RESUMO
Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo.
RESUMO
Interferon-gamma (IFN-γ) is shown to stimulate melanoma development and progression. However, the underlying mechanism has not been completely defined. Our study aimed to determine the role of neuronal nitric oxide synthase (nNOS)-mediated signaling in IFN-γ-stimulated melanoma progression and the anti-melanoma effects of novel nNOS inhibitors. Our study shows that IFN-γ markedly induced the expression levels of nNOS in melanoma cells associated with increased intracellular nitric oxide (NO) levels. Co-treatment with novel nNOS inhibitors effectively alleviated IFN-γ-activated STAT1/3. Further, reverse phase protein array (RPPA) analysis demonstrated that IFN-γ induced the expression of HIF1α, c-Myc, and programmed death-ligand 1 (PD-L1), in contrast to IFN-α. Blocking the nNOS-mediated signaling pathway using nNOS-selective inhibitors was shown to effectively diminish IFN-γ-induced PD-L1 expression in melanoma cells. Using a human melanoma xenograft mouse model, the in vivo studies revealed that IFN-γ increased tumor growth compared to control, which was inhibited by the co-administration of nNOS inhibitor MAC-3-190. Another nNOS inhibitor, HH044, was shown to effectively inhibit in vivo tumor growth and was associated with reduced PD-L1 expression levels in melanoma xenografts. Our study demonstrates the important role of nNOS-mediated NO signaling in IFN-γ-stimulated melanoma progression. Targeting nNOS using highly selective small molecular inhibitors is a unique and effective strategy to improve melanoma treatment.
Assuntos
Carcinogênese/induzido quimicamente , Carcinogênese/efeitos dos fármacos , Progressão da Doença , Inibidores Enzimáticos/administração & dosagem , Interferon gama/administração & dosagem , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Animais , Antígeno B7-H1/metabolismo , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Melanoma/patologia , Camundongos , Camundongos Nus , Óxido Nítrico Sintase Tipo I/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias Cutâneas/patologia , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO) from l-arginine. Overexpressed or dysregulated iNOS has been implicated in numerous pathologies including sepsis, cancer, neurodegeneration, and various types of pain. Extensive knowledge has been accumulated about the roles iNOS plays in different tissues and organs. Additionally, X-ray crystal and cryogenic electron microscopy structures have shed new insights on the structure and regulation of this enzyme. Many potent iNOS inhibitors with high selectivity over related NOS isoforms, neuronal NOS, and endothelial NOS, have been discovered, and these drugs have shown promise in animal models of endotoxemia, inflammatory and neuropathic pain, arthritis, and other disorders. A major issue in iNOS inhibitor development is that promising results in animal studies have not translated to humans; there are no iNOS inhibitors approved for human use. In addition to assay limitations, both the dual modalities of iNOS and NO in disease states (ie, protective vs harmful effects) and the different roles and localizations of NOS isoforms create challenges for therapeutic intervention. This review summarizes the structure, function, and regulation of iNOS, with focus on the development of iNOS inhibitors (historical and recent). A better understanding of iNOS' complex functions is necessary before specific drug candidates can be identified for classical indications such as sepsis, heart failure, and pain; however, newer promising indications for iNOS inhibition, such as depression, neurodegenerative disorders, and epilepsy, have been discovered.
Assuntos
Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Animais , Doença , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Biológicos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Topoisomerases are DNA processing enzymes that relieve supercoiling (torsional strain) in DNA, are necessary for normal cellular division, and act by nicking (and then religating) DNA strands. Type 1B topoisomerase (Top1) is overexpressed in certain tumors, and the enzyme has been extensively investigated as a target for cancer chemotherapy. Various chemical agents can act as "poisons" of the enzyme's religation step, leading to Top1-DNA lesions, DNA breakage, and eventual cellular death. In this review, agents that poison Top1 (and have thus been investigated for their anticancer properties) are surveyed, including natural products (such as camptothecins and indolocarbazoles), semisynthetic camptothecin and luotonin derivatives, and synthetic compounds (such as benzonaphthyridines, aromathecins, and indenoisoquinolines), as well as targeted therapies and conjugates. Top1 has also been investigated as a therapeutic target in certain viral and parasitic infections, as well as autoimmune, inflammatory, and neurological disorders, and a summary of literature describing alternative indications is also provided. This review should provide both a reference for the medicinal chemist and potentially offer clues to aid in the development of new Top1 poisons.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/química , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , Humanos , Neoplasias/enzimologia , Inibidores da Topoisomerase I/químicaRESUMO
Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.
