RESUMO
Targeted protein degradation has emerged as a powerful tool for therapeutic development and biological exploration. In this issue of Cell, Morreale et al. report the development of the BacPROTAC technology to enable targeted protein degradation in Gram-positive bacteria and mycobacteria via reprogramming of Clp proteases.
Assuntos
Bactérias , Endopeptidase Clp , Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Bactérias Gram-Positivas , ProteóliseRESUMO
New biological tools provide new techniques to probe fundamental biological processes. Here we describe the burgeoning field of proteolysis-targeting chimeras (PROTACs), which are capable of modulating protein concentrations at a post-translational level by co-opting the ubiquitin-proteasome system. We describe the PROTAC technology and its application to drug discovery and provide examples where PROTACs have enabled novel biological insights. Furthermore, we provide a workflow for PROTAC development and use and discuss the benefits and issues associated with PROTACs. Finally, we compare PROTAC-mediated protein-level modulation with other technologies, such as RNAi and genome editing.
Assuntos
Descoberta de Drogas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , HumanosRESUMO
The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain (ß2 microglobulin, ß2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/ß2m interface, to generate conformationally stable, peptide-receptive molecules named "open MHC-I." Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type when loaded with low- to moderate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in ß2m-interacting sites of the peptide-binding groove to long-range effects on the α2-1 helix and α3 domain. The interchain disulfide bond stabilizes MHC-I molecules in an open conformation to promote peptide exchange across multiple human leukocyte antigen (HLA) allotypes, covering representatives from five HLA-A supertypes, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structure-guided design, combined with conditional ß-peptide ligands, provides a universal platform to generate ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires covering highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules.
Assuntos
Antígenos de Histocompatibilidade Classe II , Antígenos de Histocompatibilidade , Humanos , Peptídeos/genética , Complexo Principal de Histocompatibilidade , Epitopos , DissulfetosRESUMO
Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved ß2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.
Assuntos
Apresentação de Antígeno , Galinhas , Antígenos HLA-B , Proteínas de Membrana Transportadoras , Chaperonas Moleculares , Animais , Humanos , Antígenos HLA-B/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Epitopos/metabolismo , Engenharia de ProteínasRESUMO
Chaperones tapasin and transporter associated with antigen processing (TAP)-binding protein related (TAPBPR) associate with the major histocompatibility complex (MHC)-related protein 1 (MR1) to promote trafficking and cell surface expression. However, the binding mechanism and ligand dependency of MR1/chaperone interactions remain incompletely characterized. Here in vitro, biochemical and computational studies reveal that, unlike MHC-I, TAPBPR recognizes MR1 in a ligand-independent manner owing to the absence of major structural changes in the MR1 α2-1 helix between empty and ligand-loaded molecules. Structural characterization using paramagnetic nuclear magnetic resonance experiments combined with restrained molecular dynamics simulations reveals that TAPBPR engages conserved surfaces on MR1 to induce similar adaptations to those seen in MHC-I/TAPBPR co-crystal structures. Finally, nuclear magnetic resonance relaxation dispersion experiments using 19F-labeled diclofenac show that TAPBPR can affect the exchange kinetics of noncovalent metabolites with the MR1 groove, serving as a catalyst. Our results support a role of chaperones in stabilizing nascent MR1 molecules to enable loading of endogenous or exogenous cargo.
Assuntos
Antígenos de Histocompatibilidade Classe I , Imunoglobulinas , Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/química , Imunoglobulinas/química , Ligantes , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Peptídeos/químicaRESUMO
Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.
Assuntos
Cobre/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Chaperonas Moleculares/metabolismo , Linhagem Celular , Ativação Enzimática , Humanos , Ligação ProteicaRESUMO
Since late 2019, biomedical labs all over the world have been struggling to cope with the 'new normal' and to find ways in which they can contribute to the fight against COVID-19. In this unique situation where a biomedical issue dominates people's lives and the news cycle, chemical biology has a great deal to contribute. This review will describe the importance of science at the chemistry/biology interface to both understand and combat the SARS-CoV-2 pandemic.
Assuntos
Antivirais/química , COVID-19/virologia , SARS-CoV-2/fisiologia , Animais , Antivirais/farmacologia , Desenho de Fármacos , Descoberta de Drogas , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Tratamento Farmacológico da COVID-19RESUMO
The study of epigenetics has greatly benefited from the development and application of various chemical biology approaches. In this review, we highlight the key targets for modulation and recent methods developed to enact such modulation. We discuss various chemical biology techniques to study DNA methylation and the post-translational modification of histones as well as their effect on gene expression. Additionally, we address the wealth of protein synthesis approaches to yield histones and nucleosomes bearing epigenetic modifications. Throughout, we highlight targets that present opportunities for the chemical biology community, as well as exciting new approaches that will provide additional insight into the roles of epigenetic marks.
