RESUMO
Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the LâR shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/química , Inibidores de Proteínas Quinases/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Modelos Biológicos , Nucleotídeos/química , Nucleotídeos/metabolismo , Fosforilação/efeitos dos fármacos , Estrutura Secundária de ProteínaRESUMO
Little is known about how permanent, inclusive, affordable, and supportive long-term housing may affect the health of low-income lesbian, gay, bisexual, transgender, queer, intersex, asexual and/or another identity (LGBTQIA+) older adults. Focus group interviews were conducted with 21 older adults to explore the lived experiences and potential health benefits of living in a new LGBTQIA+-welcoming senior housing. Participants reported that moving into the housing was associated with benefits for health and well-being, especially for psychological health. Community, social support, and in-house services were particularly important. However, the combined nature of LGBTQIA+-welcoming and older adult only housing evoked mixed feelings. Appropriate and accessible housing solutions are essential for LGBTQIA+ older adults and may help address health disparities for these populations.
Assuntos
Minorias Sexuais e de Gênero , Pessoas Transgênero , Idoso , Bissexualidade , Feminino , Identidade de Gênero , Habitação , HumanosRESUMO
Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Camundongos , Modelos Moleculares , Mutação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibition of the thiamine-utilizing enzyme transketolase (TK) has been linked with diminished tumor cell proliferation. Most thiamine antagonists have a permanent positive charge on the B-ring, and it has been suggested that this charge is required for diphosphorylation by thiamine pyrophosphokinase (TPPK) and binding to TK. We sought to make neutral thiazolium replacements that would be substrates for TPPK, while not necessarily needing thiamine transporters (ThTr1 and ThTr2) for cell penetration. The synthesis, SAR, and structure-based rationale for highly potent non-thiazolium TK antagonists are presented.
Assuntos
Inibidores Enzimáticos/farmacologia , Tiamina/análogos & derivados , Transcetolase/antagonistas & inibidores , Animais , Catálise , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Camundongos , Conformação Proteica , Relação Estrutura-Atividade , Tiamina/química , Tiamina/farmacologiaRESUMO
Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Tiamina/análogos & derivados , Tiamina/antagonistas & inibidores , Transcetolase/antagonistas & inibidores , Animais , Neoplasias do Colo/enzimologia , Cristalografia por Raios X , Glucosefosfato Desidrogenase/metabolismo , Humanos , Técnicas In Vitro , Complexo Cetoglutarato Desidrogenase/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Nus , Estrutura Molecular , Oxitiamina/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/enzimologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic, cellular, and animal models. EXPERIMENTAL DESIGN: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally, the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. RESULTS: The IC(50) of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP, which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor-induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate-induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells. Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line. When dosed orally, ARRY-142886 was capable of inhibiting both ERK1/2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition, tumors remained responsive to growth inhibition after a 7-day dosing holiday. CONCLUSIONS: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies.
Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase 1/efeitos dos fármacos , MAP Quinase Quinase 2/efeitos dos fármacos , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recombinant human GH is used to treat GH deficiency in children and adults and wasting in AIDS patients. GH has a circulating half-life of only a few hours in humans and must be administered to patients by daily injection for maximum effectiveness. Previous studies showed that longer-acting forms of GH could be created by modification of GH with multiple 5-kDa amine-reactive polyethylene glycols (PEGs). Eight of nine lysine residues and the N-terminal amino acid were modified to varying extents by amine PEGylation of GH. The amine-PEGylated GH product comprised a complex mixture of multiple PEGylated species that differed from one another in mass, in vitro bioactivity, and in vivo potency. In vitro bioactivity of GH was reduced 100- to 1000-fold by extensive amine PEGylation of the protein. Here we describe a