Assuntos
Aminoquinolinas/química , Aminoquinolinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Éteres Fenílicos/química , Éteres Fenílicos/farmacologia , Aminoquinolinas/farmacocinética , Células CACO-2 , Cristalografia por Raios X , Inibidores Enzimáticos/farmacocinética , Humanos , Modelos Moleculares , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Éteres Fenílicos/farmacocinética , Relação Estrutura-AtividadeRESUMO
Nitric oxide (NO) is an important signaling molecule in the human body, playing a crucial role in cell and neuronal communication, regulation of blood pressure, and in immune activation. However, overproduction of NO by the neuronal isoform of nitric oxide synthase (nNOS) is one of the fundamental causes underlying neurodegenerative disorders and neuropathic pain. Therefore, developing small molecules for selective inhibition of nNOS over related isoforms (eNOS and iNOS) is therapeutically desirable. The aims of this review focus on the regulation and dysregulation of NO signaling, the role of NO in neurodegeneration and pain, the structure and mechanism of nNOS, and the use of this information to design selective inhibitors of this enzyme. Structure-based drug design, the bioavailability and pharmacokinetics of these inhibitors, and extensive target validation through animal studies are addressed.
Assuntos
Neuralgia/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Sítios de Ligação , Calmodulina/química , Calmodulina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Neuralgia/metabolismo , Neuralgia/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Óxido Nítrico Sintase/metabolismo , Peptídeos/química , Peptídeos/uso terapêutico , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Estrutura Terciária de ProteínaRESUMO
Hydroxylated analogues of the anticancer topoisomerase I (Top1) inhibitors indotecan (LMP400) and indimitecan (LMP776) have been prepared because (1) a variety of potent Top1 poisons are known that contain strategically placed hydroxyl groups, which provides a clear rationale for incorporating them in the present case, and (2) the hydroxylated compounds could conceivably serve as synthetic standards for the identification of metabolites. Indeed, incubating LMP400 and LMP776 with human liver microsomes resulted in two major metabolites of each drug, which had HPLC retention times and mass fragmentation patterns identical to those of the synthetic standards. The hydroxylated indotecan and indimitecan metabolites and analogues were tested as Top1 poisons and for antiproliferative activity in a variety of human cancer cell cultures and in general were found to be very potent. Differences in activity resulting from the placement of the hydroxyl group are explained by molecular modeling analyses.
Assuntos
Antineoplásicos/química , Benzodioxóis/química , Indenos/química , Isoquinolinas/química , Inibidores da Topoisomerase I/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Benzodioxóis/metabolismo , Benzodioxóis/farmacologia , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidroxilação , Técnicas In Vitro , Indenos/metabolismo , Indenos/farmacologia , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Proteínas Recombinantes/química , Estereoisomerismo , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase I/farmacologiaRESUMO
The DNA-relaxing enzyme topoisomerase I (Top1) can be inhibited by heterocyclic compounds such as indolocarbazoles and indenoisoquinolines. Carbohydrate and hydroxyl-containing side chains are essential for the biological activity of indolocarbazoles. The current study investigated how similar functionalities could be "translated" to the indenoisoquinoline system and how stereochemistry and hydrogen bonding affect biological activity. Herein is described the preparation and assay of indenoisoquinolines substituted with short-chain alcohols, diols, and carbohydrates. Several compounds (including those derived from sugars) display potent Top1 poisoning and antiproliferative activities. The Top1 poisoning activity of diol-substituted indenoisoquinolines is dependent upon stereochemistry. Although the effect is striking, molecular modeling and docking studies do not indicate any reason for the difference in activity due to similar calculated interactions between the ligand and Top1-DNA complex and ambiguity about the binding mode. A stereochemical dependence was also observed for carbohydrate-derived indenoisoquinolines. Although similar trends were observed in other classes of Top1 inhibitors, the exact nature of this effect has yet to be elucidated.
Assuntos
Álcoois/síntese química , Hexoses/síntese química , Indenos/síntese química , Pentoses/síntese química , Quinolinas/síntese química , Inibidores da Topoisomerase I/síntese química , Álcoois/química , Álcoois/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Hexoses/química , Hexoses/farmacologia , Humanos , Ligação de Hidrogênio , Indenos/química , Indenos/farmacologia , Modelos Moleculares , Pentoses/química , Pentoses/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologiaRESUMO
In the search for agents with cancer chemopreventive potential, 14-chloromethyl-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (compound 1), originally synthesized as a potential topoisomerase I inhibitor, and 2,4-dibromo-1-hydroxyphenazine (compound 2), an analog of a substance found in the marine bacteria Streptomyces CNS284, were found to significantly enhance NADP(H):quinone oxidoreductase 1 (QR1), glutathione S-transferase (GST), and glutathione (GSH) levels in cell culture. However, following a short-term absorption study, analyses of livers from the treatment groups did not reveal a significant increase in QR1 or GST activity, or GSH levels. This was consistent with RT-PCR analyses of tissue samples. The compounds were absorbed, as judged by LC/MS analyses of serum and tissue samples, although levels were well below the concentrations required to mediate in vitro responses. Metabolites of compound 2 formed in vitro by human liver microzones were characterized using high resolution tandem mass spectrometry. In sum, the in vivo activity of these compounds appears to be diminished by low bioavailability, but this experimental approach indicates the importance of systematic biomarker investigation.
