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1.
Histochem Cell Biol ; 162(1-2): 161-183, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38758429

RESUMO

The nucleolus is the largest membraneless organelle and nuclear body in mammalian cells. It is primarily involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and accounts for the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the substructural mechanistic principles of the nucleolar function in preribosome biogenesis have only recently begun to emerge. Here, we provide a new perspective using advanced super-resolution microscopy and single-molecule MINFLUX nanoscopy on the mechanistic principles governing ribosomal RNA-seeded nucleolar formation and the resulting tripartite suborganization of the nucleolus driven, in part, by liquid-liquid phase separation. With recent advances in the cryogenic electron microscopy (cryoEM) structural analysis of ribosome biogenesis intermediates, we highlight the current understanding of the step-wise assembly of preribosomal subunits in the nucleolus. Finally, we address how novel anticancer drug candidates target early steps in ribosome biogenesis to exploit these essential dependencies for growth arrest and tumor control.


Assuntos
Nucléolo Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/química , Humanos , Ribossomos/metabolismo , Ribossomos/química , Microscopia , Animais
2.
Cancers (Basel) ; 15(8)2023 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-37190294

RESUMO

Triple-negative breast carcinoma (TNBC) is one of the most aggressive types of solid-organ cancers. While immune checkpoint blockade (ICB) therapy has significantly improved outcomes in certain types of solid-organ cancers, patients with immunologically cold TNBC are afforded only a modest gain in survival by the addition of ICB to systemic chemotherapy. Thus, it is urgently needed to develop novel effective therapeutic approaches for TNBC. Utilizing the 4T1 murine model of TNBC, we developed a novel combination immunotherapeutic regimen consisting of intratumoral delivery of high-mobility group nucleosome binding protein 1 (HMGN1), TLR2/6 ligand fibroblast-stimulating lipopeptide (FSL-1), TLR7/8 agonist (R848/resiquimod), and CTLA-4 blockade. We also investigated the effect of adding SX682, a small-molecule inhibitor of CXCR1/2 known to reduce MDSC trafficking to tumor microenvironment, to our therapeutic approach. 4T1-bearing mice responded with significant tumor regression and tumor elimination to our therapeutic combination regimen. Mice with complete tumor regressions did not recur and became long-term survivors. Treatment with HMGN1, FSL-1, R848, and anti-CTLA4 antibody increased the number of infiltrating CD4+ and CD8+ effector/memory T cells in both tumors and draining lymph nodes and triggered the generation of 4T1-specific cytotoxic T lymphocytes (CTLs) in the draining lymph nodes. Thus, we developed a potentially curative immunotherapeutic regimen consisting of HMGN1, FSL-1, R848, plus a checkpoint inhibitor for TNBC, which does not rely on the administration of chemotherapy, radiation, or exogenous tumor-associated antigen(s).

3.
Cells ; 12(9)2023 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-37174654

RESUMO

Activated M2-polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios, including Idiopathic Pulmonary Fibrosis (IPF). In this study, we investigated the effects of targeting the CD206 receptor in M2-like macrophages with a novel synthetic analogue of a naturally occurring Host Defense Peptide (HDP), RP-832c, to decrease profibrotic cytokines. RP-832c selectively binds to CD206 on M2-polarized bone marrow-derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression and a transient increase in M1-macrophage marker TNF-α. To elucidate the antifibrotic effects of RP-832c, we used a murine model of bleomycin (BLM)-induced early-stage pulmonary fibrosis. RP-832c significantly reduced fibrosis in a dose-dependent manner, and decreased CD206, TGF-ß1, and α-SMA expression in mouse lungs. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased lung fibrosis and significantly decreased inflammatory cytokines TNF-α, IL-6, IL-10, IFN-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with the FDA-approved drugs Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed. In summary, our findings showed that inhibiting the profibrotic alternatively activated M2-like macrophages using a novel peptide, RP-832c, could reduce BLM-induced pulmonary fibrosis in mice, warranting the therapeutic potential of this peptide for patients with pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática , Fator de Crescimento Transformador beta1 , Animais , Camundongos , Bleomicina/efeitos adversos , Citocinas , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fator de Necrose Tumoral alfa
4.
Artigo em Inglês | MEDLINE | ID: mdl-37196527

