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1.
Cell ; 161(6): 1252-65, 2015 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-26046436

RESUMEN

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.


Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , National Institutes of Health (U.S.) , Estados Unidos
2.
J Biol Chem ; 300(1): 105501, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38016516

RESUMEN

Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.


Asunto(s)
Quinasas Ciclina-Dependientes , Ciclinas , Poliadenilación , Ciclinas/metabolismo , Conformación Molecular , Humanos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/química
3.
Bioconjug Chem ; 33(6): 1192-1200, 2022 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-35584359

RESUMEN

Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11 Å deep pocket that permits site-specific conjugation of ß-diketone-, ß-lactam-, and heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here, we expand the scope of this ADC assembly strategy by mutating h38C2's reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for the precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F, h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.


Asunto(s)
Cisteína , Inmunoconjugados , Anticuerpos Monoclonales/química , Cisteína/química , Inmunoconjugados/química , Lisina/química
4.
Bioorg Med Chem Lett ; 76: 129014, 2022 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-36202189

RESUMEN

Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 µM of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.


Asunto(s)
Éteres Cíclicos , Inhibidores de la Metaloproteinasa de la Matriz , Ratas , Animales , Metaloproteinasa 13 de la Matriz/química , Metaloproteinasa 13 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/química , Quelantes/farmacología , Quelantes/química , Zinc/química
5.
Biochemistry ; 60(14): 1080-1087, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33754696

RESUMEN

Monoclonal antibody h38C2 is a humanized catalytic antibody that has been used to generate various immunoconjugate species such as chemically programmed antibodies, antibody-drug conjugates, and antibody-siRNA conjugates. Highly efficient and specific conjugation of h38C2 occurs at its uniquely reactive lysine (Lys) residue buried inside the antibody's catalytic pocket. We recently reported the rational mutation of this Lys residue at position 99 in the heavy chain variable domain to an arginine (Arg) residue. The Lys99Arg mutation can be site-selectively conjugated with molecules containing a hapten-like triazolyl-phenylglyoxal (TPG) unit. Here we show that this conjugation is facilitated by the unusual pH-sensitive reactivity of the Arg99 residue, consistent with an indirectly measured pKa of 5.2. The Arg99/TPG conjugation holds promise to further expand the versatility of the h38C2 conjugation platform, such as for the generation of antibody conjugates with dual payloads.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Arginina , Inmunoconjugados/química , Inmunoconjugados/genética , Ingeniería de Proteínas , Sitios de Unión , Concentración de Iones de Hidrógeno , Dominios Proteicos
6.
Bioorg Med Chem ; 26(18): 4984-4995, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30249495

RESUMEN

A structure-activity/structure-property relationship study based on the physicochemical as well as in vitro pharmacokinetic properties of a first generation matrix metalloproteinase (MMP)-13 inhibitor (2) was undertaken. After systematic variation of inhibitor 2, compound 31 was identified which exhibited microsomal half-life higher than 20 min, kinetic solubility higher than 20 µM, and a permeability coefficient greater than 20 × 10-6 cm/s. Compound 31 also showed excellent in vivo PK properties after IV dosing (Cmax = 56.8 µM, T1/2 (plasma) = 3.0 h, Cl = 0.23 mL/min/kg) and thus is a suitable candidate for in vivo efficacy studies in an OA animal model.


Asunto(s)
Metaloproteinasa 13 de la Matriz/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Animales , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Semivida , Humanos , Concentración 50 Inhibidora , Cinética , Inhibidores de la Metaloproteinasa de la Matriz/farmacocinética , Ratones , Microsomas Hepáticos/metabolismo , Modelos Animales , Ratas , Solubilidad , Relación Estructura-Actividad
7.
Exp Parasitol ; 188: 36-41, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29551628

RESUMEN

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. PAM occurs principally in healthy children of less than 13 years old with a history of recent exposure to warm fresh water. While as yet not a reportable disease, the Centers for Disease Control and Prevention (CDC) documents a total of 143 cases in the United States. Only four patients have survived. Infection results from water containing N. fowleri entering the nose, followed by migration of the amebae to the brain. Within the brain, N. fowleri infection results in extensive necrosis, leading to death in 3-7 days. Mortality among patients with PAM is greater than 95%. The drugs of choice in treating PAM are the antifungal amphotericin B, and the antileishmanial, miltefosine. However neither drug is FDA-approved for this indication and the use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with amphotericin B have survived PAM. Therefore, development of new, safe and effective drugs is a critical unmet need to avert future deaths of children. The molecular mechanisms underlying the pathogenesis of PAM are poorly understood but it is known that cysteine proteases of N. fowleri play a role in the progression of PAM. We therefore assessed the in vitro activity of the synthetic vinyl sulfone cysteine protease inhibitor, K11777, and 33 analogs with valine, phenylalanine or pyridylalanine at P2 position, against cysteine protease activity in the lysate of N. fowleri. Inhibitors with phenylalanine or pyridylalanine at P2 position were particularly effective in inhibiting the cysteine protease activity of N. fowleri cell lysate with IC50 ranging between 3 nM and 6.6 µM. Three of the 34 inhibitors also showed inhibitory activity against N. fowleri in a cell viability assay and were 1.6- to 2.5-fold more potent than the standard of care drug miltefosine. Our study provides the first evidence of the activity of synthetic, small molecule cysteine protease inhibitors against N. fowleri.


