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1.
ACS Med Chem Lett ; 7(11): 999-1004, 2016 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-27882198

RESUMEN

The tubulysin class of natural products has attracted much attention from the medicinal chemistry community due to its potent cytotoxicity against a wide range of human cancer cell lines, including significant activity in multidrug-resistant carcinoma models. As a result of their potency, the tubulysins have become an important tool for use in targeted therapy, being widely pursued as payloads in the development of novel small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs). A structure-based and parallel medicinal chemistry approach was applied to the synthesis of novel tubulysin analogues. These efforts led to the discovery of a number of novel and potent cytotoxic tubulysin analogues, providing a framework for our simultaneous report, which highlights the discovery of tubulysin-based ADCs, including use of site-specific conjugation to address in vivo stability of the C-11 acetate functionality.

2.
ACS Chem Biol ; 11(9): 2529-40, 2016 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-27391855

RESUMEN

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.


Asunto(s)
Hidrolasas/metabolismo , Lisofosfolipasa/antagonistas & inhibidores , Serina/metabolismo , Animales , Cristalización , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Lisofosfolipasa/química
3.
J Med Chem ; 58(6): 2678-702, 2015 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-25695670

RESUMEN

The identification of centrally efficacious ß-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) has historically been thwarted by an inability to maintain alignment of potency, brain availability, and desired absorption, distribution, metabolism, and excretion (ADME) properties. In this paper, we describe a series of truncated, fused thioamidines that are efficiently selective in garnering BACE1 activity without simultaneously inhibiting the closely related cathepsin D or negatively impacting brain penetration and ADME alignment, as exemplified by 36. Upon oral administration, these inhibitors exhibit robust brain availability and are efficacious in lowering central Amyloid ß (Aß) levels in mouse and dog. In addition, chronic treatment in aged PS1/APP mice effects a decrease in the number and size of Aß-derived plaques. Most importantly, evaluation of 36 in a 2-week exploratory toxicology study revealed no accumulation of autofluorescent material in retinal pigment epithelium or histology findings in the eye, issues observed with earlier BACE1 inhibitors.


Asunto(s)
Amidinas/química , Amidinas/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Encéfalo/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Placa Amiloide/tratamiento farmacológico , Enfermedad de Alzheimer/tratamiento farmacológico , Amidinas/farmacocinética , Amidinas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Perros , Diseño de Fármacos , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Modelos Moleculares , Placa Amiloide/metabolismo , Placa Amiloide/patología , Ratas , Ratas Wistar , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacocinética , Compuestos de Sulfhidrilo/farmacología , Compuestos de Sulfhidrilo/uso terapéutico
4.
J Med Chem ; 57(24): 10527-43, 2014 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-25431858

RESUMEN

Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.


Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Depsipéptidos/química , Depsipéptidos/farmacología , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Animales , Área Bajo la Curva , Células Cultivadas , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Ratas , Ratas Wistar , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , Tubulina (Proteína)/metabolismo
5.
Biochemistry ; 51(10): 2065-77, 2012 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-22352991

RESUMEN

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.


Asunto(s)
Hemo/química , Peroxidasa/química , Peroxidasa/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cromatografía Liquida , Cristalografía por Rayos X , Humanos , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Neutrófilos/enzimología , Oxidación-Reducción , Fragmentos de Péptidos/química , Mapeo Peptídico , Espectrometría de Masas en Tándem
6.
Biochemistry ; 48(13): 2941-9, 2009 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-19222187

