RESUMEN
C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium.
Asunto(s)
Quimiocina CCL2/biosíntesis , Receptores CCR2/metabolismo , Animales , Biomarcadores , Línea Celular , Quimiocina CCL2/sangre , Quimiocina CCL2/genética , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica , Humanos , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Receptores CCR2/antagonistas & inhibidoresRESUMEN
Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function.
Asunto(s)
Nefropatías Diabéticas/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Riñón/efectos de los fármacos , Receptores CCR2/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Nefropatías Diabéticas/genética , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Resistencia a la Insulina , Pruebas de Función Renal , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CCR2/genéticaRESUMEN
The following manuscript was published as a Fast Forward article on February 29, 2012: Sullivan TJ, Dairaghi DJ, Krasinski A, Miao Z, Wang Y, Zhao BN, Baumgart T, Berahovich R, Ertl LS, Pennell A, Seitz L, Miao S, Ungashe S, Wei Z, Johnson D, Boring L, Tsou C-L, Charo IF, Bekker P, Schall TJ, and Jaen JC, Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications. J Pharmacol Exp Ther jpet.111.190918; doi:10.1124/jpet.111.190918 It was later found that the chemical identity of a compound cited in the article, CCX140-B, was not sufficiently disclosed. The authors are unable, at this time, to provide the chemical identity of CCX140-B in accordance with the editorial policies of The Journal of Pharmacology and Experimental Therapeutics. As a result, the authors have voluntarily withdrawn this manuscript from publication. We apologize for any inconvenience this may cause JPET's readers.
RESUMEN
Focal segmental glomerulosclerosis (FSGS) comprises a group of uncommon disorders that present with marked proteinuria, nephrotic syndrome, progressive renal failure and characteristic glomerular lesions on histopathology. The current standard of care for patients with FSGS include immunosuppressive drugs such as glucocorticoids followed by calcineurin inhibitors, if needed for intolerance or inadequate response to glucocorticoids. Renin-angiotensin-aldosterone (RAAS) blockers are also used to control proteinuria, an important signature of FSGS. Existing treatments, however, achieved only limited success. Despite best care, treatment failure is common and FSGS is causal in a significant proportion of end stage renal disease. Thus, an unmet need exists for novel disease modifying treatments for FSGS. We employed two widely-used murine models of FSGS to test the hypothesis that systemic inhibition of chemokine receptor CCR2 would have therapeutic benefit. Here we report that administration CCX872, a potent and selective small molecule antagonist of CCR2, achieved rapid and sustained attenuation of renal damage as determined by urine albumin excretion and improved histopathological outcome. Therapeutic benefit was present when CCX872 was used as a single therapy, and moreover, the combination of CCX872 and RAAS blockade was statistically more effective than RAAS blockade alone. In addition, the combination of CCR2 and RAAS blockade was equally as effective as endothelin receptor inhibition. We conclude that specific inhibition of CCR2 is effective in the Adriamycin-induced and 5/6 nephrectomy murine models of FSGS, and thus holds promise as a mechanistically distinct therapeutic addition to the treatment of human FSGS.
Asunto(s)
Albuminuria , Glomeruloesclerosis Focal y Segmentaria , Glomérulos Renales , Receptores CCR2/antagonistas & inhibidores , Sistema Renina-Angiotensina/efectos de los fármacos , Albuminuria/tratamiento farmacológico , Albuminuria/patología , Albuminuria/orina , Animales , Línea Celular , Modelos Animales de Enfermedad , Glomeruloesclerosis Focal y Segmentaria/tratamiento farmacológico , Glomeruloesclerosis Focal y Segmentaria/patología , Glomeruloesclerosis Focal y Segmentaria/orina , Humanos , Glomérulos Renales/lesiones , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Ratones , Ratones Endogámicos BALB C , Receptores CCR2/metabolismoRESUMEN
Among the large variety of reversible inhibitors that bind to cholinesterases (ChE), only a few exhibit exquisitely strong binding reflected in low femtomolar to picomolar equilibrium dissociation constants. These tight binding inhibitors owe their high affinity to distinctive modes of interaction with the enzyme: naturally occurring snake toxins, the fasciculins, share a large 1000 angstroms2 complementary surface for its complex with acetylcholinesterases (AChE; EC 3.1.1.7); transition state analogs trifluoroacetophenones form a covalent bond with the active serine; disubstituted 1,2,3-triazole inhibitors formed in situ are selected by AChE for optimal interaction surface over the length of the active center gorge. All these inhibitors bind with higher affinity to AChEs than to the closely related butyrylcholinesterases (BuChE; EC 3.1.1.8). Selectivity of individual inhibitors towards BuChE increases with increasing their molecular size. Interaction kinetics for all three classes of compounds reveal very slow rates of dissociation of the AChE-inhibitor complexes or conjugates combined with very fast association rates. The influence of conformational flexibility of the active center gorge on the affinity of inhibitor binding was demonstrated by comparing binding properties of a series of disubstituted 1,2,3-triazoles having systematically varied structures. Analysis of the linear free energy relationships of binding to both mouse and Electrophorus AChE reveals independent contributions of individual structural elements of inhibitors to their binding with the triazole ring emerging as an independently contributing pharmacophore. These tight binding inhibitor interactions reveal useful information not only on the conformational flexibility of ChEs, but also on the diversity of modes of interaction that achieve inhibition.
