RESUMO
Metallo-ß-lactamases (MBLs) hydrolyze almost all ß-lactam antibiotics and are unaffected by clinically available ß-lactamase inhibitors (ßLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of ßLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive ßLIs of all MBL classes in vitro, with Kis of 6-15 µM or 36-84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26-0.36 µM) than d-BTZs (26-29 µM). Importantly, cell-based time-kill assays show BTZs restore ß-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate ß-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble ß-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Tiazolidinas/química , Inibidores de beta-Lactamases/química , beta-Lactamases/química , Domínio Catalítico , Desenho de Fármacos , Hidrólise , Cinética , Modelos MolecularesRESUMO
The most important resistance mechanism to ß-lactam antibiotics involves hydrolysis by two ß-lactamase categories: the nucleophilic serine and the metallo-ß-lactamases (SBLs and MBLs, respectively). Cyclobutanones are hydrolytically stable ß-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using 19 F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone through interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a 13 C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition-state and/or intermediate analogues as inhibitors of all ß-lactamase classes.
Assuntos
Ciclobutanos/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , beta-Lactamas/química , Catálise , Meropeném , Mimetismo Molecular , Ressonância Magnética Nuclear Biomolecular , Tienamicinas/química , Inibidores de beta-Lactamases/química , beta-Lactamases/química , beta-Lactamases/classificaçãoRESUMO
ß-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete ß-lactams. However, commercially available BLIs are not effective against metallo-ß-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R â L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki* values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E*I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Tiazolidinas/farmacologia , beta-Lactamases/química , Substituição de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Imipenem/química , Imipenem/farmacologia , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ligação Proteica , Pseudomonas aeruginosa/enzimologia , Tiazolidinas/química , Resistência beta-Lactâmica , beta-Lactamases/genéticaRESUMO
Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217.
Assuntos
Aurora Quinases/antagonistas & inibidores , Aurora Quinases/química , Imidazóis/síntese química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/síntese química , Pirazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalização , Relação Dose-Resposta a Droga , Humanos , Imidazóis/química , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Pirazóis/química , Piridinas/química , Relação Estrutura-AtividadeRESUMO
N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated ß-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3µM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the ß-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Ureia/análogos & derivados , Animais , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Glicogênio Fosforilase Muscular/química , Glicogênio Fosforilase Muscular/metabolismo , Modelos Moleculares , Coelhos , Ureia/síntese química , Ureia/química , Ureia/farmacologiaRESUMO
The production of selective protein kinase inhibitors is often frustrated by the similarity of the enzyme active sites. For this reason, it is challenging to design inhibitors that discriminate between the three Aurora kinases, which are important targets in cancer drug discovery. We have used a triple-point mutant of Aurora-A (AurAx3) which mimics the active site of Aurora-B to investigate the structural basis of MLN8054 selectivity. The bias toward Aurora-A inhibition by MLN8054 is fully recapitulated by AurAx3 in vitro. X-ray crystal structures of the complex suggest that the basis for the discrimination is electrostatic repulsion due to the T217E substitution, which we have confirmed using a single-point mutant. The activation loop of Aurora-A in the AurAx3-MLN8054 complex exhibits an unusual conformation in which Asp274 and Phe275 side chains point into the interior of the protein. There is to our knowledge no documented precedent for this conformation, which we have termed DFG-up. The sequence requirements of the DFG-up conformation suggest that it might be accessible to only a fraction of kinases. MLN8054 thus circumvents the problem of highly homologous active sites. Binding of MLN8054 to Aurora-A switches the character of a pocket within the active site from polar to a hydrophobic pocket, similar to what is observed in the structure of Aurora-A bound to a compound that induces DFG-out. We propose that targeting this pocket may be a productive route in the design of selective kinase inhibitors and describe the structural basis for the rational design of these compounds.
