RESUMO
DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , DNA Primase/metabolismo , DNA Primase/fisiologia , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Células HEK293 , Humanos , Células K562 , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/fisiologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/fisiologia , Fatores de Transcrição/genéticaRESUMO
Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.
Assuntos
Nucleotidiltransferases , Bibliotecas de Moléculas Pequenas , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Relação Estrutura-Atividade , Ligação Proteica , Estrutura Molecular , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , DNA Polimerase Dirigida por DNA/metabolismo , Camundongos , Síntese de DNA TranslesãoRESUMO
A significant unmet need for new contraceptive options for both women and men remains due to side-effect profiles, medical concerns, and the inconvenience of many currently available contraceptive products. Unfortunately, the development of novel nonsteroidal female contraceptive medicine has been stalled in the last couple of decades due to the lack of effective screening platforms. Drosophila utilizes conserved signaling pathways for follicle rupture, a final step in ovulation that is essential for female reproduction. Therefore, we explored the potential to use Drosophila as a model to screen compounds that could inhibit follicle rupture and be nonsteroidal contraceptive candidates. Using our ex vivo follicle rupture assay, we screened 1,172 Food and Drug Administration (FDA)-approved drugs and identified six drugs that could inhibit Drosophila follicle rupture in a dose-dependent manner. In addition, we characterized the molecular actions of these drugs in the inhibition of adrenergic signaling and follicle rupture. Furthermore, we validated that three of the four drugs consistently inhibited mouse follicle rupture in vitro and that two of them did not affect progesterone production. Finally, we showed that chlorpromazine, one of the candidate drugs, can significantly inhibit mouse follicle rupture in vivo. Our work suggests that Drosophila ovulation is a valuable platform for identifying lead compounds for nonsteroidal contraceptive development and highlights the potential of these FDA-approved drugs as novel nonsteroidal contraceptive agents.
Assuntos
Anticoncepcionais , Drosophila melanogaster/fisiologia , Hormônios/metabolismo , Ovulação/fisiologia , Animais , Bioensaio , Clorpromazina/farmacologia , Dexmedetomidina/farmacologia , Aprovação de Drogas , Feminino , Camundongos , Octopamina/metabolismo , Folículo Ovariano/fisiologia , Estados Unidos , United States Food and Drug AdministrationRESUMO
DNA damage tolerance (DDT) pathways enable cells to cope with a variety of replication blocks that threaten their ability to complete DNA replication. Helicase-like transcription factor (HLTF) plays a central role in the error-free DDT pathway, template switching (TS), by serving as a ubiquitin ligase to polyubiquitinate the DNA sliding clamp PCNA, which promotes TS initiation. HLTF also serves as an ATP-dependent DNA translocase facilitating replication fork remodeling. The HIP116, Rad5p N-terminal (HIRAN) domain of HLTF specifically recognizes the unmodified 3'-end of single-stranded DNA (ssDNA) at stalled replication forks to promote fork regression. Several crystal structures of the HIRAN domain in complex with ssDNA have been reported; however, optimal ssDNA sequences for high-affinity binding with the domain have not been described. Here we elucidated DNA sequence preferences of HLTF HIRAN through systematic studies of its binding to ssDNA substrates using fluorescence polarization assays and a computational analysis of the ssDNA:HIRAN interaction. These studies reveal that the HLTF HIRAN domain preferentially recognizes a (T/C)TG sequence at the 3'-hydroxyl ssDNA end, which occurs in the CTG trinucleotide repeat (TNR) regions that are susceptible to expansion and deletion mutations identified in neuromuscular and neurodegenerative disorders. These findings support a role for HLTF in maintaining the stability of difficult to replicate TNR microsatellite regions.
RESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the pork industry globally. PRRS is caused by PRRS virus (PRRSV). Currently there are no effective treatments against this swine disease. METHODS: Through artificial intelligence molecular screening, we obtained a set of small molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which is a cell surface receptor specific for PRRSV infection. These compounds were screened using a cell-based bimolecular fluorescence complementation (BiFC) assay, and the function of positive hit was further evaluated and validated by PRRSV-infection assay using porcine alveolar macrophages (PAMs). RESULTS: Using the BiFC assay, we identified one compound with previously unverified function, 4-Fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide (designated here as B7), that significantly inhibits the interaction between the PRRSV glycoprotein (GP2a or GP4) and the CD163-SRCR5 domain. We further demonstrated that compound B7 inhibits PRRSV infection of PAMs, the primary target of PRRSV in a dose-dependent manner. B7 significantly inhibited the infection caused by both type I and type II PRRSV strains. Further comparison and functional evaluation of chemical compounds structurally related to B7 revealed that the 3-(morpholinosulfonyl)aniline moiety of B7 or the 3-(piperidinylsulfonyl)aniline moiety in a B7 analogue is important for the inhibitory function against PRRSV infection. CONCLUSIONS: Our study identified a novel strategy to potentially prevent PRRSV infection in pigs by blocking the PRRSV-CD163 interaction with small molecules.
Assuntos
Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Inteligência Artificial , Linhagem Celular , Células HEK293 , Humanos , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Domínios Proteicos , SuínosRESUMO
Itraconazole (ITZ) is an FDA-approved antifungal agent that has recently been explored for novel biological properties. In particular, ITZ was identified as a potent inhibitor of the hedgehog (Hh) pathway, a cell signalling pathway that has been linked to a variety of cancers and accounts for â¼25% of paediatric medulloblastoma (MB) cases. To date, there is not a targeted therapeutic option for paediatric MB, resulting in long-term side effects such as hormone deficiency, organ damage and secondary cancers. A primary obstacle for developing targeted therapy for brain ailments is the presence of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances. Due to its size and hydrophobicity, ITZ does not penetrate the BBB. Alternatively, liposomes are being increasingly used within the clinic to increase drug bioavailability, target specificity and BBB permeability. With this in mind, we have successfully developed ITZ-containing liposomes with an optimal size for BBB penetration (<100 nm) and encapsulation efficiency (â¼95%) by utilizing a continuous manufacturing approach-turbulent coaxial jet in co-flow. Our preliminary in vitro data demonstrate that these liposomes inhibit the Hh pathway, albeit at a reduced level in comparison to free ITZ. (196/250 words).
Assuntos
Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Itraconazol/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Barreira Hematoencefálica/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Itraconazol/síntese química , Itraconazol/química , Lipossomos , Células Tumorais CultivadasRESUMO
While loss-of-function mutations in the ATRX gene have been implicated as a driving force for a variety of pediatric brain tumors, as well as pancreatic neuroendocrine tumors, the role of ATRX in gene regulation and oncogenic development is not well-characterized. The ADD domain of ATRX (ATRXADD) localizes the protein to chromatin by specifically binding to the histone H3 tail. This domain is also a primary region that is mutated in these cancers. The overall goal of our studies was to utilize a variety of techniques (experimental and computational) to probe the H3:ATRXADD protein-protein interaction (PPI). We developed two biochemical assays that can be utilized to study the interaction. These assays were utilized to experimentally validate and expand upon our previous computational results. We demonstrated that the three anchor points in the H3 tail (A1, K4, and K9) are all essential for high affinity binding and that disruption of more than one contact region will be required to develop a small molecule that disrupts the PPI. Our approach in this study could be applied to other domains of ATRX, as well as PPIs between other distinct proteins.
Assuntos
Histonas/química , Mapas de Interação de Proteínas , Bioensaio , Biotina/metabolismo , Calorimetria , Histonas/metabolismo , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Calcitriol, vitamin D3 (VD3), and structurally related VD3 analogues are inhibitors of Hh signaling in multiple contexts and are promising anti-cancer agents in Hh-dependent forms of cancer; however, the cellular mechanisms through which these compounds regulate Hh signal transmission are not clearly defined. Previous studies in this area have implicated both Smoothened, a key mediator of Hh signaling, and the vitamin D receptor (VDR) as potential mediators of Hh inhibition for this class of seco-steroids. We have performed a series of in vitro studies to more fully probe the cellular mechanisms that govern seco-steroid-mediated inhibition of Hh signaling. Our results support a role for both the Hh and VDR pathways in this process, as well as the possibility that other, as yet unidentified proteins, are also central to seco-steroid-mediated inhibition of Hh signaling.
