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1.
Mol Psychiatry ; 23(2): 271-281, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-27752082

RESUMO

The brain-specific tyrosine phosphatase, STEP (STriatal-Enriched protein tyrosine Phosphatase) is an important regulator of synaptic function. STEP normally opposes synaptic strengthening by increasing N-methyl D-aspartate glutamate receptor (NMDAR) internalization through dephosphorylation of GluN2B and inactivation of the kinases extracellular signal-regulated kinase 1/2 and Fyn. Here we show that STEP61 is elevated in the cortex in the Nrg1+/- knockout mouse model of schizophrenia (SZ). Genetic reduction or pharmacological inhibition of STEP prevents the loss of NMDARs from synaptic membranes and reverses behavioral deficits in Nrg1+/- mice. STEP61 protein is also increased in cortical lysates from the central nervous system-specific ErbB2/4 mouse model of SZ, as well as in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons and Ngn2-induced excitatory neurons, from two independent SZ patient cohorts. In these selected SZ models, increased STEP61 protein levels likely reflect reduced ubiquitination and degradation. These convergent findings from mouse and hiPSC SZ models provide evidence for STEP61 dysfunction in SZ.


Assuntos
Proteínas Tirosina Fosfatases/fisiologia , Esquizofrenia/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuregulina-1/genética , Neurônios/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/genética , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/genética , Ubiquitinação
2.
J Org Chem ; 66(26): 8772-8, 2001 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-11749605

RESUMO

tert-Butanesulfinyl aldimines and ketimines bearing an alpha-benzyloxy or alpha-silyloxy substituent serve as precursors in the synthesis of protected 1,2-amino alcohols in high yields and diastereoselectivities. General protocols are described for the addition of unbranched alkyl, branched alkyl, and aryl organometallic reagents to N-sulfinyl aldimines 1 and 2 and ketimines 5 and 6. Furthermore, the selective N- or O-deprotection of sulfinamide products 3, 4, 7, and 8 is described, enabling further synthetic transformations of the reaction products.


Assuntos
Amino Álcoois/síntese química , Iminas/química , Ácidos Sulfínicos/química , Indicadores e Reagentes , Compostos Organometálicos/química , Estereoisomerismo
3.
Org Lett ; 3(23): 3707-9, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11700118

RESUMO

[reaction--see text] A seven-step parallel solution-phase synthesis has been developed for access to ketone-containing mechanism-based cysteine protease inhibitors. The use of liquid-liquid extractions, volatile or solid-supported reagents, and resin-bound scavengers eliminates the need for intermediate column chromatographic purification during this synthesis sequence.


Assuntos
Inibidores de Cisteína Proteinase/síntese química , Cetonas/síntese química , Amidas/química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Oxirredução
8.
J Biol Chem ; 276(37): 34941-7, 2001 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-11438529

RESUMO

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.


Assuntos
Isoenzimas/metabolismo , Serina Endopeptidases/metabolismo , Humanos , Pichia/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Triptases
10.
Proc Natl Acad Sci U S A ; 97(14): 7754-9, 2000 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-10869434

RESUMO

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137, 180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.


Assuntos
Técnicas de Química Combinatória , Cumarínicos/metabolismo , Endopeptidases/metabolismo , Corantes Fluorescentes/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Cumarínicos/química , Cisteína Endopeptidases/metabolismo , Serina Endopeptidases/metabolismo , Especificidade por Substrato
12.
J Neurochem ; 74(4): 1469-77, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10737603

RESUMO

Lysosomal disturbances may be a contributing factor to Alzheimer's disease. We used novel compounds to test if suppression of the lysosomal protease cathepsin D blocks production of known precursors to neurofibrillary tangles. Partial lysosomal dysfunction was induced in cultured hippocampal slices with a selective inhibitor of cathepsins B and L. This led within 48 h to hyperphosphorylated tau protein fragments recognized by antibodies against human tangles. Potent nonpeptidic cathepsin D inhibitors developed using combinatorial chemistry and structure-based design blocked production of the fragments in a dose-dependent fashion. Threshold was in the submicromolar range, with higher concentrations producing complete suppression. The effects were selective and not accompanied by pathophysiology. Comparable results were obtained with three structurally distinct inhibitors. These results support the hypothesis that cathepsin D links lysosomal dysfunction to the etiology of Alzheimer's disease and suggest a new approach to treating the disease.


Assuntos
Catepsina D/antagonistas & inibidores , Catepsina D/metabolismo , Diazometano/análogos & derivados , Inibidores Enzimáticos/farmacologia , Hipocampo/enzimologia , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Animais , Diazometano/química , Diazometano/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Lisossomos/enzimologia , Técnicas de Cultura de Órgãos , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley
13.
Proc Natl Acad Sci U S A ; 97(6): 2419-24, 2000 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-10716979

RESUMO

A method for the rapid and efficient identification of ligands to biological targets is reported. The combinatorial method does not require structural or mechanistic information and is accomplished in four straightforward steps. (i) A set of potential binding elements is prepared wherein each molecule incorporates a common chemical linkage group. (ii) The set of potential binding elements is screened to identify all binding elements that interact even weakly with the biological target. (iii) A combinatorial library of linked binding elements is prepared whereby the binding elements are connected by the common chemical linkage groups through a set of flexible linkers. (iv) The combinatorial library is screened to identify the tightest-binding ligands. The utility of the method was demonstrated by the identification of a potent and subtype-selective small molecule inhibitor of the non-receptor tyrosine kinase c-Src (IC(50) = 64 nM). Because the method relies on connecting two distinct binding elements, the relative contributions of the two binding elements to the potency and selectivity of the inhibitor were readily determined. This information provides valuable insight into the molecular basis of inhibition.