Asunto(s)
Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/metabolismo , Colinesterasas/química , Colinesterasas/metabolismo , Acetofenonas/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Venenos Elapídicos/química , Fluoruros/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Estereoisomerismo , Triazoles/químicaRESUMEN
After revisiting earlier works reporting the regioselective synthesis of 1,5-disubstituted-1,2,3-triazoles via the addition of bromomagnesium acetylides to azides, much improved yields of the products were obtained for a wide array of azides and alkynes. The intermediates of that reaction can be trapped with different electrophiles to regioselectively form 1,4,5-trisubstituted 1,2,3-triazoles. [reaction: see text]
Asunto(s)
Alquinos/química , Magnesio/química , Triazoles/síntesis química , Acetileno/análogos & derivados , Azidas/química , Bromuros/química , Química Farmacéutica/métodos , Estructura MolecularRESUMEN
In recent years, immune-based therapies have become an increasingly attractive treatment option for patients with cancer. Cancer immunotherapy is often used in combination with conventional chemotherapy for synergistic effects. The alkylating agent cyclophosphamide (CTX) has been included in various chemoimmunotherapy regimens because of its well-known immunostimulatory effects. Paradoxically, cyclophosphamide can also induce suppressor cells that inhibit immune responses. However, the identity and biologic relevance of these suppressor cells are poorly defined. Here we report that cyclophosphamide treatment drives the expansion of inflammatory monocytic myeloid cells (CD11b(+)Ly6C(hi)CCR2(hi)) that possess immunosuppressive activities. In mice with advanced lymphoma, adoptive transfer (AT) of tumor-specific CD4(+) T cells following cyclophosphamide treatment (CTX+CD4 AT) provoked a robust initial antitumor immune response, but also resulted in enhanced expansion of monocytic myeloid cells. These therapy-induced monocytes inhibited long-term tumor control and allowed subsequent relapse by mediating functional tolerization of antitumor CD4(+) effector cells through the PD-1-PD-L1 axis. PD-1/PD-L1 blockade after CTX+CD4 AT therapy led to persistence of CD4(+) effector cells and durable antitumor effects. Depleting proliferative monocytes by administering low-dose gemcitabine effectively prevented tumor recurrence after CTX+CD4 AT therapy. Similarly, targeting inflammatory monocytes by disrupting the CCR2 signaling pathway markedly potentiated the efficacy of cyclophosphamide-based therapy. Besides cyclophosphamide, we found that melphalan and doxorubicin can also induce monocytic myeloid suppressor cells. These findings reveal a counter-regulation mechanism elicited by certain chemotherapeutic agents and highlight the importance of overcoming this barrier to prevent late tumor relapse after chemoimmunotherapy.
