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1.
Proteins ; 86(12): 1265-1276, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30168177

RESUMO

N-methyl-D-aspartate (NMDA) receptors are transmembrane glutamate-binding ion channels that mediate neurotransmission in mammals. NMDA receptor subunits are tetrameric complexes of GluN1 and GluN2A-D subunits, encoded by the GRIN gene family. Of these subunits, GluN2B is suggested to be required for normal development of the central nervous system. A mutation identified in a patient with developmental delay, E413G, resides in the GluN2B ligand-binding domain and substantially reduces glutamate potency by an unknown mechanism. GluN2B Gly413, though near the agonist, is not in van der Waals contact with glutamate. Visual analysis of the GluN2B structure with the E413G mutation modeled suggests that replacement of Glu with Gly at this position increases solvent access to the ligand-binding domain. This was confirmed by molecular modeling, which showed that the ligand is more mobile in GluN2B-E413G than WT GluN2B. Evaluation of agonist occupancy using random accelerated molecular dynamics (RAMD) simulations predicts that the glutamate exits the binding-site more rapidly for GluN2B-E413G than WT receptors. This analysis was extended to other binding-site mutations, which produced qualitative agreement between experimentally determined EC50 values, deactivation time constants, and ligand motion within the binding-site. Furthermore, long sub-microsecond molecular dynamics simulations of the bi-lobed ligand-binding domain revealed that it adopted a cleft-open ligand-free state more often for GluN2B-E413G than wild-type GluN2B. This is consistent with the idea that L-glutamate binding is altered such that the ligand-binding domain occupies the open-cleft conformation associated with the closed channel.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ácido Glutâmico/genética , Glicina/genética , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Mutação , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Solventes
2.
Mol Carcinog ; 56(1): 288-299, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27128654

RESUMO

The transcription factor NF-κB plays a central role in angiogenesis in colorectal cancer (CRC). Curcumin is a natural dietary product that inhibits NF-κB. The objective of this study is to evaluate the antiangiogenic effects of curcumin and two potent synthetic analogues (EF31 and UBS109) in CRC. IC50 values for curcumin, EF31, and UBS109 were determined in the HCT116 and HT-29 cell lines. HUVEC tube formation, egg CAM assay, and matrigel plug assays revealed decreased angiogenesis in cell lines treated with curcumin, EF31, or UBS109. Curcumin and its analogues significantly inhibited VEGF-A synthesis and secretion in both cell lines in association with loss of HIF-1α, COX-2, and p-STAT-3 expression. Nuclear NF-κB expression was inhibited by curcumin, EF31, and UBS109. Transfection of p65-NF-κB in HCT116 and HT-29 cells resulted in increased expression of HIF-1α, COX-2, STAT-3, and VEGF-A. Treatment with curcumin, EF31, or UBS109 inhibited these effects in transfected cell lines. In mice carrying HCT116 and HT-29 cell xenografts, EF31 and UBS109 inhibited subcutaneous tumor growth and potentiated the effects of oxaliplatin and 5-FU. Tumors from treated animals revealed inhibition of HIF-1α, COX-2, p-STAT-3, and VEGF expression. Our findings suggest that inhibition of NF-κB leading to decreased transcription and expression of HIF-1α, COX-2, STAT-3, and VEGF is a rational approach for antiangiogenic therapy in CRC. The distinctive properties of EF31 and UBS109 make them promising therapeutic agents for development in CRC as single agents or as part of combination chemotherapy regimens. © 2016 Wiley Periodicals, Inc.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Colo/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Curcumina/análogos & derivados , Neovascularização Patológica/tratamento farmacológico , Piperidonas/uso terapêutico , Piridinas/uso terapêutico , Reto/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Animais , Galinhas , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Curcumina/farmacologia , Curcumina/uso terapêutico , Feminino , Células HCT116 , Células HT29 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Nus , NF-kappa B/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Piperidonas/farmacologia , Piridinas/farmacologia , Ratos , Reto/metabolismo , Reto/patologia , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Biochem Biophys Res Commun ; 466(1): 28-32, 2015 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26301631

