RESUMO
Despite decades of development of treatments and the successful application of targeted therapies for multiple myeloma, clinical challenges remain for patients with relapsed/refractory disease. A drug designed for efficient delivery of an alkylating payload into tumor cells that yields a favorable therapeutic window can be an attractive choice. Herein we describe melphalan flufenamide (melflufen), a drug with a peptide carrier component conjugated to an alkylating payload, and its cellular metabolism. We further underline the fundamental role of enzymatic hydrolysis in the rapid and robust accumulation of alkylating metabolites in cancer cells and their importance for downstream effects. The formed alkylating metabolites were shown to cause DNA damage, both on purified DNA and on chromatin in cells, with both nuclear and mitochondrial DNA affected in the latter. Furthermore, the rapid intracellular enrichment of alkylating metabolites is shown to be essential for the rapid kinetics of the downstream intracellular effects such as DNA damage signaling and induction of apoptosis. To evaluate the importance of enzymatic hydrolysis for melflufen's efficacy, all four stereoisomers of the compound were studied in a systematic approach and shown to have a different pattern of metabolism. In comparison with melflufen, stereoisomers lacking intracellular accumulation of alkylating payloads showed cytotoxic activity only at significantly higher concentration, slower DNA damage kinetics, and different mechanisms of action to reach cellular apoptosis.
Assuntos
Melfalan , Mieloma Múltiplo , Humanos , Melfalan/efeitos adversos , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Fenilalanina/farmacologiaRESUMO
The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.
Assuntos
Desenho de Fármacos , Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Piridinas/administração & dosagem , Piridinas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Piridinas/farmacocinética , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
Thyroid hormone receptors (TRs) exert profound effects on development, metabolism, and multiple specific organ functions. Principally by regulating crucial genes in a variety of tissues, the thyroid hormones, 3,5,3'-triiodo-L-thyronine (L-T(3), 1) and 3,5,3',5'-tetraiodo-L-thyronine (L-T(4), 2), influence basal calorigenesis and oxygen consumption, cardiac rate and contractility, lipid metabolism, bone structure and strength, and central nervous system functions critical for normal mentation and mood. Elevated levels of circulating and tissue 1 and/or 2 result in the thyrotoxic clinical state, manifested by weight loss despite increased caloric intake; heat intolerance due to increased calorigenesis; cardiac tachyarrhythmias, systolic hypertension, and heart failure; skeletal muscle weakness; and a spectrum of neuropsychiatric symptoms ranging from anxiety to delirium and psychosis. The current standard treatments of endogenous hyperthyroidism causing thyrotoxicosis reduce the overproduction of thyroid hormones by pharmacologically inhibiting their synthesis or release (e.g., with thionamides or lithium, respectively), or by ablating thyroid tissue surgically or with radioiodine. TR-antagonists could hypothetically have significant clinical use in treating thyrotoxic states if they were capable of promptly and completely restoring euthyroid levels of thyroid-specific gene activity. No TRalpha-selective ligands have been prepared up to this date, ligands that potentially would further ameliorate the problem with cardiac disease connected with hyperthyroidism and maybe cardiac arrhythmia. Despite its significant potential use, no TR-antagonist has reached clinical application. Design of TR-antagonists ligands has been based on the attachment of a large extension group at the 5-prime position of 1 or other structurally related analogues. This extension is believed to distort folding of the C-terminal helix (helix 12) to the body of the ligand binding domain (LBD), which normally forms a coactivator site. Examples of synthetic TR antagonists based on this extension strategy are reviewed, as well as other strategies to achieve functional TR-antagonism.
Assuntos
Antitireóideos/farmacologia , Receptores dos Hormônios Tireóideos/antagonistas & inibidores , Animais , Antitireóideos/química , Humanos , Ligantes , Estrutura Molecular , Receptores dos Hormônios Tireóideos/química , Relação Estrutura-AtividadeRESUMO
A series of 4-(amido-biarylether)-quinolines was prepared as potential LXR agonists. Appropriate substitution with amide groups provided high affinity LXR ligands, some with excellent potency and efficacy in functional assays of LXR activity. Novel amide 4g had a binding IC(50)=1.9 nM for LXRbeta and EC(50)=34 nM (96% efficacy relative to T0901317) in an ABCA1 gene expression assay in mouse J774 cells, demonstrating that 4-(biarylether)-quinolines with appropriate amide substitution are potent LXR agonists.
