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1.
BMC Cancer ; 19(1): 1251, 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31881855

RESUMO

BACKGROUND: Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be "undrugable" because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription. METHODS: This study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt's lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents. RESULTS: GQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin. CONCLUSION: These results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.


Assuntos
Compostos de Anilina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Elipticinas/farmacologia , Quadruplex G/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Sulfonamidas/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Elipticinas/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , Resultado do Tratamento
2.
Cell Chem Biol ; 26(8): 1110-1121.e4, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31155510

RESUMO

Increased telomerase activity is associated with malignancy and poor prognosis in human cancer, but the development of targeted agents has not yet provided clinical benefit. Here we report that, instead of targeting the telomerase enzyme directly, small molecules that bind to the G-hairpin of the hTERT G-quadruplex-forming sequence kill selectively malignant cells without altering the function of normal cells. RG260 targets the hTERT G-quadruplex stem-loop folding but not tetrad DNAs, leading to downregulation of hTERT expression. To improve physicochemical and pharmacokinetic properties, we derived a small-molecule analog, RG1603, from the parent compound. RG1603 induces mitochondrial defects including PGC1α and NRF2 inhibition and increases oxidative stress, followed by DNA damage and apoptosis. RG1603 injected as a single agent has tolerable toxicity while achieving strong anticancer efficacy in a tumor xenograft mouse model. These results demonstrate a unique approach to inhibiting the hTERT that functions by impairing mitochondrial activity, inducing cell death.

3.
ACS Chem Biol ; 14(9): 2006-2013, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31241884

RESUMO

RNA dysregulation likely contributes to disease pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. A pathological form of the transactive response (TAR) DNA binding protein (TDP-43) binds to RNA in stress granules and forms membraneless, amyloid-like TDP-43 aggregates in the cytoplasm of ALS motor neurons. In this study, we hypothesized that by targeting the RNA recognition motif (RRM) domains of TDP-43 that confer a pathogenic interaction between TDP-43 and RNA, motor neuron toxicity could be reduced. In silico docking of 50000 compounds to the RRM domains of TDP-43 identified a small molecule (rTRD01) that (i) bound to TDP-43's RRM1 and RRM2 domains, (ii) partially disrupted TDP-43's interaction with the hexanucleotide RNA repeat of the disease-linked c9orf72 gene, but not with (UG)6 canonical binding sequence of TDP-43, and (iii) improved larval turning, an assay measuring neuromuscular coordination and strength, in an ALS fly model based on the overexpression of mutant TDP-43. Our findings provide an instructive example of a chemical biology approach pivoted to discover small molecules targeting RNA-protein interactions in neurodegenerative diseases.

4.
Pain ; 160(7): 1644-1661, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30933958

RESUMO

Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of α, ß, γ and α2δ subunits. The ß subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of CaV. We targeted the high-affinity protein-protein interface of CaVß's pocket within the CaVα subunit. Structure-based virtual screening of 50,000 small molecule library docked to the ß subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaVß and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaVα-CaVß function and ultimately be developed as a nonopioid therapeutic for chronic pain.

5.
Channels (Austin) ; 12(1): 219-227, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30081699

RESUMO

Drug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2's SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mapas de Interação de Proteínas , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação Alostérica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Canal de Sódio Disparado por Voltagem NAV1.7/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética
6.
ACS Chem Biol ; 13(10): 3000-3010, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30141626

RESUMO

Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50 000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which ( i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), ( ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner, and ( iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.


Assuntos
Modelos Animais de Doenças , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Isoquinolinas/farmacologia , Isoquinolinas/toxicidade , Atrofias Olivopontocerebelares/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Peixe-Zebra/anormalidades , Animais , Atrofia , Cerebelo/patologia , Regulação para Baixo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Técnicas de Silenciamento de Genes , Humanos , Isoquinolinas/metabolismo , Simulação de Acoplamento Molecular , Mutação , Atrofias Olivopontocerebelares/induzido quimicamente , Atrofias Olivopontocerebelares/patologia , Fenótipo , Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Curvaturas da Coluna Vertebral/induzido quimicamente , Transcriptoma/efeitos dos fármacos , Regulação para Cima
7.
Pain ; 159(10): 2115-2127, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29847471