Asunto(s)
Infecciones Protozoarias del Sistema Nervioso Central/tratamiento farmacológico , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Naegleria fowleri/efectos de los fármacos , Infecciones Protozoarias del Sistema Nervioso Central/parasitología , Niño , Proteasas de Cisteína/metabolismo , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/uso terapéutico , Dipéptidos/química , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Agua Dulce , Humanos , Concentración 50 Inhibidora , Naegleria fowleri/enzimología , Fenilalanina/análogos & derivados , Piperazinas , Temperatura , Compuestos de Tosilo , Compuestos de Vinilo/química , Compuestos de Vinilo/farmacología , Compuestos de Vinilo/uso terapéutico
8.
J Biol Chem ; 291(37): 19661-73, 2016 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-27445334

RESUMEN

Chemically programmed bispecific antibodies (biAbs) endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system. Here we report a platform of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells. Two different antibody technologies were merged together to make a novel chemically programmed biAb. This was achieved by combining the humanized anti-hapten monoclonal antibody (mAb) h38C2 with the humanized anti-human CD3 mAb v9 in a clinically investigated diabody format known as Dual-Affinity Re-Targeting (DART). We show that h38C2 × v9 DARTs can readily be equipped with tumor-targeting hapten-derivatized small molecules without causing a systemic response harming healthy tissues. As a proof of concept, we chemically programmed h38C2 × v9 with hapten-folate and demonstrated its selectivity and potency against folate receptor 1 (FOLR1)-expressing ovarian cancer cells in vitro and in vivo Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy.


Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Antineoplásicos/química , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Antineoplásicos/inmunología , Línea Celular Tumoral , Femenino , Receptor 1 de Folato/antagonistas & inhibidores , Receptor 1 de Folato/química , Receptor 1 de Folato/inmunología , Células HEK293 , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Neoplasias Ováricas/química , Neoplasias Ováricas/inmunología
9.
J Am Chem Soc ; 139(16): 5865-5869, 2017 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-28414442

RESUMEN

Stolonidiol, a marine natural product, has been reported to potentiate the activity of choline acetyltransferase (ChAT), the enzyme that produces the neurotransmitter acetylcholine. Here we report the total synthesis of stolonidiol starting from (R)-(+)-limonene. To identify the mechanism by which ChAT activity is increased, we sought to identify the biological target of stolonidiol. We show that stolonidiol binds to the phorbol ester binding site of protein kinase C (PKC), induces translocation of PKC to the cell membrane, and activates kinase activity. Furthermore, we confirmed the increase in ChAT activity observed upon treatment of cells with stolonidiol and show that this effect is mediated by PKC. Collectively, our data strongly suggest that PKC activation by stolonidiol is responsible for the resulting potentiation of ChAT activity.


Asunto(s)
Colina O-Acetiltransferasa/metabolismo , Diterpenos/farmacología , Cristalografía por Rayos X , Diterpenos/síntesis química , Diterpenos/química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad
10.
J Pharmacol Exp Ther ; 363(3): 367-376, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28928121

RESUMEN

Current knowledge regarding acute regulation of adipocyte lipolysis is largely based on receptor-mediated activation or inhibition of pathways that influence intracellular levels of cAMP, thereby affecting protein kinase A (PKA) activity. We recently identified synthetic ligands of α-ß-hydrolase domain containing 5 (ABHD5) that directly activate adipose triglyceride lipase (ATGL) by dissociating ABHD5 from its inhibitory regulator, perilipin-1 (PLIN1). In the current study, we used these novel ligands to determine the direct contribution of ABHD5 to various aspects of lipolysis control in white (3T3-L1) and brown adipocytes. ABHD5 ligands stimulated adipocyte lipolysis without affecting PKA-dependent phosphorylation on consensus sites of PLIN1 or hormone-sensitive lipase (HSL). Cotreatment of adipocytes with synthetic ABHD5 ligands did not alter the potency or maximal lipolysis efficacy of the ß-adrenergic receptor (ADRB) agonist isoproterenol (ISO), indicating that both target a common pool of ABHD5. Reducing ADRB/PKA signaling with insulin or desensitizing ADRB suppressed lipolysis responses to a subsequent challenge with ISO, but not to ABHD5 ligands. Lastly, despite strong treatment differences in PKA-dependent phosphorylation of HSL, we found that ligand-mediated activation of ABHD5 led to complete triglyceride hydrolysis, which predominantly involved ATGL, but also HSL. These results indicate that the overall pattern of lipolysis controlled by ABHD5 ligands is similar to that of isoproterenol, and that ABHD5 plays a central role in the regulation of adipocyte lipolysis. As lipolysis is critical for adaptive thermogenesis and in catabolic tissue remodeling, ABHD5 ligands may provide a means of activating these processes under conditions where receptor signaling is compromised.


Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Adipocitos Marrones/efectos de los fármacos , Adipocitos Blancos/efectos de los fármacos , Piperazinas/farmacología , Tiazepinas/farmacología , Urea/análogos & derivados , Urea/farmacología , Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Femenino , Hidrólisis , Insulina/farmacología , Ligandos , Lipólisis , Ratones , Perilipina-1/metabolismo , Fosforilación , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Esterol Esterasa/metabolismo , Triglicéridos/metabolismo
11.
Nature ; 472(7344): 491-4, 2011 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-21499262

RESUMEN

T-helper cells that produce interleukin-17 (T(H)17 cells) are a recently identified CD4(+) T-cell subset with characterized pathological roles in autoimmune diseases. The nuclear receptors retinoic-acid-receptor-related orphan receptors α and γt (RORα and RORγt, respectively) have indispensible roles in the development of this cell type. Here we present SR1001, a high-affinity synthetic ligand-the first in a new class of compound-that is specific to both RORα and RORγt and which inhibits T(H)17 cell differentiation and function. SR1001 binds specifically to the ligand-binding domains of RORα and RORγt, inducing a conformational change within the ligand-binding domain that encompasses the repositioning of helix 12 and leads to diminished affinity for co-activators and increased affinity for co-repressors, resulting in suppression of the receptors' transcriptional activity. SR1001 inhibited the development of murine T(H)17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines when added to differentiated murine or human T(H)17 cells. Finally, SR1001 effectively suppressed the clinical severity of autoimmune disease in mice. Our data demonstrate the feasibility of targeting the orphan receptors RORα and RORγt to inhibit specifically T(H)17 cell differentiation and function, and indicate that this novel class of compound has potential utility in the treatment of autoimmune diseases.


Asunto(s)
Autoinmunidad/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Sulfonamidas/farmacología , Células Th17/citología , Células Th17/inmunología , Tiazoles/farmacología , Animales , Autoinmunidad/inmunología , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Interleucinas/biosíntesis , Interleucinas/inmunología , Ligandos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Células Th17/efectos de los fármacos , Células Th17/metabolismo
12.
Bioconjug Chem ; 27(10): 2271-2275, 2016 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-27666414

RESUMEN

Bioorthogonal labeling of antibodies enables the conjugation of compounds, such as small molecules or peptides, which expand targeting capacity or enhance cytotoxicity. Taking advantage of a cyclohexene sulfonamide compound that site-selectively labels Lys64 in human serum albumin (HSA), we demonstrate that domain I of HSA can be used as a fusion protein for the preparation of antibody conjugates. Trastuzumab fusions were expressed at the N-terminus of the light chain or the C-terminus of the heavy chain enabling conjugation to small molecules. Moreover, these conjugates retained HER2 binding and proved to be highly stable in human plasma. Antibody conjugation via HSA domain I fusion should therefore have broad utility for making serum-stable antibody conjugates, particularly for antibody-drug conjugates.


Asunto(s)
Inmunoconjugados/química , Proteínas Recombinantes de Fusión/química , Albúmina Sérica/química , Anticuerpos/química , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inmunoconjugados/sangre , Inmunoconjugados/metabolismo , Lisina/química , Dominios Proteicos , Ingeniería de Proteínas/métodos , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/metabolismo , Rodaminas/química , Trastuzumab/química
13.
Org Biomol Chem ; 14(22): 5141-7, 2016 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-27184239

RESUMEN

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures that have poor pharmacokinetic properties and decreased efficacy relative to homogenous ADCs. Furthermore, ADCs that are maleimide-based often have inadequate circulatory stability, which can result in premature drug release with consequent off-target toxicities. Selenocysteine-modified antibodies have been developed that allow site-specific antibody conjugation, yielding homogeneous ADCs. Herein, we survey several electrophilic functional groups that react with selenocystine with high efficiency. Several of these result in conjugates with stabilities that are superior to maleimide conjugates. Among these, the allenamide functional group reacts with notably high efficiency, leads to conjugates with remarkable stability, and shows exquisite selectivity for selenocysteine conjugation.