homogeneously modified, mono-PEGylated GH protein that possesses near complete in vitro bioactivity, a long half-life, and increased potency in vivo. The mono-PEGylated GH was created by substituting cysteine for threonine-3 (T3C) of GH, followed by modification of the added cysteine residue with a single 20-kDa cysteine-reactive PEG. The PEG-T3C protein has an approximate 8-fold longer half-life than GH after sc administration to rats. Every other day or every third day administration of PEG-T3C stimulates increases in body weight and tibial epiphysis growth comparable with that produced by daily administration of GH in hypophysectomized rats. Long-acting, mono-PEGylated GH analogs such as PEG-T3C are promising candidates for future testing in humans.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Hormônio do Crescimento Humano/análogos & derivados , Hormônio do Crescimento Humano/farmacocinética , Hipofisectomia , Aumento de Peso/efeitos dos fármacos , Animais , Preparações de Ação Retardada , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Hormônio do Crescimento Humano/química , Masculino , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Ratos , Ratos Sprague-Dawley , Tíbia/efeitos dos fármacos , Resultado do TratamentoRESUMO
OBJECTIVE: Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimide-PEGs to engineered cysteine analogs of Epo, and to determine whether the pegylated Epo cysteine analogs have improved pharmacokinetic properties in vivo. MATERIALS AND METHODS: Thirty-four Epo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monopegylated derivatives of 12 cysteine analogs were prepared using 20-kDa maleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild-type Epo (BV Epo) and a pegylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats. RESULTS: Biological activities of 17 purified Epo cysteine analogs and 10 purified pegylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only pegylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The pegylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation. CONCLUSIONS: Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monopegylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed Epo proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific pegylation appears to improve the pharmacokinetic properties of Epo.
Assuntos
Substituição de Aminoácidos , Cisteína/química , Eritropoetina/química , Etilenoglicóis/química , Maleimidas/química , Animais , Bioensaio , Linhagem Celular , Cisteína/genética , Eritropoetina/administração & dosagem , Eritropoetina/genética , Eritropoetina/farmacocinética , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas RecombinantesRESUMO
Younger and older physically active and sedentary adults participated in a task switching paradigm in which they performed a task repeatedly or switched between two different tasks, while measures of response speed, response accuracy, P3 amplitude, and P3 latency were recorded. Overall, response times were faster and midline P3 amplitudes were larger for the active than for the sedentary participants. P3 latencies discriminated between active and sedentary individuals on trials in which multiple task sets were maintained in memory and task switches occurred unpredictably but not in blocks of trials in which a single task was repeatedly performed. Results are discussed in terms of the specificity and generality of physical activity effects on cognition.
Assuntos
Envelhecimento/fisiologia , Atenção/fisiologia , Encéfalo/fisiologia , Potenciais Evocados P300/fisiologia , Atividade Motora/fisiologia , Tempo de Reação/fisiologia , Idoso , Análise de Variância , Estudos Transversais , Eletroencefalografia/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes NeuropsicológicosRESUMO
We compared emotional reactivity to affective pictures for 32 older (60-71 years) and 34 younger (18-23 years) adults. We collected the startle-blink reflex, N1 and P3 components of the probe-evoked event-related brain potential, corrugator electromyogram, heart rate, and self-report measures of pleasure and arousal. Self-report findings indicated that older, compared with younger, adults reported greater overall pleasure and arousal. Older adults also exhibited decreased N1 and P3 amplitude, corrugator activity, and heart rate deceleration compared with younger adults. The startle-blink reflex revealed that older adults exhibited increased startle-blink magnitude compared with younger adults during unpleasant pictures, with no age differences observed for pleasant and neutral contents. These age differences suggest that older adults have differential reactivity to affective picture viewing, and they indicate that age-related changes in emotion are not unitary across response systems.
