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1.
Angew Chem Int Ed Engl ; : e202401782, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38818649

RESUMO

gem-Diborylalkanes are highly valuable building blocks in organic synthesis and pharmaceutical chemistry due to their ability to participate in multi-step cross-coupling transformations, allowing for the rapid generation of molecular complexity. While progress has been made in their synthetic metholodology, the construction of ß-tertiary and C(sp3)-rich gem-diborylalkanes remains a synthetic challenge due to substrate limitations and steric hindrance issues. An approach is presented that utilizes synergistic photoredox and copper catalysis to achieve efficient C(sp3)-C(sp3) cross-coupling of alkyl N-hydroxyphthalimide esters, which can easily be obtained from alkyl carboxylic acids, with diborylmethyl species, providing a series of C(sp3)-rich gem-diborylalkanes with 1°, 2°, and even 3° ß positions. Furthermore, this approach can also be applied to complex medicinal compounds and natural products, offering rapid access to molecular complexity and late-stage functionalization of C(sp3)-rich drug candidates. Mechanistic experiments revealed that diborylmethyl Cu(I) species participated in both the photoredox process and the key C(sp3)-C(sp3) bond-forming step.

2.
Nucleic Acids Res ; 52(W1): W132-W139, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38647044

RESUMO

The possible effects of mutations on stability and function of a protein can only be understood in the context of protein 3D structure. The MutationExplorer webserver maps sequence changes onto protein structures and allows users to study variation by inputting sequence changes. As the user enters variants, the 3D model evolves, and estimated changes in energy are highlighted. In addition to a basic per-residue input format, MutationExplorer can also upload an entire replacement sequence. Previously the purview of desktop applications, such an upload can back-mutate PDB structures to wildtype sequence in a single step. Another supported variation source is human single nucelotide polymorphisms (SNPs), genomic coordinates input in VCF format. Structures are flexibly colorable, not only by energetic differences, but also by hydrophobicity, sequence conservation, or other biochemical profiling. Coloring by interface score reveals mutation impacts on binding surfaces. MutationExplorer strives for efficiency in user experience. For example, we have prepared 45 000 PDB depositions for instant retrieval and initial display. All modeling steps are performed by Rosetta. Visualizations leverage MDsrv/Mol*. MutationExplorer is available at: http://proteinformatics.org/mutation_explorer/.


Assuntos
Internet , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas , Software , Proteínas/genética , Proteínas/química , Humanos , Gráficos por Computador , Termodinâmica
3.
bioRxiv ; 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38464310

RESUMO

The possible effects of mutations on stability and function of a protein can only be understood in the context of protein 3D structure. The MutationExplorer webserver maps sequence changes onto protein structures and allows users to study variation by inputting sequence changes. As the user enters variants, the 3D model evolves, and estimated changes in energy are highlighted. In addition to a basic per-residue input format, MutationExplorer can also upload an entire replacement sequence. Previously the purview of desktop applications, such an upload can back-mutate PDB structures to wildtype sequence in a single step. Another supported variation source is human single nucelotide polymorphisms (SNPs), genomic coordinates input in VCF format.

4.
Cell Rep ; 43(1): 113640, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38180839

RESUMO

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.


Assuntos
Drosophila , Hiperpotassemia , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Larva/metabolismo , Potássio/metabolismo , Taquicardia , Adesão Celular
5.
Trends Biochem Sci ; 48(8): 726-739, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37349240

RESUMO

Adhesion-type G protein-coupled receptors (aGPCRs) have long resisted approaches to resolve the structural details of their heptahelical transmembrane (7TM) domains. Single-particle cryogenic electron microscopy (cryo-EM) has recently produced aGPCR 7TM domain structures for ADGRD1, ADGRG1, ADGRG2, ADGRG3, ADGRG4, ADGRG5, ADGRF1, and ADGRL3. We review the unique properties, including the position and conformation of their activating tethered agonist (TA) and signaling motifs within the 7TM bundle, that the novel structures have helped to identify. We also discuss questions that the kaleidoscope of novel aGPCR 7TM domain structures have left unanswered. These concern the relative positions, orientations, and interactions of the 7TM and GPCR autoproteolysis-inducing (GAIN) domains with one another. Clarifying their interplay remains an important goal of future structural studies on aGPCRs.


Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Adesão Celular , Relação Estrutura-Atividade , Receptores Acoplados a Proteínas G/química , Membrana Celular , Domínios e Motivos de Interação entre Proteínas
6.
SLAS Discov ; 28(5): 211-222, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37001588

RESUMO

The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. This class of enzymes catalyzes the interconversion between the cis and trans configuration of proline-containing peptide bonds. Mip has been shown to be important for the virulence of a wide range of pathogenic microorganisms, including the Gram-negative bacterium Burkholderia pseudomallei. Small molecules derived from the natural product rapamycin, lacking its immunosuppression-inducing moiety, inhibit Mip's peptidyl-prolyl cis-trans isomerase (PPIase) activity and lead to a reduction in pathogen load in vitro. Here, a fluorescence polarization assay (FPA) to enable the screening and effective development of BpMip inhibitors was established. A fluorescent probe was prepared, derived from previous pipecolic scaffold Mip inhibitors labeled with fluorescein. This probe showed moderate affinity for BpMip and enabled a highly robust FPA suitable for screening large compound libraries with medium- to high-throughput (Z factor ∼ 0.89) to identify potent new inhibitors. The FPA results are consistent with data from the protease-coupled PPIase assay. Analysis of the temperature dependence of the probe's binding highlighted that BpMip's ligand binding is driven by enthalpic rather than entropic effects. This has considerable consequences for the use of low-temperature kinetic assays.