Asunto(s)
Antígeno B7-H1/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfoma/inmunología , Células Mieloides/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Combinada , Ciclofosfamida/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Doxorrubicina/farmacología , Femenino , Terapia de Inmunosupresión , Inmunosupresores/farmacología , Inmunoterapia Adoptiva , Activación de Linfocitos/inmunología , Linfoma/tratamiento farmacológico , Melfalán/farmacología , Ratones , Ratones Endogámicos BALB C , Receptores CCR2/antagonistas & inhibidores , GemcitabinaRESUMEN
OBJECTIVE: CCR2 inhibition has produced promising experimental and clinical anti-hyperglycemic effects. These results support the thesis that insulin resistance and Type 2 diabetes (T2D) are associated with chronic unresolved inflammation. The aim of this study was to provide a broad analysis of the various physiological changes occurring in mouse models of T2D in connection with pharmacological CCR2 inhibition. MATERIALS/METHODS: A mouse-active chemical analogue of the clinical candidate CCX140-B was tested in diet-induced obese (DIO) mice and db/db mice. Measurements included: adipose tissue inflammatory macrophage counts; peripheral blood glucose levels at steady-state and after glucose and insulin challenges; peripheral blood insulin and adiponectin levels; 24-h urine output and urinary glucose levels; pancreatic islet number and size; hepatic triglyceride and glycogen content; and hepatic glucose-6-phosphatase levels. RESULTS: In DIO mice, the CCR2 antagonist completely blocked the recruitment of inflammatory macrophages to visceral adipose tissue. The mice exhibited reduced hyperglycemia and insulinemia, improved insulin sensitivity, increased circulating adiponectin levels, decreased pancreatic islet size and increased islet number. It also reduced urine output, glucose excretion, hepatic glycogen and triglyceride content and glucose 6-phosphatase levels. Similar effects were observed in the db/db diabetic mice. CONCLUSIONS: These data indicate that pharmacological inhibition of CCR2 in models of T2D can reduce inflammation in adipose tissue, alter hepatic metabolism and ameliorate multiple diabetic parameters. These mechanisms may contribute to the promising anti-diabetic effects seen in humans with at least one CCR2 antagonist.
Asunto(s)
Tejido Adiposo/patología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Macrófagos , Obesidad/metabolismo , Receptores CCR2/antagonistas & inhibidores , Adiponectina/sangre , Animales , Biomarcadores/sangre , Glucemia/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa , Relación Dosis-Respuesta a Droga , Glucosa-6-Fosfatasa/metabolismo , Glucógeno/metabolismo , Glucosuria/diagnóstico , Hipoglucemiantes/uso terapéutico , Inflamación/metabolismo , Insulina/administración & dosificación , Insulina/sangre , Células Secretoras de Insulina/patología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/complicaciones , Obesidad/etiología , Receptores CCR2/metabolismo , Triglicéridos/metabolismoRESUMEN
The target-guided, in situ click chemistry approach to lead discovery has been successfully employed for discovering acetylcholinesterase (AChE) inhibitors by incubating a selected enzyme/tacrine azide combination with a variety of acetylene reagents that were not previously known to interact with the enzyme's peripheral binding site. The triazole products, formed by the enzyme, were identified by HPLC-mass spectrometry analysis of the crude reaction mixtures. The target-guided lead discovery search was also successful when performed with reagent mixtures containing up to 10 components. From 23 acetylene reagents, the enzyme selected two phenyltetrahydroisoquinoline (PIQ) building blocks that combined with the tacrine azide within the active center gorge to form multivalent inhibitors that simultaneously associate with the active and peripheral binding sites. These new inhibitors are up to 3 times as potent as our previous phenylphenanthridinium-derived compounds, and with dissociation constants as low as 33 femtomolar, they are the most potent noncovalent AChE inhibitors known. In addition, the new compounds lack a permanent positive charge and aniline groups and possess fewer fused aromatic rings. Remarkably, despite the high binding affinity, the enzyme displayed a surprisingly low preference for one PIQ enantiomer over the other.
Asunto(s)
Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Fenantridinas/química , Fenantridinas/farmacología , Tacrina/análogos & derivados , Tacrina/farmacología , Animales , Sitios de Unión , Técnicas Químicas Combinatorias/métodos , Ratones , Estereoisomerismo , Triazoles/química , Triazoles/farmacologíaRESUMEN
The in situ click chemistry approach to lead discovery employs the biological target itself for assembling inhibitors from complementary building block reagents via irreversible connection chemistry. The present publication discusses the optimization of this target-guided strategy using acetylcholinesterase (AChE) as a test system. The application of liquid chromatography with mass spectroscopic detection in the selected ion mode for product identification greatly enhanced the sensitivity and reliability of this method. It enabled the testing of multicomponent mixtures, which may dramatically increase the in situ screening throughput. In addition to the previously reported in situ product syn-TZ2PA6, we discovered three new inhibitors, syn-TZ2PA5, syn-TA2PZ6, and syn-TA2PZ5, derived from tacrine and phenylphenanthridinium azides and acetylenes, in the reactions with Electrophorus electricus and mouse AChE. All in situ-generated compounds were extremely potent AChE inhibitors, because of the presence of multiple sites of interaction, which include the newly formed triazole nexus as a significant pharmacophore.