RESUMO

We previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Receptoras Sensoriais/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/fisiopatologia , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Proteínas do Tecido Nervoso/genética , Neuralgia/tratamento farmacológico , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Teste de Desempenho do Rota-Rod , Sódio/metabolismo , Transdução Genética , Enzimas de Conjugação de Ubiquitina/genética
8.
Br J Pharmacol ; 175(12): 2244-2260, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-28161890

RESUMO

BACKGROUND AND PURPOSE: N-type voltage-gated calcium (Cav 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Cav 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. EXPERIMENTAL APPROACH: Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Cav 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. KEY RESULTS: The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. CONCLUSIONS AND IMPLICATIONS: These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.


Assuntos
Analgésicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Dor/tratamento farmacológico , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Analgésicos/química , Animais , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Dor/metabolismo , Fragmentos de Peptídeos/química , Ratos , Ratos Sprague-Dawley
9.
Nucleic Acids Res ; 46(2): 886-896, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29156002

RESUMO

Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5' splice site (5'SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5'SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5'SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5'SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.


Assuntos
Processamento Alternativo/genética , Quadruplex G , Precursores de RNA/genética , Proteína bcl-X/genética , Processamento Alternativo/efeitos dos fármacos , Sequência de Bases , Elipticinas/farmacologia , Humanos , Ligantes , Mutação , Precursores de RNA/química , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética
10.
Aging Cell ; 16(5): 1146-1154, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28779511

RESUMO

There is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual-specificity tyrosine phosphorylation-regulated kinase-1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD-like pathology developed by 3xTg-AD mice, a widely used animal model of AD. We dosed 10-month-old 3xTg-AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1-inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg-AD mice. These effects were associated with a reduction in amyloid-ß (Aß) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Aß levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Disfunção Cognitiva/prevenção & controle , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Benzimidazóis/farmacologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Locomoção/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas tau/antagonistas & inibidores , Proteínas tau/metabolismo
11.
F1000Res ; 62017.
Artigo em Inglês | MEDLINE | ID: mdl-28690829

RESUMO

There many possible types of drug-target interactions, because there are a surprising number of ways in which drugs and their targets can associate with one another.  These relationships are expressed as polypharmacology and polyspecificity.  Polypharmacology is the capability of a given drug to exhibit activity with respect to multiple drug targets, which are not necessarily in the same activity class. Adverse drug reactions ('side effects') are its principal manifestation, but polypharmacology is also playing a role in the repositioning of existing drugs for new therapeutic indications.  Polyspecificity, on the other hand, is the capability of a given target to exhibit activity with respect to multiple, structurally dissimilar drugs.  That these concepts are closely related to one another is, surprisingly, not well known.  It will be shown in this work that they are, in fact, mathematically related to one another and are in essence 'two sides of the same coin'.  Hence, information on polypharmacology provides equivalent information on polyspecificity, and vice versa. Networks are playing an increasingly important role in biological research. Drug-target networks, in particular, are made up of drug nodes that are linked to specific target nodes if a given drug is active with respect to that target.  Such networks provide a graphic depiction of polypharmacology and polyspecificity.  However, by their very nature they can obscure information that may be useful in their interpretation and analysis.  This work will show how such latent information can be used to determine bounds for the degrees of polypharmacology and polyspecificity, and how to estimate other useful features associated with the lack of completeness of most drug-target datasets.

12.
J Med Chem ; 60(15): 6587-6597, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28605593

RESUMO

Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.


Assuntos
Antineoplásicos/uso terapêutico , Quadruplex G , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Apoptose/efeitos dos fármacos , Benzoxazinas/uso terapêutico , Caspase 3/metabolismo , Linhagem Celular , Ciclofosfamida/uso terapêutico , Sistemas de Liberação de Medicamentos , Elipticinas/uso terapêutico , Técnicas de Silenciamento de Genes , Humanos , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Pregnanos/uso terapêutico , RNA Mensageiro/metabolismo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Am Chem Soc ; 139(22): 7456-7475, 2017 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-28471683

RESUMO

The platelet-derived growth factor receptor ß (PDGFR-ß) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-ß promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-ß NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-ß pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-ß is the 3'-end G-quadruplex, which has a GGA sequence at the 3'-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-ß NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-ß NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-ß promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3'-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-ß i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-ß promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-ß-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.


