RESUMO
Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.
Assuntos
Proteômica , Fatores de Transcrição , Humanos , Proteômica/métodos , Cisteína/metabolismo , LigantesRESUMO
The peptide natural product nisin has been used as a food preservative for 6 decades with minimal development of resistance. Nisin contains the unusual amino acids dehydroalanine and dehydrobutyrine, which are posttranslationally installed by class I lanthipeptide dehydratases (LanBs) on a linear peptide substrate through an unusual glutamyl-tRNA-dependent dehydration of Ser and Thr. To date, little is known about how LanBs catalyze the transfer of glutamate from charged tRNAGlu to the peptide substrate, or how they carry out the subsequent elimination of the peptide-glutamyl adducts to afford dehydro amino acids. Here, we describe the synthesis of inert analogs that mimic substrate glutamyl-tRNAGlu and the glutamylated peptide intermediate, and determine the crystal structures of 2 LanBs in complex with each of these compounds. Mutational studies were used to characterize the function of the glutamylation and glutamate elimination active-site residues identified through the structural analysis. These combined studies provide insights into the mechanisms of substrate recognition, glutamylation, and glutamate elimination by LanBs to effect a net dehydration reaction of Ser and Thr.
Assuntos
Ácido Glutâmico/química , Hidroliases/química , Aminoacil-RNA de Transferência/química , Alanina/análogos & derivados , Alanina/química , Alanina/genética , Cristalografia por Raios X , Ácido Glutâmico/genética , Hidroliases/genética , Nisina/química , Domínios Proteicos , Aminoacil-RNA de Transferência/genética , Proteínas RecombinantesRESUMO
Photoacoustic (PA) imaging has emerged as a reliable in vivo technique for diverse biomedical applications ranging from disease screening to analyte sensing. Most contemporary PA imaging agents employ NIR-I light (650-900 nm) to generate an ultrasound signal; however, there is significant interference from endogenous biomolecules such as hemoglobin that are PA active in this window. Transitioning to longer excitation wavelengths (i.e., NIR-II) reduces the background and facilitates the detection of low abundance targets (e.g., nitric oxide, NO). In this study, we employed a two-phase tuning approach to develop APNO-1080, a NIR-II NO-responsive probe for deep-tissue PA imaging. First, we performed Hammett and Brønsted analyses to identify a highly reactive and selective aniline-based trigger that reacts with NO via N-nitrosation chemistry. Next, we screened a panel of NIR-II platforms to identify chemical structures that have a low propensity to aggregate since this can diminish the PA signal. In a head-to-head comparison with a NIR-I analogue, APNO-1080 was 17.7-fold more sensitive in an in vitro tissue phantom assay. To evaluate the deep-tissue imaging capabilities of APNO-1080 in vivo, we performed PA imaging in an orthotopic breast cancer model and a heterotopic lung cancer model. Relative to control mice not bearing tumors, the normalized turn-on response was 1.3 ± 0.12 and 1.65 ± 0.07, respectively.
Assuntos
Desenvolvimento de Medicamentos , Corantes Fluorescentes/química , Óxido Nítrico/análise , Imagem Óptica , Técnicas Fotoacústicas , Células A549 , Animais , Corantes Fluorescentes/síntese química , Humanos , Raios Infravermelhos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagemRESUMO
Until recently, there were no generalizable methods for assessing the effects of post-translational regulation on enzymatic activity. Activity-based sensing (ABS) has emerged as a powerful approach for monitoring small-molecule and enzyme activities within living systems. Initial examples of ABS were applied for measuring general enzymatic activity; however, a recent focus has been placed on increasing the selectivity to monitor a single enzyme or isoform. The highest degree of selectivity is required for differentiating between isoforms, where the targets display significant structural similarities as a result of a gene duplication or alternative splicing. This Minireview highlights key examples of small-molecule isoform-selective probes with a focus on the relevance of isoform differentiation, design strategies to achieve selectivity, and applications in basic biology or in the clinic.
Assuntos
Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Isoenzimas/análise , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/metabolismo , Humanos , Isoenzimas/metabolismo , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
Ascorbate is an important biological reductant and enzyme cofactor. Although direct detection through ascorbate-mediated reduction is possible, this approach suffers from poor selectivity due to the wide range of cellular reducing agents. To overcome this limitation, we leverage reduction potential of ascorbate to mediate a copper-mediated oxidative bond cleavage of ether-caged fluorophores. The copper(II) complexes supported by a {bis(2-pyridylmethyl)}benzylamine or a {bis(2-pyridylmethyl)}(2-methoxybenzyl)amine ligand were identified as an ascorbate responsive unit and their reaction with ascorbate yields a copper-based oxidant that enables rapid benzylic oxidation and the release of an ether-caged dye (coumarin or fluorescein). The copper-mediated bond cleavage is specific to ascorbate and the trigger can be readily derivatized for tuning photophysical properties of the probes. The probes were successfully applied for the fluorometric detection of ascorbate in commercial food samples, human plasma, and serum, and within live cells by using confocal microscopy and flow cytometry.
