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1.
Anal Chem ; 84(10): 4373-82, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22519810

RESUMO

Although liquid chromatography/mass spectrometry using selected reaction monitoring (LC/SRM-MS) holds great promise for targeted protein analysis, quantification of therapeutic monoclonal antibody (mAb) in tissues represents a daunting challenge due to the extremely low tissue levels, complexity of tissue matrixes, and the absence of an efficient strategy to develop an optimal LC/SRM-MS method. Here we describe a high-throughput, streamlined strategy for the development of sensitive, selective, and reliable quantitative methods of mAb in tissue matrixes. A sensitive nano-LC/nanospray-MS method was employed to achieve a low lower limit of quantification (LOQ). For selection of signature peptides (SP), the SP candidates were identified by a high-resolution Orbitrap and then optimal SRM conditions for each candidate were obtained using a high-throughput, on-the-fly orthogonal array optimization (OAO) strategy, which is capable of optimizing a large set of SP candidates within a single nano-LC/SRM-MS run. Using the optimized conditions, the candidates were experimentally evaluated for both sensitivity and stability in the target matrixes, and SP selection was based on the results of the evaluation. Two unique SP, respectively from the light and heavy chain, were chosen for quantification of each mAb. The use of two SP improves the quantitative reliability by gauging possible degradation/modification of the mAb. Standard mAb proteins with verified purities were utilized for calibration curves, to prevent the quantitative biases that may otherwise occur when synthesized peptides were used as calibrators. We showed a proof of concept by rapidly developing sensitive nano-LC/SRM-MS methods for quantifying two mAb (8c2 and cT84.66) in multiple preclinical tissues. High sensitivity was achieved for both mAb with LOQ ranged from 0.156 to 0.312 µg/g across different tissues, and the overall procedure showed a wide dynamic range (≥500-fold) and good accuracy [relative error (RE) < 18.8%] and precision [interbatch relative standard deviation (RSD) < 18.1%, intrabatch RSD < 17.2%]. The quantitative method was applied to a comprehensive investigation of the steady-state tissue distribution of 8c2 in wild-type mice versus those deficient in FcRn α-chain, FcγIIb, and FcγRI/FcγRIII, following a chronic dosing regimen. This work represents the first extensive quantification of mAb in tissues by an LC/MS-based method.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Nanotecnologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Camundongos , Peptídeos/análise , Distribuição Tecidual
2.
J Med Chem ; 65(1): 343-368, 2022 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-34982568

RESUMO

Inhibition of oxidative phosphorylation (OXPHOS) is a promising therapeutic strategy for select cancers that are dependent on aerobic metabolism. Here, we report the discovery, optimization, and structure-activity relationship (SAR) study of a series of novel OXPHOS inhibitors. The hit compound, benzene-1,4-disulfonamide 1, was discovered in a phenotypic screen selective for cytotoxicity in a galactose-containing medium. Our multi-parameter optimization campaign led to the discovery of 65 (DX3-235), showing nanomolar inhibition of complex I function and adenosine triphosphate (ATP) production in a galactose-containing medium resulting in significant cytotoxicity. Importantly, 64 (DX3-234), a close analogue of 65, is well tolerated in mice and shows significant single agent efficacy in a Pan02 syngeneic pancreatic cancer model, suggesting that highly potent and selective OXPHOS inhibitors can be useful for the treatment of pancreatic cancer.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Sulfonamidas/química , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/química , Apoptose , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Med Chem ; 65(4): 3404-3419, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35167303

RESUMO

Targeting oxidative phosphorylation (OXPHOS) complexes is an emerging strategy to disrupt the metabolism of select cancer subtypes and to overcome resistance to targeted therapies. Here, we describe our lead optimization campaign on a series of benzene-1,4-disulfonamides as novel OXPHOS complex I inhibitors. This effort led to the discovery of compound 23 (DX3-213B) as one of the most potent complex I inhibitors reported to date. DX3-213B disrupts adenosine triphosphate (ATP) generation, inhibits complex I function, and results in the growth inhibition of pancreatic cancer cells in the low nanomolar range. Importantly, the oral administration of DX3-213B resulted in significant in vivo efficacy in a pancreatic cancer syngeneic model without obvious toxicity. Our data clearly demonstrate that OXPHOS inhibition can be a safe and efficacious strategy to treat pancreatic cancer.