RESUMO

CXCR4 is a GPCR involved in leukocyte trafficking. Small molecule antagonists of the receptor may treat inflammatory disease, cancer and HIV. Here we probe the binding of a tetrahydroisoquinoline-based antagonist (TIQ-10) to CXCR4 using saturation transfer double-difference (STDD) NMR. STDD spectra were acquired using extracts from Chinese Hamster Ovary cells expressing membrane-embedded CXCR4. The experiments demonstrate competitive binding between TIQ-10 and established antagonists and provide the TIQ-10 - CXCR4 binding epitope. Molecular modeling of TIQ-10 into the binding pocket provides a pose consistent with STDD-derived interactions. This study paves the way for future investigations of GPCR-ligand interactions in a biological milieu for use in chemical biology, biochemistry, structural biology, and rational drug design.


Assuntos
Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologia , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Receptores CXCR4/química
4.
Acc Chem Res ; 47(8): 2682-91, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25052294

RESUMO

Paclitaxel (PTX), introduced into the clinic in 1991, has revealed itself as an effective antimicrotubule drug for treatment of a range of otherwise intractable cancers. Along with docetaxel (DTX) and in combination with other agents such as cisplatin, it has proven to be a first-line therapy. Unfortunately, PTX and DTX carry severe liabilities such as debilitating side effects, rapid onset of resistance, and rather complex molecular structures offering substantial challenges to ease of synthetic manipulation. Consequently, the past 15 years has witnessed many efforts to synthesize and test highly modified analogs based on intuitive structural similarity relationships with the PTX molecular skeleton, as well as efforts to mimic the conformational profile of the ligand observed in the macromolecular tubulin-PTX complex. Highly successful improvements in potency, up to 50-fold increases in IC50, have been achieved by constructing bridges between distal centers in PTX that imitate the conformer of the electron crystallographic binding pose. Much less successful have been numerous attempts to truncate PTX by replacing the baccatin core with simpler moieties to achieve PTX-like potencies and applying a wide range of flexible synthesis-based chemistries. Reported efforts, characterized by a fascinating array of baccatin substitutes, have failed to surpass the bioactivities of PTX in both microtubule disassembly assays and cytotoxicity measurements against a range of cell types. Most of the structures retain the main elements of the PTX C13 side chain, while seeking a smaller rigid bicycle as a baccatin replacement adorned with substituents to mimic the C2 benzoyl moiety and the oxetane ring. We surmise that past studies have been handicapped by solubility and membrane permeability issues, but primarily by the existence of an expansive taxane binding pocket and the discrepancy in molecular size between PTX and the pruned analogs. A number of these molecules offer molecular volumes 50-60% that of PTX, fewer contacts with the tubulin protein, severe mismatches with the PTX pharmacophore, lessened capacity to dispel binding site waters contributing to ΔGbind, and unanticipated binding poses. The latter is a critical drawback if molecular designs of simpler PTX structures are based on a perceived or known PTX binding conformation. We conclude that design and synthesis of a highly cytotoxic tubulin-assembly agent based on the paclitaxel pharmacophore remains an unsolved challenge, but one that can be overcome by focus on the architecture of the taxane binding site independent of the effective, but not unique, hand-in-glove match represented by the PTX-tubulin complex.


Assuntos
Antineoplásicos/química , Paclitaxel/análogos & derivados , Antineoplásicos/metabolismo , Sítios de Ligação , Docetaxel , Paclitaxel/metabolismo , Ligação Proteica , Taxoides/química , Taxoides/metabolismo , Termodinâmica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Água/química , Água/metabolismo
5.
Mol Cell Biochem ; 401(1-2): 1-10, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25416449