RESUMO
Cyclooxygenase-2 (COX-2) overexpression is prominent in inflammatory diseases, neurodegenerative disorders, and cancer. Directly monitoring COX-2 activity within its native environment poses an exciting approach to account for and illuminate the effect of the local environments on protein activity. Herein, we report the development of CoxFluor, the first activity-based sensing approach for monitoring COX-2 within live cells with confocal microscopy and flow cytometry. CoxFluor strategically links a natural substrate with a dye precursor to engage both the cyclooxygenase and peroxidase activities of COX-2. This catalyzes the release of resorufin and the natural product, as supported by molecular dynamics and ensemble docking. CoxFluor enabled the detection of oxygen-dependent changes in COX-2 activity that are independent of protein expression within live macrophage cells.
Assuntos
Técnicas Biossensoriais/métodos , Ciclo-Oxigenase 2/química , Humanos , Simulação de Dinâmica MolecularRESUMO
Photoacoustic (PA) imaging is an emerging, non-invasive imaging modality that encompasses attributes of both optical and ultrasound imaging. Because of the combination of optical excitation and acoustic detection, PA imaging enables high contrast and high resolution within deep tissue (centimeter depths). Recent advances in PA probe development have allowed for stimulus-responsive imaging in a variety of biological models with implications for basic, translational, and clinical sciences. This perspective highlights recent progress in the development of PA probes and their application to live-animal molecular imaging.
Assuntos
Imagem Molecular/métodos , Técnicas Fotoacústicas/métodos , Ativação Metabólica , Animais , Fenômenos Eletromagnéticos , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Estrutura Molecular , Pró-Fármacos/farmacocinética , Temperatura , TransdutoresRESUMO
Photoacoustic (PA) imaging is an emerging imaging modality that utilizes optical excitation and acoustic detection to enable high resolution at centimeter depths. The development of activatable PA probes can expand the utility of this technology to allow for detection of specific stimuli within live-animal models. Herein, we report the design, development, and evaluation of a series of Acoustogenic Probe(s) for Nitric Oxide (APNO) for the ratiometric, analyte-specific detection of nitric oxide (NO) in vivo. The best probe in the series, APNO-5, rapidly responds to NO to form an N-nitroso product with a concomitant 91 nm hypsochromic shift. This property enables ratiometric PA imaging upon selective irradiation of APNO-5 and the corresponding product, tAPNO-5. Moreover, APNO-5 displays the requisite photophysical characteristics for in vivo PA imaging (e.g., high absorptivity, low quantum yield) as well as high biocompatibility, stability, and selectivity for NO over a variety of biologically relevant analytes. APNO-5 was successfully applied to the detection of endogenous NO in a murine lipopolysaccharide-induced inflammation model. Our studies show a 1.9-fold increase in PA signal at 680 nm and a 1.3-fold ratiometric turn-on relative to a saline control.
Assuntos
Óxido Nítrico/análise , Técnicas Fotoacústicas/métodos , Animais , Modelos Animais de Doenças , Inflamação/diagnóstico , Inflamação/diagnóstico por imagem , Camundongos , Compostos Nitrosos/químicaRESUMO
We report pH rate profiles for kcat and Km for the isomerization reaction of glyceraldehyde 3-phosphate catalyzed by wildtype triosephosphate isomerase (TIM) from three organisms and by ten mutants of TIM; and, for Ki for inhibition of this reaction by phosphoglycolate trianion (I3-). The pH profiles for Ki show that the binding of I3- to TIM (E) to form EH·I3- is accompanied by uptake of a proton by the carboxylate side-chain of E165, whose function is to abstract a proton from substrate. The complexes for several mutants exist mainly as E-·I3- at high pH, in which cases the pH profiles define the p Ka for deprotonation of EH·I3-. The linear free energy correlation, with slope of 0.73 ( r2 = 0.96), between kcat/ Km for TIM-catalyzed isomerization and the disassociation constant of PGA trianion for TIM shows that EH·I3- and the transition state are stabilized by similar interactions with the protein catalyst. Values of p Ka = 10-10.5 were estimated for deprotonation of EH·I3- for wildtype TIM. This p Ka decreases to as low as 6.3 for the severely crippled Y208F mutant. There is a correlation between the effect of several mutations on kcat/ Km and on p Ka for EH·I3-. The results support a model where the strong basicity of E165 at the complex to the enediolate reaction intermediate is promoted by side-chains from Y208 and S211, which serve to clamp loop 6 over the substrate; I170, which assists in the creation of a hydrophobic environment for E165; and P166, which functions in driving the carboxylate side-chain of E165 toward enzyme-bound substrate.