Assuntos
Antineoplásicos/uso terapêutico , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Trifosfato de Adenosina/biossíntese , Animais , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Acta Pharm Sin B ; 12(5): 2462-2478, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35646532

RESUMO

Drug optimization, which improves drug potency/specificity by structure‒activity relationship (SAR) and drug-like properties, is rigorously performed to select drug candidates for clinical trials. However, the current drug optimization may overlook the structure‒tissue exposure/selectivity-relationship (STR) in disease-targeted tissues vs. normal tissues, which may mislead the drug candidate selection and impact the balance of clinical efficacy/toxicity. In this study, we investigated the STR in correlation with observed clinical efficacy/toxicity using seven selective estrogen receptor modulators (SERMs) that have similar structures, same molecular target, and similar/different pharmacokinetics. The results showed that drug's plasma exposure was not correlated with drug's exposures in the target tissues (tumor, fat pad, bone, uterus), while tissue exposure/selectivity of SERMs was correlated with clinical efficacy/safety. Slight structure modifications of four SERMs did not change drug's plasma exposure but altered drug's tissue exposure/selectivity. Seven SERMs with high protein binding showed higher accumulation in tumors compared to surrounding normal tissues, which is likely due to tumor EPR effect of protein-bound drugs. These suggest that STR alters drug's tissue exposure/selectivity in disease-targeted tissues vs. normal tissues impacting clinical efficacy/toxicity. Drug optimization needs to balance the SAR and STR in selecting drug candidate for clinical trial to improve success of clinical drug development.

5.
JCI Insight ; 6(3)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33373329

RESUMO

Ginger is known to have antiinflammatory and antioxidative effects and has traditionally been used as an herbal supplement in the treatment of various chronic diseases. Here, we report antineutrophil properties of 6-gingerol, the most abundant bioactive compound of ginger root, in models of lupus and antiphospholipid syndrome (APS). Specifically, we demonstrate that 6-gingerol attenuates neutrophil extracellular trap (NET) release in response to lupus- and APS-relevant stimuli through a mechanism that is at least partially dependent on inhibition of phosphodiesterases. At the same time, administration of 6-gingerol to mice reduces NET release in various models of lupus and APS, while also improving other disease-relevant endpoints, such as autoantibody formation and large-vein thrombosis. In summary, this study is the first to our knowledge to demonstrate a protective role for ginger-derived compounds in the context of lupus. Importantly, it provides a potential mechanism for these effects via phosphodiesterase inhibition and attenuation of neutrophil hyperactivity.


Assuntos
Catecóis/farmacologia , Álcoois Graxos/farmacologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Animais , Anticorpos Antifosfolipídeos/biossíntese , Síndrome Antifosfolipídica/tratamento farmacológico , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Catecóis/sangue , Catecóis/farmacocinética , Modelos Animais de Doenças , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/imunologia , Álcoois Graxos/sangue , Álcoois Graxos/farmacocinética , Feminino , Humanos , Técnicas In Vitro , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Inibidores de Fosfodiesterase/sangue , Inibidores de Fosfodiesterase/farmacocinética , Inibidores de Fosfodiesterase/farmacologia , Fitoterapia , Espécies Reativas de Oxigênio/metabolismo , Trombose Venosa/tratamento farmacológico , Trombose Venosa/patologia
6.
J Med Chem ; 63(17): 9838-9855, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32809827