Assuntos
Proteínas Hedgehog/metabolismo , Receptores de Calcitriol/metabolismo , Secoesteroides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , CamundongosRESUMO
Translesion synthesis (TLS) is a mechanism of replication past damaged DNA through which multiple forms of human cancer survive and acquire resistance to first-line genotoxic chemotherapies. As such, TLS is emerging as a promising target for the development of a new class of anticancer agents. The C-terminal domain of the DNA polymerase Rev1 (Rev1-CT) mediates assembly of the functional TLS complex through protein-protein interactions (PPIs) with Rev1 interacting regions (RIRs) of several other TLS DNA polymerases. Utilizing structural knowledge of the Rev1-CT/RIR interface, we have identified the phenazopyridine scaffold as an inhibitor of this essential TLS PPI. We demonstrate direct binding of this scaffold to Rev1-CT, and the synthesis and evaluation of a small series of analogues have provided important structure-activity relationships for further development of this scaffold. Furthermore, we utilized the umbrella sampling method to predict the free energy of binding to Rev1-CT for each of our analogues. Binding energies calculated through umbrella sampling correlated well with experimentally determined IC50 values, validating this computational tool as a viable approach to predict the biological activity for inhibitors of the Rev1-CT/RIR PPI.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Descoberta de Drogas , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Fenazopiridina/análogos & derivados , Fenazopiridina/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , TermodinâmicaRESUMO
Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows replicative bypass of DNA lesions, including DNA adducts formed by cancer chemotherapeutics. Previous studies demonstrated that suppression of TLS can increase sensitivity of cancer cells to first-line chemotherapeutics and decrease mutagenesis linked to the onset of chemoresistance, marking the TLS pathway as an emerging therapeutic target. TLS is mediated by a heteroprotein complex consisting of specialized DNA polymerases, including the Y-family DNA polymerase Rev1. Previously, we developed a screening assay to identify the first small molecules that disrupt the protein-protein interaction between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) present in multiple DNA polymerases involved in TLS. Herein we report additional hit scaffolds that inhibit this key TLS PPI. In addition, through a series of biochemical, computational, and cellular studies we have identified preliminary structure-activity relationships and determined initial pharmacokinetic parameters for our original hits.
Assuntos
Antineoplásicos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Nucleotidiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Tiofenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
BACKGROUND: Multiple oxysterols (OHCs) have demonstrated the ability to act as agonists or antagonists of the hedgehog (Hh) signaling pathway, a developmental signaling pathway that has been implicated as a potential therapeutic target in a variety of human diseases. These OHCs are known to modulate Hh signaling through direct binding interactions with the N-terminal cysteine rich domain (CRD) of Smoothened, a key regulator of Hh signal transduction. METHODS: Homology modeling, molecular dynamics simulations, and MM/GBSA energy calculations were utilized to explore binding interactions between the OHC scaffold and the human Smoothened CRD. Follow-up cellular assays explored the in vitro activity of potential Hh pathway modulators. RESULTS: Structural features that govern key molecular interactions between the Smoothened CRD and the OHC scaffold were identified. Orientation of the iso-octyl side chain as well as the overall entropy of the OHC-CRD complex are important for determining activity against the Hh pathway. OHC 9, which was previously thought to be inactive because it was not an Hh agonist, was identified as an inhibitor of Hh signal transmission. CONCLUSIONS: Calculated MM/GBSA binding energies for OHCs in complex with the CRD of Smoothened correlate well with in vitro Hh modulatory activity. Compounds with high affinity stabilize Smoothened and are antagonists, whereas compounds with reduced affinity allow a conformational change in Smoothened that results in pathway activation. GENERAL SIGNIFICANCE: Computational modeling and molecular dynamics simulations can be used to predict whether a small molecule that binds the Smoothened CRD will be an agonist or antagonist of the pathway.
Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Oxisteróis/antagonistas & inibidores , Oxisteróis/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Cisteína/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica/fisiologia , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Previous structure-activity relationship studies have provided potent and selective analogues of vitamin D3 as inhibitors of the Hedgehog (Hh) signaling pathway. These analogues contain both modified A- and seco-B ring motifs, and have been evaluated for anticancer therapeutic potential. To continue our studies on this scaffold, a new series of compounds were synthesized to explore additional interactions and spatial constraints. These compounds incorporate functional groups of varying size and hydrophobicity at the C-11 position. While large hydrophobic moieties (9c-e) resulted in significant loss of Hh inhibition, smaller or more flexible moieties (9a, 11) maintain anti-Hh activity. These results call for additional and continued studies to identify the binding pocket to better understand these structure-activity relationships.