Assuntos
Benzazepinas/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Mimetismo Molecular , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Estrutura Molecular , Conformação Proteica , Proteínas Serina-Treonina Quinases/genética , Relação Estrutura-AtividadeRESUMO
Co-crystallisation of the imidazo[1,2-a]pyrazine derivative 15 (3-chloro-N-(4-morpholinophenyl)-6-(pyridin-3-yl)imidazo[1,2-a]pyrazin-8-amine) with Aurora-A provided an insight into the interactions of this class of compound with Aurora kinases. This led to the design and synthesis of potent Aurora-A inhibitors demonstrating up to 70-fold selectivity in cell-based Aurora kinase pharmacodynamic biomarker assays.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazinas/química , Pirazinas/farmacologia , Antineoplásicos/síntese química , Aurora Quinases , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Pirazinas/síntese química , Relação Estrutura-AtividadeRESUMO
Metallo-ß-Lactamases (MBLs) protect bacteria from almost all ß-lactam antibiotics. Verona integron-encoded MBL (VIM) enzymes are among the most clinically important MBLs, with VIM-1 increasing in carbapenem-resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are among the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here, we present crystal structures of VIM-1 and its complexes with a substrate-mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM-1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water-mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221-bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs. ENZYMES: EC 3.5.2.6 DATABASES: Co-ordinates and structure factors for protein structures described in this manuscript have been deposited in the Protein Data Bank (www.rcsb.org/pdb) with accession codes 5N5G (VIM-1), 5N5H (VIM-1:ML302F complex) and 5N5I (VIM-1-hydrolysed meropenem complex).
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/enzimologia , Meropeném/química , Meropeném/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.
Assuntos
Inibidores Enzimáticos/metabolismo , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Músculos/enzimologia , Conformação Proteica/efeitos dos fármacos , Ureia/metabolismo , Monofosfato de Adenosina/metabolismo , Sítio Alostérico , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Hepática/metabolismo , Glicogênio Fosforilase Muscular/química , Glicogênio Fosforilase Muscular/metabolismo , Humanos , Hipoglicemiantes , Cinética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Coelhos , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of beta-D-glucopyranose, 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc-1-P) with K(i) values of 145.2 (+/-11.6), 76 (+/-4.8), and 8.6 (+/-0.7) muM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb-methyl-oxadiazole complex, 1.92 A; GPb-benzothiazole, 2.10 A; GPb-benzimidazole, 1.93 A). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (approximately 32 A) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.
Assuntos
Benzimidazóis/química , Inibidores Enzimáticos/química , Glucosídeos/química , Oxidiazóis/química , Fosforilase b/química , Tiazóis/química , Benzimidazóis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Glucosídeos/metabolismo , Cinética , Modelos Moleculares , Oxidiazóis/metabolismo , Fosforilase b/metabolismo , Tiazóis/metabolismoRESUMO
CP320626 has been identified as a potent inhibitor, synergistic with glucose, of human liver glycogen phosphorylase a (LGPa), a possible target for type 2 diabetes therapy. CP320626 is also a potent inhibitor of human muscle GPa. In order to elucidate the structural basis of the mechanism of CP320626 inhibition, the structures of T state rabbit muscle GPa (MGPa) in complex with glucose and in complex with both glucose and CP320626 were determined at 2.0 A resolution, and refined to crystallographic R values of 0.179 (R(free)=0.218) and 0.207 (R(free)=0.235), respectively. CP320626 binds at the new allosteric site, some 33 A from the catalytic site, where glucose binds. The binding of CP320626 to MGPa does not promote extensive conformational changes except for small shifts of the side chain atoms of residues R60, V64, and K191. Both CP320626 and glucose promote the less active T state, while structural comparisons of MGPa-glucose-CP320626 complex with LGPa complexed with a related compound (CP403700) and a glucose analogue inhibitor indicate that the residues of the new allosteric site, conserved in the two isozymes, show no significant differences in their positions.