Assuntos
Proteínas de Bactérias , Burkholderia pseudomallei , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/metabolismo , Corantes Fluorescentes/metabolismo , Peptidilprolil Isomerase/metabolismo , Macrófagos/metabolismo
7.
Molecules ; 28(3)2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36771089

RESUMO

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.


Assuntos
Cisteína , Imageamento por Ressonância Magnética , Humanos , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Xenônio/química , Neuropeptídeo Y
8.
J Antimicrob Chemother ; 77(6): 1625-1634, 2022 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-35245364

RESUMO

BACKGROUND: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. OBJECTIVES: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. METHODS: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. RESULTS: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. CONCLUSIONS: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.


Assuntos
Proteínas de Bactérias , Bactérias Gram-Negativas , Leishmania major , Peptidilprolil Isomerase , Proteínas de Protozoários , Proteínas de Bactérias/antagonistas & inibidores , Bactérias Gram-Negativas/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Macrófagos/metabolismo , Neisseria meningitidis , Peptidilprolil Isomerase/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Proteínas Recombinantes
9.
Biosens Bioelectron ; 192: 113506, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34325320

RESUMO

An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(ß-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.


Assuntos
Biomimética , Técnicas Biossensoriais , Estradiol , Estrogênios , Receptores de Estrogênio
10.
Mol Cell ; 81(5): 905-921.e5, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33497605

RESUMO

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.


Assuntos
Antígenos CD/química , Proteínas do Tecido Nervoso/química , Peptídeos/química , Receptores Acoplados a Proteínas G/química , Receptores de Peptídeos/química , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Expressão Gênica , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteólise , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais
11.
Bioorg Med Chem ; 24(21): 5134-5147, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27591009

RESUMO

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.


Assuntos
Burkholderia pseudomallei/enzimologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Legionella pneumophila/enzimologia , Peptidilprolil Isomerase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptidilprolil Isomerase/metabolismo , Relação Estrutura-Atividade
12.
Int J Antimicrob Agents ; 48(4): 401-8, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27516227

RESUMO

The pathogenic bacteria Chlamydia trachomatis, Neisseria gonorrhoeae and Neisseria meningitidis express the surface-exposed macrophage infectivity potentiator (MIP)-like protein, which plays a role in their pathogenicity. MIP exhibits a peptidyl-prolyl isomerase (PPIase) activity that is inhibited by rapamycin and FK506. In this study, pipecolic acid derivatives were tested for their activity against the chlamydial and neisserial MIP. Two MIP inhibitors were identified, PipN3 and PipN4, that affected the developmental cycle of C. trachomatis in HeLa cells. Furthermore, we could show that deletion of neisserial MIP or addition of the two MIP inhibitors affected the survival of N. gonorrhoeae in the presence of neutrophils. Furthermore, both compounds inhibited the adherence, invasion and/or survival of N. meningitidis in epithelial cells. These results confirm the importance of MIP-like proteins in infection and indicate the relevance of pipecolic acid derivatives as antimicrobials against C. trachomatis, N. gonorrhoeae and N. meningitidis.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Chlamydia trachomatis/patogenicidade , Neisseria gonorrhoeae/patogenicidade , Neisseria meningitidis/patogenicidade , Fatores de Virulência/antagonistas & inibidores , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Chlamydia trachomatis/imunologia , Chlamydia trachomatis/metabolismo , Técnicas de Inativação de Genes , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/fisiologia , Neisseria meningitidis/imunologia , Neisseria meningitidis/metabolismo , Neisseria meningitidis/fisiologia , Virulência/efeitos dos fármacos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
13.
Molecules ; 17(12): 14685-99, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23519247

RESUMO

The well-known Ugi reaction of aldehydes with amines, carboxylic acids and isocyanides leads to the formation of acyclic α-acylaminocarboxamides. Replacement of the carboxylic acid derivatives with ß-acyl substituted acrylic acids gives access to highly substituted 2,5-diketopiperazines in one single reaction-step without additives or complex reaction procedures. The obtained diketopiperazines show anti-proliferative effects on activated T cells and represent therefore potential candidates for targeting unwanted T cell-mediated immune responses.


Assuntos
Proliferação de Células/efeitos dos fármacos , Dicetopiperazinas/síntese química , Aldeídos/química , Aminas/química , Apoptose/efeitos dos fármacos , Cristalografia por Raios X , Células Dendríticas/metabolismo , Humanos , Linfócitos T/metabolismo
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