Assuntos
Transtornos do Humor/psicologia , Percepção Visual , Adolescente , Afeto , Fatores Etários , Idoso , Envelhecimento/psicologia , Nível de Alerta/fisiologia , Encéfalo/fisiologia , Eletromiografia , Potenciais Evocados/fisiologia , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos do Humor/diagnóstico , Reflexo de Sobressalto/fisiologia , Autoavaliação (Psicologia)RESUMO
OBJECTIVE: The aim of this study was to determine whether fusion proteins comprising human granulocyte colony-stimulating factor (G-CSF) joined to human immunoglobulin G1 and G4 (IgG1 and IgG4) Fc and C(H) domains are biologically active and have improved pharmacokinetic and hematopoietic properties in vivo. MATERIAL AND METHODS: Chimeric genes encoding human G-CSF fused to the N-termini of the Fc and C(H) domains of human IgG1 and IgG4 were constructed and used to transfect monkey COS cells. The fusion proteins were purified from the conditioned media by protein A affinity chromatography. Bioactivities of the proteins were measured in a G-CSF-dependent in vitro bioassay. Pharmacokinetic and granulopoietic properties of the G-CSF/IgG1-Fc fusion protein were measured in normal rats. RESULTS: The G-CSF/IgG-Fc and G-CSF/IgG-C(H) fusion proteins were secreted from transfected COS cells primarily as disulfide-linked homodimers. On a molar basis, the purified G-CSF/IgG-Fc fusion proteins were as active as G-CSF in in vitro bioassays, whereas bioactivities of the purified G-CSF/IgG-C(H) fusion proteins were decreased 3- to 4-fold. The G-CSF/IgG1-Fc fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating neutrophils and white blood cells than G-CSF following intravenous and subcutaneous administration to rats. CONCLUSION: Fusion of G-CSF to human IgG domains results in homodimeric fusion proteins possessing high in vitro bioactivities, long circulating half-lives, and enhanced hematopoietic properties in vivo.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese/efeitos dos fármacos , Imunoglobulina G/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Contagem de Células Sanguíneas , Exame de Medula Óssea , Células COS , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/genética , Meia-Vida , Humanos , Imunoglobulina G/genética , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
OBJECTIVE: Thousands of Americans are prescribed cervical orthoses each year. These orthoses restrict motion, which may influence the patient's driving performance. No legal restrictions exist that prohibit patients from wearing cervical orthoses while driving. No study addressing this issue has been published to date. Thus, we sought to assess the effects of wearing a restrictive neck brace on driver performance on the open road. METHODS: We conducted a prospective, randomized block design study in 23 volunteers. Twenty-three adult licensed drivers from the state of Iowa were recruited. Evaluation of neck motion was performed with and without the rigid cervical orthosis. On-road performance testing was conducted with the use of a state-of-the-art mobile laboratory. Drivers were randomly assigned to one of two testing groups. Each driver was evaluated during two separate drives. Volunteers in Group A (n = 11) wore a neck brace for the first drive but not during the second. Participants in Group B (n = 12) did not wear a neck brace in the first drive but did for the second. The assessment included velocity, acceleration, cervical axial rotation, and evaluation of the driver's blind spot. RESULTS: Driving performance measures were collected and analyzed for both drives. Wearing a cervical orthosis resulted in decreased velocity (P < 0.05), decreased lateral acceleration (P < 0.05), decreased axial rotation (P < 0.05), inadequate evaluation of intersection traffic, and an increase in the blind spot. CONCLUSION: A rigid cervical orthosis alters driver performance.
Assuntos
Condução de Veículo , Braquetes/efeitos adversos , Vértebras Cervicais/fisiologia , Análise e Desempenho de Tarefas , Adulto , Feminino , Humanos , Imobilização/fisiologia , Masculino , Movimento/fisiologia , Estudos Prospectivos , Amplitude de Movimento Articular/fisiologia , Valores de ReferênciaRESUMO
Thirty-six participants (18 female, 18 male) viewed affective pictures to investigate the coupling between emotional reactions and motivated behavior. Framed within the biphasic theory of emotion, the three systems approach was employed by collecting measures of subjective report, expressive physiology, and motivated behavior. Postural adjustments associated with viewing affective pictures were measured. Results indicated sex-differences for postural responses to unpleasant pictures; an effect not found for pleasant and neutral picture contents. Females exhibited increased postural movement in the posterior direction, and males exhibited increased movement in the anterior direction, for unpleasant pictures. Subjective report of valence and arousal using the self-assessment manikin (SAM), and the startle eye-blink reflex were collected during a separate session, which replicated previous picture-viewing research. Specifically, participants rated pleasant pictures higher in valence and exhibited smaller startle responses compared to unpleasant pictures. Females also reported lower valence ratings compared to males across all picture contents. These findings extend our knowledge of motivated engagement with affective stimuli and indicate that postural responses may provide insight into sex-related differences in withdrawal behavior.
