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1.
J Biol Chem ; 299(4): 104586, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36889589

RESUMO

MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors) are synaptic cell surface molecules that regulate the formation of trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs), which promote synaptic development. Mutations in MDGAs are implicated in various neuropsychiatric diseases. MDGAs bind NLGNs in cis on the postsynaptic membrane and physically block NLGNs from binding to NRXNs. In crystal structures, the six immunoglobulin (Ig) and single fibronectin III domains of MDGA1 reveal a striking compact, triangular shape, both alone and in complex with NLGNs. Whether this unusual domain arrangement is required for biological function or other arrangements occur with different functional outcomes is unknown. Here, we show that WT MDGA1 can adopt both compact and extended 3D conformations that bind NLGN2. Designer mutants targeting strategic molecular elbows in MDGA1 alter the distribution of 3D conformations while leaving the binding affinity between soluble ectodomains of MDGA1 and NLGN2 intact. In contrast, in a cellular context, these mutants result in unique combinations of functional consequences, including altered binding to NLGN2, decreased capacity to conceal NLGN2 from NRXN1ß, and/or suppressed NLGN2-mediated inhibitory presynaptic differentiation, despite the mutations being located far from the MDGA1-NLGN2 interaction site. Thus, the 3D conformation of the entire MDGA1 ectodomain appears critical for its function, and its NLGN-binding site on Ig1-Ig2 is not independent of the rest of the molecule. As a result, global 3D conformational changes to the MDGA1 ectodomain via strategic elbows may form a molecular mechanism to regulate MDGA1 action within the synaptic cleft.


Assuntos
Moléculas de Adesão de Célula Nervosa , Sinapses , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Sinapses/metabolismo , Sítios de Ligação , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Conformação Molecular , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo
2.
Nucleic Acids Res ; 50(16): 9548-9567, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-36039764

RESUMO

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.


Assuntos
Cisteína , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Cisteína/genética , Cisteína/metabolismo , Regulação da Expressão Gênica , DNA/genética , DNA/metabolismo , Oxirredução , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
3.
Structure ; 29(7): 664-678.e6, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33535026

RESUMO

Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Osteonectina/química , Osteonectina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Mapas de Interação de Proteínas
4.
J Mol Biol ; 432(19): 5287-5303, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32710982

RESUMO

Neuronal growth regulator 1 (NEGR1) and neurotrimin (NTM) are abundant cell-surface proteins found in the brain and form part of the IgLON (Immunoglobulin LSAMP, OBCAM, Neurotrimin) family. In humans, NEGR1 is implicated in obesity and mental disorders, while NTM is linked to intelligence and cognitive function. IgLONs dimerize homophilically and heterophilically, and they are thought to shape synaptic connections and neural circuits by acting in trans (spanning cellular junctions) and/or in cis (at the same side of a junction). Here, we reveal homodimeric structures of NEGR1 and NTM. They assemble into V-shaped complexes via their Ig1 domains, and disruption of the Ig1-Ig1 interface abolishes dimerization in solution. A hydrophobic ridge from one Ig1 domain inserts into a hydrophobic pocket from the opposing Ig1 domain producing an interaction interface that is highly conserved among IgLONs but remarkably plastic structurally. Given the high degree of sequence conservation at the interaction interface, we tested whether different IgLONs could elicit the same biological effect in vivo. In a small-scale study administering different soluble IgLONs directly into the brain and monitoring feeding, only NEGR1 altered food intake significantly. Taking NEGR1 as a prototype, our studies thus indicate that while IgLONs share a conserved mode of interaction and are able to bind each other as homomers and heteromers, they are structurally plastic and can exert unique biological action.


Assuntos
Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão de Célula Nervosa/química , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Cristalografia por Raios X , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Modelos Moleculares , Moléculas de Adesão de Célula Nervosa/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ratos Sprague-Dawley
5.
Curr Res Struct Biol ; 2: 1-13, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32542236

RESUMO

ΔFosB is a highly stable transcription factor that accumulates in specific brain regions upon chronic exposure to drugs of abuse, stress, or seizures, and mediates lasting behavioral responses. ΔFosB reportedly heterodimerizes with JunD forming a canonical bZIP leucine zipper coiled coil that clamps onto DNA. However, the striking accumulation of ΔFosB protein in brain upon chronic insult has brought its molecular status into question. Here, we demonstrate through a series of crystal structures that the ΔFosB bZIP domain self-assembles into stable oligomeric assemblies that defy the canonical arrangement. The ΔFosB bZIP domain also self-assembles in solution, and in neuron-like Neuro 2a cells it is trapped into molecular arrangements that are consistent with our structures. Our data suggest that, as ΔFosB accumulates in brain in response to chronic insult, it forms non-canonical assemblies. These species may be at the root of ΔFosB's striking protein stability, and its unique transcriptional and behavioral consequences.

6.
Nucleic Acids Res ; 45(19): 11425-11436, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28981703

RESUMO

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a 'redox switch' centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the 'OFF' state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.