RESUMO

UBS109 is a curcumin analog that possesses antitumor properties has been shown to stimulate osteoblastogenesis and suppress osteoclastogenesis in vitro. This study was undertaken to determine whether UBS109 might alleviate the inhibitory activity of breast cancer cells on osteoblastic mineralization and stimulatory effects on osteoclastogenesis. Mouse bone marrow cells were cocultured with breast cancer MDA-MB-231 bone metastatic cells in vitro. UBS109 stimulated osteoblastic mineralization and suppressed adipogenesis and osteoclastogenesis in bone marrow culture. Coculture with MDA-MB-231 cells suppressed osteoblastic mineralization and enhanced osteoclastogenesis in bone marrow culture. Effects that were reserved by UBS109 (50-200 nM). Mineralization in preosteoblastic MC3T3-E1 cells was suppressed by coculture with MDA-MB-231 cells, while MDA-MB-231 cells did not have effects on osteoclastogenesis of RAW267.4 cells in vitro. UBS109 (500 nM) revealed toxic effects on MDA-MB-231 bone metastatic cells. This study demonstrates that UBS109, which is an antitumor agent, reveals restorative effects on bone marrow cell differentiation disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro. This in vitro model may be a useful tool to evaluate the mechanism of breast cancer cell bone metastasis.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/patologia , Neoplasias da Mama/patologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Piperidonas/farmacologia , Piridinas/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Neoplasias Ósseas/secundário , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Humanos , Camundongos , Osteoblastos/patologia , Osteoclastos/patologia
6.
Mol Pharmacol ; 85(4): 618-29, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24452473

RESUMO

Three residues within the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluA1 C terminus (Ser818, Ser831, Thr840) can be phosphorylated by Ca(2+)/phospholipid-dependent protein kinase (PKC). Here, we show that PKC phosphorylation of GluA1 Ser818 or Thr840 enhances the weighted mean channel conductance without altering the response time course or agonist potency. These data support the idea that these residues constitute a hyper-regulatory domain for the AMPA receptor. Introduction of phosphomimetic mutations increases conductance only at these three sites within the proximal C terminus, consistent with a structural model with a flexible linker connecting the distal C-terminal domain to the more proximal domain containing a helix bracketed by Ser831 and Thr840. NMR spectra support this model and raise the possibility that phosphorylation can alter the configuration of this domain. Our findings provide insight into the structure and function of the C-terminal domain of GluA1, which controls AMPA receptor function and trafficking during synaptic plasticity in the central nervous system.


Assuntos
Proteína Quinase C/metabolismo , Receptores de AMPA/metabolismo , Serina/metabolismo , Treonina/metabolismo , Animais , Feminino , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Camundongos , Modelos Moleculares , Mutação , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Cultura Primária de Células , Conformação Proteica , Ratos , Receptores de AMPA/agonistas , Receptores de AMPA/genética
7.
Mol Pharmacol ; 86(5): 548-60, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25205677

RESUMO

NMDA receptors are tetrameric complexes of GluN1, GluN2A-D, and GluN3A-B subunits and are involved in normal brain function and neurologic disorders. We identified a novel class of stereoselective pyrrolidinone (PYD) positive allosteric modulators for GluN2C-containing NMDA receptors, exemplified by methyl 4-(3-acetyl-4-hydroxy-1-[2-(2-methyl-1H-indol-3-yl)ethyl]-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate. Here we explore the site and mechanism of action of a prototypical analog, PYD-106, which at 30 µM does not alter responses of NMDA receptors containing GluN2A, GluN2B, and GluN2D and has no effect on AMPA [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] and kainate receptors. Coapplication of 50 µM PYD-106 with a maximally effective concentration of glutamate and glycine increases the response of GluN1/GluN2C NMDA receptors in HEK-293 cells to 221% of that obtained in the absence of PYD (taken as 100%). Evaluation of the concentration dependence of this enhancement revealed an EC50 value for PYD of 13 µM. PYD-106 increased opening frequency and open time of single channel currents activated by maximally effective concentrations of agonist but only had modest effects on glutamate and glycine EC50. PYD-106 selectively enhanced the responses of diheteromeric GluN1/GluN2C receptors but not triheteromeric GluN1/GluN2A/GluN2C receptors. Inclusion of residues encoded by GluN1-exon 5 attenuated the effects of PYD. Three GluN2C residues (Arg194, Ser470, Lys470), at which mutagenesis virtually eliminated PYD function, line a cavity at the interface of the ligand binding and the amino terminal domains in a homology model of GluN1/GluN2C built from crystallographic data on GluN1/GluN2B. We propose that this domain interface constitutes a new allosteric modulatory site on the NMDA receptor.