Assuntos
Proteínas de Ligação a DNA/agonistas , Quinolinas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Animais , Linhagem Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Ligantes , Receptores X do Fígado , Camundongos , Modelos Moleculares , Receptores Nucleares Órfãos , Quinolinas/síntese química , Quinolinas/química , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
A series of substituted 2-benzyl-3-aryl-7-trifluoromethylindazoles were prepared as LXR modulators. These compounds were partial agonists in transactivation assays when compared to 1 (T0901317) and were slightly weaker with respect to potency and efficacy on LXRalpha than on LXRbeta. Lead compounds in this series 12 (WAY-252623) and 13 (WAY-214950) showed less lipid accumulation in HepG2 cells than potent full agonists 1 and 3 (WAY-254011) but were comparable in efficacy to 1 and 3 with respect to cholesterol efflux in THP-1 foam cells, albeit weaker in potency. Compound 13 reduced aortic lesion area in LDLR knockout mice equivalently to 3 or positive control 2 (GW3965). In a 7-day hamster model, compound 13 showed a lesser propensity for plasma TG elevation than 3, when the compounds were compared at doses in which they elevated ABCA1 and ABCG1 gene expression in duodenum and liver at equal levels. In contrast to results previously published for 2, the lack of TG effect of 13 correlated with its inability to increase liver fatty acid synthase (FAS) gene expression, which was up-regulated 4-fold by 3. These results suggest indazoles such as 13 may have an improved profile for potential use as a therapeutic agent.
Assuntos
Arteriosclerose/tratamento farmacológico , Proteínas de Ligação a DNA/agonistas , Indazóis/farmacologia , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Triglicerídeos/biossíntese , Animais , Arteriosclerose/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligação de Hidrogênio , Indazóis/síntese química , Indazóis/química , Ligantes , Fígado/efeitos dos fármacos , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Modelos Moleculares , Estrutura Molecular , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Triglicerídeos/sangueRESUMO
A new high-affinity thyroid hormone antagonist 6 with druglike properties was designed and synthesized. The compound behaved as an antagonist in a cell transactivation assay, and in a first in vivo experiment in rats.
Assuntos
Fenilpropionatos/síntese química , Piridinas/síntese química , Receptores dos Hormônios Tireóideos/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Ligantes , Modelos Moleculares , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Piridinas/química , Piridinas/farmacologia , Ratos , Receptores alfa dos Hormônios Tireóideos/antagonistas & inibidores , Receptores beta dos Hormônios Tireóideos/antagonistas & inibidores , Receptores beta dos Hormônios Tireóideos/química , Ativação Transcricional/efeitos dos fármacosRESUMO
A structure-based approach was used to optimize our new class of quinoline LXR modulators leading to phenyl acetic acid substituted quinolines 15 and 16. Both compounds displayed good binding affinity for LXRbeta and LXRalpha and were potent activators in LBD transactivation assays. The compounds also increased expression of ABCA1 and stimulated cholesterol efflux in THP-1 cells. Quinoline 16 showed good oral bioavailability and in vivo efficacy in a LDLr knockout mouse model for lesions.
