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1.
Mol Pharmacol ; 95(2): 196-209, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30514721

RESUMO

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein-coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography-mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P-NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P-binding receptor via liquid chromatography-tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.


Assuntos
Diazometano/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , Cromatografia Líquida/métodos , Células HEK293 , Humanos , Ligantes , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Espectrometria de Massas em Tandem/métodos
2.
Org Biomol Chem ; 16(29): 5254-5274, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-29892733

RESUMO

Syntheses of novel semi-orthogonally protected CycloTriVeratrilene (CTV) analogues with enhanced water solubility, that is 3 and 4, derived from the previously described CTV scaffold derivative 2 are described here. These scaffolds 2-4 enabled a sequential introduction of three different complementarity determining region (CDR) mimics via Cu(i)-catalysed azide-alkyne cycloaddition towards medium-sized protein mimics denoted as "synthetic antibodies". The highly optimised sequential introduction enabled selective attachment of three different CDR mimics in a one-pot fashion. This approach of obtaining synthetic antibodies, demonstrated by the synthesis of paratope mimics of monoclonal antibody infliximab (Remicade®), provided a facile access to a range of (highly) pre-organised molecules bearing three different (cyclic) peptide segments and may find a wide range of applications in the field of protein-protein interaction disruptors as well as in the development of synthetic vaccines or lectin mimics. The prepared synthetic antibodies were tested for their affinity towards tumour necrosis factor alpha using surface plasmon resonance and synthetic antibodies with micromolar affinities were uncovered.

3.
J Med Chem ; 61(12): 5395-5411, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29782167

RESUMO

A unique category of basic side chain containing amino acid derived sulfonyl fluorides (SFs) has been synthesized for incorporation into new proteasome inhibitors targeting the trypsin-like site of the 20S proteasome. Masking the former α-amino functionality of the amino acid starting derivatives as an azido functionality allowed an elegant conversion to the corresponding amino acid derived sulfonyl fluorides. The inclusion of different SFs at the P1 site of a proteasome inhibitor resulted in 14 different peptidosulfonyl fluorides (PSFs) having a high potency and an excellent selectivity for the proteolytic activity of the ß2 subunit over that of the ß5 subunit. The results of this study strongly indicate that a free N-terminus of PSFs inhibitors is crucial for high selectivity toward the trypsin-like site of the 20S proteasome. Nevertheless, all compounds are slightly more selective for inhibition of the constitutive over the immunoproteasome.


Assuntos
Aminoácidos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Ácidos Sulfínicos/química , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Complexo de Endopeptidases do Proteassoma/química , Relação Estrutura-Atividade , Tripsina/química , Tripsina/metabolismo
4.
Bioconjug Chem ; 29(4): 1091-1101, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29382188

RESUMO

Mimicry of the binding interface of antibody-antigen interactions using peptide-based modulators (i.e., epitope mimics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward fashion, thereby allowing for high-throughput analysis. The design of epitope mimics is highly influenced by their spatial configuration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be implemented. This paper describes the synthesis of bromide derivatives functionalized with a flexible TEG linker equipped with a thiol-moiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugated via the free thiol-moiety on maleimide-activated plate surfaces. The resulting covalent, uniform, and oriented coated surface of cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry of an antigen-antibody interaction of the HCV E2 glycoprotein. To the best of our knowledge, the benefit of cyclized peptides over linear peptides has been clearly demonstrated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated specificity toward their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of epitope mimics shows potential for high-throughput screening of key binding residues by simply changing the amino acid sequences within synthetic peptides. In this way, leucine-438 has been identified as a key binding residue for binding monoclonal antibody V3.2.


Assuntos
Hepacivirus/metabolismo , Mimetismo Molecular , Peptídeos Cíclicos/química , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Alquilação , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Ensaios de Triagem em Larga Escala , Polietilenoglicóis/química , Homologia de Sequência de Aminoácidos
5.
Bioorg Med Chem ; 25(19): 5055-5063, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28734665

RESUMO

Peptido sulfonyl fluoride derivatives were designed and synthesized containing a substituent on the alpha position (αPSFs) with respect to the sulfonyl fluoride electrophilic trap. The chemical reactivity of these α-substituted amino sulfonyl fluorides was studied and compared with the previously described ß-substituted amino sulfonyl fluorides in order to get a deeper insight into the importance of the immediate structural environment of the sulfonyl fluoride moiety. Unfortunately, the poor solubility of the resulting αPSFs precluded a proper evaluation of their biological activity.