Assuntos
Afeto , Emoções , Modelos Teóricos , Motivação , Postura , Adolescente , Adulto , Nível de Alerta , Piscadela , Feminino , Humanos , Masculino , Fatores Sexuais , Percepção VisualRESUMO
Previously we showed that granulocyte colony-stimulating factor (G-CSF) in vitro bioactivity is preserved when the protein is joined via a flexible 7 amino acid linker to an immunoglobulin-1 (IgG1)-Fc domain and that the G-CSF/IgG1-Fc fusion protein possessed a longer circulating half-life and improved hematopoietic properties compared to G-CSF in normal rats. We have extended this analysis by comparing the relative hematopoietic potencies of G-CSF/IgG1-Fc to G-CSF in normal mice and to G-CSF and polyethylene glycol (PEG) -modified G-CSF in neutropenic rats. Mice were treated for 5 days using different doses and dosing regimens of G-CSF/IgG1-Fc or G-CSF and circulating neutrophil levels in the animals measured on Day 6. G-CSF/IgG1-Fc stimulated greater increases in blood neutrophils than comparable doses of G-CSF when administered using daily, every other day or every third day dosing regimens. In rats made neutropenic with cyclophosphamide, G-CSF/IgG1-Fc accelerated recovery of blood neutrophils to normal levels (from Day 9 to Day 5) when administered as 5 daily injections or as a single injection on Day 1. By contrast, G-CSF accelerated neutrophil recovery when administered as 5 daily injections, but not when administered as a single injection. G-CSF/IgG1-Fc was as effective as PEG-G-CSF at accelerating neutrophil recovery following a single injection in neutropenic rats. G-CSF/IgG1-Fc and G-CSF/IgG4-Fc fusion proteins in which the 7 amino acid linker was deleted also were effective at accelerating neutrophil recovery following a single injection in neutropenic rats. These studies confirm the enhanced in vivo hematopoietic properties of G-CSF/IgG-Fc fusion proteins.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/farmacologia , Neutropenia/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Animais , Contagem de Células Sanguíneas , Modelos Animais de Doenças , Feminino , Expressão Gênica , Ordem dos Genes , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/genética , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Contagem de Leucócitos , Camundongos , Neutropenia/etiologia , Neutrófilos/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Fatores de TempoRESUMO
Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.
Assuntos
MAP Quinase Quinase 1/química , Proteínas Proto-Oncogênicas B-raf/química , Domínio Catalítico , Cristalografia por Raios X , Células HCT116 , Células HEK293 , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Mutação Puntual , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Proteínas ras/genéticaRESUMO
A series of 2,3,4,4a,10,10a-hexahydropyrano[3,2-b]chromene analogs was developed that demonstrated high selectivity (>2000-fold) for BACE1 vs Cathepsin D (CatD). Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Guided by structure based design, changes to P2' and P3 moieties were explored. A conformationally restricted P2' methyl group provided inhibitors with excellent cell potency (37-137 nM) and selectivity (435 to >2000-fold) for BACE1 vs CatD. These efforts lead to compound 59, which demonstrated a 69% reduction in rat CSF Aß1-40 at 60 mg/kg (PO).