Assuntos
Regulação Alostérica/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Células HEK293 , Humanos , Ratos , Relação Estrutura-Atividade , Xenopus laevis
8.
Chembiochem ; 15(11): 1614-20, 2014 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-24990206

RESUMO

The CXC chemokine receptor 4 (CXCR4) is involved in chemotaxis and serves as a coreceptor for T-tropic HIV-1 viral entry, thus making this receptor an attractive drug target. Recently, crystal structures of CXCR4 were reported as complexes with the small molecule IT1t and the CVX15 peptide. Follow-up efforts to model different antagonists into the small molecule CXCR4:IT1t crystal structure did not generate poses consistent with either the X-ray crystal structure or site-directed mutagenesis (SDM). Here, we compare the binding pockets of the two CXCR4 crystal structures, revealing differences in helices IV, V, VI, and VII, with major differences for the His203 residue buried in the binding pocket. The small molecule antagonist AMD11070 was docked into both CXCR4 crystal structures. An AMD11070 pose identified from the CXCR4:CVX15 model presented interactions with Asp171, Glu288, Trp94, and Asp97, consistent with published SDM data, thus suggesting it is the bioactive pose. A CXCR4 receptor model was optimized around this pose of AMD11070, and the resulting model correlated HIV-1 inhibition with MM-GBSA docking scores for a congeneric AMD11070-like series. Subsequent NAMFIS NMR results successfully linked the proposed binding pose to an independent experimental structure. These results strongly suggest that not all small molecules will bind to CXCR4 in a similar manner as IT1t. Instead, the CXCR4:CVX15 crystal structure may provide a binding locus for small organic molecules that is more suitable than the secondary IT1t site. This work is expected to provide modeling insights useful for future CXCR4 antagonist and X4-tropic HIV-1 based drug design efforts.


Assuntos
Fármacos Anti-HIV/farmacologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Peptídeos/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Aminoquinolinas , Fármacos Anti-HIV/química , Benzimidazóis , Sítios de Ligação/efeitos dos fármacos , Butilaminas , Cristalografia por Raios X , Compostos Heterocíclicos com 1 Anel/química , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Peptídeos/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
9.
Mol Genet Metab ; 113(3): 161-70, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24863970

RESUMO

The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. Demonstrating a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Receptores de N-Metil-D-Aspartato/genética , Síndrome de Smith-Magenis/diagnóstico , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Síndrome de Smith-Magenis/genética , Transativadores
10.
Cell Tissue Res ; 357(1): 245-52, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24723227

RESUMO

Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10(6) cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150 mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20 mg/kg body weight) once daily for 5 days per week for 7 weeks. After UBS109 administration for 7 weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150 mg/kg body weight) and i.p. treatment of UBS109 (10 and 20 mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow cells obtained from normal nude mice in vitro. These findings suggest that the novel curcumin analogue UBS109 prevents breast cancer cell bone metastasis-induced bone loss by stimulating osteoblastic mineralization and suppressing osteoclastogenesis.


Assuntos
Neoplasias Ósseas/prevenção & controle , Neoplasias Ósseas/secundário , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Piperidonas/farmacologia , Piridinas/farmacologia , Animais , Reabsorção Óssea/patologia , Reabsorção Óssea/prevenção & controle , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Osteoblastos/patologia , Osteoclastos/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Org Biomol Chem ; 12(44): 8952-65, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25277547

RESUMO

A structure-based design campaign for non-covalent small molecule inhibitors of human granzyme B was carried out by means of a virtual screening strategy employing three constraints and probe site-mapping with FTMAP to identify ligand "hot spots". In addition, new scaffolds of diverse structures were subsequently explored with ROCS shape-based superposition methods, following by Glide SP docking, induced fit docking and analysis of QikProp molecular properties. Novel classes of moderately active small molecule blockers (≥25 µM IC50 values) from commercially available libraries were identified, and three novel scaffolds have been synthesized by multi-step procedures. Furthermore, we provide an example of a comprehensive structure-based drug discovery approach to non-covalent inhibitors that relies on the X-ray structure of a covalently bound ligand and suggest that the design path may be compromised by alternative and unknown binding poses.