RESUMO

Redox modulators have been developed as an attractive approach to treat cancer. Herein, we report the synthesis, identification, and biological evaluation of a quinazolinedione reactive oxygen species (ROS) inducer, QD394, with significant cytotoxicity in pancreatic cancer cells. QD394 shows a transcriptomic profile remarkably similar to napabucasin, a cancer stemness inhibitor. Both small molecules inhibit STAT3 phosphorylation, increase cellular ROS, and decrease the GSH/GSSG ratio. Moreover, QD394 causes an iron- and ROS-dependent, GPX4 mediated cell death, suggesting ferroptosis as a major mechanism. Importantly, QD394 decreases the expression of LRPPRC and PNPT1, two proteins involved in mitochondrial RNA catabolic processes and both negatively correlated with the overall survival of pancreatic cancer patients. Pharmacokinetics-guided lead optimization resulted in the derivative QD394-Me, which showed improved plasma stability and reduced toxicity in mice compared to QD394. Overall, QD394 and QD394-Me represent novel ROS-inducing drug-like compounds warranting further development for the treatment of pancreatic cancer.


Assuntos
Antineoplásicos/farmacologia , Ferro/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Quinazolinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Benzofuranos/farmacologia , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Sinergismo Farmacológico , Feminino , Ferroptose/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Naftoquinonas/farmacologia , Quinazolinonas/síntese química , Quinazolinonas/farmacocinética , Quinazolinonas/toxicidade
7.
Sci Rep ; 10(1): 2990, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076076

RESUMO

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661 W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.


Assuntos
Apoptose/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Células Fotorreceptoras/efeitos dos fármacos , Piridazinas/farmacologia , Pirróis/farmacologia , Piruvato Quinase/metabolismo , Doenças Retinianas/tratamento farmacológico , Animais , Cegueira/etiologia , Cegueira/prevenção & controle , Linhagem Celular , Modelos Animais de Doenças , Ativadores de Enzimas/uso terapêutico , Glicólise/efeitos dos fármacos , Humanos , Injeções Intravítreas , Masculino , Camundongos , Camundongos Knockout , Células Fotorreceptoras/patologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Piridazinas/uso terapêutico , Pirróis/uso terapêutico , Piruvato Quinase/genética , Coelhos , Ratos , Doenças Retinianas/complicações , Doenças Retinianas/patologia
8.
J Med Chem ; 62(24): 11280-11300, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31747516

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor and an attractive therapeutic target for cancer and other human diseases. Despite 20 years of persistent research efforts, targeting STAT3 has been very challenging. We report herein the structure-based discovery of potent small-molecule STAT3 degraders based upon the proteolysis targeting chimera (PROTAC) concept. We first designed SI-109 as a potent, small-molecule inhibitor of the STAT3 SH2 domain. Employing ligands for cereblon/cullin 4A E3 ligase and SI-109, we obtained a series of potent PROTAC STAT3 degraders, exemplified by SD-36. SD-36 induces rapid STAT3 degradation at low nanomolar concentrations in cells and fails to degrade other STAT proteins. SD-36 achieves nanomolar cell growth inhibitory activity in leukemia and lymphoma cell lines with high levels of phosphorylated STAT3. A single dose of SD-36 results in complete STAT3 protein degradation in xenograft tumor tissue and normal mouse tissues. SD-36 achieves complete and long-lasting tumor regression in the Molm-16 xenograft tumor model at well-tolerated dose-schedules. SD-36 is a potent, selective, and efficacious STAT3 degrader.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Azocinas/química , Desenho de Fármacos , Descoberta de Drogas , Indóis/química , Indóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Organofosfonatos/química , Proteólise/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Animais , Antineoplásicos/farmacocinética , Apoptose , Azocinas/farmacocinética , Azocinas/farmacologia , Proliferação de Células , Feminino , Humanos , Indóis/farmacocinética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos SCID , Estrutura Molecular , Organofosfonatos/farmacocinética , Organofosfonatos/farmacologia , Conformação Proteica , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/química , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Curr Drug Metab ; 16(10): 894-910, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26652257

