RESUMO
PURPOSE: PI3K signaling is a common feature of B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL), and PI3K inhibitors have been introduced into the clinic. However, there remains a clear need to develop new strategies to target PI3K signaling. PI3K activity is countered by Src homology domain 2-containing inositol-5'-phosphatase 1 (SHIP1) and, here, we have characterized the activity of a novel SHIP1 activator, AQX-435, in preclinical models of B-cell malignancies. EXPERIMENTAL DESIGN: In vitro activity of AQX-435 was evaluated using primary CLL cells and DLBCL-derived cell lines. In vivo activity of AQX-435, alone or in combination with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, was assessed using DLBCL cell line and patient-derived xenograft models. RESULTS: Pharmacologic activation of SHIP1 using AQX-435 was sufficient to inhibit anti-IgM-induced PI3K-mediated signaling, including induction of AKT phosphorylation and MYC expression, without effects on upstream SYK phosphorylation. AQX-435 also cooperated with the BTK inhibitor ibrutinib to enhance inhibition of anti-IgM-induced AKT phosphorylation. AQX-435 induced caspase-dependent apoptosis of CLL cells preferentially as compared with normal B cells, and overcame in vitro survival-promoting effects of microenvironmental stimuli. Finally, AQX-435 reduced AKT phosphorylation and growth of DLBCL in vivo and cooperated with ibrutinib for tumor growth inhibition. CONCLUSIONS: Our results using AQX-435 demonstrate that SHIP1 activation may be an effective novel therapeutic strategy for treatment of B-cell neoplasms, alone or in combination with ibrutinib.
Assuntos
Antineoplásicos/farmacologia , Ativadores de Enzimas/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Sesquiterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: The phosphatase SHIP1 negatively regulates the PI3K pathway, and its predominant expression within cells of the haematopoietic compartment makes SHIP1 activation a novel strategy to limit inflammatory signalling generated through PI3K. AQX-1125 is the only clinical-stage, orally administered, SHIP1 activator. Here, we demonstrate the prophylactic and therapeutic effects of AQX-1125, in a mouse model of bleomycin-induced lung fibrosis. EXPERIMENTAL APPROACH: For prophylactic evaluation, AQX-1125 (3, 10 or 30 mg·kg-1 ·d-1 , p.o.) or dexamethasone (1 mg·kg-1 ·d-1 , i.p.) were given to CD-1 mice starting 3 days before intratracheal administration of bleomycin (0.1 IU per mouse) and continued daily for 7 or 21 days. Therapeutic potentials of AQX-1125 (3, 10 or 30 mg·kg-1 ·d-1 , p.o.) or pirfenidone (90 mg·kg-1 ·d-1 , p.o.) were assessed by initiating treatment 13 days after bleomycin instillation and continuing until day 28. KEY RESULTS: Given prophylactically, AQX-1125 (10 and 30 mg·kg-1 ) reduced histopathological changes in lungs, 7 and 21 days following bleomycin-induced injury. At the same doses, AQX-1125 reduced the number of total leukocytes, neutrophil activity, TGF-ß immunoreactivity and soluble collagen in lungs. Administered therapeutically, AQX-1125 (10 and 30 mg·kg-1 ) improved lung histopathology, cellular infiltration and reduced lung collagen content. At 30 mg·kg-1 , the effects of AQX-1125 were similar to those of pirfenidone (90 mg·kg-1 ) with corresponding improvements in disease severity. CONCLUSIONS AND IMPLICATIONS: AQX-1125 prevented bleomycin-induced lung injury during the inflammatory and fibrotic phases. AQX-1125, given therapeutically, modified disease progression and improved survival, as effectively as pirfenidone.