Assuntos
Aminoácidos/metabolismo , Ácido Glutâmico/metabolismo , Triose-Fosfato Isomerase/metabolismo , Aminoácidos/química , Domínio Catalítico , Cristalografia por Raios X , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Triose-Fosfato Isomerase/químicaRESUMO
Photoacoustic (PA) tomography is a noninvasive technology that utilizes near-infrared (NIR) excitation and ultrasonic detection to image biological tissue at centimeter depths. While several activatable small-molecule PA sensors have been developed for various analytes, the use of PA molecules for deep-tissue analyte delivery and monitoring remains an underexplored area of research. Herein, we describe the synthesis, characterization, and in vivo validation of photoNOD-1 and photoNOD-2, the first organic, NIR-photocontrolled nitric oxide (NO) donors that incorporate a PA readout of analyte release. These molecules consist of an aza-BODIPY dye appended with an aryl N-nitrosamine NO-donating moiety. The photoNODs exhibit chemostability to various biological stimuli, including redox-active metals and CYP450 enzymes, and demonstrate negligible cytotoxicity in the absence of irradiation. Upon single-photon NIR irradiation, photoNOD-1 and photoNOD-2 release NO as well as rNOD-1 or rNOD-2, PA-active products that enable ratiometric monitoring of NO release. Our in vitro studies show that, upon irradiation, photoNOD-1 and photoNOD-2 exhibit 46.6-fold and 21.5-fold ratiometric turn-ons, respectively. Moreover, unlike existing NIR NO donors, the photoNODs do not require encapsulation or multiphoton activation for use in live animals. In this study, we use PA tomography to monitor the local, irradiation-dependent release of NO from photoNOD-1 and photoNOD-2 in mice after subcutaneous treatment. In addition, we use a murine model for breast cancer to show that photoNOD-1 can selectively affect tumor growth rates in the presence of NIR light stimulation following systemic administration.
Assuntos
Neoplasias Mamárias Animais/tratamento farmacológico , Doadores de Óxido Nítrico/farmacologia , Técnicas Fotoacústicas , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Raios Infravermelhos , Injeções Subcutâneas , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Óxido Nítrico/análise , Doadores de Óxido Nítrico/administração & dosagem , Doadores de Óxido Nítrico/química , Distribuição TecidualRESUMO
Kinetic parameters are reported for the reactions of whole substrates (kcat/Km, M(-1) s(-1)) (R)-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) and for the substrate pieces [(kcat/Km)E·HPi/Kd, M(-2) s(-1)] glycolaldehyde (GA) and phosphite dianion (HPi) catalyzed by the I172A/L232A mutant of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM). A comparison with the corresponding parameters for wild-type, I172A, and L232A TbbTIM-catalyzed reactions shows that the effect of I172A and L232A mutations on ΔG(⧧) for the wild-type TbbTIM-catalyzed reactions of the substrate pieces is nearly the same as the effect of the same mutations on TbbTIM previously mutated at the second side chain. This provides strong evidence that mutation of the first hydrophobic side chain does not affect the functioning of the second side chain in catalysis of the reactions of the substrate pieces. By contrast, the effects of I172A and L232A mutations on ΔG(⧧) for wild-type TbbTIM-catalyzed reactions of the whole substrate are different from the effect of the same mutations on TbbTIM previously mutated at the second side chain. This is due to the change in the rate-determining step that determines the barrier to the isomerization reaction. X-ray crystal structures are reported for I172A, L232A, and I172A/L232A TIMs and for the complexes of these mutants to the intermediate analogue phosphoglycolate (PGA). The structures of the PGA complexes with wild-type and mutant enzymes are nearly superimposable, except that the space opened by replacement of the hydrophobic side chain is occupied by a water molecule that lies â¼3.5 Å from the basic side chain of Glu167. The new water at I172A mutant TbbTIM provides a simple rationalization for the increase in the activation barrier ΔG(⧧) observed for mutant enzyme-catalyzed reactions of the whole substrate and substrate pieces. By contrast, the new water at the L232A mutant does not predict the decrease in ΔG(⧧) observed for the mutant enzyme-catalyzed reactions of the substrate piece GA.