Assuntos
Anticolesterolemiantes/síntese química , Aterosclerose/tratamento farmacológico , Proteínas de Ligação a DNA/agonistas , Fenilacetatos/síntese química , Quinolinas/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Anticolesterolemiantes/química , Anticolesterolemiantes/farmacologia , Sítios de Ligação , Disponibilidade Biológica , Linhagem Celular , Colesterol/metabolismo , Proteínas de Ligação a DNA/genética , Estabilidade de Medicamentos , Feminino , Humanos , Técnicas In Vitro , Ligantes , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Receptores Nucleares Órfãos , Fenilacetatos/química , Fenilacetatos/farmacologia , Estrutura Terciária de Proteína , Quinolinas/química , Quinolinas/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Relação Estrutura-Atividade , Ativação TranscricionalRESUMO
Ribonucleotide reductase is an indispensable enzyme for all cells, since it catalyses the biosynthesis of the precursors necessary for both building and repairing DNA. The ribonucleotide reductase class I enzymes, present in all mammals as well as in many prokaryotes and DNA viruses, are composed mostly of two homodimeric proteins, R1 and R2. The reaction involves long-range radical transfer between the two proteins. Here, we present the first crystal structure of a ribonucleotide reductase R1/R2 holocomplex. The biological relevance of this complex is based on the binding of the R2 C terminus in the hydrophobic cleft of R1, an interaction proven to be crucial for enzyme activity, and by the fact that all conserved amino acid residues in R2 are facing the R1 active sites. We suggest that the asymmetric R1/R2 complex observed in the 4A crystal structure of Salmonella typhimurium ribonucleotide reductase represents an intermediate stage in the reaction cycle, and at the moment of reaction the homodimers transiently form a tight symmetric complex.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estrutura Quaternária de Proteína , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Salmonella typhimurium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Complexos Multiproteicos , Ribonucleotídeo Redutases/genética , Alinhamento de SequênciaRESUMO
Based on the examination of the crystal structure of rat TRbeta complexed with 3,5,3'-triiodo-l-thyronine (2) a novel TRbeta-selective indole derivative 6b was prepared and tested in vitro. This compound was found to be 14 times selective for TRbeta over TRalpha in binding and its beta-selectivity could be rationalized through the comparison of the X-ray crystallographic structures of 6b complexed with TRalpha and TRbeta.
Assuntos
Indóis/química , Indóis/farmacologia , Receptores beta dos Hormônios Tireóideos/agonistas , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Cristalografia por Raios X , Ciclização , Humanos , Indóis/metabolismo , Concentração Inibidora 50 , Ligantes , Estrutura Molecular , Ratos , Especificidade por Substrato , Receptores beta dos Hormônios Tireóideos/química , Tiroxina/síntese química , Tiroxina/químicaRESUMO
Members of the nuclear receptor gene family act as biological rheostats to maintain metabolic homeostasis in response to endocrine and nutritional changes. The liver X (LXR) and thyroid hormone (TR) receptors have been shown to regulate overlapping but distinct metabolic pathways important for overall lipid homeostasis. Dyslipidemia is one out of four key determinants for cardiovascular risk and both LXRs and TRs may provide attractive targets for intervention of cardiovascular disease. In this review we will compare the two receptor systems to highlight similarities and differences in structure and function with implications for development of novel treatments for dyslipidemia and atherosclerosis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Metabolismo dos Lipídeos , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores dos Hormônios Tireóideos/fisiologia , Animais , Arteriosclerose/fisiopatologia , Homeostase/fisiologia , Humanos , Hiperlipidemias/fisiopatologia , Receptores X do Fígado , Receptores Nucleares ÓrfãosRESUMO
Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decreases circulating glucose levels, but systemic glucocorticoid antagonism can produce adrenal insufficiency and other undesirable side effects. These hepatic and systemic responses might be dissected, leading to liver-selective pharmacology, when a GR antagonist is linked to a bile acid in an appropriate manner. Bile acid conjugation can be accomplished with a minimal loss of binding affinity for GR. The resultant conjugates remain potent in cell-based functional assays. A novel in vivo assay has been developed to simultaneously evaluate both hepatic and systemic GR blockade; this assay has been used to optimize the nature and site of the linker functionality, as well as the choice of the GR antagonist and the bile acid. This optimization led to the identification of A-348441, which reduces glucose levels and improves lipid profiles in an animal model of diabetes.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/síntese química , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/síntese química , Fígado/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Ácidos e Sais Biliares/química , Sítios de Ligação , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Células CHO , Células Cultivadas , Simulação por Computador , Cricetinae , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Glucose/biossíntese , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , Camundongos , Modelos Moleculares , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-AtividadeRESUMO
A set of thyromimetics having improved selectivity for TR-beta1 were prepared by replacing the 3'-isopropyl group of 2 and 3 with substituents having increased steric bulk. From this limited SAR study, the most potent and selective compounds identified were derived from 2 and contained a 3'-phenyl moiety bearing small hydrophobic groups meta to the biphenyl link. X-ray crystal data of 15c complexed with TR-beta1 LBD shows methionine 442 to be displaced by the bulky R3' phenyl ethyl amide side chain. Movement of this amino acid side chain provides an expanded pocket for the bulky side chain while the ligand-receptor complex retains full agonist activity.