Assuntos
Desenho de Fármacos , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Ácidos Sulfínicos/química , Ácidos Sulfínicos/farmacologia , Aminoácidos/síntese química , Aminoácidos/química , Aminoácidos/farmacologia , Humanos , Peptidomiméticos/síntese química , Inibidores de Proteassoma/síntese química , Solubilidade , Ácidos Sulfínicos/síntese química
6.
Bioorg Med Chem ; 25(18): 5008-5015, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28583807

RESUMO

The synthesis of a semi-orthogonally protected CycloTriVeratrilene (CTV) scaffold derivative as well as the sequential introduction of three different peptide loops onto this molecular scaffold via Cu(I)-catalyzed azide alkyne cycloaddition towards a medium-sized protein mimic is described. This approach for the construction of medium-sized protein mimics is illustrated by the synthesis of a paratope mimic of the monoclonal antibody Infliximab (Remicade®) and provides access to a range of highly pre-organized molecular constructs bearing three different peptide segments. This approach may find wide applications for development of protein-protein interaction disruptors as well as synthetic vaccines.


Assuntos
Peptídeos Cíclicos/síntese química , Compostos Policíclicos/química , Alcinos/química , Azidas/química , Catálise , Cobre/química , Reação de Cicloadição , Infliximab/química , Infliximab/metabolismo , Peptídeos Cíclicos/química
7.
Chembiochem ; 18(4): 387-395, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982494

RESUMO

Two polar hinges for cyclization of peptides have been developed, leading to bicyclic peptides and cyclized peptides with improved solubility and biological activity. Increasingly, we note that a good aqueous solubility of peptides is an absolute prerequisite, not only to allow handling and purification of our target peptides but also being crucial for biological activity characteristics. Compared to earlier hinges, the 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB) and 2,4,6-tris(bromomethyl)-s-triazine (TBMT), each containing three nitrogen atoms are structurally similar but chemically very different. Both were accessible in a one-step fashion from bromoacetonitrile. TATB and TBMT are very suitable for the preparation of more soluble bicyclic peptides. Azide-modified TATB and TBMT derivatives provide hinges for the preparation of cyclized peptides for incorporation on scaffolds to afford protein mimics.


Assuntos
Biomimética , Peptídeos Cíclicos/química , Conformação Molecular , Peptídeos Cíclicos/síntese química
8.
Bioorg Med Chem ; 24(16): 3429-35, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27316540

RESUMO

The success of inhibition of the proteasome by formation of covalent bonds is a major victory over the long held-view that this would lead to binding the wrong targets and undoubtedly lead to toxicity. Great challenges are now found in uncovering ensembles of new moieties capable of forming long lasting ties. We have introduced peptido sulfonyl fluorides for this purpose. Tuning the reactivity of this electrophilic trap may be crucial for modulating the biological action. Here we describe incorporation of a vinyl moiety into a peptido sulfonyl fluoride backbone, which should lead to a combined attack of the proteasome active site threonine on the double bond and the sulfonyl fluoride. Although this led to strong proteasome inhibitors, in vitro studies did not unambiguously demonstrate the formation of the proposed seven-membered ring structure. Possibly, formation of a seven-membered covalent adduct with the proteosomal active site threonine can only be achieved within the context of the enzyme. Nevertheless, this dual warhead concept may provide exclusive possibilities for duration and selectivity of proteasome inhibition.


Assuntos
Fluoretos/química , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Sulfonas/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cromatografia Líquida de Alta Pressão , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray
9.
Org Lett ; 16(11): 3106-9, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24856258

RESUMO

The synthesis of a new triazacyclophane scaffold (TACO scaffold) containing three selectively deprotectable amines is described. The TACO scaffold is conformationally more constrained than our frequently used TAC scaffold, due to introduction of a substituent on the para position of the benzoic acid hinge, which prevents ring flipping and makes it more attractive than the TAC scaffold for preparation of artificial receptor molecules or for mimicking discontinuous epitopes toward protein mimics when more preorganization is required.


