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The first-line treatment of advanced and metastatic human epidermal growth factor receptor type 2 (HER2+) breast cancer requires two HER2-targeting antibodies (trastuzumab and pertuzumab) and a taxane (docetaxel or paclitaxel). The three-drug regimen costs over $320,000 per treatment course, requires a 4 h infusion time, and has many adverse side effects, while achieving only 18 months of progression-free survival. To replace this regimen, reduce infusion time, and enhance efficacy, a single therapeutic is developed based on trastuzumab-conjugated nanoparticles for co-delivering docetaxel and siRNA against HER2 (siHER2). The optimal nanoconstruct has a hydrodynamic size of 100 nm and specifically treats HER2+ breast cancer cells over organ-derived normal cells. In a drug-resistant orthotopic HER2+ HCC1954 tumor mouse model, the nanoconstruct inhibits tumor growth more effectively than the docetaxel and trastuzumab combination. When coupled with microbubble-assisted focused ultrasound that transiently disrupts the blood brain barrier, the nanoconstruct inhibits the growth of trastuzumab-resistant HER2+ BT474 tumors residing in the brains of mice. The nanoconstruct has a favorable safety profile in cells and in mice. Combination therapies have become the cornerstone of cancer treatment and this versatile nanoparticle platform can co-deliver multiple therapeutic types to ensure that they reach the target cells at the same time to realize their synergy.
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Neoplasias Encefálicas , Neoplasias da Mama , Nanopartículas , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , RNA Interferente Pequeno , Receptor ErbB-2/genética , Taxoides/farmacologia , Taxoides/uso terapêutico , Trastuzumab/efeitos adversos , Trastuzumab/uso terapêuticoRESUMO
The prevailing view on post-translational modifications (PTMs) is that amino acid side chains in proteins are modified with a single PTM at any given time. However, a growing body of work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation, where specialized E3 ligases ubiquitylate targets for proteasomal degradation in an ADP-ribosylation-dependent manner. More recently, the DELTEX family of E3 ligases was reported to catalyze ubiquitylation of the 3'- hydroxy group of the adenine-proximal ribose of free NAD + and ADP-ribose in vitro , generating a non-canonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester (MARUbe). We term this process m ono- A DP-ribosyl ub iquit ylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that PARP10 MARUbylation is extended with K11-linked polyubiquitin chains. Finally, mechanistic studies using proteasomal and ubiquitin-activating enzyme inhibitors demonstrated that PARP10 MARUbylation leads to its proteasomal degradation, providing a functional role for this new PTM in regulating protein turnover.
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ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nico-tinamide adenine dinucleotide (NAD+) groups to proteins, is a conserved post-translational modification (PTM) that occurs most prominently in response to DNA damage. ADP-ribosylation is a dynamic PTM regulated by writers (PARPs), erasers (ADPr hy-drolases), and readers (ADPR binders). PARP1 is the primary DNA damage-response writer responsible for adding a polymer of ADPR to proteins (PARylation). Real-time monitoring of PARP1-mediated PARylation, especially in live cells, is critical for under-standing the spatial and temporal regulation of this unique PTM. Here, we describe a genetically encoded FRET probe (pARS) for semi-quantitative monitoring of PARylation dynamics. pARS feature a PAR-binding WWE domain flanked with turquoise and Venus. With a ratiometric readout and excellent signal-to-noise characteristics, we show that pARS can monitor PARP1-dependent PARylation temporally and spatially in real-time. pARS provided unique insights into PARP1-mediated PARylation kinetics in vitro and high-sensitivity detection of PARylation in live cells, even under mild DNA damage. We also show that pARS can be used to determine the potency of PARP inhibitors in vitro and, for the first time, in live cells in response to DNA damage. The robustness and ease of use of pARS make it an important tool for the PARP field.
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PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses.
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The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.
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Antineoplásicos , Interferon Tipo I , Ácidos Nucleicos , Animais , Camundongos , Fibroblastos , Transdução de SinaisRESUMO
PARP16-the sole ER-resident PARP family member-is gaining attention as a potential therapeutic target for cancer treatment. Nevertheless, the precise function of the catalytic activity of PARP16 is poorly understood. This is primarily due to the lack of inhibitors that are selective for PARP16 over other PARP family members. Herein, we describe a structure-guided strategy for generating a selective PARP16 inhibitor by incorporating two selectivity determinants into a phthalazinone pan-PARP inhibitor scaffold: (i) an acrylamide-based inhibitor (DB008) designed to covalently react with a non-conserved cysteine (Cys169, human numbering) in the NAD+ binding pocket of PARP16 and (ii) a dual-purpose ethynyl group designed to bind in a unique hydrophobic cavity adjacent to the NAD+ binding pocket as well as serve as a click handle. DB008 exhibits good selectivity for PARP16 versus other PARP family members. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) confirmed that covalent labeling of PARP16 by DB008 in cells is dependent on Cys169. DB008 exhibits excellent proteome-wide selectivity at concentrations required to achieve saturable labeling of endogenous PARP16. In-cell competition labeling experiments using DB008 provided a facile strategy for evaluating putative PARP16 inhibitors. Lastly, we found that PARP16 is sequestered into a detergent-insoluble fraction under prolonged amino acid starvation, and surprisingly, treatment with PARP16 inhibitors prevented this effect. These results suggest that the catalytic activity of PARP16 regulates its solubility in response to nutrient stress.