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Cromanos/síntese química , Inibidores de Proteases/síntese química , Compostos de Espiro/síntese química , Animais , Encéfalo/metabolismo , Catepsina D , Cromanos/farmacocinética , Cromanos/farmacologia , Células HEK293 , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Modelos Moleculares , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In an attempt to increase selectivity vs Cathepsin D (CatD) in our BACE1 program, a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromene analogues was developed. Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Using structure-based design, substitutions to improve binding to both the S3 and S2' sites of BACE1 were explored. An acyl guanidine moiety provided the most potent analogues. These compounds demonstrated 10-420 fold selectivity for BACE1 vs CatD, and were highly potent in a cell assay measuring Aß1-40 production (5-99 nM). They also suffered from high efflux. Despite this undesirable property, two of the acyl guanidines achieved free brain concentrations (Cfree,brain) in a guinea pig PD model sufficient to cover their cell IC50s. Moreover, a significant reduction of Aß1-40 in guinea pig, rat, and cyno CSF (58%, 53%, and 63%, respectively) was observed for compound 62.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Encéfalo/metabolismo , Cromanos/síntese química , Piranos/síntese química , Compostos de Espiro/síntese química , Animais , Células CHO , Linhagem Celular Tumoral , Cromanos/farmacocinética , Cromanos/farmacologia , Cricetinae , Cricetulus , Cristalografia por Raios X , Cobaias , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Modelos Moleculares , Piranos/farmacocinética , Piranos/farmacologia , Ratos , Ratos Sprague-Dawley , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aß), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aß is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aß in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aß lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aß in rodents and in monkey.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores de Proteases/síntese química , Compostos de Espiro/síntese química , Animais , Barreira Hematoencefálica/metabolismo , Cromanos/síntese química , Cromanos/farmacocinética , Cromanos/farmacologia , Cobaias , Células HEK293 , Humanos , Hidantoínas/síntese química , Hidantoínas/farmacocinética , Hidantoínas/farmacologia , Masculino , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
Recombinant interferon alpha-2 (IFN-alpha2) is used clinically to treat a variety of viral diseases and cancers. IFN-alpha2 has a short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of IFN-alpha2 by modifying lysine residues of the protein with amine-reactive poly(ethylene glycol) (PEG) reagents. However, amine-PEGylated IFN-alpha2 comprises a heterogeneous product mixture with low specific activity due to the large number and critical locations of lysine residues in IFN-alpha2. In an effort to overcome these problems we determined the feasibility of creating site-specific, mono-PEGylated IFN-alpha2 analogues by introducing a free (unpaired) cysteine residue into the protein, followed by modification of the added cysteine residue with a maleimide-PEG reagent. IFN-alpha2 cysteine analogues were expressed in Escherichia coli and purified, and their in vitro bioactivities were measured in the human Daudi cell line growth inhibition assay. Several cysteine analogues were identified that do not significantly affect in vitro biological activity of IFN-alpha2. Certain of the cysteine analogues, but not wild-type IFN-alpha2, reacted with maleimide-PEG to produce mono-PEGylated proteins. The PEG-Q5C analogue retained high in vitro bioactivity (within 3- to 4-fold of wild-type IFN-alpha2) even when modified with 20- and 40-kDa PEGs. Pharmacokinetic experiments indicated that the 20-kDa PEG-Q5C and 40-kDa PEG-Q5C proteins have 20-fold and 40-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. These studies demonstrate the feasibility of using site-specific PEGylation technology to create a long-acting, mono-PEGylated IFN-alpha2 protein with high specific activity.
Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Interferon-alfa/farmacologia , Polietilenoglicóis/química , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cisteína/química , Relação Dose-Resposta a Droga , Escherichia coli/genética , Humanos , Interferon-alfa/química , Lisina/química , Maleimidas/química , Maleimidas/farmacologia , Peso Molecular , Proteínas/química , RatosRESUMO
Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates proliferation of hematopoietic cells of the macrophage and granulocyte lineages and is used clinically to treat neutropenia and other myeloid disorders. Because of its short circulating half-life, GM-CSF is administered to patients by daily injection. We describe here the engineering of highly potent, long-acting human GM-CSF proteins through site-specific modification of GM-CSF cysteine analogues with a cysteine-reactive poly(ethylene glycol) (PEG) reagent. Thirteen cysteine analogues of GM-CSF were constructed, primarily in nonhelical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogues were properly processed but insoluble following secretion into the Escherichia coli periplasm. The proteins were refolded and purified by column chromatography. Ten of the cysteine analogues could be modified with a 5-kDa maleimide PEG, and seven of the mono-PEGylated proteins were purified by ion-exchange column chromatography. Biological activities of the 13 cysteine analogues and 7 PEGylated cysteine analogues were comparable to that of wild-type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analogue was modified with larger 10-, 20-, and 40-kDa PEGs, with only minimal loss of in vitro bioactivity. Pharmacokinetic experiments in rats demonstrated that the PEGylated proteins had up to 47-fold longer circulating half-lives than wild-type GM-CSF. These data demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting GM-CSF analogues and provide further evidence that the nonhelical regions of human GM-CSF examined are largely nonessential for biological activity of the protein.