Assuntos
Desenho de Fármacos , Granzimas/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Algoritmos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Granzimas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Relação Estrutura-Atividade
12.
J Chem Inf Model ; 54(8): 2214-23, 2014 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-25058809

RESUMO

Biologically active organic molecules characterized by a high single bond torsional barrier generate isolable isomers (atropisomers) and offer a unique stereochemical component to the design of selective therapeutic agents. The present work presents a nanomolar active inhibitor of myxoviruses, which most likely acts by blocking one or more cellular host proteins but also, serendipitously, exhibits axial chirality with an energy barrier of ΔG((++)) ≥30 kcal/mol. The latter has been probed by variable temperature NMR and microwave irradiation and by high level DFT transition state analysis and force field calculations. Full conformational profiles of the corresponding (aR,S) and (aS,S) atropisomers at ambient temperature were derived by conformer deconvolution with NAMFIS (NMR Analysis by Molecular Flexibility In Solution) methodology to generate seven and eight individual conformations, each assigned a % population. An accurate evaluation of a key torsion angle at the center of the molecules associated with a (3)JC-S-C-H coupling constant was obtained by mapping the S-C bond rotation with the MPW1PW91/6-31G-d,p DFT method followed by fitting the resulting dihedral angles and J-values to a Karplus expression. Accordingly, we have developed a complete conformational profile of diastereomeric atropisomers consistent with both high and low rotational barriers. We expect this assessment to assist the rationalization of the selectivity of the two (aR,S) and (aS,S) forms against host proteins, while offering insights into their divergent toxicity behavior.


Assuntos
Antivirais/química , Benzimidazóis/química , Fatores Celulares Derivados do Hospedeiro/antagonistas & inibidores , Orthomyxoviridae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Animais , Antivirais/síntese química , Antivirais/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Cristalografia por Raios X , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/metabolismo , Células Eucarióticas/patologia , Células Eucarióticas/virologia , Fatores Celulares Derivados do Hospedeiro/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Conformação Molecular , Orthomyxoviridae/fisiologia , Ligação Proteica , Teoria Quântica , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Estereoisomerismo , Termodinâmica
13.
Molecules ; 20(1): 249-92, 2014 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-25547726

RESUMO

Curcumin is a natural product with several thousand years of heritage. Its traditional Asian application to human ailments has been subjected in recent decades to worldwide pharmacological, biochemical and clinical investigations. Curcumin's Achilles heel lies in its poor aqueous solubility and rapid degradation at pH ~ 7.4. Researchers have sought to unlock curcumin's assets by chemical manipulation. One class of molecules under scrutiny are the monocarbonyl analogs of curcumin (MACs). A thousand plus such agents have been created and tested primarily against cancer and inflammation. The outcome is clear. In vitro, MACs furnish a 10-20 fold potency gain vs. curcumin for numerous cancer cell lines and cellular proteins. Similarly, MACs have successfully demonstrated better pharmacokinetic (PK) profiles in mice and greater tumor regression in cancer xenografts in vivo than curcumin. The compounds reveal limited toxicity as measured by murine weight gain and histopathological assessment. To our knowledge, MAC members have not yet been monitored in larger animals or humans. However, Phase 1 clinical trials are certainly on the horizon. The present review focuses on the large and evolving body of work in cancer and inflammation, but also covers MAC structural diversity and early discovery for treatment of bacteria, tuberculosis, Alzheimer's disease and malaria.