Assuntos
Colecalciferol/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Colecalciferol/análogos & derivados , Colecalciferol/síntese química , Relação Dose-Resposta a Droga , Proteínas Hedgehog/metabolismo , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Vitamin D3 (VD3) is a moderately potent and non-selective inhibitor of the Hedgehog (Hh) signaling cascade. Previous studies have established that the CD-ring region of VD3 serves as the Hh inhibitory pharmacophore. Subsequently, compound 3, an ester linked aromatic A-ring and CD-ring derivative was identified as an improved and selective Hh inhibitor. Herein, we report modifications of the CD-ring side chain that afford enhancement of selectivity for Hh modulation thereby diminishing the detrimental effects of concomitant vitamin D receptor activation. In general, linear or moderately branched alkyl chains of five or six carbons were optimal for potent and selective inhibition of Hh signaling. Moreover, hybrid VD3 side chain derivative 20 demonstrated 4-fold improvement in Hh antagonistic activity over VD3(IC50=1.1-1.6 µM) while gaining greater than a 1000-fold selectivity for Hh signaling over canonical activation of the vitamin D receptor pathway.
Assuntos
Colecalciferol/análogos & derivados , Colecalciferol/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Colecalciferol/síntese química , Desenho de Fármacos , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C3H , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Epitranscriptomics, the field of post-translational RNA modifications, is a burgeoning domain of research that has recently received significant attention for its role in multiple diseases, including cancer. N6-methyladenosine (m6A) is the most prominent post-translational RNA modification and plays a critical role in RNA transcription, processing, translation, and metabolism. The m6A modification is controlled by three protein classes known as writers (methyltransferases), erasers (demethylases), and readers (m6A-binding proteins). Each class of m6A regulatory proteins has been implicated in cancer initiation and progression. As such, many of these proteins have been identified as potential targets for anti-cancer chemotherapeutics. In this work, we provide an overview of the role m6A-regulating proteins play in cancer and discuss the current state of small molecule therapeutics targeting these proteins.
Assuntos
Adenosina , Antineoplásicos , Neoplasias , Bibliotecas de Moléculas Pequenas , Humanos , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacologia , Adenosina/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , RNA/metabolismo , Animais , Estrutura Molecular , Processamento Pós-Transcricional do RNA/efeitos dos fármacosRESUMO
The anti-proliferative activity of a series of ester- and amide-linked Inhoffen-Lythgoe side chain analogues is reported. Whereas the Inhoffen-Lythgoe diol was inactive in these studies, a number of aromatic and aliphatic ester-linked side chains demonstrated modest in vitro growth inhibition in two human cancepar cell lines, U87MG (glioblastoma) and HT-29 (colorectal adenocarcinoma). Structure-activity relationship (SAR) studies demonstrated the most active aromatic (13) and aliphatic (25 and 29) substituted analogues were approximately equipotent in U87MG and HT-29 cells. Further evaluation of 13, 25, and 29 indicated these analogues do not activate canonical vitamin D signaling nor antagonize Hedgehog (Hh) signaling. Thus, the cellular mechanism(s) that govern the anti-proliferative activity for this class of truncated vitamin D-based structures appears to be different from classical mechanisms previously identified for these scaffolds.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Indanos/química , Indanos/farmacologia , Amidas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ésteres/química , Células HT29 , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Vitamina D/químicaRESUMO
The Enterovirus (EV) genus includes several important human and animal pathogens. EV-A71, EV-D68, poliovirus (PV), and coxsackievirus (CV) outbreaks have affected millions worldwide, causing a range of upper respiratory, skin, and neuromuscular diseases, including acute flaccid myelitis, and hand-foot-and-mouth disease. There are no FDA-approved antiviral therapeutics for these enteroviruses. This study describes novel antiviral compounds targeting the conserved non-structural viral protein 2C with low micromolar to nanomolar IC50 values. The selection of resistant mutants resulted in amino acid substitutions in the viral capsid protein, implying these compounds may play a role in inhibiting the interaction of 2C and the capsid protein. The assembly and encapsidation stages of the viral life cycle still need to be fully understood, and the inhibitors reported here could be useful probes in understanding these processes.