Assuntos
Amidas/química , Inibidores Enzimáticos/química , Glicogênio Fosforilase Muscular/química , Hipoglicemiantes/química , Indóis/química , Regulação Alostérica , Animais , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Glucose/química , CoelhosRESUMO
Using a focused screening approach, acyl ureas have been discovered as a new class of inhibitors of human liver glycogen phosphorylase (hlGPa). The X-ray structure of screening hit 1 (IC50 = 2 microM) in a complex with rabbit muscle glycogen phosphorylase b reveals that 1 binds at the AMP site, the main allosteric effector site of the dimeric enzyme. A first cycle of chemical optimization supported by X-ray structural data yielded derivative 21, which inhibited hlGPa with an IC50 of 23 +/- 1 nM, but showed only moderate cellular activity in isolated rat hepatocytes (IC50 = 6.2 microM). Further optimization was guided by (i) a 3D pharmacophore model that was derived from a training set of 24 compounds and revealed the key chemical features for the biological activity and (ii) the 1.9 angstroms crystal structure of 21 in complex with hlGPa. A second set of compounds was synthesized and led to 42 with improved cellular activity (hlGPa IC50 = 53 +/- 1 nM; hepatocyte IC50 = 380 nM). Administration of 42 to anaesthetized Wistar rats caused a significant reduction of the glucagon-induced hyperglycemic peak. These findings are consistent with the inhibition of hepatic glycogenolysis and support the use of acyl ureas for the treatment of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/síntese química , Monofosfato de Adenosina/química , Sítio Alostérico , Animais , Sítios de Ligação , Cristalografia por Raios X , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Muscular/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Técnicas In Vitro , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Coelhos , Ratos , Ureia/químicaRESUMO
Metallo-ß-lactamases (MBLs) hydrolyze almost all classes of ß-lactam antibiotic, including carbapenems-currently first choice drugs for opportunistic infections by Gram-negative bacterial pathogens. MBL inhibitor development is complicated by the diversity within this group of enzymes, and by the appearance of new enzymes that continue to be identified both as chromosomal genes and on mobile genetic elements. One such newly discovered MBL is DIM-1, a mobile enzyme originally discovered in the opportunist pathogen Pseudomonas stutzeri but subsequently identified in other species and locations. DIM-1 is a subclass B1 MBL more closely related to the TMB-1, GIM-1 and IMP enzymes than to other clinically encountered MBLs such as VIM and NDM; and possesses Arg, rather than the more usual Lys, at position 224 in the putative substrate binding site. Here we report the crystallization and structure determination of DIM-1. DIM-1 possesses a binuclear metal center with a 5 (rather than the more usual 4) co-ordinate tri-histidine (Zn1) site and both 4- and 5-co-ordinate Cys-His-Asp- (Zn2) sites observed in the two molecules of the crystallographic asymmetric unit. These data indicate a degree of variability in metal co-ordination geometry in the DIM-1 active site, as well as facilitating inclusion of DIM-1 in structure-based MBL inhibitor discovery programmes.
Assuntos
Proteínas de Bactérias/química , Conformação Proteica , Pseudomonas stutzeri/enzimologia , beta-Lactamases/química , Sítios de Ligação , Cristalografia , Histidina/química , Isoformas de Proteínas/química , Zinco/químicaRESUMO
Pathogenic Gram-negative bacteria resistant to almost all ß-lactam antibiotics are a major public health threat. Zn(II)-dependent or metallo-ß-lactamases (MBLs) produced by these bacteria inactivate most ß-lactam antibiotics, including the carbapenems, which are "last line therapies" for life-threatening Gram-negative infections. NDM-1 is a carbapenemase belonging to the MBL family that is rapidly spreading worldwide. Regrettably, inhibitors of MBLs are not yet developed. Here we present the bisthiazolidine (BTZ) scaffold as a structure with some features of ß-lactam substrates, which can be modified with metal-binding groups to target the MBL active site. Inspired by known interactions of MBLs with ß-lactams, we designed four BTZs that behave as in vitro NDM-1 inhibitors with Ki values in the low micromolar range (from 7 ± 1 to 19 ± 3 µM). NMR spectroscopy demonstrated that they inhibit hydrolysis of imipenem in NDM-1-producing Escherichia coli. In vitro time kill cell-based assays against a variety of bacterial strains harboring blaNDM-1 including Acinetobacter baumannii show that the compounds restore the antibacterial activity of imipenem. A crystal structure of the most potent heterocycle (L-CS319) in complex with NDM-1 at 1.9 Å resolution identified both structural determinants for inhibitor binding and opportunities for further improvements in potency.
RESUMO
The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3',5'-ADP, 2',5'-ADP, 5'-ADP, U-2'-p and U-3'-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5'-ADP (Ki = 1.2 microM), adopts a syn conformation (in contrast to 3',5'-ADP and 2',5'-ADP, which adopt an anti), and it is the beta- rather than the alpha-phosphate group that binds to P1. 3',5'-ADP binds with the 5'-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2',5'-ADP. This inhibitor binds with either the 3' or the 5' phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2' versus 3') has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies.