Assuntos
Curcumina/química , Mimetismo Molecular , Animais , Cristalografia por Raios X , Curcumina/farmacocinética , Curcumina/farmacologia , Curcumina/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Camundongos , Neoplasias/tratamento farmacológico
14.
Beilstein J Org Chem ; 9: 197-203, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23400228

RESUMO

High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC(50) values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

15.
Mol Pharmacol ; 82(2): 344-59, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22596351

RESUMO

We have used recent structural advances in our understanding of the N-methyl-d-aspartate (NMDA) receptor amino terminal domain to explore the binding mode of multiple diaryl GluN2B-selective negative allosteric modulators at the interface between the GluN1 and GluN2B amino-terminal domains. We found that interaction of the A ring within the binding pocket seems largely invariant for a variety of structurally distinct ligands. In addition, a range of structurally diverse linkers between the two aryl rings can be accommodated by the binding site, providing a potential opportunity to tune interactions with the ligand binding pocket via changes in hydrogen bond donors, acceptors, as well as stereochemistry. The most diversity in atomic interactions between protein and ligand occur in the B ring, with functional groups that contain electron donors and acceptors providing additional atomic contacts within the pocket. A cluster of residues distant to the binding site also control ligand potency, the degree of inhibition, and show ligand-induced increases in motion during molecular dynamics simulations. Mutations at some of these residues seem to distinguish between structurally distinct ligands and raise the possibility that GluN2B-selective ligands can be divided into multiple classes. These results should help facilitate the development of well tolerated GluN2B subunit-selective antagonists.


Assuntos
Piperidinas/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Feminino , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Piperidinas/farmacologia , Ligação Proteica/genética , Mapeamento de Interação de Proteínas/métodos , Ratos , Receptores de N-Metil-D-Aspartato/deficiência , Xenopus laevis
16.
J Chem Inf Model ; 52(12): 3190-9, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23157456

RESUMO

We describe the application of different methods in the development of QSAR models for IKKA and IKKB inhibition. The results show that the best QSAR models provide highly accurate predictions for existing IkB-kinase (IKK) inhibitors. The exceptions, corresponding to 5% of the known collection of inhibitors, are five classes of compounds incorporating the nitrile or sulfonamide moieties, small compounds with molecular weights of less than 300, and two classes of blockers considered to be type II kinase inhibitors. Comparison of our novel IKKB homology model and the recently reported IKKB crystal structure implies that a predictive protein-antagonist complex structure is more likely to exist as an inactive form in the crystalline state as observed in the recent protein X-ray structure.


Assuntos
Descoberta de Drogas/métodos , Quinase I-kappa B/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Relação Quantitativa Estrutura-Atividade , Cristalografia por Raios X , Bases de Dados de Produtos Farmacêuticos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo , Homologia de Sequência de Aminoácidos
17.
J Neurosci ; 30(7): 2741-54, 2010 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-20164358

RESUMO

We have studied relative efficacies of NR1 agonists glycine and d-cycloserine (DCS), and found efficacy to be dependent on the NR2 subunit. DCS shows partial agonism at NR1/NR2B but has higher relative efficacy than glycine at NR1/NR2C receptor. Molecular dynamics (MD) simulations of the NR1/NR2B and NR1/NR2C agonist binding domain dimer suggest only subtle differences in the interactions of DCS with NR1 binding site residues relative to glycine. The most pronounced differences were observed in the NR1/NR2C simulation between the orientation of helices F and G of the NR1 subunit. Interestingly, Helix F was previously proposed to influence receptor gating and to adopt an orientation depending on agonist efficacy. MD simulations and site-directed mutagenesis further suggest a role for residues at the agonist binding domain dimer interface in regulating DCS efficacy. To relate the structural rearrangements to receptor gating, we recorded single-channel currents from outside-out patches containing a single active NR1/NR2C receptor. DCS increased the mean open time and open probability of NR1/NR2C receptors compared with glycine. Maximum likelihood fitting of a gating model for NR1/NR2C receptor activation to the single-channel data suggests that DCS specifically accelerates the rate constant governing a fast gating step and reduces the closing rate. These changes appear to reflect a decreased activation energy for a pregating step and increased stability of the open states. We suggest that the higher efficacy of DCS at NR1/NR2C receptors involves structural rearrangements at the dimer interface and an effect on NR1/NR2C receptor pregating conformational changes.


