.

• MeSH
• antimetabolity antitumorózní mozkomíšní mok   terapeutické užití   MeSH
• diazooxonorleucin mozkomíšní mok   terapeutické užití   MeSH
• glioblastom farmakoterapie   metabolismus   MeSH
• glutamin metabolismus   MeSH
• Haplorrhini MeSH
• lidé MeSH
• myši nahé MeSH
• myši MeSH
• nádory mozku farmakoterapie   metabolismus   MeSH
• prekurzory léčiv farmakokinetika   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antimetabolity antitumorózní MeSH
• diazooxonorleucin MeSH
• glutamin MeSH
• prekurzory léčiv MeSH

Inhibition of glutamate carboxypeptidase II (GCPII) is effective in preclinical models of neurological disorders associated with excessive activation of glutamatergic systems. Here we report synthesis, structural characterization, and biological activity of new hydroxamic acid-based inhibitors with nanomolar affinity for human GCPII. Crystal structures of GCPII/hydroxamate complexes revealed an unprecedented binding mode in which the putative P1' glutarate occupies the spacious entrance funnel rather than the conserved glutamate-binding S1' pocket. This unique binding mode provides a mechanistic explanation for the structure-activity relationship data, most notably the lack of enantiospecificity and the tolerance for bulky/hydrophobic functions as substituents of a canonical glutarate moiety. The in vivo pharmacokinetics profile of one of the inhibitors will be presented along with analgesic efficacy data from the rat chronic constrictive injury model of neuropathic pain.

• MeSH
• antigeny povrchové metabolismus   MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   metabolismus   MeSH
• inhibitory enzymů chemická syntéza   chemie   farmakologie   MeSH
• kyseliny hydroxamové chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• inhibitory enzymů MeSH
• kyseliny hydroxamové MeSH