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1.
Nat Chem Biol ; 19(6): 731-739, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36759751

RESUMO

Bioluminescence imaging (BLI) allows non-invasive visualization of cells and biochemical events in vivo and thus has become an indispensable technique in biomedical research. However, BLI in the central nervous system remains challenging because luciferases show relatively poor performance in the brain with existing substrates. Here, we report the discovery of a NanoLuc substrate with improved brain performance, cephalofurimazine (CFz). CFz paired with Antares luciferase produces greater than 20-fold more signal from the brain than the standard combination of D-luciferin with firefly luciferase. At standard doses, Antares-CFz matches AkaLuc-AkaLumine/TokeOni in brightness, while occasional higher dosing of CFz can be performed to obtain threefold more signal. CFz should allow the growing number of NanoLuc-based indicators to be applied to the brain with high sensitivity. Using CFz, we achieve video-rate non-invasive imaging of Antares in brains of freely moving mice and demonstrate non-invasive calcium imaging of sensory-evoked activity in genetically defined neurons.


Assuntos
Diagnóstico por Imagem , Medições Luminescentes , Camundongos , Animais , Medições Luminescentes/métodos , Encéfalo/diagnóstico por imagem , Luciferina de Vaga-Lumes , Luciferinas
2.
Mol Pharmacol ; 2024 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-39322411

RESUMO

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish where expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disrpution. We transfected HEK-293 cells with both NanoLuc and human ABCB1 or ABCG2, or their zebrafish homologs Abcb4 and Abcg2a, which are functionally homologous to human P-gp and ABCG2, respectively, and expressed at the zebrafish BBB. We evaluated the brightness of ten NanoLuc substrates, then screened the eight brightest for their ability to be effluxed by the ABC transporters. We identified one ABCB1 substrate, two Abcb4 substrates, six ABCG2 substrates, and four Abcg2a substrates. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

3.
Nat Methods ; 17(8): 852-860, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661427

RESUMO

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.


Assuntos
Furanos/química , Luciferases/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Animais , Ensaios Enzimáticos/métodos , Especificidade por Substrato
4.
Int J Mol Sci ; 21(16)2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32824188

RESUMO

Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.


Assuntos
Furanos/farmacocinética , Imidazóis/farmacocinética , Substâncias Luminescentes/farmacocinética , Proteínas Luminescentes/genética , Imagem Óptica/métodos , Pirazinas/farmacocinética , Viroses/diagnóstico por imagem , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Furanos/administração & dosagem , Células HEK293 , Humanos , Imidazóis/administração & dosagem , Injeções Intraperitoneais , Substâncias Luminescentes/administração & dosagem , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Vírus Oncolíticos/genética , Pirazinas/administração & dosagem , Proteínas Recombinantes/genética
5.
Org Biomol Chem ; 15(40): 8559-8567, 2017 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-28972606

RESUMO

We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc. Finally, we show that this new bioluminescent system is capable of efficient BRET to far-red fluorophores. We anticipate these new principles of NanoLuc substrate design will impact applications that depend on shifting the colour of emission to the red, most notably in vivo bioluminescent imaging.


Assuntos
Imidazóis/química , Luciferases/química , Substâncias Luminescentes/química , Pirazinas/química , Imidazóis/metabolismo , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Estrutura Molecular , Pirazinas/metabolismo
6.
Chemistry ; 22(30): 10369-75, 2016 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-27305599

RESUMO

The growing popularity of bioluminescent assays has highlighted the need for coelenterazine analogues possessing properties tuned for specific applications. However, the structural diversity of known coelenterazine analogues has been limited by current syntheses. Known routes for the preparation of coelenterazine analogues employ harsh reaction conditions that limit access to many substituents and functional groups. Novel synthetic routes reported here establish simple and robust methods for synthesis and investigation of structurally diverse marine luciferase substrates. Specifically, these new routes allow synthesis of coelenterazine analogues containing various heterocyclic motifs and substituted aromatic groups with diverse electronic substituents at the R(2) position. Interesting analogues described herein were characterized by their physicochemical properties, bioluminescent half-life, light output, polarity and cytotoxicity. Some of the analogues represent leads that can be utilized in the development of improved bioluminescent systems.

