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1.
Sci Rep ; 12(1): 15494, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109543

RESUMO

In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between "good" and "damaged" virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.


Assuntos
Ácidos Nucleicos , Vacinas , Reatores Biológicos
2.
Sci Rep ; 11(1): 7432, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795759

RESUMO

Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD's Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage. Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.


Assuntos
Citometria de Fluxo/métodos , Vacinas Atenuadas/normas , Vacinologia/métodos , Vacinologia/normas , Vacinas Virais/normas , Animais , Chlorocebus aethiops , Vacinas contra Ebola/normas , Humanos , Temperatura , Vacinas Sintéticas/normas , Células Vero , Vírion/ultraestrutura
3.
Mol Ther Nucleic Acids ; 16: 194-205, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-30901578

RESUMO

Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c+ cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology.

4.
Mol Imaging Biol ; 15(4): 431-40, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23440602

RESUMO

PURPOSE: Visualization of the cell cycle in living subjects has long been a big challenge. The present study aimed to noninvasively visualize mitotic arrest of the cell cycle with an optical reporter in living subjects. PROCEDURES: An N-terminal cyclin B1-luciferase fusion construct (cyclin B-Luc) controlled by the cyclin B promoter, as a mitosis reporter, was generated. HeLa or HCT116 cells stably expressing cyclin B-Luc reporter were used to evaluate its cell cycle-dependent regulation and ubiquitination-mediated degradation. We also evaluated its feasibility to monitor the mitotic arrest caused by Taxotere both in vitro and in vivo. RESULTS: We showed that the cyclin B-Luc fusion protein was regulated in a cell cycle-dependent manner and accumulated in the mitotic phase (M phase) in cellular assays. The regulation of cyclin B-Luc reporter was mediated by proteasome ubiquitination. In the present study, in vitro imaging showed that antimitotic reagents like Taxotere upregulated the reporter through cell cycle arrest in the M phase. Noninvasive longitudinal bioluminescence imaging further demonstrated an upregulation of the reporter consistent with mitotic arrest induced in tumor xenograft models. Induction of this reporter was also observed with a kinesin spindle protein inhibitor, which causes cell cycle blockage in the M phase. CONCLUSIONS: Our results demonstrate that the cyclin B-Luc reporter can be used to image whether compounds are capable, in vivo, of causing an M phase arrest and/or altering cyclin B turnover. This reporter can also be potentially used in high-throughput screening efforts aimed at discovering novel molecules that will cause cell cycle arrest at the M phase in cultivated cell lines and animal models.


Assuntos
Pontos de Checagem do Ciclo Celular , Luminescência , Mitose , Imagem Molecular/métodos , Animais , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciclina B1/metabolismo , Docetaxel , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Luciferases/metabolismo , Camundongos , Mitose/efeitos dos fármacos , Nocodazol/farmacologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tela Subcutânea/efeitos dos fármacos , Taxoides/farmacologia , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Artigo em Inglês | MEDLINE | ID: mdl-23366734

RESUMO

Transgenic mice with Tie2- green fluorescent protein (GFP) are used as a model to study the kinetic distribution of the Cy5-siRNA delivered by lipid nanoparticles (LNP) into the liver. After the mouse is injected with the LNP, it undergoes a procedure of intra-vital multi-photon microscopy imaging over a period of two hours, during which the process for the nanoparticle to diffuse into the hepatocytes from the vasculature system is monitored. Since the images are obtained in-vivo, the quantification of Cy5 kinetics suffers from the moving field of view (FOV). A method is proposed to register the sequence of images through template matching. Based on the semi-automatic segmentations of the vessels in the common FOV, the registered images are segmented into three regions of interest (ROI) in which the Cy5 signals are quantified. Computation of the percentage signal strength in the ROIs over time allows for the analysis of the diffusion of Cy5-siRNA into the hepatocytes, and helps demonstrate the effectiveness of the Cy5-siRNA delivery vehicle.


Assuntos
Carbocianinas/metabolismo , Imageamento Tridimensional , Microscopia de Fluorescência por Excitação Multifotônica/métodos , RNA Interferente Pequeno/metabolismo , Processamento de Sinais Assistido por Computador , Animais , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos
6.
Mol Ther ; 19(3): 567-75, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21179008