Asunto(s)
Selenocisteína/química , Estabilidad de Medicamentos , Maleimidas/química
14.
Mol Pharmacol ; 87(2): 296-304, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25473120

RESUMEN

The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1-expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1.


Asunto(s)
Proliferación Celular/fisiología , Pirimidinas/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células HEK293 , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos C57BL , Pirimidinas/química , Pirimidinas/farmacología
15.
J Biol Chem ; 289(27): 18893-903, 2014 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-24817118

RESUMEN

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.


Asunto(s)
Quinasa Idelta de la Caseína/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Secuencia de Aminoácidos , Animales , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/química , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica/efectos de los fármacos , Proteínas Tirosina Quinasas/química , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología
16.
Bioconjug Chem ; 26(11): 2243-8, 2015 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-26161903

RESUMEN

Site-specific conjugation technologies enable the production of homogeneous antibody-drug conjugates (ADCs) with improved therapeutic indices compared to conventional ADCs. However, current site-specific conjugation methods can only attach one type of drug to a single antibody. Given the emergence of acquired resistance to current ADCs, arming single antibodies with different drugs may provide an attractive option in the development of next-generation ADCs. Here, we describe a site-specific dual conjugation strategy as a platform for dual warhead ADCs.


Asunto(s)
Cisteína/química , Inmunoconjugados/química , Selenocisteína/química , Trastuzumab/química , Línea Celular Tumoral , Humanos
17.
Chembiochem ; 15(8): 1111-20, 2014 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-24771705

RESUMEN

Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4-aminopyridyl-based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub-nanomolar compound 3, have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X-ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground-state conformation of CYP51 drug-target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template.


Asunto(s)
Inhibidores de 14 alfa Desmetilasa/farmacología , Esterol 14-Desmetilasa/metabolismo , Sulfonamidas/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Inhibidores de 14 alfa Desmetilasa/síntesis química , Inhibidores de 14 alfa Desmetilasa/química , Animales , Relación Dosis-Respuesta a Droga , Ratones , Modelos Moleculares , Estructura Molecular , Mioblastos/efectos de los fármacos , Mioblastos/parasitología , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Tripanocidas/síntesis química , Tripanocidas/química , Trypanosoma cruzi/enzimología
18.
J Am Chem Soc ; 135(25): 9512-7, 2013 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-23758559

RESUMEN

Kinetically controlled hydroboration of allenylboronate 5 followed by double allylboration with the resulting allylborane (Z)-7 gave (Z)-2-methyl-1,5-anti-pentenediols 6 in good yield and high enantioselectivity in the presence of 10% BF3·OEt2 as the catalyst in the second allylboration step. Under thermodynamically controlled isomerization conditions, (Z)-7 can readily isomerize to (E)-7. Double allylboration of representative aldehydes with allylborane (E)-7 gave (E)-2-methyl-1,5-anti-pentenediols 4 in good yield and high enantioselectivity without requiring use of the BF3·OEt2 catalyst. Thus, 2-methyl-1,5-anti-pentenediols with either olefin geometry can be synthesized from the same allenylboronate precursor 5. Furthermore, 1,5-pentenediols 4 and 6 can be easily converted to 1,3,5-triols with excellent diastereoselectivity in one step.


Asunto(s)
Alcadienos/química , Boranos/química , Pentanoles/síntesis química , Boranos/síntesis química , Estructura Molecular , Pentanoles/química , Estereoisomerismo
19.
J Am Chem Soc ; 135(14): 5340-3, 2013 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-23530855

RESUMEN

A highly diastereoselective synthesis of N-acetyl dihydrotetrafibricin methyl ester (34) is described. The synthesis features three enantioselective double allylboration reactions and an intramolecular hydrosilylation/Fleming-Tamao oxidation sequence to establish seven of the hydroxy-bearing stereocenters of 34. Especially noteworthy is the fragment-assembly double allyboration reaction of 2 and 7 using reagent 3, which provides the advanced intermediate 6 with >20:1 diastereoselectivity.


Asunto(s)
Macrólidos/síntesis química , Macrólidos/química , Estructura Molecular , Estereoisomerismo , Streptomyces/química
20.
J Org Chem ; 78(1): 3-8, 2013 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-22703288

RESUMEN

Syntheses of the C(15)-C(27) fragments of chaxamycins A/D, rifamycin S, and the C(12)-C(24) fragment of salinisporamycin have been accomplished in 10 steps from commercially available starting materials. Three crotylboron reagents were utilized to construct the seven contiguous stereocenters in these fragments with excellent stereoselectivity.


Asunto(s)
Antibacterianos/síntesis química , Rifamicinas/síntesis química , Antibacterianos/química , Indicadores y Reactivos/química , Rifamicinas/química , Estereoisomerismo
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