RESUMO
Adduction of an electrophile to privileged sensor proteins and the resulting phenotypically dominant responses are increasingly appreciated as being essential for metazoan health. Functional similarities between the biological electrophiles and electrophilic pharmacophores commonly found in covalent drugs further fortify the translational relevance of these small-molecule signals. Genetically encodable or small-molecule-based fluorescent reporters and redox proteomics have revolutionized the observation and profiling of cellular redox states and electrophile-sensor proteins, respectively. However, precision mapping between specific redox-modified targets and specific responses has only recently begun to be addressed, and systems tractable to both genetic manipulation and on-target redox signaling in vivo remain largely limited. Here we engineer transgenic Caenorhabditis elegans expressing functional HaloTagged fusion proteins and use this system to develop a generalizable light-controlled approach to tagging a prototypical electrophile-sensor protein with native electrophiles in vivo. The method circumvents issues associated with low uptake/distribution and toxicity/promiscuity. Given the validated success of C. elegans in aging studies, this optimized platform offers a new lens with which to scrutinize how on-target electrophile signaling influences redox-dependent life span regulation.
Assuntos
Proteínas de Caenorhabditis elegans/análise , Caenorhabditis elegans/metabolismo , Aldeídos/química , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Eletroquímica , Hidrolases/química , Hidrolases/genética , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Longevidade , Proteínas Luminescentes/análise , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Oxirredução , Fotoquímica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transgenes , Proteína Vermelha FluorescenteRESUMO
Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.
Assuntos
Inibidores Enzimáticos , Receptores ErbB , Reprodutibilidade dos Testes , Inibidores Enzimáticos/farmacologia , Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/químicaRESUMO
Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.
RESUMO
Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.
RESUMO
It is now clear that some cysteines on some proteins are highly tuned to react with electrophiles. Based on numerous studies, it is also established that electrophile sensing underpins rewiring of several critical signaling processes. These electrophile-sensing proteins, or privileged first responders (PFRs), are likely critically relevant for drug design. However, identifying PFRs remains a challenging and unsolved problem, despite the development of several high-throughput methods to ID proteins that react with electrophiles. More importantly, we remain unable to rank how different PFRs identified under different conditions relate to one another, in terms of sensing or signaling capacity. Here we evaluate different methods to assay sensing functions of proteins and discuss these methods in the context of developing a "ranking scheme." Based on theoretical and experimental evidence, we propose that T-REX-the only targeted-electrophile delivery tool presently available-is a reliable method to rank PFRs. Finally, we address to what extent electrophile sensing and downstream signaling are correlated. Based on our current data, we observe that such behaviors are indeed correlated. It is our hope that through this manuscript researchers from various arms of the stress signaling fields will focus on developing a quantitative understanding of precision electrophile labeling.
Assuntos
Elementos de Resposta Antioxidante , Proteínas , Transdução de Sinais , Cisteína , Desenho de Fármacos , Oxirredução , Proteínas/genética , Proteínas/metabolismoRESUMO
T-REX (targetable reactive electrophiles and oxidants) enables electrophile targeting in living systems with high spatiotemporal precision and at single-protein-target resolution. T-REX allows functional consequences of individual electrophile signaling events to be directly linked to on-target modifications. T-REX is accomplished by expressing a HaloTagged protein of interest (POI) and introducing a Halo-targetable bioinert photocaged precursor to a reactive electrophilic signal (RES). Light exposure releases the unfettered RES on demand, enabling precision modification of the POI due to proximity. Using alkyne-functionalized 4-hydroxynonenal (HNE) as a representative RES, this protocol delineates optimized strategies to (1) execute T-REX in live human cells and C. elegans, (2) quantitate the POI's RES-sensitivity by either azido-fluorescent-dye conjugation or (3) enrich using biotin-azide/streptavidin pulldown procedure in both model systems, and (4) identify the site of RES-labeling on the POI using proteomics. Built-in T-REX controls that allow users to directly confirm on-target/on-site specificity of RES-sensing are also described. © 2018 by John Wiley & Sons, Inc.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Estrutura Molecular , Oxirredução , Proteínas/química , Especificidade da EspécieRESUMO
Small heat shock protein (sHSP)-B7 (HSPB7) is a muscle-specific member of the non-ATP-dependent sHSPs. The precise role of HSPB7 is enigmatic. Here, we disclose that zebrafish Hspb7 is a kinetically privileged sensor that is able to react rapidly with native reactive electrophilic species (RES), when only substoichiometric amounts of RES are available in proximity to Hspb7 expressed in living cells. Among the two Hspb7-cysteines, this RES sensing is fulfilled by a single cysteine (C117). Purification and characterizations in vitro reveal that the rate for RES adduction is among the most efficient reported for protein-cysteines with native carbonyl-based RES. Covalent-ligand binding is accompanied by structural changes (increase in ß-sheet-content), based on circular dichroism analysis. Among the two cysteines, only C117 is conserved across vertebrates; we show that the human ortholog is also capable of RES sensing in cells. Furthermore, a cancer-relevant missense mutation reduces this RES-sensing property. This evolutionarily conserved cysteine-biosensor may play a redox-regulatory role in cardioprotection.