Assuntos
Bleomicina/antagonistas & inibidores , Cicloexanóis/farmacologia , Indanos/farmacologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cicloexanóis/administração & dosagem , Cicloexanóis/química , Relação Dose-Resposta a Droga , Indanos/administração & dosagem , Indanos/química , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/patologia , Masculino , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
PURPOSE: In this 6-week, randomized, double-blind, placebo controlled, multicenter trial we assessed the effect of the novel SHIP1 (SH2-containing inositol-5'-phosphatase 1) activator AQX-1125 on bladder pain and urinary symptoms in patients with interstitial cystitis/bladder pain syndrome. MATERIALS AND METHODS: Women with interstitial cystitis/bladder pain syndrome and a mean pain score of 5 or greater on an 11-point scale despite treatment were randomized to AQX-1125 or placebo orally once daily for 6 weeks. Average and maximum pain scores (daily) and urinary frequency (before visits) were recorded by e-diary and at clinic visits. The O'Leary-Sant ICSI (Interstitial Cystitis Symptom Index) and ICPI (Interstitial Cystitis Problem Index), BPIC-SS (Bladder Pain Interstitial Cystitis Symptom Score) and SF-12v2® questionnaires were administered. Safety was monitored through 6 weeks of treatment and 4 weeks of followup. RESULTS: A total of 37 patients received oral AQX-1125 and 32 received placebo. At 6 weeks average daily pain on an e-diary decreased by 2.4 points for AQX-1125 vs 1.4 for placebo (p = 0.061), while average pain at clinic decreased by 2.6 vs 1.1 (p = 0.008), maximum daily pain on e-diary diary decreased by 2.6 vs 1.4 (p = 0.030) and maximum pain at clinic decreased by 2.8 vs 1.1 (p = 0.028). AQX-1125 reduced ICSI by 3.8 points vs 1.4 for placebo (p = 0.005), ICPI by 3.6 points vs 1.6 (p = 0.014) and BPIC-SS by 8.8 points vs 4.0 (p = 0.011). Urinary frequency decreased on AQX-1125 by 3.6 voids per 24 hours vs 0.8 for placebo (p = 0.040). Adverse event rates were similar for AQX-1125 and placebo (51.4% and 78.1%, respectively). No serious adverse events were reported. CONCLUSIONS: Women with moderate to severe interstitial cystitis/bladder pain syndrome who were treated with the oral SHIP1 activator AQX-1125 reported significantly reduced bladder pain and improved urinary symptoms at 6 weeks. AQX-1125 was well tolerated. AQX-1125 may be a potential new treatment for interstitial cystitis/bladder pain syndrome. It warrants further investigation.
Assuntos
Cicloexanóis/administração & dosagem , Cistite Intersticial/tratamento farmacológico , Indanos/administração & dosagem , Dor Pélvica/tratamento farmacológico , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/efeitos dos fármacos , Bexiga Urinária/diagnóstico por imagem , Administração Oral , Adolescente , Adulto , Idoso , Cistite Intersticial/complicações , Cistite Intersticial/diagnóstico , Cistoscopia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Dor Pélvica/diagnóstico , Dor Pélvica/etiologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Estudos Retrospectivos , Síndrome , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/metabolismo , Celulose/química , Teoria Quântica , Domínio Catalítico , Cristalografia por Raios X , Glicosilação , Hidrólise , Estrutura Molecular , TermodinâmicaRESUMO
BACKGROUND: The efficacy of AQX-1125, a small-molecule SH2-containing inositol-5'-phosphatase 1 (SHIP1) activator and clinical development candidate, is investigated in rodent models of inflammation. EXPERIMENTAL APPROACH: AQX-1125 was administered orally in a mouse model of passive cutaneous anaphylaxis (PCA) and a number of rodent models of respiratory inflammation including: cigarette smoke, LPS and ovalbumin (OVA)-mediated airway inflammation. SHIP1 dependency of the AQX-1125 mechanism of action was investigated by comparing the efficacy in wild-type and SHIP1-deficient mice subjected to an intrapulmonary LPS challenge. RESULTS: AQX-1125 exerted anti-inflammatory effects in all of the models studied. AQX-1125 decreased the PCA response at all doses tested. Using bronchoalveolar lavage (BAL) cell counts as an end point, oral or aerosolized AQX-1125 dose dependently decreased the LPS-mediated pulmonary neutrophilic infiltration at 3-30 mg kg⻹ and 0.15-15 µg kg⻹ respectively. AQX-1125 suppressed the OVA-mediated airway inflammation at 0.1-10 mg kg⻹. In the smoke-induced airway inflammation model, AQX-1125 was tested at 30 mg kg⻹ and significantly reduced the