RESUMO
Life is orchestrated by biomolecules interacting in complex networks of biological circuitry with emerging function. Progress in different areas of chemistry has made the design of systems that can recapitulate elements of such circuitry possible. Herein we review prominent examples of networks, the methodologies available to translate an input into various outputs, and speculate on potential applications and directions for the field. The programmability of nucleic acid hybridization has inspired applications beyond its function in heredity. At the circuitry level, DNA provides a powerful platform to design dynamic systems that respond to nucleic acid input sequences with output sequences through diverse logic gates, enabling the design of ever more complex circuitry. In order to interface with more diverse biomolecular inputs and yield outputs other than oligonucleotide sequences, an array of nucleic acid conjugates have been reported that can engage proteins as their input and yield a turn-on of enzymatic activity, a bioactive small molecule, or morphological changes in nanoobjects. While the programmability of DNA makes it an obvious starting point to design circuits, other biosupramolecular interactions have also been demonstrated, and harnessing progress in protein design is bound to deliver further integration of macromolecules in artificial circuits.
Assuntos
DNA/química , Bibliotecas de Moléculas Pequenas/química , Substâncias Macromoleculares/químicaRESUMO
Dimeric proteins are prominent in biology, and receptor dimerization (homo- or heterodimerization) is central to signal transduction. Herein, we report a network that responds to a membrane-associated dimeric protein with the uncaging of a powerful cytotoxic. The network is based on two ligands functionalized with peptide nucleic acids (PNAs) (templating strand and catalyst-functionalized strand, respectively) and a substrate with the caged cytotoxic (monomethyl auristatin E: MMAE; a high-affinity tubulin ligand). In the presence of the dimeric protein, the network yields a cooperative supramolecular assembly with a hybridization architecture that enhances the templated reaction and enables the uncaging of a substrate. The network was tested on cells that express a cancer biomarker, carbonic anhydrase IX, in response to hypoxia. The output of the network correlates with the expression of carbonic anhydrase IX, and this biomarker was harnessed to uncage a potent cytotoxic agent.
Assuntos
Antígenos de Neoplasias/química , Biomarcadores Tumorais/química , Anidrase Carbônica IX/química , Proteínas de Membrana/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estrutura Molecular , Oligopeptídeos/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , Células Tumorais CultivadasRESUMO
DNA-based circuitry empowers logic gated operations and amplifications but is restricted to nucleic acid output. Templated reactions enable the translation of nucleic acid cues into diverse small-molecule outputs but are more limited in their amplification. Herein, we demonstrate the coupling of a DNA circuit to templated reactions in order to achieve high levels of amplification in the output of small molecules, in response to nucleic acid input. We demonstrate that the coupling of the DNA circuit to templated reactions allows for the detection of the fM concentration of analyte and can respond with the release of a cytotoxic drug.
Assuntos
DNA/química , Moldes Genéticos , DNA/metabolismo , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Peptídeos/síntese químicaRESUMO
The development of bioorthogonal reactions have had a transformative impact in chemical biology and the quest to expand this toolbox continues. Herein we review recent applications of ruthenium-catalyzed photoredox reactions used in chemical biology.
Assuntos
Complexos de Coordenação/química , Luz , Rutênio/química , Animais , Catálise , Complexos de Coordenação/metabolismo , Morfolinos/química , Oxirredução , Espécies Reativas de Oxigênio/química , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Bioluminescence resonance energy transfer (BRET) has been widely used for studying dynamic processes in biological systems such as protein-protein interactions and other signaling events. Aside from acting as a reporter, BRET can also turn on functions in living systems. Herein, we report the application of BRET to performing a biorthogonal reaction in living cells; namely, releasing functional molecules through energy transfer to a coumarin molecule, a process termed bioluminolysis. An efficient BRET from Nanoluc-Halotag chimera protein (H-Luc) to a coumarin substrate yields the excited state of coumarin, which in turn triggers hydrolysis to uncage a target molecule. Compared to the conventional methods, this novel uncaging system requires no external light source and shows fast kinetics (t1/2 <2â min). We applied this BRET uncaging system to release a potent kinase inhibitor, ibrutinib, in living cells, highlighting its broad utility in controlling the supply of bioactive small molecules inâ vivo.
Assuntos
Cumarínicos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Luciferases/metabolismo , Medições Luminescentes/métodos , Proteínas Luminescentes/metabolismo , Pirazóis/metabolismo , Pirimidinas/metabolismo , Adenina/análogos & derivados , Células HeLa , Humanos , Piperidinas , Ligação ProteicaRESUMO
Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin αV ß3 . Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin αV ß3 receptor (IC50 =11.0â nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin αV ß3 expression: human glioblastoma U87 (αV ß3 +) and U87 ß3 -KO (αV ß3 -). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4).
Assuntos
Oligopeptídeos/química , Paclitaxel/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Peptidomiméticos/química , Peptidomiméticos/toxicidadeRESUMO
Drugs are administered at a dosing schedule set by their therapeutic index, and termination of action is achieved by clearance and metabolism of the drug. In some cases, such as anticoagulant drugs or immunotherapeutics, it is important to be able to quickly reverse the drug's action. Here, we report a general strategy to achieve on-demand reversibility by designing a supramolecular drug (a noncovalent assembly of two cooperatively interacting drug fragments held together by transient hybridization of peptide nucleic acid (PNA)) that can be reversed with a PNA antidote that outcompetes the hybridization between the fragments. We demonstrate the approach with thrombin-inhibiting anticoagulants, creating very potent and reversible bivalent direct thrombin inhibitors (Ki = 74 pM). The supramolecular inhibitor effectively inhibited thrombus formation in mice in a needle injury thrombosis model, and this activity could be reversed by administration of the PNA antidote. This design is applicable to therapeutic targets where two binding sites can be identified.
RESUMO
The HCR represents a powerful tool for amplification in DNA-based circuitry and sensing applications, yet requires the use of long DNA sequences to grant hairpin metastability. Here we describe a minimal HCR system based on peptide nucleic acids (PNAs). A system comprising a 5-mer stem and 5-mer loop/toehold hairpins was found to be suitable to achieve rapid amplification. These hairpins were shown to yield >10-fold amplification in 2 h and be suitable for the detection of a cancer biomarker on live cells. The use of γ-peg-modified PNA was found to be beneficial.
RESUMO
Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Trifosfato de Adenosina , Animais , Sítios de Ligação , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Camundongos , Paclitaxel , Transporte Proteico , Células RAW 264.7RESUMO
Bioluminescence resonance energy transfer (BRET) is extensively used to study dynamic systems and has been utilized in sensors for studying protein proximity, metabolites, and drug concentrations. Herein, we demonstrate that BRET can activate a ruthenium-based photocatalyst which performs bioorthogonal reactions. BRET from luciferase to the ruthenium photocatalyst is used to uncage effector molecules with up to 64 turnovers of the catalyst, achieving concentrations >0.6 µM effector with 10 nM luciferase construct. Using a BRET sensor, we further demonstrate that the catalysis can be modulated in response to an analyte, analogous to allosterically controlled enzymes. The BRET-induced reaction is used to uncage small-molecule drugs (ibrutinib and duocarmycin) at biologically effective concentrations in cellulo.