RESUMEN

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) on target cells and lowers the level of receptor by impeding its recycling. PCSK9 is self-processed to a complex of its prodomain and catalytic domain like a typical protein convertase, but it does not develop normal proteolytic activity. Instead, its propeptide remains complexed with the catalytic domain, and the C-terminal Gln152 of the prodomain occupies the active site like a substrate for peptide synthesis. To probe its latent catalytic activity, PCSK9 and its complex with the soluble LDLR extracellular domain were separately transferred into H218O, and time point samples were analyzed by peptide mapping with mass spectrometry to measure the rate and extent of incorporation of 18O into the Gln152 carboxylate. In free wild-type or D374Y mutant PCSK9, the t1/2 for exchange of 18O for both oxygens was near 5 min. This slow process progressed to completion, with the distribution of oxygen isotopes in the Gln152 carboxylate finally matching that in solvent. In contrast, exchange reached its final state in <30 s in LDLR-complexed D374Y mutant PCSK9, but approximately 40% of the molecules gave data indicating the presence of only one 18O atom in Gln152. With support from further experiments, this was attributed to hydrolysis of acylenzyme in H216O during preparations for digestion and indicated that PCSK9 complexed with LDLR contains approximately 40% intramolecular acylenzyme at equilibrium. The synthetic EGF-A domain of LDLR induced similar effects as the full-length receptor. The data suggest the existence of distinct conformational states in free and receptor-bound PCSK9.


Asunto(s)
Biocatálisis , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Glutamina/metabolismo , Humanos , Isótopos , Espectrometría de Masas , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Péptidos/química , Péptidos/metabolismo , Proproteína Convertasa 9 , Proproteína Convertasas , Serina Endopeptidasas/química , Ciclo del Sustrato
7.
J Pharmacol Exp Ther ; 326(3): 801-8, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18577702

RESUMEN

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 microM PF-429242 (Bioorg Med Chem Lett 17:4411-4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC(50) of 0.5 microM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.


Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Lipogénesis/fisiología , Proproteína Convertasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Células CHO , Línea Celular , Células Cultivadas , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipogénesis/efectos de los fármacos , Masculino , Ratones , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Inhibidores de Proteasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/biosíntesis
9.
Nat Struct Mol Biol ; 14(5): 413-9, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17435765

RESUMEN

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.


Asunto(s)
Hipercolesterolemia/genética , Mutación Missense/fisiología , Serina Endopeptidasas/metabolismo , Sitios de Unión , Humanos , Concentración de Iones de Hidrógeno , Hipercolesterolemia/etiología , Proproteína Convertasa 9 , Proproteína Convertasas , Unión Proteica/genética , Conformación Proteica , Receptores de LDL/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética
10.
Brain Res ; 985(2): 113-26, 2003 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-12967715

RESUMEN

PDE10A is a newly identified cAMP/cGMP phosphodiesterase for which mRNA is highly expressed in the mammalian striatum. In the present study, PDE10A protein and mRNA expression throughout the rat brain were determined, using a monoclonal antibody (24F3.F11) for Western blot and immunohistochemical analyses and an antisense riboprobe for in situ hybridization. High levels of mRNA are observed in most of the neuronal cell bodies of striatal complex (caudate n, n. accumbens and olfactory tubercle), indicating that PDE10A is expressed by the striatal medium spiny neurons. PDE10A-like immunoreactivity is dense throughout the striatal neuropil, as well as in the internal capsule, globus pallidus, and substantia nigra. These latter regions lack significant expression of PDE10A mRNA. Thus, PDE10A is transported throughout the dendritic tree and down the axons to the terminals of the medium spiny neurons. These data suggest a role for PDE10A in regulating activity within both the striatonigral and striatopallidal pathways. In addition, PDE10A immunoreactivity and mRNA are found at lower levels in the hippocampal pyramidal cell layer, dentate granule cell layer and throughout the cortex and cerebellar granule cell layer. Immunoreactivity is detected only in cell bodies in these latter regions. This more restricted subcellular localization of PDE10A outside the striatum suggests a second, distinct function for the enzyme in these regions.


Asunto(s)
Encéfalo/citología , Encéfalo/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Autorradiografía , Western Blotting , Química Encefálica , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas , ADN Complementario/química , ADN Complementario/genética , Inmunohistoquímica/métodos , Hibridación in Situ , Insectos , Masculino , Ratones , Ratones Endogámicos BALB C , Neuronas/citología , Neuronas/metabolismo , Oligodesoxirribonucleótidos Antisentido , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/inmunología , Isótopos de Fósforo/farmacocinética , Pruebas de Precipitina , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección
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