RESUMO

Metarrestin is a first-in-class small molecule inhibitor targeting the perinucleolar compartment, a subnuclear body associated with metastatic capacity. Promising preclinical results led to the clinical translation of the compound into a first-in-human phase I trial (NCT04222413). To characterize metarrestin's pharmacokinetic profile in humans, a uHPLC-MS/MS assay was developed and validated to determine the disposition of the drug in human plasma. Efficient sample preparation was accomplished through one-step protein precipitation paired with elution through a phospholipid filtration plate. Chromatographic separation was achieved with gradient elution through an Acuity UPLC® BEH C18 column (50 × 2.1 mm, 1.7 µm). Tandem mass spectrometry facilitated the detection of metarrestin and tolbutamide, the internal standard. The effective calibration range spanned 1-5000 ng/mL and was both accurate (range -5.9 % to 4.9 % deviation) and precise (≤9.0 %CV). Metarrestin proved stable (≤4.9 % degradation) under various assay-imposed conditions. Matrix effects, extraction efficiency, and process efficiency were assessed. Further, the assay was successfully able to determine the disposition of orally administered metarrestin in patients from the lowest dose cohort (1 mg) for 48 h post-administration. Thus, the validated analytical method detailed in this work is simple, sensitive, and clinically applicable.


Assuntos
Pirimidinas , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Pirimidinas/farmacocinética , Pirróis/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Reprodutibilidade dos Testes
5.
Mol Cancer Res ; 21(4): 316-331, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36790955

RESUMO

Combinatorial molecular therapy in pancreatic ductal adenocarcinoma (PDAC) has yielded largely disappointing results in clinical testing to-date as a multitude of adaptive resistance mechanisms is making selection of patients via molecular markers that capture essential, intersecting signaling routes challenging. Here, we report the scaffolding protein connector enhancer of kinase suppressor of Ras 1 (CNKSR1) as mediator of resistance to MAPK (MEK) inhibition. MEK inhibition in CNKSR1high cancer cells induces translocation of CNKSR1 to the plasma membrane where the scaffolding protein interacts with and stabilizes the phosphorylated form of AKT. CNKSR1-mediated AKT activation following MEK inhibition was associated with increased cellular p-PRAS40 levels and reduced nuclear translocation and cellular levels of FoxO1, a negative regulator of AKT signaling. In clinical PDAC specimens, high cytoplasmatic CNKSR1 levels correlated with increased cellular phospho-AKT and mTOR levels. Pharmacological co-blockade of AKT and MEK ranked top in induced synergies with MEK inhibition in CNKSR1high pancreas cancer cells among other inhibitor combinations targeting known CNKSR1 signaling. In vivo, CNKSR1high pancreatic tumors treated with AKT and MEK inhibitors showed improved outcome in the combination arm compared with single-agent treatment, an effect not observed in CNKSR1low models.Our results identify CNKSR1 as regulator of adaptive resistance to MEK inhibition by promoting crosstalk to AKT signaling via a scaffolding function for the phosphorylated form of AKT. CNSKR1 expression might be a possible molecular marker to enrich patients for future AKT-MEK inhibitor precision medicine studies. IMPLICATIONS: The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , Linhagem Celular Tumoral , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pancreáticas
6.
Expert Opin Emerg Drugs ; 27(3): 345-368, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36250721

RESUMO

INTRODUCTION: Pancreatic cancer is one of the deadliest solid organ cancers. In the absence of specific warning symptoms pancreatic cancer is diagnosed notoriously late. Current systemic chemotherapy regimens extend survival by a mere few months. With the advances in genetic, proteomic, and immunological profiling there is strong rationale to test kinase inhibitors to improve outcome. AREAS COVERED: This review article provides a comprehensive summary of approved treatments and past, present, and future developments of kinase inhibitors in pancreatic cancer. Emerging roles of protein kinase inhibitors are discussed in the context of the unique stroma, the lack of high-prevalence therapeutic targets and rapid emergence of acquired resistance, novel immuno-oncology kinase targets, and recent medicinal chemistry advances. EXPERT OPINION: Due to the to-date frequent failure of protein kinase inhibitors indiscriminately administered to unselected pancreatic cancer patients, there is a shift toward the development of these agents in molecularly defined subgroups which are more likely to respond. The development of accurate biomarkers to select patients who are the best candidates based on a detailed understanding of mechanism of action, pro-survival roles, and mediation of resistance of targeted kinases will be critical for the future development of protein kinase inhibitors in this disease.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteômica , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Pancreáticas
7.
J Med Chem ; 65(12): 8303-8331, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35696646

RESUMO

The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. Structure-activity relationship exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human phase 1 clinical trial.


