Neurosteroids are endogenous steroidal compounds that can modulate neuronal receptors. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that are of particular interest, as they participate in synaptic transmission and are implicated in various processes, such as learning, memory, or long-term neuronal potentiation. Positive allosteric modulators that increase the activity of NMDARs may provide a therapeutic aid for patients suffering from neuropsychiatric disorders where NMDAR hypofunction is thought to be involved, such as intellectual disability, autism spectrum disorder, or schizophrenia. We recently described a new class of pregn-5-ene and androst-5-ene 3beta-dicarboxylic acid hemiesters (2-24) as potent positive modulators of NMDARs. Considering the recommended guidelines for the early stage development of new, potent compounds, we conducted an in vitro safety assessment and plasma stability screening to evaluate their druglikeness. First, compounds were screened for their hepatotoxicity and mitochondrial toxicity in a HepG2 cell line. Second, toxicity in primary rat postnatal neurons was estimated. Next, the ability of compounds 2-24 to cross a Caco-2 monolayer was also studied. Finally, rat and human plasma stability screening revealed an unforeseen high stability of the C-3 hemiester moiety. In summary, by using potency/efficacy towards NMDARs data along with toxicity profile, Caco-2 permeability and plasma stability, compounds 14 and 15 were selected for further in vivo animal studies.
- MeSH
- androstenoly chemie farmakologie krev MeSH
- buňky Hep G2 MeSH
- cholesterol chemie farmakologie krev MeSH
- estery chemie farmakologie krev MeSH
- kyseliny dikarboxylové chemie farmakologie krev MeSH
- lidé MeSH
- poruchy autistického spektra farmakoterapie metabolismus MeSH
- stabilita léku MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Neurosteroids are endogenous steroidal compounds that can modulate neuronal receptors. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that are of particular interest, as they participate in synaptic transmission and are implicated in various processes, such as learning, memory, or long-term neuronal potentiation. Positive allosteric modulators that increase the activity of NMDARs may provide a therapeutic aid for patients suffering from neuropsychiatric disorders where NMDAR hypofunction is thought to be involved, such as intellectual disability, autism spectrum disorder, or schizophrenia. We recently described a new class of pregn-5-ene and androst-5-ene 3β-dicarboxylic acid hemiesters (2-24) as potent positive modulators of NMDARs. Considering the recommended guidelines for the early stage development of new, potent compounds, we conducted an in vitro safety assessment and plasma stability screening to evaluate their druglikeness. First, compounds were screened for their hepatotoxicity and mitochondrial toxicity in a HepG2 cell line. Second, toxicity in primary rat postnatal neurons was estimated. Next, the ability of compounds 2-24 to cross a Caco-2 monolayer was also studied. Finally, rat and human plasma stability screening revealed an unforeseen high stability of the C-3 hemiester moiety. In summary, by using potency/efficacy towards NMDARs data along with toxicity profile, Caco-2 permeability and plasma stability, compounds 14 and 15 were selected for further in vivo animal studies.
- MeSH
- androstenoly krev chemie farmakologie MeSH
- buňky Hep G2 MeSH
- cholesterol krev chemie farmakologie MeSH
- estery krev chemie farmakologie MeSH
- krysa rodu rattus MeSH
- kyseliny dikarboxylové krev chemie farmakologie MeSH
- lidé MeSH
- mentální retardace farmakoterapie metabolismus MeSH
- mitochondrie účinky léků metabolismus MeSH
- molekulární struktura MeSH
- nádorové buňky kultivované MeSH
- neurony účinky léků metabolismus MeSH
- neuroprotektivní látky krev chemie farmakologie MeSH
- poruchy autistického spektra farmakoterapie metabolismus MeSH
- potkani Wistar MeSH
- pregnenolon analogy a deriváty krev farmakologie MeSH
- receptory N-methyl-D-aspartátu antagonisté a inhibitory metabolismus MeSH
- schizofrenie farmakoterapie metabolismus MeSH
- stabilita léku MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) - a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 μM) than PAG (IC50 = 51.7 μM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 μM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 μM and 276.4 ± 178.7 μM, respectively). In addition, compound 6 (10 μM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5-7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.
- Publikační typ
- časopisecké články MeSH
The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.
- MeSH
- cirkulární dichroismus MeSH
- databáze proteinů MeSH
- datové soubory jako téma MeSH
- molekulární modely * MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- peptidová knihovna * MeSH
- proteinové agregáty MeSH
- rekombinantní proteiny chemie izolace a purifikace toxicita MeSH
- rozpustnost MeSH
- sbalování proteinů MeSH
- sekundární struktura proteinů * MeSH
- sekvence aminokyselin MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).
- MeSH
- dipeptidylpeptidasa 4 metabolismus MeSH
- endopeptidasy metabolismus MeSH
- glutamátkarboxypeptidasa II chemie genetika metabolismus MeSH
- krysa rodu rattus MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- regulace genové exprese enzymů MeSH
- sekvence aminokyselin MeSH
- střeva enzymologie MeSH
- terciární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
72 stran : tabulky ; 18 cm
Brožura a příručka, která se zaměřuje na možnosti laboratorní diagnostiky dědičných metabolických poruch. Určeno odborné veřejnosti.
- MeSH
- klinické laboratorní techniky MeSH
- vrozené poruchy metabolismu diagnóza MeSH
- Publikační typ
- příručky MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- vnitřní lékařství
- genetika, lékařská genetika
- NLK Publikační typ
- brožury
