RESUMEN
A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (Kd=0.52nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC50=0.098µM, NCI-N87 IC50=0.066µM) and also displayed high oral bioavailability in mice (F=44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition=136%).
Asunto(s)
Benzopiranos/farmacología , Diseño de Fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias Experimentales/tratamiento farmacológico , Triazinas/farmacología , Administración Oral , Animales , Benzopiranos/administración & dosificación , Benzopiranos/síntesis química , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Triazinas/administración & dosificación , Triazinas/síntesis químicaRESUMEN
We conducted a high throughput screening for glyoxalase I (GLO1) inhibitors and identified 4,6-diphenyl-N-hydroxypyridone as a lead compound. Using a binding model of the lead and public X-ray coordinates of GLO1 enzymes complexed with glutathione analogues, we designed 4-(7-azaindole)-substituted 6-phenyl-N-hydroxypyridones. 7-Azaindole's 7-nitrogen was expected to interact with a water network, resulting in an interaction with the protein. We validated this inhibitor design by comparing its structure-activity relationship (SAR) with that of corresponding indole derivatives, by analyzing the binding mode with X-ray crystallography and by evaluating its thermodynamic binding parameters.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Indoles/química , Lactoilglutatión Liasa/antagonistas & inhibidores , Piridonas/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Lactoilglutatión Liasa/metabolismo , Modelos Moleculares , Estructura Molecular , Piridonas/síntesis química , Piridonas/química , Relación Estructura-ActividadRESUMEN
Macrocyclic compounds bearing a 2-amino-6-arylpyrimidine moiety were identified as potent heat shock protein 90 (Hsp90) inhibitors by modification of 2-amino-6-aryltriazine derivative (CH5015765). We employed a macrocyclic structure as a skeleton of new inhibitors to mimic the geldanamycin-Hsp90 interactions. Among the identified inhibitors, CH5164840 showed high binding affinity for N-terminal Hsp90α (K(d)=0.52nM) and strong anti-proliferative activity against human cancer cell lines (HCT116 IC(50)=0.15µM, NCI-N87 IC(50)=0.066µM). CH5164840 displayed high oral bioavailability in mice (F=70.8%) and potent antitumor efficacy in a HCT116 human colorectal cancer xenograft model (tumor growth inhibition=83%).
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Compuestos Macrocíclicos/farmacología , Pirimidinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Compuestos Macrocíclicos/administración & dosificación , Compuestos Macrocíclicos/síntesis química , Ratones , Ratones Desnudos , Ratones SCID , Modelos Moleculares , Estructura Molecular , Pirimidinas/administración & dosificación , Pirimidinas/síntesis química , Estereoisomerismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Heat shock protein 90 (Hsp90) is a molecular chaperone which regulates maturation and stabilization of its substrate proteins, known as client proteins. Many client proteins of Hsp90 are involved in tumor progression and survival and therefore Hsp90 can be a good target for developing anticancer drugs. With the aim of efficiently identifying a new class of orally available inhibitors of the ATP binding site of this protein, we conducted fragment screening and virtual screening in parallel against Hsp90. This approach quickly identified 2-aminotriazine and 2-aminopyrimidine derivatives as specific ligands to Hsp90 with high ligand efficiency. In silico evaluation of the 3D X-ray Hsp90 complex structures of the identified hits allowed us to promptly design CH5015765, which showed high affinity for Hsp90 and antitumor activity in human cancer xenograft mouse models.
Asunto(s)
Antineoplásicos/síntesis química , Benzopiranos/química , Benzopiranos/síntesis química , Simulación por Computador , Diseño de Fármacos , Descubrimiento de Drogas/métodos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Triazinas/química , Triazinas/síntesis química , Adenosina Trifosfatasas/metabolismo , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Benzopiranos/metabolismo , Benzopiranos/farmacocinética , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Neoplasias/tratamiento farmacológico , Reproducibilidad de los Resultados , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Triazinas/metabolismo , Triazinas/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, we identified CH5424802, a potent, selective, and orally available ALK inhibitor with a unique chemical scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as nonsmall cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. Our results support the potential for clinical evaluation of CH5424802 for the treatment of patients with ALK-driven tumors.
Asunto(s)
Antineoplásicos/farmacología , Carbazoles/farmacología , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Administración Oral , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Carbazoles/administración & dosificación , Carbazoles/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Modelos Moleculares , Mutación , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Piperidinas/administración & dosificación , Piperidinas/química , Conformación Proteica , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A novel quinoline derivative that selectively inhibits c-Met kinase was identified. The molecular design is based on a result of the analysis of a PF-2341066 (1)/c-Met cocrystal structure (PDB code: 2wgj). The kinase selectivity of the derivatives is discussed from the view point of the sequence homology of the kinases, the key interactions found in X-ray cocrystal structures, and the structure-activity relationship (SAR) obtained in this work.
Asunto(s)
Oxiquinolina/síntesis química , Piperidinas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Piridinas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Crizotinib , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Oxiquinolina/química , Oxiquinolina/farmacología , Piperidinas/farmacología , Pirazoles , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
A series of benzofuran-based farnesyltransferase inhibitors have been designed and synthesized as antitumor agents. Among them, 11f showed the most potent enzyme inhibitory activity (IC(50)=1.1nM) and antitumor activity in human cancer xenografts in mice.
