RESUMO
Natural proteins are highly optimized for function but are often difficult to produce at a scale suitable for biotechnological applications due to poor expression in heterologous systems, limited solubility, and sensitivity to temperature. Thus, a general method that improves the physical properties of native proteins while maintaining function could have wide utility for protein-based technologies. Here, we show that the deep neural network ProteinMPNN, together with evolutionary and structural information, provides a route to increasing protein expression, stability, and function. For both myoglobin and tobacco etch virus (TEV) protease, we generated designs with improved expression, elevated melting temperatures, and improved function. For TEV protease, we identified multiple designs with improved catalytic activity as compared to the parent sequence and previously reported TEV variants. Our approach should be broadly useful for improving the expression, stability, and function of biotechnologically important proteins.
Assuntos
Endopeptidases , Temperatura , Endopeptidases/metabolismo , Proteínas Recombinantes de FusãoRESUMO
Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor and a validated target for immune and inflammatory diseases. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified garcinoic acid as a selective and efficient PXR agonist. The properties of this natural molecule as a specific PXR agonist were demonstrated by the screening on a panel of nuclear receptors, the assessment of the physical and thermodynamic binding affinity, and the determination of the PXR-garcinoic acid complex crystal structure. Cytotoxicity, transcriptional, and functional properties were investigated in human liver cells, and compound activity and target engagement were confirmed in vivo in mouse liver and gut tissue. In conclusion, garcinoic acid is a selective natural agonist of PXR and a promising lead compound toward the development of new PXR-regulating modulators.
Assuntos
Benzopiranos/farmacologia , Receptor de Pregnano X/agonistas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Benzopiranos/metabolismo , Benzopiranos/toxicidade , Linhagem Celular Tumoral , Cristalografia por Raios X , Citocromo P-450 CYP3A/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X/metabolismoRESUMO
Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.
Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Glucuronidase/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/farmacologia , Bactérias/efeitos dos fármacos , Modelos Animais de Doenças , Disbiose/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Feminino , Glucuronidase/metabolismo , Humanos , Irinotecano/farmacologia , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológicoRESUMO
It is increasingly clear that interindividual variability in human gut microbial composition contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not observed in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial ß-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the molecular basis for differential gut bacterial GUS inhibition observed between human fecal samples. Taken together, this work provides an unprecedented technical and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.
Assuntos
Proteínas de Bactérias/metabolismo , Cicloexanóis/química , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Inibidores Enzimáticos/química , Microbioma Gastrointestinal/fisiologia , Glucuronidase/metabolismo , Irinotecano/química , Animais , Biomarcadores/metabolismo , Biologia Computacional , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/microbiologia , Inibidores Enzimáticos/metabolismo , Fezes/química , Feminino , Glucuronídeos/metabolismo , Humanos , Hidrólise , Irinotecano/metabolismo , Cinética , Masculino , Metaboloma , Camundongos , Modelos Moleculares , Medicina de Precisão , Ligação Proteica , Conformação ProteicaRESUMO
The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct ß-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.
Assuntos
Bacteroides/enzimologia , Microbioma Gastrointestinal , Glucuronidase/química , Glucuronidase/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Ácido Glucárico/análogos & derivados , Glucuronidase/antagonistas & inibidores , Humanos , Conformação ProteicaRESUMO
Microbial ß-glucuronidases (GUSs) cause severe gut toxicities that limit the efficacy of cancer drugs and other therapeutics. Selective inhibitors of bacterial GUS have been shown to alleviate these side effects. Using structural and chemical biology, mass spectrometry, and cell-based assays, we establish that piperazine-containing GUS inhibitors intercept the glycosyl-enzyme catalytic intermediate of these retaining glycosyl hydrolases. We demonstrate that piperazine-based compounds are substrate-dependent GUS inhibitors that bind to the GUS-GlcA catalytic intermediate as a piperazine-linked glucuronide (GlcA, glucuronic acid). We confirm the GUS-dependent formation of inhibitor-glucuronide conjugates by LC-MS and show that methylated piperazine analogs display significantly reduced potencies. We further demonstrate that a range of approved piperazine- and piperidine-containing drugs from many classes, including those for the treatment of depression, infection, and cancer, function by the same mechanism, and we confirm through gene editing that these compounds selectively inhibit GUS in living bacterial cells. Together, these data reveal a unique mechanism of GUS inhibition and show that a range of therapeutics may impact GUS activities in the human gut.
RESUMO
The selective inhibition of bacterial ß-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative ß-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a ß-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the ß-glucuronidase active site. Finally, we establish that ß-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial ß-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.