Assuntos
Núcleo Celular , Neoplasias , Biomarcadores/metabolismo , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Núcleo Celular/metabolismo , Humanos , Neoplasias/metabolismo , Pirimidinas , Pirróis
8.
J Clin Transl Sci ; 6(1): e66, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35754433

RESUMO

There are numerous examples of translational science innovations addressing challenges in the translational process, accelerating progress along the translational spectrum, and generating solutions relevant to a wide range of human health needs. Examining these successes through an education lens can identify core principles and effective practices that lead to successful translational outcomes. The National Center for Advancing Translational Sciences (NCATS) is identifying and teaching these core principles and practices to a broad audience via online courses in translational science which teach from case studies of NCATS-led or supported research initiatives. In this paper, we share our approach to the design of these courses and offer a detailed description of our initial course, which focused on a preclinical drug discovery and development project spanning academic and government settings. Course participants were from a variety of career stages and institutions. Participants rated the course high in overall value to them and in providing a unique window into the translational science process. We share our model for course development as well as initial findings from the course evaluation with the goal of continuing to stimulate development of novel education activities teaching foundational principles in translational science to a broad audience.

9.
Mol Cancer Ther ; 20(10): 1926-1940, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376576

RESUMO

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFß antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFß inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFß-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFß inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFß inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFß inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFß inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/imunologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Células Estromais/imunologia , Microambiente Tumoral , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Terapia Combinada , Desoxicitidina/farmacologia , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
10.
J Cancer Treatment Diagn ; 5(1): 30-35, 2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-34079928

RESUMO

Cancer stem cells (CSCs) are subpopulations of tumor cells that possess abilities for self-renewal, differentiation, and tumor initiation. These rare but therapy-recalcitrant cells are assumed to repopulate tumors following administration of systemic chemotherapy driving therapy failure, tumor recurrence, and disease progression. In early clinical trials, anti-CSC therapies have found limited success to-date possibly due to the inherent heterogeneity and plasticity of CSCs and the incomplete characterization of essential CSC targets. Here, we review the role of 3-phosphoinositide dependent protein kinase-1 (PDPK1) as an emerging CSC target. While most previous studies have relied on CSC models which are based on lineage and tissue-specific marker profiles to define the relationships between putative target and CSC traits, this review discusses PDPK1 and its role in CSC biology with an emphasis on CSC systems which are based on proposed function like label-retaining cancer cells (LRCCs).

11.
Invest New Drugs ; 39(3): 821-828, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33405090

RESUMO

Background Preclinical evidence has suggested that a subset of pancreatic cancers with the G12R mutational isoform of the KRAS oncogene is more sensitive to MAPK pathway blockade than pancreatic tumors with other KRAS isoforms. We conducted a biomarker-driven trial of selumetinib (KOSELUGO™; ARRY-142886), an orally active, allosteric mitogen-activated protein kinase 1 and 2 (MEK1/2) inhibitor, in pancreas cancer patients with somatic KRASG12R mutations. Methods In this two-stage, phase II study (NCT03040986) patients with advanced pancreas cancer harboring somatic KRASG12R variants who had received at least one standard-of-care systemic therapy regimen received 75 mg selumetinib orally twice a day until disease progression or unacceptable toxicity occurred. The primary outcome of the study was best objective response (BOR). Results From August 2017 to February 2018 a total of 8 patients with confirmed somatic KRASG12R mutations and a median age of 61.5 years were treated with selumetinib. Seven out of eight (87.5%) had received two or more lines of prior systemic chemotherapy. After a median follow-up period of 8.5 months (range 2 to 20), three patients had stable disease for more than 6 months while receiving selumetinib. No patients achieved an objective partial response. Median progression-free survival (PFS) was 3.0 months (95% CI, 0.8-8.2) and median overall survival (OS) 9 months (95% CI, 2.5-20.9). Conclusion This study in heavily pre-treated pancreatic adenocarcinoma patients suggests alternative strategies beyond single agent MEK inhibition are required for this unique, molecular subset of pancreatic cancer patients. The trial was registered on February 2nd, 2017 under identifier NCT03040986 with ClinicalTrials.gov .