Asunto(s)
Antineoplásicos/síntesis química , Benzofuranos/síntesis química , Química Farmacéutica/métodos , Farnesiltransferasa/antagonistas & inhibidores , Quinolonas/síntesis química , Animales , Antineoplásicos/farmacología , Benzofuranos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/química , Humanos , Concentración 50 Inhibidora , Ratones , Trasplante de Neoplasias , Quinolonas/farmacología , Relación Estructura-ActividadRESUMEN
Uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosylphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5'-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP). This is an S(N)2 reaction; the non-esterified oxygen atom of the GlcNAc-1-P phosphate group attacks the alpha-phosphate group of UTP. We determined crystal structures of UAP from Candida albicans (CaUAP1) without any ligands and also complexed with its substrate or with its product. The series of structures in different forms shows the induced fit movements of CaUAP1. Three loops approaching the ligand molecule close the active site when ligand is bound. In addition, Lys-421, instead of the metal ion in prokaryotic UAPs, is coordinated by both phosphate groups of UDP-Glc-NAc and acts as a cofactor. However, a magnesium ion enhances the enzymatic activity of CaUAP1, and thus we propose that the magnesium ion increases the affinity between UTP and the enzyme by coordinating to the alpha- and gamma-phosphate group of UTP.
Asunto(s)
Candida albicans/enzimología , Nucleotidiltransferasas/química , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Nucleotidiltransferasas/metabolismo , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 A resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 A, alpha = 90.01, beta = 97.72, gamma = 92.88 degrees, whereas those of the product complex belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 A.
Asunto(s)
Candida albicans/enzimología , Nucleotidiltransferasas/química , Cristalización , Cristalografía por Rayos X , Nucleotidiltransferasas/aislamiento & purificaciónRESUMEN
N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the alpha-D-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional beta-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180 degrees on the axis linking C-4 and the midpoint of the C-5-O-5 bond in the reaction.
Asunto(s)
Candida albicans/enzimología , Transferasas Intramoleculares/química , Secuencia de Aminoácidos , Catálisis , Cristalización , Cristalografía por Rayos X , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the alpha-D-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P2(1), with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 angstroms, beta = 106.7 degrees. The crystals diffract X-rays to beyond 1.8 angstroms resolution using synchrotron radiation.
Asunto(s)
Candida albicans/enzimología , Fosfotransferasas (Fosfomutasas)/química , Cristalización , Fosfotransferasas (Fosfomutasas)/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Sincrotrones , Difracción de Rayos XRESUMEN
The (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase (LVR) of the soil isolate bacterium Corynebacterium aquaticum M-13 is a NAD(H)-linked enzyme that catalyzes reversible oxidoreduction between (4R)-hydroxy-(6R)-2,2,6-trimethylcyclohexanone (actinol) and levodione. Here the crystal structure of a ternary complex of LVR with NADH and its inhibitor 2-methyl-2,4-pentanediol has been determined by molecular replacement and refined at 1.6-A resolution with a crystallographic R factor of 0.199. The overall structure is similar to those of other short-chain alcohol dehydrogenase/reductase enzymes. The positions of NADH and 2-methyl-2,4-pentanediol indicate the binding site of the substrate and identify residues that are likely to be important in the catalytic reaction. Modeling of the substrate binding in the active site suggests that the specificity of LVR is determined by electrostatic interactions between the negatively charged surface of Glu-103 of LVR and the positively charged surface on the re side of levodione. Mutant LVR enzymes in which Glu-103 is substituted with alanine (E103A), glutamine (E103Q), asparagines (E103N), or aspartic acid (E103D) show a 2-6-fold increase in Km values as compared with wild-type LVR and a much lower enantiomeric excess of the reaction products (60%) than the wild-type enzyme (95%). Together, these data indicate that Glu-103 has an important role in determining the stereospecificity of LVR.
Asunto(s)
Corynebacterium/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Sitios de Unión , Catálisis , Dicroismo Circular , Cristalización , Cristalografía , Inhibidores Enzimáticos/metabolismo , Escherichia coli/genética , Expresión Génica , Ácido Glutámico , Glicoles/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Mutación , NAD/metabolismo , Oxidorreductasas/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Electricidad Estática , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a monomeric enzyme that catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine residue of a variety of eukaryotic and viral proteins. Genetic and biochemical studies have established that Nmt is an attractive target for antifungal drugs. We present here crystal structures of C. albicans Nmt complexed with two classes of inhibitor competitive for peptide substrates. One is a peptidic inhibitor designed from the peptide substrate; the other is a nonpeptidic inhibitor having a benzofuran core. Both inhibitors are bound into the same binding groove, generated by some structural rearrangements of the enzyme, with the peptidic inhibitor showing a substrate-like binding mode and the nonpeptidic inhibitor binding differently. Further, site-directed mutagenesis for C. albicans Nmt has been utilized in order to define explicitly which amino acids are critical for inhibitor binding. The results suggest that the enzyme has some degree of flexibility for substrate binding and provide valuable information for inhibitor design.