RESUMO

A major hurdle for harnessing small interfering RNA (siRNA) for therapeutic application is an effective and safe delivery of siRNA to target tissues and cells via systemic administration. While lipid nanoparticles (LNPs) composed of a cationic lipid, poly-(ethylene glycol) lipid and cholesterol, are effective in delivering siRNA to hepatocytes via systemic administration, they may induce multi-faceted toxicities in a dose-dependent manner, independently of target silencing. To understand the underlying mechanism of toxicities, pharmacological probes including anti-inflammation drugs and specific inhibitors blocking different pathways of innate immunity were evaluated for their abilities to mitigate LNP-siRNA-induced toxicities in rodents. Three categories of rescue effects were observed: (i) pretreatment with a Janus kinase (Jak) inhibitor or dexamethasone abrogated LNP-siRNA-mediated lethality and toxicities including cytokine induction, organ impairments, thrombocytopenia and coagulopathy without affecting siRNA-mediated gene silencing; (ii) inhibitors of PI3K, mammalian target of rapamycin (mTOR), p38 and IκB kinase (IKK)1/2 exhibited a partial alleviative effect; (iii) FK506 and etoricoxib displayed no protection. Furthermore, knockout of Jak3, tumor necrosis factor receptors (Tnfr)p55/p75, interleukin 6 (IL-6) or interferon (IFN)-γ alone was insufficient to alleviate LNP-siRNA-associated toxicities in mice. These indicate that activation of innate immune response is a primary trigger of systemic toxicities and that multiple innate immune pathways and cytokines can mediate toxic responses. Jak inhibitors are effective in mitigating LNP-siRNA-induced toxicities.


Assuntos
Inibidores Enzimáticos/metabolismo , Janus Quinases/antagonistas & inibidores , Lipídeos , Nanopartículas , RNA Interferente Pequeno/toxicidade , Animais , Citocinas/sangue , Dexametasona/metabolismo , Etoricoxib , Feminino , Técnicas de Inativação de Genes , Quinase I-kappa B/antagonistas & inibidores , Interferon gama/genética , Interleucina-6/genética , Janus Quinases/genética , Lipídeos/química , Lipídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Tipo II do Fator de Necrose Tumoral/genética , Sulfonas/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tacrolimo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
7.
Mol Ther ; 18(9): 1657-66, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20628357

RESUMO

Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.


Assuntos
Lipídeos/química , Fígado/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Animais , Imunofluorescência , Inativação Gênica/fisiologia , Fígado/enzimologia , Luciferases/genética , Camundongos
8.
Mol Ther ; 18(1): 171-80, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19738601

RESUMO

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.


Assuntos
Dexametasona/uso terapêutico , Nanopartículas/efeitos adversos , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Animais , Feminino , Inativação Gênica , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Camundongos , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Receptores de Glucocorticoides/agonistas
9.
J Med Chem ; 51(14): 4239-52, 2008 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-18578472

RESUMO

Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Cinesinas/antagonistas & inibidores , Neoplasias/enzimologia , Piperidinas/farmacologia , Pirróis/farmacologia , Taxoides/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Taxoides/uso terapêutico
10.
Bioorg Med Chem Lett ; 17(19): 5390-5, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17761419

RESUMO

3,5-diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Careful adjustment of amine basicity was essential for preserving cellular potency in a multidrug resistant cell line while maintaining good aqueous solubility.


Assuntos
Amidas/síntese química , Amidas/farmacologia , Antimitóticos/síntese química , Antimitóticos/farmacologia , Cinesinas/antagonistas & inibidores , Mitose/efeitos dos fármacos , Pirazóis/síntese química , Pirazóis/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Fenômenos Químicos , Físico-Química , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Genes MDR/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Solubilidade , Estereoisomerismo , Relação Estrutura-Atividade
11.
Bioorg Med Chem Lett ; 17(20): 5671-6, 2007 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17804233

RESUMO

Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.


Assuntos
Benzoxazinas/química , Benzoxazinas/farmacologia , Desenho de Fármacos , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Mitose/efeitos dos fármacos , Pirazóis/química , Animais , Benzoxazinas/síntese química , Benzoxazinas/farmacocinética , Linhagem Celular , Cães , Humanos , Hidrogênio/química , Estrutura Molecular , Relação Estrutura-Atividade
13.
Mol Cell Biol ; 27(2): 689-98, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17101792

RESUMO

The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.


Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Apoptose/fisiologia , Caspase 3/metabolismo , Cinesinas/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Proteína Supressora de Tumor p53/fisiologia , Proteína X Associada a bcl-2/biossíntese , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Humanos , Cinesinas/antagonistas & inibidores , Paclitaxel/farmacologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Fuso Acromático
14.
Cancer Cell ; 8(1): 49-59, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16023598

RESUMO

The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.


Assuntos
Apoptose , Genes cdc/fisiologia , Cinesinas/antagonistas & inibidores , Mitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fuso Acromático/fisiologia , Caspase 3 , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Resistência a Medicamentos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Estrutura Molecular , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Pirróis/farmacologia , Proteína X Associada a bcl-2
16.
J Biomol Screen ; 8(4): 430-8, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-14567795

