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1.
Chem Sci ; 10(3): 694-700, 2019 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-30774870

RESUMO

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that utilize the specificity of antibodies to selectively deliver highly potent cytotoxins to target cells. Although recent years have witnessed significant interest in ADCs, problems remain with the standard linkage chemistries used for cytotoxin-antibody bioconjugation. These typically (1) generate unstable constructs, which may lead to premature cytotoxin release, (2) often give a wide variance in drug-antibody ratios (DAR) and (3) have poor control of attachment location on the antibody, resulting in a variable pharmacokinetic profile. Herein, we report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation via covalent re-bridging of reduced disulfide bonds on native antibodies. Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads (including cytotoxins) in an efficient and site-selective manner with precise control over DAR. DVP linkers were also used to efficiently re-bridge both monomeric and dimeric protein systems, demonstrating their potential utility for general protein modification, protein stabilisation or the development of other protein-conjugate therapeutics.

3.
ACS Chem Biol ; 14(3): 526-533, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30702850

RESUMO

Stapled peptides have great potential as modulators of protein-protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a "toolbox" of diversified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure-activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.


Assuntos
Peptídeos/química , Alcinos/química , Sequência de Aminoácidos , Linhagem Celular , Permeabilidade da Membrana Celular , Humanos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismo
4.
Chem Sci ; 8(5): 3871-3878, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28966779

RESUMO

Transition metal catalysis has emerged as a powerful strategy to expand synthetic flexibility of protein modification. Herein, we report a cationic Ru(ii) system that enables the first example of alkyne hydrosilylation between dimethylarylsilanes and O-propargyl-functionalized proteins using a substoichiometric amount or low-loading of Ru(ii) catalyst to achieve the first C-Si bond formation on full-length substrates. The reaction proceeds under physiological conditions at a rate comparable to other widely used bioorthogonal reactions. Moreover, the resultant gem-disubstituted vinylsilane linkage can be further elaborated through thiol-ene coupling or fluoride-induced protodesilylation, demonstrating its utility in further rounds of targeted modifications.

5.
Science ; 346(6209): 638-641, 2014 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-25323695

RESUMO

Small molecules are useful tools for probing the biological function and therapeutic potential of individual proteins, but achieving selectivity is challenging when the target protein shares structural domains with other proteins. The Bromo and Extra-Terminal (BET) proteins have attracted interest because of their roles in transcriptional regulation, epigenetics, and cancer. The BET bromodomains (protein interaction modules that bind acetyl-lysine) have been targeted by potent small-molecule inhibitors, but these inhibitors lack selectivity for individual family members. We developed an ethyl derivative of an existing small-molecule inhibitor, I-BET/JQ1, and showed that it binds leucine/alanine mutant bromodomains with nanomolar affinity and achieves up to 540-fold selectivity relative to wild-type bromodomains. Cell culture studies showed that blockade of the first bromodomain alone is sufficient to displace a specific BET protein, Brd4, from chromatin. Expansion of this approach could help identify the individual roles of single BET proteins in human physiology and disease.


Assuntos
Sondas Moleculares/química , Proteínas Nucleares/química , Engenharia de Proteínas/métodos , Fatores de Transcrição/química , Sequência de Aminoácidos , Azepinas/química , Azepinas/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/química , Cristalografia por Raios X , Humanos , Leucina/genética , Modelos Moleculares , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis/química , Triazóis/farmacologia
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