Assuntos
Técnicas de Química Combinatória , Proteínas Tirosina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Proteína Tirosina Quinase CSK , Desenho de Fármacos , Concentração Inibidora 50 , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Químicos , Biblioteca de Peptídeos , Fosforilação , Ligação Proteica , Quinases da Família src
14.
Nat Biotechnol ; 18(2): 187-93, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10657126

RESUMO

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.


Assuntos
Técnicas de Química Combinatória , Fibrinolisina/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Simulação por Computador , Corantes Fluorescentes , Modelos Moleculares , Especificidade por Substrato
15.
Bioorg Med Chem Lett ; 10(24): 2731-3, 2000 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-11133079

RESUMO

Constrained analogues 5-7 of the potent and subtype selective somatostatin mimetic 1 were prepared by incorporating conformational constraints into the nine-membered heterocyclic scaffold. Each constrained peptidomimetic showed an altered activity profile relative to lead compound 1, with compound 7 exhibiting a 25-fold and 2-fold binding enhancement against somatostatin receptor subtypes sst4 and sst5, respectively.


Assuntos
Mimetismo Molecular , Somatostatina/análogos & derivados , Ligação Competitiva , Humanos , Concentração Inibidora 50 , Ligantes , Receptores de Somatostatina/antagonistas & inibidores , Somatostatina/síntese química , Somatostatina/metabolismo , Relação Estrutura-Atividade
16.
J Med Chem ; 42(21): 4380-7, 1999 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-10543881

RESUMO

A library of 951 compounds based upon the beta-turn motif were examined for their ability to stimulate the melanocortin-1 receptor. From this screening process, we have identified two compounds possessing low micromolar agonist activity at the mMC1R. The compound EL1 with racemic Nal(2') in the i + 1 position, DPro in the i + 2 position, and Trp in the i + 3 position possesses an EC(50) of 42.5 +/- 6.9 microM. Compound EL2 with Trp in the i + 1 position, DLys in the i + 2 position, and Phe in the i + 3 position possesses an EC(50) value of 63.4 +/- 26.9 microM. The results of the library screening process are consistent with a hypothesis dating back to the 1980s proposing that a beta-turn conformation involving the melanocortin "Phe-Arg-Trp" core amino acids provides the key recognition element. Additionally, these compounds represent the first nonpeptidic heterocyclic molecules reported to date that are able to activate the MC1R, a melanocyte receptor involved in skin pigmentation and animal coat coloration.


Assuntos
Oligopeptídeos/química , Receptores da Corticotropina/química , Animais , Células Cultivadas , Desenho de Fármacos , Ligantes , Camundongos , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Estrutura Secundária de Proteína , Receptores da Corticotropina/agonistas , Receptores da Corticotropina/biossíntese , Receptores de Melanocortina , Relação Estrutura-Atividade
17.
J Med Chem ; 42(8): 1428-40, 1999 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-10212129

RESUMO

A number of single-digit nanomolar, low-molecular-weight plasmepsin II aspartyl protease inhibitors have been identified using combinatorial chemistry and structure-based design. By identifying multiple, small-molecule inhibitors using the parallel synthesis of several focused libraries, it was possible to select for compounds with desirable characteristics including enzyme specificity and minimal binding to serum proteins. The best inhibitors identified have Ki's of 2-10 nM, molecular weights between 594 and 650 Da, between 3- and 15-fold selectivity toward plasmepsin II over cathepsin D, the most closely related human protease, good calculated log P values (2.86-4.56), and no apparent binding to human serum albumin at 1 mg/mL in an in vitro assay. These compounds represent the most potent non-peptide plasmepsin II inhibitors reported to date.


Assuntos
Antimaláricos/síntese química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Plasmodium falciparum/enzimologia , Animais , Antimaláricos/química , Antimaláricos/metabolismo , Catepsina D/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Humanos , Ligação Proteica , Proteínas de Protozoários , Albumina Sérica/metabolismo , Relação Estrutura-Atividade
18.
J Comb Chem ; 1(6): 534-9, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10748730

RESUMO

The first demonstration of the rapid parallel synthesis of diverse prostaglandin derivatives is reported. Upper (alpha-) side chain diversity was introduced to core 1 via the parallel Suzuki coupling of hydroborated alkenes. Conversion to the enones 3 and 9 was followed by the addition of the lower (omega-) side chains as higher-order cuprates 4. Upper side chains incorporating an N-acylsulfonamide protecting group were further transformed into prostaglandin amide analogues. Cleavage from support with HF/pyridine followed by scavenging provided 26 prostaglandin E1 analogues in high purity.


Assuntos
Alprostadil/análogos & derivados , Alprostadil/síntese química , Alprostadil/química , Alprostadil/metabolismo , Amidas , Técnicas de Química Combinatória/métodos , Desenho de Fármacos , Indicadores e Reagentes , Cinética , Conformação Molecular , Estrutura Molecular , Receptores de Prostaglandina E/metabolismo , Relação Estrutura-Atividade
20.
Bioorg Med Chem Lett ; 8(17): 2297-302, 1998 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-9873531
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