RESUMO
As an emerging approach to protein perturbation, small molecule-induced protein degradation has gained significant attention as both a chemical tool and a potential therapeutic. To enable discrete control over its function, we have developed a broadly applicable approach for the optical activation of small molecule-induced protein degradation. By installing two different photolabile protecting groups, so-called caging groups, onto two different ligands recruiting Von Hippel-Lindau (VHL) and cereblon (CRBN) E3 ubiquitin ligases, our strategy enables light-triggered protein degradation for any small molecule warhead.
Assuntos
Proteólise , Bibliotecas de Moléculas Pequenas/química , Ligantes , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
MicroRNAs (miRNAs) are short, non-coding RNA molecules estimated to regulate expression of a large number of protein-coding genes and are implicated in a variety of biological processes such as development, differentiation, proliferation, and cell survival. Dysregulation of miRNAs has been attributed to the onset and progression of various human diseases, including cancer. MicroRNA-21 (miR-21), one of the most established oncogenic miRNAs, is found to be upregulated in a wide range of cancers making it an attractive therapeutic target. Employment of a luciferase-based live-cell reporter assay in a high-throughput screen of >300,000 small molecules led to the discovery of a new class of ether-amide miR-21 inhibitors. Following a structure-activity relationship study, an optimized lead molecule was found to inhibit miR-21 transcription. Furthermore, the inhibitor demonstrated cytotoxicity in a cervical cancer cell line via induction of apoptosis and was capable of reducing microtumor formation in a long-term clonogenic assay. Altogether, this work reports the discovery of a new small molecule inhibitor of miR-21 and demonstrates its potential as an alternative approach in cancer therapy.
Assuntos
MicroRNAs/metabolismo , Sobrevivência Celular , Humanos , Relação Estrutura-AtividadeRESUMO
A diverted total synthesis effort is described that is designed to prepare potent cytotoxins based on the actin-binding natural product bistramideâ A. The major focus of this study is the preparation of analogues that contain oxygenation at the C29 position, which is necessary for a key reaction in the sequence but is not present in the natural product. This process showed that C29 ketone analogues are accessed more readily and show similar potency compared to the natural product. The ability to incorporate C29 oxygenation and to replace a secondary alcohol by a primary alcohol allowed for the development of a more convergent approach that provides a potent analogue in just eight steps in its longest linear sequence.
RESUMO
Biological processes are naturally regulated with high spatial and temporal control, as is perhaps most evident in metazoan embryogenesis. Chemical tools have been extensively utilized in cell and developmental biology to investigate cellular processes, and conditional control methods have expanded applications of these technologies toward resolving complex biological questions. Light represents an excellent external trigger since it can be controlled with very high spatial and temporal precision. To this end, several optically regulated tools have been developed and applied to living systems. In this review we discuss recent developments of optochemical tools, including small molecules, peptides, proteins, and nucleic acids that can be irreversibly or reversibly controlled through light irradiation, with a focus on applications in cells and animals.
Assuntos
Fenômenos Fisiológicos Celulares/efeitos da radiação , Optogenética/métodos , Fotoquímica/métodos , Animais , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Descoberta de Drogas/métodos , Humanos , Luz , Simulação de Acoplamento Molecular , Ácidos Nucleicos/genética , Ácidos Nucleicos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Processos Fotoquímicos , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos da radiação , Proteínas/genética , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
α-Boryl ethers, carbonates, and acetals, readily prepared from the corresponding alcohols that are accessed through ketone diboration, react rapidly with hydrogen peroxide to release alcohols, aldehydes, and ketones through the collapse of hemiacetal intermediates. Experiments with α-boryl acetals containing a latent fluorophore clearly demonstrate that cargo can be released inside cells in the presence of exogenous or endogenous hydrogen peroxide. These experiments show that this protocol can be used for drug activation in an oxidative environment without generating toxic byproducts.
Assuntos
Álcoois/química , Aldeídos/química , Compostos de Boro/química , Éter/química , Peróxido de Hidrogênio/química , Espaço Intracelular/metabolismo , Cetonas/química , Acetais/química , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismoRESUMO
MicroRNAs (miRNAs) are single stranded RNA molecules of â¼22 nucleotides that negatively regulate gene expression. MiRNAs are involved in fundamental cellular processes, such as development, differentiation, proliferation, and survival. MiRNA misregulation has been linked to various human diseases, most notably cancer. MicroRNA-21 (miR-21), a well-established oncomiR, is significantly overexpressed in many types of human cancers, thus rendering miR-21 a potential therapeutic target. Using a luciferase-based reporter assay under the control of miR-21 expression, a high-throughput screen of >300,000 compounds led to the discovery of a new aryl amide class of small-molecule miR-21 inhibitors. Structure-activity relationship (SAR) studies resulted in the development of four aryl amide derivatives as potent and selective miR-21 inhibitors. The intracellular levels of various miRNAs in HeLa cells were analyzed by qRT-PCR revealing specificity for miR-21 inhibition over other miRNAs. Additionally, preliminary mechanism of action studies propose a different mode of action compared to previously reported miR-21 inhibitors, thus affording a new chemical probe for future studies.
Assuntos
Amidas/farmacologia , MicroRNAs/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Amidas/síntese química , Amidas/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células HeLa , Humanos , MicroRNAs/genética , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology.
Assuntos
Sondas Moleculares , Fótons , Proteínas/fisiologia , Cromatografia Líquida , Fluorescência , Células HEK293 , Humanos , Methanosarcina barkeri/química , Proteínas/química , Espectrometria de Massas em TandemRESUMO
We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.
Assuntos
Cisteína/química , Cisteína/genética , Escherichia coli/genética , Código Genético/genética , Homocisteína/química , Homocisteína/genética , Células Cultivadas , Cisteína/síntese química , Escherichia coli/citologia , Células HEK293 , Homocisteína/síntese química , Humanos , Luciferases de Renilla/química , Luciferases de Renilla/metabolismo , Modelos Moleculares , Estrutura MolecularRESUMO
Chemical probes of microRNA (miRNA) function are potential tools for understanding miRNA biology that also provide new approaches for discovering therapeutics for miRNA-associated diseases. MicroRNA-21 (miR-21) is an oncogenic miRNA that is overexpressed in most cancers and has been strongly associated with driving chemoresistance in cancers such as renal cell carcinoma (RCC). Using a cell-based luciferase reporter assay to screen small molecules, we identified a novel inhibitor of miR-21 function. Following structure-activity relationship studies, an optimized lead compound demonstrated cytotoxicity in several cancer cell lines. In a chemoresistant-RCC cell line, inhibition of miR-21 via small molecule treatment rescued the expression of tumor-suppressor proteins and sensitized cells to topotecan-induced apoptosis. This resulted in a >10-fold improvement in topotecan activity in cell viability and clonogenic assays. Overall, this work reports a novel small molecule inhibitor for perturbing miR-21 function and demonstrates an approach to enhancing the potency of chemotherapeutics specifically for cancers derived from oncomir addiction.