Assuntos
DNA/química , Domínios Proteicos , Proteínas Proto-Oncogênicas c-jun/química , Fator de Transcrição AP-1/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Oxirredução , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
7.
Neuron ; 94(6): 1132-1141.e4, 2017 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-28641112

RESUMO

Neuroligins and neurexins promote synapse development and validation by forming trans-synaptic bridges spanning the synaptic cleft. Select pairs promote excitatory and inhibitory synapses, with neuroligin 2 (NLGN2) limited to inhibitory synapses and neuroligin 1 (NLGN1) dominating at excitatory synapses. The cell-surface molecules, MAM domain-containing glycosylphosphatidylinositol anchor 1 (MDGA1) and 2 (MDGA2), regulate trans-synaptic adhesion between neurexins and neuroligins, impacting NLGN2 and NLGN1, respectively. We have determined the molecular mechanism of MDGA action. MDGA1 Ig1-Ig2 is sufficient to bind NLGN2 with nanomolar affinity; its crystal structure reveals an unusual locked rod-shaped array. In the crystal structure of the complex, two MDGA1 Ig1-Ig2 molecules each span the entire NLGN2 dimer. Site-directed mutagenesis confirms the observed interaction interface. Strikingly, Ig1 from MDGA1 binds to the same region on NLGN2 as neurexins do. Thus, MDGAs regulate the formation of neuroligin-neurexin trans-synaptic bridges by sterically blocking access of neurexins to neuroligins.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Adesão Celular/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular , Cristalografia , Humanos , Mutagênese Sítio-Dirigida , Mutação , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Estrutura Quaternária de Proteína
8.
Proc Natl Acad Sci U S A ; 114(10): E2053-E2062, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28137883

RESUMO

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Proteínas de Plantas/química , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Sítios de Ligação , Morte Celular/genética , Morte Celular/imunologia , Cristalografia por Raios X , Erwinia/patogenicidade , Erwinia/fisiologia , Interações Hospedeiro-Patógeno , Modelos Moleculares , Mutação , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Pseudomonas syringae/patogenicidade , Pseudomonas syringae/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transdução de Sinais , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/microbiologia , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
9.
ChemMedChem ; 12(3): 207-213, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28032464

RESUMO

Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure-based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)-specific inhibitors. An enzyme-linked immunosorbent assay (ELISA) in 384-well format was employed to evaluate the inhibitory activity of macrocycles in a cell-based assay assessing tyrosine phosphorylation of MerTK. Through structure-activity relationship (SAR) studies, analogue 11 [UNC2541; (S)-7-amino-N-(4-fluorobenzyl)-8-oxo-2,9,16-triaza-1(2,4)-pyrimidinacyclohexadecaphane-1-carboxamide] was identified as a potent and MerTK-specific inhibitor that exhibits sub-micromolar inhibitory activity in the cell-based ELISA. In addition, an X-ray structure of MerTK protein in complex with 11 was resolved to show that these macrocycles bind in the MerTK ATP pocket.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirimidinas/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Compostos Macrocíclicos/química , Simulação de Acoplamento Molecular , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Pirimidinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , c-Mer Tirosina Quinase
10.
ACS Med Chem Lett ; 7(12): 1044-1049, 2016 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-27994735

RESUMO

Mer tyrosine kinase (MerTK) is aberrantly elevated in various tumor cells and has a normal anti-inflammatory role in the innate immune system. Inhibition of MerTK may provide dual effects against these MerTK-expressing tumors through reducing cancer cell survival and redirecting the innate immune response. Recently, we have designed novel and potent macrocyclic pyrrolopyrimidines as MerTK inhibitors using a structure-based approach. The most active macrocycles had an EC50 below 40 nM in a cell-based MerTK phosphor-protein ELISA assay. The X-ray structure of macrocyclic analogue 3 complexed with MerTK was also resolved and demonstrated macrocycles binding in the ATP binding pocket of the MerTK protein as anticipated. In addition, the lead compound 16 (UNC3133) had a 1.6 h half-life and 16% oral bioavailability in a mouse PK study.

11.
Mol Hum Reprod ; 22(6): 410-26, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26921398

RESUMO

STUDY HYPOTHESIS: Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. STUDY FINDING: This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. WHAT IS KNOWN ALREADY: Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. MAIN RESULTS AND THE ROLE OF CHANCE: Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 µM compared with an IC50 of 38.5 µM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 µM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions). LIMITATIONS, REASONS FOR CAUTION: The chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development. WIDER IMPLICATIONS OF THE FINDINGS: This study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets. LARGE SCALE DATA: None. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.


Assuntos
Inibidores Enzimáticos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , Glicólise/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Motilidade dos Espermatozoides/efeitos dos fármacos
12.
Protein Sci ; 24(4): 434-45, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25287625

RESUMO

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 Å) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Sequência de Aminoácidos , Clonagem Molecular , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
J Med Chem ; 56(23): 9693-700, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24219778

RESUMO

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.