Assuntos
Antibióticos Antituberculose/farmacologia , Ciclosserina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Biofísica , Linhagem Celular Transformada , Simulação por Computador , Relação Dose-Resposta a Droga , Estimulação Elétrica/métodos , Feminino , Glicina/farmacologia , Humanos , Ativação do Canal Iônico/genética , Microinjeções/métodos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese/genética , Oócitos , Técnicas de Patch-Clamp/métodos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevis
18.
Mol Pharmacol ; 80(5): 782-95, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21807990

RESUMO

The compound 4-(5-(4-bromophenyl)-3-(6-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-oxobutanoic acid (DQP-1105) is a representative member of a new class of N-methyl-d-aspartate (NMDA) receptor antagonists. DQP-1105 inhibited GluN2C- and GluN2D-containing receptors with IC(50) values that were at least 50-fold lower than those for recombinant GluN2A-, GluN2B-, GluA1-, or GluK2-containing receptors. Inhibition was voltage-independent and could not be surmounted by increasing concentrations of either coagonist, glutamate or glycine, consistent with a noncompetitive mechanism of action. DQP-1105 inhibited single-channel currents in excised outside-out patches without significantly changing mean open time or single-channel conductance, suggesting that DQP inhibits a pregating step without changing the stability of the open pore conformation and thus channel closing rate. Evaluation of DQP-1105 inhibition of chimeric NMDA receptors identified two key residues in the lower lobe of the GluN2 agonist binding domain that control the selectivity of DQP-1105. These data suggest a mechanism for this new class of inhibitors and demonstrate that ligands can access, in a subunit-selective manner, a new site located in the lower, membrane-proximal portion of the agonist-binding domain.


Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Pirazóis/farmacologia , Quinolonas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Células Cultivadas , Cricetinae , DNA Complementar , Antagonistas de Aminoácidos Excitatórios/química , Humanos , Técnicas de Patch-Clamp , Pirazóis/química , Quinolonas/química , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Relação Estrutura-Atividade
19.
J Am Chem Soc ; 133(8): 2427-36, 2011 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-21299225

RESUMO

Dictyostatin (DCT, 1) is a complex, flexible polyketide macrolide that demonstrates potent microtubule-polymerization activity. Both a solution structure (2a) and a possible binding mode for DCT (Conf-1) have been proposed by earlier NMR experiments. In the present study, the conformational landscape of DCT in DMSO-d(6) and methanol-d(4) was explored using extensive force-field-based conformational searches combined with geometric parameters derived from solution NMR data. The results portray a diversity of conformations for dictyostatin that illustrates the molecule's flexibility and excludes the previously suggested dominant solution conformation 2a. One conformation present in DMSO-d(6) with a 7% population (Conf-2, 0.6 kcal/mol above the global minimum at 298°) also satisfies the TR-NOESY NMR parameters of Canales et al. that characterize the taxane binding-site interaction between DCT and assembled microtubules in water. Application of several docking methods (Glide, Autodock, and RosettaLigand) has identified a low-energy binding model of the DCT/ß-tubulin complex (Pose-2/Conf-2) that is gratifyingly compatible with the emerging DCT structure-activity data.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Macrolídeos/química , Tubulina (Proteína)/química , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Soluções
20.
Chemistry ; 17(52): 14792-804, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22127984

RESUMO

A series of conformationally restrained epothilone analogues with a short bridge between the methyl groups at C6 and C8 was designed to mimic the binding pose assigned to our recently reported EpoA-microtubule binding model. A versatile synthetic route to these bridged epothilone analogues has been successfully devised and implemented. Biological evaluation of the compounds against A2780 human ovarian cancer and PC3 prostate cancer cell lines suggested that the introduction of a bridge between C6-C8 reduced potency by 25-1000 fold in comparison with natural epothilone D. Tubulin assembly measurements indicate these bridged epothilone analogues to be mildly active, but without significant microtubule stabilization capacity. Molecular mechanics and DFT energy evaluations suggest the mild activity of the bridged epo-analogues may be due to internal conformational strain.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Epotilonas/química , Epotilonas/síntese química , Epotilonas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Modelos Moleculares , Ligação Proteica , Relação Estrutura-Atividade
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