RESUMO

The liposome delivery system has been intensively explored as novel drug delivery system (DDS) for antitumor drugs, due to its safety, selective cytotoxicity, long circulation and slow elimination in blood, which is favorable for cancer therapy. The liposome-based chemotherapeutics are used to treat a variety of cancers to enhance the therapeutic index of antitumor drugs. Here, the author reviewed the important targets for cancer therapy and the pharmacokinetic behavior of liposomal drugs in vivo, as well as the application of the targeting liposomal system in cancer therapy. Considering further application for clinical use, the great challenges of the liposome-based delivery system were also proposed as follows: 1) prepare stealth liposome with steric stabilization and further enhance the therapeutic effects and safety; 2) explore more safe clinical targets and complementary or different types of targeting liposome; 3) thirdly, more investment is needed on the research of pharmacokinetics of the elements such as the ligands (antibody), PEG and lipids of liposome delivery system as well as safety evaluation. Considering the complex process of the liposomal encapsulation drugs in vivo, the author inferred that there are maybe different forms of the encapsulation drug to be internalized by the tumor tissues at the same time and space, although there are little reports on it.


Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Humanos , Ligantes , Lipídeos/química , Lipossomos , Polietilenoglicóis/química
10.
J Chromatogr A ; 1251: 63-73, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22770385

RESUMO

Therapeutic monoclonal antibodies (mAbs) constitute a group of highly effective agents for treating various refractory diseases. Nonetheless it is challenging to achieve selective and accurate quantification of mAb in pharmaceutical matrices, which is required by PK studies. Liquid chromatography/mass spectrometry under selected reaction monitoring mode (LC/SRM-MS) is emerging as an attractive alternative to immunoassays because of the high specificity and multiplexing capacity it provides, but may fall short in terms of sensitivity, reliability and quantitative accuracy. Moreover, the strategy for optimization of the MS conditions for many candidates of signature peptides (SP) and the selection of the optimal SP for quantification remains elusive. In this study, we employed a suite of technical advances to overcome these difficulties, which include: (i) a nano-LC/SRM-MS approach to achieve high analytical sensitivity, (ii) a high-resolution nano-LC/LTQ/Orbitrap for confident identification of candidate peptides, (iii) an on-the-fly orthogonal array optimization (OAO) method for the high-throughput, accurate and reproducible optimization for numerous candidate peptides in a single LC/MS run without using synthesized peptides, (iv) a comprehensive evaluation of stability of candidates in matrix using the optimized SRM parameters, (v) the use of two unique SP for quantification of one mAb to gauge possible degradation/modification in biological system and thus enhancing data reliability (e.g. rejection of data if the deviation between the two SP is greater than 25%) and (vi) the utilization of purified target protein as the calibrator to eliminate the risk of severe negative biases that could occur when a synthesized peptide is used as calibrator. To show a proof of concept, this strategy is applied in the quantification of cT84.66, a chimeric, anti-CEA antibody, in preclinical mouse models. A low detection limit of the mAb down to 3.2 ng/mL was achieved, which is substantially more sensitive than established immunoassay methods for anti-CEA antibodies. The quantitative method showed good linearity (within the range of 12.9 ng/mL to 32.3 µg/mL in plasma), accuracy and precision. Additionally, the ultra-low sample consumption (2 µL plasma per preparation) permits the acquisition of an entire set of time course data from the same mouse, which represents a prominent advantage for PK study using small-animal models. The developed method enabled an accurate PK investigation of cT84.66 in mice following intravenous and subcutaneous administrations at relatively low doses over an extended period of time. The strategy employed in this study can be easily adapted to the sensitive and accurate analysis of other mAb and therapeutic proteins.


Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/farmacocinética , Cromatografia Líquida/instrumentação , Modelos Lineares , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Estabilidade Proteica , Reprodutibilidade dos Testes , Projetos de Pesquisa , Sensibilidade e Especificidade
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