Assuntos
Receptores beta dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/química , Tri-Iodotironina/química , 2-Propanol/química , Aminoácidos/química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Concentração Inibidora 50 , Ligantes , Relação Estrutura-Atividade , Receptores beta dos Hormônios Tireóideos/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/farmacologia , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
The nrdE gene product R1E, the large subunit of the class 1b Salmonella typhimurium ribonucleotide reductase, has been overexpressed, purified and crystallized. Initially, the protein crystallized in two orthorhombic space groups, C222(1) and P2(1)2(1)2, using tartrate and PEG 6000 as precipitants, respectively. Better diffracting crystals belonging to the tetrahedral space group P4(3)2(1)2 were obtained using sodium malonate as precipitant. The P4(3)2(1)2 crystals could only be obtained after seeding from a drop containing C222(1) crystals grown in sodium tartrate. Thus, streak-seeding resulted in crystals of a supergroup to C222(1). Data to 2.8 A resolution have been collected on the P4(3)2(1)2 crystals which contained one R1E subunit in the asymmetric unit.
Assuntos
Ribonucleotídeo Redutases/química , Salmonella typhimurium/química , Cristalização , Genes Bacterianos/genética , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Difração de Raios XRESUMO
The structures of the liver X receptor LXRbeta (NR1H2) have been determined in complexes with two synthetic ligands, T0901317 and GW3965, to 2.1 and 2.4 A, respectively. Together with its isoform LXRalpha (NR1H3) it regulates target genes involved in metabolism and transport of cholesterol and fatty acids. The two LXRbeta structures reveal a flexible ligand-binding pocket that can adjust to accommodate fundamentally different ligands. The ligand-binding pocket is hydrophobic but with polar or charged residues at the two ends of the cavity. T0901317 takes advantage of this by binding to His-435 close to H12 while GW3965 orients itself with its charged group in the opposite direction. Both ligands induce a fixed "agonist conformation" of helix H12 (also called the AF-2 domain), resulting in a transcriptionally active receptor.
Assuntos
Receptores Citoplasmáticos e Nucleares/química , Alanina/química , Sítios de Ligação , Colesterol/metabolismo , Proteínas de Ligação a DNA , Dimerização , Elétrons , Escherichia coli/metabolismo , Histidina/química , Humanos , Ligantes , Receptores X do Fígado , Modelos Químicos , Modelos Moleculares , Modelos Estatísticos , Receptores Nucleares Órfãos , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Raios XRESUMO
The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind.
Assuntos
Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Salmonella typhimurium/enzimologia , Regulação Alostérica/fisiologia , Sítio Alostérico , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/metabolismo , Dimerização , Escherichia coli/enzimologia , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismoRESUMO
Here we describe the three-dimensional crystal structures of human glucocorticoid receptor ligand-binding domain (GR-LBD) in complex with the antagonist RU-486 at 2.3 A resolution and with the agonist dexamethasone ligand together with a coactivator peptide at 2.8 A. The RU-486 structure was solved in several different crystal forms, two with helix 12 intact (GR1 and GR3) and one with a protease-digested C terminus (GR2). In GR1, part of helix 12 is in a position that covers the co-activator pocket, whereas in the GR3, domain swapping is seen between the crystallographically identical subunits in the GR dimer. An arm consisting of the end of helix 11 and beyond stretches out from one molecule, and helix 12 binds to the other LBD, partly blocking the coactivator pocket of that molecule. This type of GR-LBD dimer has not been described before but might be an artifact from crystallization. Furthermore, the subunits of the GR3 dimers are covalently connected via a disulfide bond between the Cys-736 residues in the two molecules. All three RU-486 GR-LBD structures show that GR has a very flexible region between the end of helix 11 and the end of helix 12.