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Adenocarcinoma/mortalidade , Administração Oral , Idoso , Antineoplásicos/efeitos adversos , Benzimidazóis/efeitos adversos , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/mortalidade , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/efeitos adversos
12.
bioRxiv ; 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32766584

RESUMO

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro , resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-ß1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.

13.
Regul Toxicol Pharmacol ; 116: 104716, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32619635

RESUMO

Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50 mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3 h (overall median Tmax) across all doses with a mean t1/2 over 168 h of 55.5 h. Dose dependent increase in systemic exposure (Cmax and AUClast) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female Cmax = 82.5 ng/mL and AUClast = 2521 h*ng/mL, on Day 27.


Assuntos
Antineoplásicos , Pirimidinas , Pirróis , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Cães , Avaliação Pré-Clínica de Medicamentos , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Nível de Efeito Adverso não Observado , Neoplasias Pancreáticas/tratamento farmacológico , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Pirimidinas/toxicidade , Pirróis/administração & dosagem , Pirróis/farmacocinética , Pirróis/toxicidade
14.
Cancer Chemother Pharmacol ; 85(4): 805-816, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32185484

RESUMO

PURPOSE: Metarrestin is a first-in-class pyrrolo-pyrimidine-derived small molecule targeting a marker of genome organization associated with metastasis and is currently in preclinical development as an anti-cancer agent. Here, we report the in vitro ADME characteristics and in vivo pharmacokinetic behavior of metarrestin. METHODS: Solubility, permeability, and efflux ratio as well as in vitro metabolism of metarrestin in hepatocytes, liver microsomes and S9 fractions, recombinant cytochrome P450 (CYP) enzymes, and potential for CYP inhibition were evaluated. Single dose pharmacokinetic profiles after intravenous and oral administration in mice, rat, dog, monkey, and mini-pig were obtained. Simple allometric scaling was applied to predict human pharmacokinetics. RESULTS: Metarrestin had an aqueous solubility of 150 µM at pH 7.4, high permeability in PAMPA and moderate efflux ratio in Caco-2 assays. The compound was metabolically stable in liver microsomes, S9 fractions, and hepatocytes from six species, including human. Metarrestin is a CYP3A4 substrate and, in mini-pigs, is also directly glucuronidated. Metarrestin did not show cytochrome P450 inhibitory activity. Plasma concentration-time profiles showed low to moderate clearance, ranging from 0.6 mL/min/kg in monkeys to 48 mL/min/kg in mice and moderate to high volume of distribution, ranging from 1.5 L/kg in monkeys to 17 L/kg in mice. Metarrestin has greater than 80% oral bioavailability in all species tested. The excretion of unchanged parent drug in urine was < 5% in dogs and < 1% in monkeys over collection periods of ≥ 144 h; in bile-duct cannulated rats, the excretion of unchanged drug was < 1% in urine and < 2% in bile over a collection period of 48 h. CONCLUSIONS: Metarrestin is a low clearance compound which has good bioavailability and large biodistribution after oral administration. Biotransformation appears to be the major elimination process for the parent drug. In vitro data suggest a low drug-drug interaction potential on CYP-mediated metabolism. Overall favorable ADME and PK properties support metarrestin's progression to clinical investigation.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Microssomos Hepáticos/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Pirróis/administração & dosagem , Pirróis/farmacocinética , Administração Oral , Animais , Biotransformação , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Feminino , Haplorrinos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Suínos , Porco Miniatura , Distribuição Tecidual
15.
Sci Transl Med ; 12(530)2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051227

RESUMO

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.


Assuntos
Lectinas de Ligação a Manose , Macrófagos Associados a Tumor , Animais , Linhagem Celular Tumoral , Humanos , Imunidade Inata , Lectinas Tipo C , Receptor de Manose , Camundongos , Receptores de Superfície Celular
16.
J Pharm Biomed Anal ; 181: 113093, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31931447

RESUMO

Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1 × 100 mm, 3.5 µm XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1 ng to 1000 ng in mouse plasma with the lower limit of detection for RP-182 at 1 ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract.