Assuntos
Infecções por Enterovirus , Enterovirus , Doenças Neuromusculares , Animais , Humanos , Antivirais/farmacologia , Antivirais/metabolismo , Proteínas do Capsídeo/metabolismo , Infecções por Enterovirus/tratamento farmacológicoRESUMO
A structure-activity relationship study to elucidate the structural basis for hedgehog (Hh) signaling inhibition by vitamin D3 (VD3) was performed. Functional and non-functional regions of VD3 and VD2 were obtained through straightforward synthetic means and their biological activity was determined in a variety of cell-based assays. Several of these compounds inhibited Hh signaling at levels comparable to the parent VD3 with no effects on canonical vitamin D signaling. Most notably, compounds 5 and 9, demonstrated potent inhibition of the Hh pathway, exhibited no binding affinity for the vitamin D receptor (VDR), and did not activate VDR in cell culture. In addition, several compounds exhibited anti-proliferative activity against two human cancer cell lines through a mechanism distinct from the Hh or VDR pathways, suggesting a new cellular mechanism of action for this class of compounds.
Assuntos
Colecalciferol/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A direct route to analogs of the naturally occurring tropolone ß-thujaplicin has been developed in just four steps from furan. Using this method, a series of derivatives were synthesized and evaluated. Several of these compounds demonstrated very high levels of potency against bacterial and fungal pathogens with good selectivity over mammalian cells.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Furanos/química , Monoterpenos/farmacologia , Tropolona/análogos & derivados , Tropolona/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antifúngicos/síntese química , Antifúngicos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Candida glabrata/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Enterococcus faecalis/efeitos dos fármacos , Humanos , Células MCF-7 , Estrutura Molecular , Monoterpenos/síntese química , Monoterpenos/química , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Tropolona/síntese química , Tropolona/químicaRESUMO
Vitamin D3 (VD3) is a seco-steroid that inhibits the Hedgehog (Hh) signaling pathway. Initial studies suggested its anti-Hh activity results from direct inhibition of Smoothened, a seven-transmembrane cell surface receptor that is a key regulator of the Hh signaling cascade. More recently, a role for the Vitamin D Receptor in mediating inhibition of Hh-signaling by seco-steroid has been suggested. Herein, an affinity-based protein profiling study was carried out to better understand the cellular proteins that govern VD3-mediated anti-Hh activity. We synthesized a novel biotinylated VD3 analogue (8) for use as a chemical probe to explore cellular binding targets of the seco-steroidal scaffold. Through a series of pull-down experiments and follow up mass spectrum analyses, heat shock protein 70 (Hsp70) was identified as a primary binding protein of VD3. Hsp70 was validated as a binding target of VD3 through a series of biochemical and cellular assays. VD3 bound with micromolar affinity to Hsp70. In addition, both selective knockdown of Hsp70 expression and pharmacological inhibition of its activity with known Hsp70 inhibitors suppressed Hh-signaling transduction in murine basal cell carcinoma cells, suggesting that Hsp70 regulates proper Hh-signaling. Additional cellular assays suggest that VD3 and its seco-steroidal metabolites inhibit Hh-signaling through different mechanisms.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Basocelular/tratamento farmacológico , Colecalciferol/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas Hedgehog/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colecalciferol/síntese química , Colecalciferol/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Hedgehog/metabolismo , Camundongos , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
In small cell lung cancer (SCLC), acquired resistance to DNA-damaging therapy is challenging to study because rebiopsy is rarely performed. We used patient-derived xenograft models, established before therapy and after progression, to dissect acquired resistance to olaparib plus temozolomide (OT), a promising experimental therapy for relapsed SCLC. These pairs of serial models reveal alterations in both cell cycle kinetics and DNA replication and demonstrate both inter- and intratumoral heterogeneity in mechanisms of resistance. In one model pair, up-regulation of translesion DNA synthesis (TLS) enabled tolerance of OT-induced damage during DNA replication. TLS inhibitors restored sensitivity to OT both in vitro and in vivo, and similar synergistic effects were seen in additional SCLC cell lines. This represents the first described mechanism of acquired resistance to DNA damage in a patient with SCLC and highlights the potential of the serial model approach to investigate and overcome resistance to therapy in SCLC.