neutrophil infiltration of the BAL fluid. AQX-1125 (10 mg kg⻹) decreased LPS-induced pulmonary neutrophilia in wild-type mice but not in SHIP1-deficient mice. CONCLUSIONS: The SHIP1 activator, AQX-1125, suppresses leukocyte accumulation and inflammatory mediator release in rodent models of pulmonary inflammation and allergy. As shown in the mouse model of LPS-induced lung inflammation, the efficacy of the compound is dependent on the presence of SHIP1. Pharmacological SHIP1 activation may have clinical potential for the treatment of pulmonary inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Asma/tratamento farmacológico , Cicloexanóis/uso terapêutico , Dermatite Alérgica de Contato/tratamento farmacológico , Ativadores de Enzimas/uso terapêutico , Indanos/uso terapêutico , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacocinética , Asma/sangue , Asma/imunologia , Asma/metabolismo , Cicloexanóis/sangue , Cicloexanóis/metabolismo , Cicloexanóis/farmacocinética , Dermatite Alérgica de Contato/sangue , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/metabolismo , Modelos Animais de Doenças , Ativadores de Enzimas/sangue , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacocinética , Feminino , Indanos/sangue , Indanos/metabolismo , Indanos/farmacocinética , Inositol Polifosfato 5-Fosfatases , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Doenças Respiratórias/sangue , Doenças Respiratórias/imunologia , Doenças Respiratórias/metabolismo , Doenças Respiratórias/prevenção & controleRESUMO
BACKGROUND: The SH2-containing inositol-5'-phosphatase 1 (SHIP1) metabolizes PI(3,4,5)P3 to PI(3,4)P2. SHIP1-deficient mice exhibit progressive inflammation. Pharmacological activation of SHIP1 is emerging as a potential therapy for pulmonary inflammatory diseases. Here we characterize the efficacy of AQX-1125, a small-molecule SHIP1 activator currently in clinical development. EXPERIMENTAL APPROACH: The effects of AQX-1125 were tested in several in vitro assays: on enzyme catalytic activity utilizing recombinant human SHIP1, on Akt phosphorylation in SHIP1-proficient and SHIP1-deficient cell lines, on cytokine release in murine splenocytes, on human leukocyte chemotaxis using modified Boyden chambers and on ß-hexosaminidase release from murine mast cells. In addition, pharmacokinetic and drug distribution studies were performed in rats and dogs. RESULTS: AQX-1125 increased the catalytic activity of human recombinant SHIP1, an effect, which was absent after deletion of the C2 region. AQX-1125 inhibited Akt phosphorylation in SHIP1-proficient but not in SHIP1-deficient cells, reduced cytokine production in splenocytes, inhibited the activation of mast cells and inhibited human leukocyte chemotaxis. In vivo, AQX-1125 exhibited >80% oral bioavailability and >5 h terminal half-life. CONCLUSIONS: Consistent with the role of SHIP1 in cell activation and chemotaxis, the SHIP1 activator AQX-1125 inhibits Akt phosphorylation, inflammatory mediator production and leukocyte chemotaxis in vitro. The in vitro effects and the pharmacokinetic properties of the compound make it a suitable candidate for in vivo testing in various models of inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Cicloexanóis/farmacologia , Ativadores de Enzimas/farmacologia , Indanos/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacocinética , Linhagem Celular , Células Cultivadas , Cicloexanóis/sangue , Cicloexanóis/metabolismo , Cicloexanóis/farmacocinética , Cães , Ativadores de Enzimas/sangue , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacocinética , Feminino , Humanos , Indanos/sangue , Indanos/metabolismo , Indanos/farmacocinética , Inositol Polifosfato 5-Fosfatases , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismoRESUMO
CD59 is a membrane-bound glycoprotein that protects host cells from lysis by inhibiting the terminal pathway of complement, preventing the formation and insertion of the membrane attack complex (MAC). Crystals of bacterially expressed and nonglycosylated recombinant soluble human CD59 have been obtained from three crystallization conditions, each of which gave rise to a distinct crystal form. Each crystal form led to a crystal structure at high resolution (1.15, 1.35 and 1.8 A). In one of these structures the electron-density map shows an as yet unidentified small molecule in the predicted C8/C9-binding site. The presence/absence of this ligand is linked to alternate conformations of the amino acids implicated in C8/C9 binding.