Assuntos
Anticarcinógenos/farmacologia , Fluorenos/farmacologia , Fenazinas/farmacologia , Animais , Anticarcinógenos/química , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Fluorenos/química , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Absorção Intestinal/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fenazinas/química , Ligação ProteicaRESUMO
Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of several heteroaromatic systems, namely the camptothecins and indenoisoquinolines, and they possess notable Top1 inhibition and cytotoxicity when substituted at position 14. The SAR of these compounds overlaps with indenoisoquinolines, suggesting that they may intercalate into the Top1-DNA complex similarly. Nonetheless, the proposed binding mode for aromathecins is purely hypothetical, as an X-ray structure is unavailable. In the present communication, we have synthesized eight novel series of A-ring-substituted (positions 1-3) aromathecins, through a simple, modular route, as part of a comprehensive SAR study. Certain groups (such as 2,3-ethylenedioxy) moderately improve Top1 inhibition, and, often, antiproliferative activity, whereas other groups (2,3-dimethoxy and 3-substituents) attenuate bioactivity. Strikingly, these trends are very similar to those previously observed for the A-ring of camptothecins, and this considerable SAR overlap lends further support (in the absence of crystallographic data) to the hypothesis that aromathecins bind in the Top1 cleavage complex as interfacial inhibitors in a 'camptothecin-like' pose.
Assuntos
Antineoplásicos/química , Camptotecina/química , Inibidores Enzimáticos/química , Inibidores da Topoisomerase I , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Camptotecina/síntese química , Camptotecina/toxicidade , DNA Topoisomerases Tipo I/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/toxicidade , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Isoquinolinas/toxicidade , Modelos Moleculares , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The aromathecin topoisomerase I (top1) inhibitors offer promising scaffolds for the development of novel cancer chemotherapeutics. They are 'composites' of the camptothecin and indenoisoquinoline top1 inhibitors. Interestingly, some structure-activity relationship (SAR) overlap between the aromathecins and the indenoisoquinolines has been observed. For both classes, placement of certain polar groups in similar regions of the heteroaromatic system improves top1 inhibitory and antiproliferative activities. A series of water-soluble aromathecins substituted at position 14 with diaminoalkanes of various lengths has been prepared. These compounds all possess similar antiproliferative potency, but a general trend is observed: aromathecins with longer diaminoalkane substituents (>6 carbons) possess lower anti-top1 activity than their smaller counterparts (2-4 carbons), presumably as a result of unfavorable hydrophobic interactions. This trend is also noted with the indenoisoquinolines, revealing additional SAR overlap that supports the hypothesis that there is a 'universal' top1 inhibitor SAR.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores da Topoisomerase I , Antineoplásicos/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
The aromathecin or "rosettacin" class of topoisomerase I (top1) inhibitors is effectively a "composite" of the natural products camptothecin and luotonin A and the synthetic indenoisoquinolines. The aromathecins have aroused considerable interest following the isolation and total synthesis of 22-hydroxyacuminatine, a rare cytotoxic natural product containing the 12 H-5,11a-diazadibenzo[ b, h]fluoren-11-one system. We have developed two novel syntheses of this system and prepared a series of 14-substituted aromathecins as novel antiproliferative topoisomerase I poisons. These inhibitors are proposed to act via an intercalation and "poisoning" mechanism identical to camptothecin and the indenoisoquinolines. Many of these compounds possess greater antiproliferative activity and anti-top1 activity than the parent unsubstituted compound (rosettacin) and previously synthesized aromathecins, as well as greater top1 inhibitory activity than 22-hydroxyacuminatine. In addition to potentially aiding solubility and localization to the DNA-enzyme complex, nitrogenous substituents located at the 14-position of the aromathecin system have been proposed to project into the major groove of the top1-DNA complex and hydrogen-bond to major-groove amino acids, thereby stabilizing the ternary complex.