Assuntos
Fosfato de Di-Hidroxiacetona/metabolismo , Ácido Glutâmico/química , Gliceraldeído 3-Fosfato/metabolismo , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo , Trypanosoma brucei brucei/enzimologia , Catálise , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutação/genética , Relação Estrutura-Atividade , Triose-Fosfato Isomerase/genéticaRESUMO
The kinetic parameters for activation of yeast triosephosphate isomerase (ScTIM), yeast orotidine monophosphate decarboxylase (ScOMPDC), and human liver glycerol 3-phosphate dehydrogenase (hlGPDH) for catalysis of reactions of their respective phosphodianion truncated substrates are reported for the following oxydianions: HPO3(2-), FPO3(2-), S2O3(2-), SO4(2-) and HOPO3(2-). Oxydianions bind weakly to these unliganded enzymes and tightly to the transition state complex (E·S()), with intrinsic oxydianion Gibbs binding free energies that range from -8.4 kcal/mol for activation of hlGPDH-catalyzed reduction of glycolaldehyde by FPO3(2-) to -3.0 kcal/mol for activation of ScOMPDC-catalyzed decarboxylation of 1-ß-d-erythrofuranosyl)orotic acid by HOPO3(2-). Small differences in the specificity of the different oxydianion binding domains are observed. We propose that the large -8.4 kcal/mol and small -3.8 kcal/mol intrinsic oxydianion binding energy for activation of hlGPDH by FPO3(2-) and S2O3(2-), respectively, compared with activation of ScTIM and ScOMPDC reflect stabilizing and destabilizing interactions between the oxydianion -F and -S with the cationic side chain of R269 for hlGPDH. These results are consistent with a cryptic function for the similarly structured oxydianion binding domains of ScTIM, ScOMPDC and hlGPDH. Each enzyme utilizes the interactions with tetrahedral inorganic oxydianions to drive a conformational change that locks the substrate in a caged Michaelis complex that provides optimal stabilization of the different enzymatic transition states. The observation of dianion activation by stabilization of active caged Michaelis complexes may be generalized to the many other enzymes that utilize substrate binding energy to drive changes in enzyme conformation, which induce tight substrate fits.
Assuntos
Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Saccharomyces cerevisiae/enzimologia , Triose-Fosfato Isomerase/metabolismo , Sítios de Ligação , Biocatálise , Descarboxilação , Humanos , Fígado/enzimologia , Conformação Molecular , Prótons , Especificidade por SubstratoRESUMO
Pioneer transcription factors (TFs) exhibit a specialized ability to bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic small molecules that stereoselectively and site-specifically bind the pioneer TF, FOXA1, at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the covalent ligands relax the canonical DNA binding preference of FOXA1 by strengthening interactions with suboptimal ancillary sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.
RESUMO
Nitric oxide (NO) plays a critical role in acute and chronic inflammation. NO's contributions to cancer are of particular interest due to its context-dependent bioactivities. For example, immune cells initially produce cytotoxic quantities of NO in response to the nascent tumor. However, it is believed that this fades over time and reaches a concentration that supports the tumor microenvironment (TME). These complex dynamics are further complicated by other factors, such as diet and oxygenation, making it challenging to establish a complete picture of NO's impact on tumor progression. Although many activity-based sensing (ABS) probes for NO have been developed, only a small fraction have been employed in vivo, and fewer yet are practical in cancer models where the NO concentration is <200 nM. To overcome this outstanding challenge, we have developed BL660-NO, the first ABS probe for NIR bioluminescence imaging of NO in cancer. Owing to the low intrinsic background, high sensitivity, and deep tissue imaging capabilities of our design, BL660-NO was successfully employed to visualize endogenous NO in cellular systems, a human liver metastasis model, and a murine breast cancer model. Importantly, its exceptional performance facilitated two dietary studies which examine the impact of fat intake on NO and the TME. BL660-NO provides the first direct molecular evidence that intratumoral NO becomes elevated in mice fed a high-fat diet, which became obese with larger tumors, compared to control animals on a low-fat diet. These results indicate that an inflammatory diet can increase NO production via recruitment of macrophages and overexpression of inducible nitric oxide synthase which in turn can drive tumor progression.
RESUMO
In this chapter, we motivate the need for photoactivatable NO donor molecules and give a brief survey of the existing chemical tools in the field. We then provide detailed protocols for the synthesis and validation of a near-infrared light-activated NO donor molecule, photoNOD-1, developed in our research group. With this tool, NO can be released in vivo in a radiation-dependent manner that can be monitored using photoacoustic imaging.