Assuntos
Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Animais , Peptídeos Catiônicos Antimicrobianos/sangue , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Ácidos Carboxílicos/química , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Grelina/sangue , Grelina/química , Grelina/isolamento & purificação , Limite de Detecção , Fenômenos Magnéticos , Camundongos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
17.
Cancer Discov ; 10(1): 104-123, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31649109

RESUMO

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. SIGNIFICANCE: We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Carcinoma Ductal Pancreático/patologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Mutação , Neoplasias Pancreáticas/patologia , Pinocitose , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Mol Cancer Ther ; 18(11): 2097-2110, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31395684

RESUMO

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell-like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine-based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.


Assuntos
Imidazóis/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis/química , Imidazóis/farmacologia , Camundongos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/enzimologia , Neoplasias Pancreáticas/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Med Genet ; 56(6): 370-379, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30745422

RESUMO

INTRODUCTION: Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome associated with variants in E-cadherin (CDH1), diffuse gastric cancer and lobular breast cancer. There is considerable heterogeneity in its clinical manifestations. This study aimed to determine associations between CDH1 germline variant status and clinical phenotypes of HDGC. METHODS: One hundred and fifty-two HDGC families, including six previously unreported families, were identified. CDH1 gene-specific guidelines released by the Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel were applied for pathogenicity classification of truncating, missense and splice site CDH1 germline variants. We evaluated ORs between location of truncating variants of CDH1 and incidence of colorectal cancer, breast cancer and cancer at young age (gastric cancer at <40 or breast cancer <50 years of age). RESULTS: Frequency of truncating germline CDH1 variants varied across functional domains of the E-cadherin receptor gene and was highest in linker (0.05785 counts/base pair; p=0.0111) and PRE regions (0.10000; p=0.0059). Families with truncating CDH1 germline variants located in the PRE-PRO region were six times more likely to have family members affected by colorectal cancer (OR 6.20, 95% CI 1.79 to 21.48; p=0.004) compared with germline variants in other regions. Variants in the intracellular E-cadherin region were protective for cancer at young age (OR 0.2, 95% CI 0.06 to 0.64; p=0.0071) and in the linker regions for breast cancer (OR 0.35, 95% CI 0.12 to 0.99; p=0.0493). Different CDH1 genotypes were associated with different intracellular signalling activation levels including different p-ERK, p-mTOR and ß-catenin levels in early submucosal T1a lesions of HDGC families with different CDH1 variants. CONCLUSION: Type and location of CDH1 germline variants may help to identify families at increased risk for concomitant cancers that might benefit from individualised surveillance and intervention strategies.


Assuntos
Antígenos CD/genética , Caderinas/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Fenótipo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Alelos , Processamento Alternativo , Antígenos CD/química , Antígenos CD/metabolismo , Caderinas/química , Caderinas/metabolismo , Éxons , Família , Humanos , Imuno-Histoquímica , Mutação de Sentido Incorreto , Razão de Chances , Linhagem , Transdução de Sinais , Neoplasias Gástricas/metabolismo
20.
Clin Exp Gastroenterol ; 11: 447-459, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30584346

RESUMO

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is a recently described, rare gastric polyposis syndrome. It is characterized by extensive involvement of the fundus and body of the stomach with fundic gland polyps sparing the antrum and lesser curvature, an autosomal dominant inheritance pattern with incomplete penetrance, and a significant predisposition for the development of gastric adenocarcinoma. Due to the recent discovery of APC promotor IB mutations (c.-191T>C, c.-192A>G, and c.-195A>C), which reduce binding of the transcription factor Yin Yang 1 (YY1) and transcriptional activity of the promotor, as its underlying genetic perturbation, GAPPS has been added to the growing molecular class of APC-associated disorders. Recent reports on family members afflicted by gastric polyposis due to GAPPS have described the development of metastatic cancer or the presence of invasive gastric adenocarcinoma in total gastrectomy specimens after variable periods of endoscopic surveillance emphasizing the need for an improved understanding of the to-date poorly characterized natural history of the syndrome. There are, however, currently no guidelines on screening, timing of prophylactic gastrectomy, or endoscopic surveillance for GAPPS available. In this review, we summarize the clinical, pathological, and genetic aspects of GAPPS as well as management approaches to this rare cancer predisposition syndrome, highlighting the need for early recognition, a multidisciplinary approach, and the creation of prospective family registries and consensus guidelines in the near future.

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