RESUMO

Farnesyl:protein transferase (FPTase) catalyzes the covalent addition of the isoprenyl moiety of farnesylpyrophosphate to the C-terminus of the Ras oncoprotein and other cellular proteins. Inhibitors of FPTase (FTIs) have been developed as potential anticancer agents, and several compounds have been evaluated in clinical trials. To facilitate the identification of cell-active FTIs with high potency, the authors developed a method that uses a radiolabeled FTI that serves as a ligand in competitive displacement assays. Using high-affinity [(3)H]-labeled or [(125)I]-labeled FTI radioligands, they show that specific binding to FPTase can be detected in intact cells. Binding of these labeled FTI radioligands can be competed with a variety of structurally diverse FTIs, and the authors show that inhibition of FTI radioligand binding correlates well with inhibition of FPTase substrate prenylation in cells. This method provides a rapid and quantitative means of assessing FTI potency in cells and is useful for guiding the discovery of potent, novel inhibitors of FPTase. Similar methods could be employed in the optimization of inhibitors for other intracellular drug targets.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/farmacologia , Ensaio Radioligante , Alquil e Aril Transferases/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Transformada , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Farnesiltranstransferase , Humanos , Células KB , Ratos , Sensibilidade e Especificidade
17.
Mol Cancer Ther ; 1(9): 747-58, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12479371

RESUMO

Farnesyl:protein transferase (FPTase) inhibitors were developed as anti-Ras drugs, but they fail to inhibit Ki-Ras activity because Ki-Ras can be modified by geranylgeranyl:protein transferase type-I (GGPTase-I). L-778,123, an inhibitor of FPTase and GGPTase-I, was developed in part because it can completely inhibit Ki-Ras prenylation. To support the clinical development of L-778,123, we developed pharmacodynamic assays using peripheral blood mononuclear cells (PBMCs) to measure the inhibition of prenylation of HDJ2 and Rap1A, proteins that are FPTase- and GGPTase-I substrates, respectively. We validated these assays in animal models and show that inhibition of HDJ2 prenylation in mouse PBMCs correlates with the concentration of FPTase inhibitors in blood. In dogs, continuous infusion of L-778,123 inhibited both HDJ2 and Rap1A prenylation in PBMCs, but we did not detect inhibition of Ki-Ras prenylation. We reported previously results from the first L-778,123 Phase I trial that showed a dose-dependent inhibition of HDJ2 farnesylation in PBMCs. In this report, we present additional analysis of patient samples from this trial and a second Phase I trial of L-778,123, and demonstrate the inhibition of both HDJ2 and Rap1A prenylation in PBMC samples. This study represents the first demonstration of GGPTase-I inhibition in humans. However, no inhibition of Ki-Ras prenylation by L-778,123 was detected in patient samples. These results confirm the pharmacologic profile of L-778,123 in humans as a dual inhibitor of FPTase and GGPTase-I, but indicate that the intended target of the drug, Ki-Ras, was not inhibited.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Animais , Cães , Relação Dose-Resposta a Droga , Humanos , Immunoblotting , Leucócitos/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Modelos Químicos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Tempo , Proteínas rap1 de Ligação ao GTP/metabolismo
18.
Bioorg Med Chem Lett ; 12(15): 2027-30, 2002 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-12113834

RESUMO

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Sítios de Ligação , Ligação Competitiva , Concentração Inibidora 50 , Ligação Proteica , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteínas rap de Ligação ao GTP/efeitos dos fármacos , Proteínas rap de Ligação ao GTP/metabolismo
19.
J Med Chem ; 45(12): 2388-409, 2002 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-12036349

RESUMO

A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side effects. In dogs, oral bioavailability was good to excellent, and increases in plasma half-life were due to attenuated clearance. It was observed that in vivo clearance correlated with the flexibility of the molecules and this concept proved useful in the design of FTIs that exhibited low clearance, such as FTI 78. X-ray crystal structures of compounds 49 and 66 complexed with farnesyltransferase (FTase)-farnesyl diphosphate (FPP) were determined, and they provide details of the key interactions in such ternary complexes. Optimization of this 3-aminopyrrolidinone series of compounds led to significant increases in potency, providing 83 and 85, the most potent inhibitors of FTase in cells described to date.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases , Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Inibidores Enzimáticos/síntese química , Naftalenos/síntese química , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Pirrolidinas/síntese química , Transativadores , Animais , Linhagem Celular , Cromatografia Líquida , Cristalografia por Raios X , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Cães , Canal de Potássio ERG1 , Eletrocardiografia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Canais de Potássio Éter-A-Go-Go , Farnesiltranstransferase , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Estrutura Molecular , Naftalenos/química , Naftalenos/farmacocinética , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Canais de Potássio/metabolismo , Ligação Proteica , Pirrolidinas/química , Pirrolidinas/farmacocinética , Estereoisomerismo , Relação Estrutura-Atividade , Regulador Transcricional ERG
20.
Bioorg Med Chem Lett ; 12(9): 1269-73, 2002 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-11965368

RESUMO

Compound 1 has been shown to be a dual prenylation inhibitor with FPTase (IC50=2 nM) and GGPTase-I (IC50=95 nM). Analogues of 1, which replaced the cyanophenyl group with various biaryls, led to the discovery of highly potent dual FPTase/GGPTase-I inhibitors. 4-trifluoromethylphenyl, trifluoropentynyl, and trifluoropentyl were identified as good p-cyano replacements.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química
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