Assuntos
Cicloexanóis/síntese química , Fibrinolíticos/síntese química , Fibrinolíticos/uso terapêutico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Cicloexanóis/uso terapêutico , Desenho de Fármacos , Humanos , Modelos Moleculares , Pirimidinas/química , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , c-Mer Tirosina Quinase
14.
J Med Chem ; 56(23): 9683-92, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24195762

RESUMO

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.


Assuntos
Antineoplásicos/síntese química , Cicloexanóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Piridinas/síntese química , Pirimidinas/síntese química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ciclização , Cicloexanóis/farmacologia , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-Atividade , c-Mer Tirosina Quinase
15.
Structure ; 21(1): 65-75, 2013 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-23260656

RESUMO

G protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and guanosine triphosphatase-accelerating protein, EhRGS-RhoGEF, in a nucleotide state-selective fashion. Coexpression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the Dbl homology domain Rho-binding surface and the pleckstrin homology domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical "regulator of G protein signaling" domain rather than a RhoGEF-RGS ("rgRGS") domain, suggesting a convergent evolution toward heterotrimeric and small G protein cross-talk.


Assuntos
Entamoeba histolytica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas de Protozoários/química , Transdução de Sinais , Trofozoítos/fisiologia , Substituição de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular , Linhagem Celular , Forma Celular , Sobrevivência Celular , Quimiotaxia , Cristalografia por Raios X , Drosophila melanogaster , Entamoeba histolytica/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/química , Interações Hospedeiro-Parasita , Hidrólise , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Trofozoítos/metabolismo
16.
PLoS Pathog ; 8(11): e1003040, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23166501

RESUMO

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGßγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.


Assuntos
Entamoeba histolytica/imunologia , Entamebíase/imunologia , Subunidades alfa de Proteínas de Ligação ao GTP/imunologia , Proteínas de Protozoários/imunologia , Fatores de Virulência/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Entamebíase/enzimologia , Entamebíase/genética , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/imunologia , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Humanos , Células Jurkat , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Transcrição Gênica/imunologia , Fatores de Virulência/biossíntese , Fatores de Virulência/química
17.
J Bacteriol ; 194(24): 6771-81, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23042995

RESUMO

Metal ion homeostasis is a critical function of many integral and peripheral membrane proteins. The genome of the etiologic agent of syphilis, Treponema pallidum, is compact and devoid of many metabolic enzyme genes. Nevertheless, it harbors genes coding for homologs of several enzymes that typically require either iron or zinc. The product of the tp0971 gene of T. pallidum, designated Tp34, is a periplasmic lipoprotein that is thought to be tethered to the inner membrane of this organism. Previous work on a water-soluble (nonacylated) recombinant version of Tp34 established that this protein binds to Zn(2+), which, like other transition metal ions, stabilizes the dimeric form of the protein. In this study, we employed analytical ultracentrifugation to establish that four transition metal ions (Ni(2+), Co(2+), Cu(2+), and Zn(2+)) readily induce the dimerization of Tp34; Cu(2+) (50% effective concentration [EC(50)] = 1.7 µM) and Zn(2+) (EC(50) = 6.2 µM) were the most efficacious of these ions. Mutations of the crystallographically identified metal-binding residues hindered the ability of Tp34 to dimerize. X-ray crystallography performed on crystals of Tp34 that had been incubated with metal ions indicated that the binding site could accommodate the metals examined. The findings presented herein, coupled with bioinformatic analyses of related proteins, point to Tp34's likely role in metal ion homeostasis in T. pallidum.


Assuntos
Lipoproteínas/química , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Metais/metabolismo , Treponema pallidum/metabolismo , Sítios de Ligação/genética , Transporte Biológico , Cobalto/química , Cobalto/metabolismo , Biologia Computacional , Cobre/química , Cobre/metabolismo , Cristalografia por Raios X , Dimerização , Genes Bacterianos , Lipoproteínas/genética , Metais/química , Família Multigênica , Mutação , Níquel/química , Níquel/metabolismo , Ligação Proteica , Multimerização Proteica , Treponema pallidum/genética , Zinco/química , Zinco/metabolismo
18.
ACS Med Chem Lett ; 3(2): 129-134, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22662287

RESUMO

Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.

19.
Structure ; 20(6): 1086-96, 2012 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-22632833

RESUMO

Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helix bundle protein. Only small perturbations to the backbone, 1-2 Å, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140°C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 Å).


Assuntos
Proteínas de Bactérias/química , Engenharia de Proteínas , Algoritmos , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Método de Monte Carlo , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Thermotoga maritima , Temperatura de Transição
20.
J Am Chem Soc ; 134(1): 375-85, 2012 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-22092237

RESUMO

Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 µM. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (Cα rmsd = 1.4 Å).


Assuntos
Desenho de Fármacos , Multimerização Proteica , Proteínas/química , Zinco , Modelos Moleculares , Estrutura Quaternária de Proteína , Proteínas/metabolismo
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