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Allosteric coupling between the DNA binding site to the NAD+-binding pocket drives PARP-1 activation. This allosteric communication occurs in the reverse direction such that NAD+ mimetics can enhance PARP-1's affinity for DNA, referred to as type I inhibition. The cellular effects of type I inhibition are unknown, largely because of the lack of potent, membrane-permeable type I inhibitors. Here we identify the phthalazinone inhibitor AZ0108 as a type I inhibitor. Unlike the structurally related inhibitor olaparib, AZ0108 induces replication stress in tumorigenic cells. Synthesis of analogs of AZ0108 revealed features of AZ0108 that are required for type I inhibition. One analog, Pip6, showed similar type I inhibition of PARP-1 but was â¼90-fold more cytotoxic than AZ0108. Washout experiments suggest that the enhanced cytotoxicity of Pip6 compared with AZ0108 is due to prolonged target residence time on PARP-1. Pip6 represents a new class of PARP-1 inhibitors that may have unique anticancer properties.
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Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/química , Regulação Alostérica , NAD/metabolismo , Antineoplásicos/farmacologia , Sítios de LigaçãoRESUMO
Immune checkpoint inhibitors (ICIs) targeting PD-L1 and PD-1 have improved survival in a subset of patients with advanced non-small cell lung cancer (NSCLC). However, only a minority of NSCLC patients respond to ICIs, highlighting the need for superior immunotherapy. Herein, we report on a nanoparticle-based immunotherapy termed ARAC (Antigen Release Agent and Checkpoint Inhibitor) designed to enhance the efficacy of PD-L1 inhibitor. ARAC is a nanoparticle co-delivering PLK1 inhibitor (volasertib) and PD-L1 antibody. PLK1 is a key mitotic kinase that is overexpressed in various cancers including NSCLC and drives cancer growth. Inhibition of PLK1 selectively kills cancer cells and upregulates PD-L1 expression in surviving cancer cells thereby providing opportunity for ARAC targeted delivery in a feedforward manner. ARAC reduces effective doses of volasertib and PD-L1 antibody by 5-fold in a metastatic lung tumor model (LLC-JSP) and the effect is mainly mediated by CD8+ T cells. ARAC also shows efficacy in another lung tumor model (KLN-205), which does not respond to CTLA-4 and PD-1 inhibitor combination. This study highlights a rational combination strategy to augment existing therapies by utilizing our nanoparticle platform that can load multiple cargo types at once.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Nanopartículas , Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Receptor de Morte Celular Programada 1RESUMO
PARP inhibitors (PARPis) display single-agent anticancer activity in small cell lung cancer (SCLC) and other neuroendocrine tumors independent of BRCA1/2 mutations. Here, we determine the differential efficacy of multiple clinical PARPis in SCLC cells. Compared with the other PARPis rucaparib, olaparib, and niraparib, talazoparib displays the highest potency across SCLC, including SLFN11-negative cells. Chemical proteomics identifies PARP16 as a unique talazoparib target in addition to PARP1. Silencing PARP16 significantly reduces cell survival, particularly in combination with PARP1 inhibition. Drug combination screening reveals talazoparib synergy with the WEE1/PLK1 inhibitor adavosertib. Global phosphoproteomics identifies disparate effects on cell-cycle and DNA damage signaling thereby illustrating underlying mechanisms of synergy, which is more pronounced for talazoparib than olaparib. Notably, silencing PARP16 further reduces cell survival in combination with olaparib and adavosertib. Together, these data suggest that PARP16 contributes to talazoparib's overall mechanism of action and constitutes an actionable target in SCLC.
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Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Idoso , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Ftalazinas/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Proteínas Tirosina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
The success of immunotherapy with immune checkpoint inhibitors (ICIs) in a subset of individuals has been very exciting. However, in many cancers, responses to current ICIs are modest and are seen only in a small subsets of patients. Herein, a widely applicable approach that increases the benefit of ICIs is reported. Intratumoral administration of augmenting immune response and inhibiting suppressive environment of tumors-AIRISE-02 nanotherapeutic that co-delivers CpG and STAT3 siRNA-results in not only regression of the injected tumor, but also tumors at distant sites in multiple tumor model systems. In particular, three doses of AIRISE-02 in combination with systemic ICIs completely cure both treated and untreated aggressive melanoma tumors in 63% of mice, while ICIs alone do not cure any mice. A long-term memory immune effect is also reported. AIRISE-02 is effective in breast and colon tumor models as well. Lastly, AIRISE-02 is well tolerated in mice and nonhuman primates. This approach combines multiple therapeutic agents into a single nanoconstruct to create whole-body immune responses across multiple cancer types. Being a local therapeutic, AIRISE-02 circumvents regulatory challenges of systemic nanoparticle delivery, facilitating rapid translation to the clinic. AIRISE-02 is under investigational new drug (IND)-enabling studies, and clinical trials will soon follow.