Assuntos
Motivos de Aminoácidos , Desoxirribonucleases/química , Sistemas de Liberação de Medicamentos , Quadruplex G , Receptor beta de Fator de Crescimento Derivado de Plaquetas/química , Sequência de Bases , Regulação para Baixo , Quadruplex G/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Mutação , Regiões Promotoras Genéticas
14.
J Am Chem Soc ; 138(41): 13673-13692, 2016 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-27643954

RESUMO

Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wild-type (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here, we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing. We show that promoter mutations exert a detrimental effect on the folding of one of these G-quadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression produces cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and induction of senescence by decreasing telomerase activity and telomere length. We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.

16.
Oncotarget ; 7(17): 23715-29, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27009859

RESUMO

The anaplastic lymphoma kinase (ALK) protein drives tumorigenesis in subsets of several tumors through chromosomal rearrangements that express and activate its C-terminal kinase domain. In addition, germline predisposition alleles and acquired mutations are found in the full-length protein in the pediatric tumor neuroblastoma. ALK-specific tyrosine kinase inhibitors (TKIs) have become important new drugs for ALK-driven lung cancer, but acquired resistance via multiple mechanisms including kinase-domain mutations eventually develops, limiting median progression-free survival to less than a year. Here we assess the impact of several kinase-domain mutations that arose during TKI resistance selections of ALK+ anaplastic large-cell lymphoma (ALCL) cell lines. These include novel variants with respect to ALK-fusion cancers, R1192P and T1151M, and with respect to ALCL, F1174L and I1171S. We assess the effects of these mutations on the activity of six clinical inhibitors in independent systems engineered to depend on either the ALCL fusion kinase NPM-ALK or the lung-cancer fusion kinase EML4-ALK. Our results inform treatment strategies with a likelihood of bypassing mutations when detected in resistant patient samples and highlight differences between the effects of particular mutations on the two ALK fusions.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Mutação , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Mutagênese Sítio-Dirigida , Células Tumorais Cultivadas
17.
PLoS One ; 10(6): e0130515, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26111161

RESUMO

The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.


Assuntos
Pneumopatias/genética , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Relação Estrutura-Atividade , Grupo com Ancestrais do Continente Africano/genética , Humanos , Pneumopatias/patologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Quinase de Cadeia Leve de Miosina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Polimorfismo de Nucleotídeo Único/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética
18.
J Biol Chem ; 290(3): 1623-38, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25477506

RESUMO

Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Apoptose , Ácido Aurintricarboxílico/análogos & derivados , Proteínas de Choque Térmico/metabolismo , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Ácido Aurintricarboxílico/química , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Glutationa/metabolismo , Resposta ao Choque Térmico/genética , Humanos , Indolquinonas/química , Concentração Inibidora 50 , Queratinócitos/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Modelos Moleculares , Estresse Oxidativo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/metabolismo , Regulação para Cima
19.
J Am Chem Soc ; 136(11): 4161-71, 2014 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-24559410

RESUMO

It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.


Assuntos
DNA/efeitos dos fármacos , Piperidinas/farmacologia , Pregnanodiol/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Termodinâmica , Animais , DNA/química , DNA/genética , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Camundongos SCID , Conformação de Ácido Nucleico/efeitos dos fármacos , Piperidinas/química , Pregnanodiol/análogos & derivados , Pregnanodiol/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
20.
Bioorg Med Chem Lett ; 23(2): 528-31, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23237838

RESUMO

This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE(2) reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R(1)=H, R(2)=p-CH(3)O) exhibited the most potent activity in cells (EC(50)=0.02 µM) and minimal inhibition of COX-2 activity (3% at 5 µM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.


Assuntos
Aminopiridinas/síntese química , Aminopiridinas/farmacologia , Dinoprostona/antagonistas & inibidores , Ftalazinas/síntese química , Ftalazinas/farmacologia , Aminopiridinas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dinoprostona/biossíntese , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Estrutura Molecular , Ftalazinas/química , Transplante Heterólogo
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