7.
iScience ; 27(9): 110655, 2024 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-39252965

RESUMO

The ability to visualize and track multiple biological processes in vivo in real time is highly desirable. Bioluminescence imaging (BLI) has emerged as an attractive modality for non-invasive cell tracking, with various luciferase reporters enabling parallel monitoring of several processes. However, simultaneous multiplexed imaging in vivo is challenging due to suboptimal reporter intensities and the need to image one luciferase at a time. We report a multiplexed BLI approach using a single substrate that leverages bioluminescence resonance energy transfer (BRET)-based reporters with distinct spectral profiles for triple-color BLI. These luciferase-fluorophore fusion reporters address light transmission challenges and use optimized coelenterazine substrates. Comparing BRET reporters across two substrate analogs identified a green-yellow-orange combination that allows simultaneous imaging of three distinct cell populations in vitro and in vivo. These tools provide a template for imaging other biological processes in vivo during a single BLI session using a single reporter substrate.

8.
bioRxiv ; 2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37986908

RESUMO

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, and expressed at the zebrafish BBB. We evaluated the luminescence of ten NanoLuc substrates, then screened the eight brightest to determine which are most efficiently effluxed by the ABC transporters. We identified one substrate efficiently pumped out by ABCB1, two by Abcb4, six by ABCG2, and four by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter.

9.
ACS Cent Sci ; 9(4): 719-732, 2023 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-37122464

RESUMO

Aberrant kinase activity contributes to the pathogenesis of brain cancers, neurodegeneration, and neuropsychiatric diseases, but identifying kinase inhibitors that function in the brain is challenging. Drug levels in blood do not predict efficacy in the brain because the blood-brain barrier prevents entry of most compounds. Rather, assessing kinase inhibition in the brain requires tissue dissection and biochemical analysis, a time-consuming and resource-intensive process. Here, we report kinase-modulated bioluminescent indicators (KiMBIs) for noninvasive longitudinal imaging of drug activity in the brain based on a recently optimized luciferase-luciferin system. We develop an ERK KiMBI to report inhibitors of the Ras-Raf-MEK-ERK pathway, for which no bioluminescent indicators previously existed. ERK KiMBI discriminates between brain-penetrant and nonpenetrant MEK inhibitors, reveals blood-tumor barrier leakiness in xenograft models, and reports MEK inhibitor pharmacodynamics in native brain tissues and intracranial xenografts. Finally, we use ERK KiMBI to screen ERK inhibitors for brain efficacy, identifying temuterkib as a promising brain-active ERK inhibitor, a result not predicted from chemical characteristics alone. Thus, KiMBIs enable the rapid identification and pharmacodynamic characterization of kinase inhibitors suitable for treating brain diseases.

10.
Adv Genet (Hoboken) ; 3(1): 2100055, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36619349

RESUMO

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. This study focuses on a novel method to image EV subtypes and their biodistribution in vivo. A red-shifted bioluminescence resonance energy transfer (BRET) EV reporter is developed, called PalmReNL, which allows for highly sensitive EV tracking in vitro and in vivo. PalmReNL enables the authors to study the common surface molecules across EV subtypes that determine EV organotropism and their functional differences in cancer progression. Regardless of injection routes, whether retro-orbital or intraperitoneal, PalmReNL positive EVs, isolated from murine mammary carcinoma cells, localized to the lungs. The early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple intraperitoneal injections of the medium and large EV (m/lEV)-enriched fraction derived from mammary carcinoma cells is demonstrated. In addition, the results presented here show that tumor cell-derived m/lEVs act on distant tissues through upregulating LC3 expression within the lung.

11.
Nat Commun ; 13(1): 2073, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440107

RESUMO

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12F36V (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.


Assuntos
Benchmarking , Proteína 1A de Ligação a Tacrolimo , Luciferases , Proteólise , Proteína 1A de Ligação a Tacrolimo/genética
12.
Viruses ; 14(3)2022 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-35336942

RESUMO

Animal models recapitulating COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Intranasally inoculated transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. We evaluated the clinical and virological dynamics of SARS-CoV-2 using two intranasal doses (104 and 106 PFUs), with a detailed spatiotemporal pathologic analysis of the 106 dose cohort. Despite generally mild-to-moderate pneumonia, clinical decline resulting in euthanasia or death was commonly associated with hypothermia and viral neurodissemination independent of inoculation dose. Neuroinvasion was first observed at 4 days post-infection, initially restricted to the olfactory bulb suggesting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. Absence of viremia suggests neuroinvasion occurs independently of transport across the blood-brain barrier. SARS-CoV-2 tropism was neither restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), nor inclusive of some ACE2-positive cell lineages (e.g., bronchiolar epithelium and brain vasculature). Absence of detectable ACE2 protein expression in neurons but overexpression in neuroepithelium suggest this as the most likely portal of neuroinvasion, with subsequent ACE2 independent lethal neurodissemination. A paucity of epidemiological data and contradicting evidence for neuroinvasion and neurodissemination in humans call into question the translational relevance of this model.


Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Animais , Humanos , Queratina-18 , Melfalan , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , Tropismo Viral , gama-Globulinas
13.
J Photochem Photobiol B ; 216: 112128, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33529963

RESUMO

NanoLuc luciferase recently gained popularity due to its small size and superior bioluminescence performance. For in vivo imaging applications, NanoLuc has been limited by its substrate furimazine, which has low solubility and bioavailability. Herein, we compared the performances of recently reported NanoLuc luciferase substrates for in vivo imaging in mice. Two substrates with improved aqueous solubility, hydrofurimazine and fluorofurimazine, were evaluated along with three stabilized O-acetylated furimazine analogues, the hikarazines. All 5 analogues, when tested in vitro, displayed greater signal intensity and reaction duration, in comparison to the standard NanoLuc substrate, furimazine. The two best-performing analogues from the in vitro study were selected for further in vivo testing. The NanoLuc/fluorofurimazine pair demonstrated the highest bioluminescence intensity, post intravenous administration. It was found to be around 9-fold brighter compared to the NanoLuc/furimazine and 11-fold more intense than the NanoLuc/hikarazine-003 pair, with an average of 3-fold higher light emission when the substrate was injected intraperitoneally, in a subcutaneous model. Excitingly, despite the fact that NanoLuc/fluorofurimazine emits mostly blue light, we prove that cells trapped in mice lungs vasculature could be visualised via the NanoLuc/fluorofurimazine pair and compare the results to the AkaLuc/AkaLumine system. Therefore, among the tested analogues, fluorofurimazine enables higher substrate loading and improved optical imaging sensitivity in small animals, upgrading the use of NanoLuc derived bioluminescent systems for deep tissue imaging.


Assuntos
Luciferases/química , Substâncias Luminescentes/química , Pulmão/diagnóstico por imagem , Vasos Retinianos/diagnóstico por imagem , Animais , Furanos/química , Células HEK293 , Humanos , Imidazóis/química , Infecções por Lentivirus , Luz , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Óptica , Pirazinas/química , Solubilidade , Relação Estrutura-Atividade
14.
Adv Sci (Weinh) ; 8(13): 2100424, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34540558

RESUMO

Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.


Assuntos
Morte Celular , Optogenética , Animais , Escherichia coli , Células HeLa/metabolismo , Células HeLa/transplante , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia , Luz , Camundongos , Nanopartículas , Necroptose , Optogenética/métodos , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Quinases/metabolismo , Piroptose
15.
bioRxiv ; 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-33469581

RESUMO

Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

16.
J Med Chem ; 62(10): 4884-4901, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31013090

RESUMO

Novel pyridine- and pyrimidine-based allosteric inhibitors are reported that achieve PDE4D subtype selectivity through recognition of a single amino acid difference on a key regulatory domain, known as UCR2, that opens and closes over the catalytic site for cAMP hydrolysis. The design and optimization of lead compounds was based on iterative analysis of X-ray crystal structures combined with metabolite identification. Selectivity for the activated, dimeric form of PDE4D provided potent memory enhancing effects in a mouse model of novel object recognition with improved tolerability and reduced vascular toxicity over earlier PDE4 inhibitors that lack subtype selectivity. The lead compound, 28 (BPN14770), has entered midstage, human phase 2 clinical trials for the treatment of Fragile X Syndrome.