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Imunoterapia , Nanopartículas , RNA Interferente Pequeno , Animais , Camundongos , VacinaçãoRESUMO
Gadolinium based contrast agents (GBCAs) have been linked to toxicity in patients, regardless of having impaired or normal renal function. Currently, no therapy is considered highly effective for removing gadolinium (Gd) from the body. We propose a new strategy to reduce blood Gd content that facilitates whole body removal of Gd using a hemoperfusion system consisting of a cartridge of porous silica beads (Davisil®) functionalized with 1,2-hydroxypyridinone (1,2-HOPO). Herein, we report optimization of the hemoperfusion system using an ex vivo blood and an in vivo rat model of chronic kidney disease (CKD). In our ex vivo system, 1,2-HOPO-Davisil outperformed Gambro activated charcoal (AC), which is commonly used in clinical hemoperfusion of aqueous toxins, in terms of Gd capture capacity and rate. In the CKD rat model, the 1,2-HOPO-Davisil hemoperfusion system removed Gd by 3.4 times over the Gambro AC system. 1,2-HOPO-Davisil did not change complete blood counts and common blood biochemistry. Thus, this strategy has great potential for clinical translation to manage GBCAs after magnetic resonance imaging (MRI), before Gd can deposit in the body and cause long-term toxicity. Although gadodiamide was used as a proof of concept model for GBCAs in this study, 1,2-HOPO functionalized mesoporous silica could also capture dissociated Gd and other GBCAs.
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Adenina/toxicidade , Meios de Contraste/isolamento & purificação , Modelos Animais de Doenças , Hipersensibilidade a Drogas/prevenção & controle , Hemoperfusão/métodos , Compostos Organometálicos/isolamento & purificação , Insuficiência Renal Crônica/prevenção & controle , Animais , Meios de Contraste/efeitos adversos , Gadolínio DTPA/efeitos adversos , Gadolínio DTPA/isolamento & purificação , Testes de Função Renal , Imageamento por Ressonância Magnética , Masculino , Compostos Organometálicos/efeitos adversos , Ratos , Ratos Wistar , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/patologia , Dióxido de Silício/químicaRESUMO
Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR + NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR + cancer types such as colorectal and breast cancer is also demonstrated.
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Carcinoma Pulmonar de Células não Pequenas/terapia , Cetuximab/administração & dosagem , Neoplasias Pulmonares/terapia , RNA Interferente Pequeno/administração & dosagem , Radiossensibilizantes/administração & dosagem , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/farmacologia , Receptores ErbB/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pulmonares/genética , Masculino , Terapia de Alvo Molecular , Nanopartículas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Radiossensibilizantes/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
INTRODUCTION: Long-term stability of therapeutic candidates is necessary toward their clinical applications. For most nanoparticle systems formulated in aqueous solutions, lyophilization or freeze-drying is a common method to ensure long-term stability. While lyophilization of lipid, polymeric, or inorganic nanoparticles have been studied, little has been reported on lyophilization and stability of hybrid nanoparticle systems, consisting of polymers, inorganic particles, and antibody. Lyophilization of complex nanoparticle systems can be challenging with respect to preserving physicochemical properties and the biological activities of the materials. We recently reported an effective small-interfering RNA (siRNA) nanoparticle carrier consisting of 50-nm mesoporous silica nanoparticles decorated with a copolymer of polyethylenimine and polyethyleneglycol, and antibody. MATERIALS AND METHODS: Toward future personalized medicine, the nanoparticle carriers were lyophilized alone and loaded with siRNA upon reconstitution by a few minutes of simple mixing in phosphate-buffered saline. Herein, we optimize the lyophilization of the nanoparticles in terms of buffers, lyoprotectants, reconstitution, and time and temperature of freezing and drying steps, and monitor the physical and chemical properties (reconstitution, hydrodynamic size, charge, and siRNA loading) and biological activities (gene silencing, cancer cell killing) of the materials after storing at various temperatures and times. RESULTS: The material was best formulated in Tris-HCl buffer with 5% w/w trehalose. Freezing step was performed at -55°C for 3 h, followed by a primary drying step at -40°C (100 µBar) for 24 h and a secondary drying step at 20°C (20 µBar) for 12 h. The lyophilized material can be stored stably for 2 months at 4°C and at least 6 months at -20°C. CONCLUSION: We successfully developed the lyophilization process that should be applicable to other similar nanoparticle systems consisting of inorganic nanoparticle cores modified with cationic polymers, PEG, and antibodies.