Assuntos
Aminas/química , Compostos Aza/síntese química , Compostos Heterocíclicos com 2 Anéis/síntese química , Peptídeos Cíclicos/química , Proteínas/química , Receptores de Peptídeos/química , Sequência de Aminoácidos , Compostos Aza/química , Compostos Heterocíclicos com 2 Anéis/química , Estrutura Molecular
10.
Org Lett ; 16(8): 2138-41, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24708093

RESUMO

Synthetic mimics of protein surfaces have the potential to become inhibitors of protein-protein interactions or even synthetic vaccines. However, the synthesis of these complicated molecular constructs is still difficult. Here we describe an efficient and versatile synthesis of protein mimics containing up to three different cyclic peptides. Using a sequential native chemical ligation strategy, peptide loops containing a thioester handle were introduced onto a triazacyclophane scaffold bearing orthogonal protected cysteine residues.


Assuntos
Compostos Aza/química , Compostos Heterocíclicos com 2 Anéis/química , Peptídeos Cíclicos/síntese química , Cisteína/química , Estrutura Molecular , Peptídeos Cíclicos/química
11.
J Pept Sci ; 20(4): 235-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24599619

RESUMO

Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein-protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides. Using an orthogonal alkyne protection strategy, peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)-catalyzed azide alkyne cycloaddition. This method provides rapid access to protein mimics requiring different peptide segments for their interaction and activity.


Assuntos
Azidas/química , Proteínas da Membrana Bacteriana Externa/química , Bordetella pertussis/química , Cobre/química , Reação de Cicloadição , Mimetismo Molecular , Peptídeos Cíclicos/síntese química , Fatores de Virulência de Bordetella/química , Alcinos/química , Espectrometria de Massas por Ionização por Electrospray
12.
J Org Chem ; 77(22): 10058-64, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23078179

RESUMO

The synthesis of cyclic peptides containing a thioester handle using a sulfo-click linker is reported. These cyclic peptides can be coupled to N-terminal cysteine-containing constructs via native chemical ligation. A successful application of a cyclic peptide bearing a thioester handle in native chemical ligation is shown by a high yielding ligation.


Assuntos
Cisteína/análogos & derivados , Cisteína/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/síntese química , Compostos de Enxofre/química , Compostos de Enxofre/síntese química , Ligadura , Dados de Sequência Molecular
13.
Mol Microbiol ; 61(4): 940-7, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16879647

RESUMO

The role of isocitrate lyase (ICL) in the glyoxylate cycle and its necessity for persistence and virulence of Mycobacterium tuberculosis has been well described. Recent reports have alluded to an additional role for this enzyme in M. tuberculosis metabolism, specifically for growth on propionate. A product of beta-oxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the by-products of its metabolism via the methylcitrate cycle is vital due to their potentially toxic effects. Although the genome of M. tuberculosis encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methylisocitrate lyase (MCL). Detailed structural analysis of the MCL from Escherichia coli suggested that the differences in substrate specificity between MCLs and ICLs could be attributed to three conserved amino acid substitutions in the active site, suggesting an MCL signature. However, here we provide enzymatic evidence that shows that despite the absence of the MCL signature, ICL1 from M. tuberculosis can clearly function as a MCL. Furthermore, the crystal structure of ICL1 with pyruvate and succinate bound demonstrates that the active site can accommodate the additional methyl group without significant changes to the structure.


Assuntos
Proteínas de Bactérias/química , Citratos/metabolismo , Glioxilatos/metabolismo , Isocitrato Liase/química , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/isolamento & purificação , Carbono-Carbono Liases/metabolismo , Citrato (si)-Sintase/metabolismo , Cristalografia , Ácidos Graxos/metabolismo , Isocitrato Liase/isolamento & purificação , Modelos Moleculares , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Conformação Proteica , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismo
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