Assuntos
Encefalopatias/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Desenho de Fármacos , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Inibidores da Fosfodiesterase 4/síntese química , Regulação Alostérica/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Encefalopatias/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Síndrome do Cromossomo X Frágil/enzimologia , Humanos , Concentração Inibidora 50 , Masculino , Camundongos Endogâmicos ICR , Estrutura Molecular , Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/farmacologia , Relação Estrutura-Atividade
17.
J Am Chem Soc ; 130(14): 4906-13, 2008 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-18345677

RESUMO

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction; whereas, the type II enzyme (IDI-2) requires reduced flavin. Epoxy, diene, and fluorinated substrate analogues, irreversible inhibitors of IDI-1, were analyzed as mechanistic probes for IDI-2. 3,4-Oxido-3-methyl-1-butyl diphosphate (eIPP), 3-methylene-4-penten-1-yl diphosphate (vIPP), and 3-(fluoromethyl)-3-buten-1-yl diphosphate (fmIPP) inactivate IDI-2 through formation of covalent adducts with the reduced flavin. UV-visible spectra of the inactivated complexes are consistent with modification of the isoalloxazine ring at position N5. vIPP and fmIPP are also alternate substrates with isomerization competing with alkylation of the flavin cofactor. (Z)-3-(Fluoromethyl)-2-buten-1-yl diphosphate ((Z)-fmDMAPP) and (Z)-3-(difluoromethyl)-2-buten-1-yl diphosphate ((Z)-dfmDMAPP) are alternate substrates, which are isomerized to the corresponding IPP derivatives. The rates of isomerization of fmIPP and (Z)-fmDMAPP are approximately 50-fold less than IPP and DMAPP, respectively. dfmIPP is not an irreversible inhibitor. These studies indicate that the irreversible inhibitors inactivate the reduced flavin required for catalysis by electrophilic alkylation and are consistent with a protonation-deprotonation mechanism for the isomerization catalyzed by IDI-2.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Mononucleotídeo de Flavina/metabolismo , Ligação Competitiva , Isomerases de Ligação Dupla Carbono-Carbono/antagonistas & inibidores , Isomerases de Ligação Dupla Carbono-Carbono/química , Ativação Enzimática , Mononucleotídeo de Flavina/química , Cromatografia Gasosa-Espectrometria de Massas , Hemiterpenos , Isoenzimas , Cinética , Espectrofotometria Ultravioleta , Thermus thermophilus/enzimologia
18.
ACS Chem Biol ; 12(4): 1028-1037, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28195704

RESUMO

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.


Assuntos
Permeabilidade da Membrana Celular , Inibidores Enzimáticos/farmacologia , Luciferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Relação Estrutura-Atividade
19.
J Carbohydr Chem ; 35(5): 249-260, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28626297

RESUMO

Improvements in the synthesis of carbon-linked glucuronide/glucoside conjugates of cancer chemopreventive retinoids have been achieved starting with 2,3,4,6-tetra-O-benzyl-D-glucopyranose. The revised approach demonstrates better yields, eliminates the use of an expensive, carcinogenic protecting group reagent, and avoids much painstaking chromatography. The new approach should allow synthesis of larger quantities of the agents for detailed animal and mechanistic studies.

20.
Anticancer Res ; 25(3c): 2391-8, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16082771

RESUMO

Previous studies from our laboratory suggest that 4-HPROG, the O-glucuronide derivative of 4-HPR, has improved mammary cancer chemopreventive/ antitumor activities as well as reduced toxicity, as compared to 4-HPR. This O-linked glucuronide derivative is a substrate to the P-glucuronidase enzyme and may also undergo hydrolysis in vivo to the vitamin A metabolite, retinoic acid, that is toxic at high concentrations. In an effort to improve analog potency relative to its toxicity, the 4-HPROG's phenolic oxygen was replaced with a methylene group, thus preventing biological cleavage of the glucuronide moiety. The resulting C-linked analog, 4-HPR-C-glucuronide (4-HPRCG), cannot be hydrolyzed to 4-HPR. The results of this study show that 4-HPRCG is an effective chemotherapeutic agent that caused 49% regression of DMBA-induced mammary tumors in rats, while showing almost no side-effects that are often observed with other natural or synthetic retinoids, such as a reduction in blood retinol level, elevation in blood triglyceride (TG) level, and decrease in bone mineral content (BMC). These results suggest that 4-HPRCG should be considered as a better candidate for breast cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Fenretinida/análogos & derivados , Glucuronatos/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , 9,10-Dimetil-1,2-benzantraceno , Animais , Antineoplásicos/efeitos adversos , Carcinógenos , Processos de Crescimento Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Feminino , Fenretinida/efeitos adversos , Fenretinida/farmacologia , Glucuronatos/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Ácido Retinoico 4 Hidroxilase , Tretinoína/efeitos adversos , Tretinoína/farmacologia
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