Assuntos
Difosfato de Adenosina/química , Inibidores Enzimáticos/química , Ribonuclease Pancreático/química , Nucleotídeos de Uracila/química , Difosfato de Adenosina/análogos & derivados , Animais , Sítios de Ligação , Proteínas Sanguíneas/antagonistas & inibidores , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Bovinos , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Proteínas Granulares de Eosinófilos , Neurotoxina Derivada de Eosinófilo , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/metabolismo , Ribonucleases/antagonistas & inibidores , Ribonucleases/química , Ribonucleases/metabolismo , Nucleotídeos de Uracila/metabolismoRESUMO
Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand-Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibitors. Guided by a computational modeling approach, appropriate C7-imidazo[4,5-b]pyridine derivatization led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in cells.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Aurora Quinase B/antagonistas & inibidores , Desenho de Fármacos , Imidazóis/química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Aurora Quinase A/química , Aurora Quinase A/metabolismo , Aurora Quinase B/química , Aurora Quinase B/metabolismo , Domínio Catalítico , Estabilidade de Medicamentos , Células HCT116 , Humanos , Imidazóis/metabolismo , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/metabolismo , Especificidade por SubstratoRESUMO
Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children.
Assuntos
Antineoplásicos/síntese química , Imidazóis/síntese química , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Purinas/síntese química , Pirazóis/síntese química , Piridinas/síntese química , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Aurora Quinase A , Aurora Quinase B , Aurora Quinases , Disponibilidade Biológica , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Feminino , Humanos , Imidazóis/química , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Mutação , Transplante de Neoplasias , Purinas/química , Purinas/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transplante Heterólogo , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss.
Assuntos
Imidazóis/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/síntese química , Administração Oral , Animais , Aurora Quinase A , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Feminino , Humanos , Imidazóis/farmacocinética , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Transplante de Neoplasias , Ligação Proteica , Piridinas/farmacocinética , Piridinas/farmacologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor site has remained unexplored. We report the chemical synthesis and bioactivity in hepatocytes of four new olefin derivatives of flavopiridol (1-4) that bind to the purine site. Flavopiridol and 1-4 counteracted the activation of phosphorylase in hepatocytes caused by AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolised to an AMP analogue. Unlike an indole carboxamide inhibitor, the analogues 1 and 4 suppressed the basal rate of glycogenolysis in hepatocytes by allosteric inhibition rather than by inactivation of phosphorylase, and accordingly caused negligible stimulation of glycogen synthesis. However, they counteracted the stimulation of glycogenolysis by dibutyryl cAMP by both allosteric inhibition and inactivation of phosphorylase. Cumulatively, the results show key differences between purine site and indole carboxamide site inhibitors in terms of (i) relative roles of dephosphorylation of phosphorylase-a as compared with allosteric inhibition, (ii) counteraction of the efficacy of the inhibitors on glycogenolysis by dibutyryl-cAMP and (iii) stimulation of glycogen synthesis.
Assuntos
Glicogênio Fosforilase/antagonistas & inibidores , Nucleosídeos de Purina/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Alcenos/síntese química , Alcenos/farmacologia , Regulação Alostérica , Sítios de Ligação , Inibidores Enzimáticos/metabolismo , Flavonoides/síntese química , Flavonoides/farmacologia , Glicogênio/biossíntese , Glicogenólise/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Piperidinas/síntese química , Piperidinas/farmacologiaRESUMO
Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of glycogen phosphorylase b (GPb), N-acyl derivatives of beta-D-glucopyranosylamine, that bind at the catalytic site of the enzyme. The first good inhibitor in this class of compounds, N-acetyl-beta-D-glucopyranosylamine (NAG) (K(i) = 32 microM), has been previously characterized by biochemical, biological and crystallographic experiments at 2.3 angstroms resolution. Bioisosteric replacement of the acetyl group by trifluoroacetyl group resulted in an inhibitor, N-trifluoroacetyl-beta-D-glucopyranosylamine (NFAG), with a K(i) = 75 microM. To elucidate the structural basis of its reduced potency, we determined the ligand structure in complex with GPb at 1.8 angstroms resolution. To compare the binding mode of N-trifluoroacetyl derivative with that of the lead molecule, we also determined the structure of GPb-NAG complex at a higher resolution (1.9 angstroms). NFAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilize the T-state conformation of the 280 s loop by making several favourable contacts to Asn284 of this loop. The difference observed in the K(i) values of the two analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interaction, and desolvation effects.