Assuntos
Epigênese Genética/genética , Histonas/genética , Proteínas/metabolismo , Metilação de DNA , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Proteínas/genéticaRESUMO
Control of protein levels by nucleic-acid-based technologies has proven to be a useful research tool but lacks the advantages of small molecules with respect to cell permeability, temporal control, and the potential generation of therapeutics. In this Review, we discuss the technologies available for the control of intracellular protein levels with small molecules and compare the various systems available.
Assuntos
Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Humanos , Imunossupressores/química , Imunossupressores/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Bibliotecas de Moléculas Pequenas/metabolismo , Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismoRESUMO
The receptor tyrosine kinase FLT-3 is frequently mutated in acute myeloid leukemia; however, current small molecule inhibitors suffer from limited efficacy in the clinic. Conversion of a FLT-3 inhibitor (quizartinib) into a proteolysis targeting chimera (PROTAC) results in a compound that induces degradation of FLT-3 ITD mutant at low nanomolar concentrations. Furthermore, the PROTAC is capable of inhibiting cell growth more potently than the warhead alone while inhibiting fewer off-target kinases. This enhanced antiproliferative activity occurs, despite a slight reduction in the PROTAC's kinase inhibitory activity, via an increased level of apoptosis induction suggesting nonkinase roles for the FLT-3 ITD protein. Additionally, the PROTAC is capable of inducing FLT-3 ITD degradation in vivo. These results suggest that degradation of FLT-3 ITD may provide a useful method for therapeutic intervention.
Assuntos
Antineoplásicos/uso terapêutico , Benzotiazóis/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Benzotiazóis/farmacocinética , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos Nus , Compostos de Fenilureia/farmacocinética , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
α-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helix mediated protein-protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamers have been widely and successfully used. This manuscript describes alternative syntheses of these compounds that can be used to access mimetics that are challenging to synthesize using previously described methodologies, permitting access to compounds functionalized with multiple sensitive side chains and accelerated library assembly through late stage derivatisation.
Assuntos
Benzamidas/síntese química , Benzamidas/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Biomimética/métodos , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
An innovative and simple expeditious synthesis of 3,4-unsubstituted isoquinolones and isocoumarins starting from safe and easy to handle two-carbon acetylene equivalent was developed. The synthetic potential of this new method was further demonstrated in the facile total synthesis of two naturally occurring alkaloids: corydaldine and doryanine.
RESUMO
The exploitation of multivalent ligands for the inhibition of protein-protein interactions has not yet been explored as a supramolecular design strategy. This is despite the fact that protein-protein interactions typically occur within the context of multi-protein complexes and frequently exploit avidity effects or co-operative binding interactions to achieve high affinity interactions. In this paper we describe preliminary studies on the use of a multivalent N-alkylated aromatic oligoamide helix mimetic for inhibition of p53/hDM2 and establish that protein dimerisation is promoted, rather than enhanced binding resulting from a higher effective concentration of the ligand.
Assuntos
Amidas/química , Amidas/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Alquilação , Amidas/síntese química , Materiais Biomiméticos/síntese química , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteína Supressora de Tumor p53/químicaRESUMO
The application of [4+2] cycloadditions between alkenes and an N-benzoyl iminium species, generated in situ under acidic conditions, is described in the synthesis of diverse molecular scaffolds. The key reaction led to the formation of cyclic imidates in good yield and with high regioselectivity. It was demonstrated that the cyclic imidates may be readily converted into 1,3-amino alcohols. Incorporation of orthogonally-reactive functionality, such as aryl and alkyl bromides, into the cycloaddition substrates enabled the synthesis of additional scaffolds. For one scaffold, the synthesis of exemplar screening compounds was undertaken to demonstrate potential value in small molecule library production.
Assuntos
Alcenos/química , Reação de Cicloadição , Descoberta de Drogas , Imidoésteres/química , Bibliotecas de Moléculas Pequenas/síntese química , Catálise , Estrutura Molecular , Paládio/química , EstereoisomerismoRESUMO
The therapeutically relevant hypoxia inducible factor HIF-1α-p300 protein-protein interaction can be orthosterically inhibited with α-helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C-terminal helix of the HIF-1α C-TAD (C-terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side-chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF-1α-p300 PPI inhibitors and the first examples of small-molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF-1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42-residue HIF-1α C-TAD from which it is derived.
Assuntos
Amidas/farmacologia , Materiais Biomiméticos/farmacologia , Proteína p300 Associada a E1A/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Amidas/síntese química , Amidas/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Biomimética , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Proteína p300 Associada a E1A/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
α-Helix mediated protein-protein interactions are of major therapeutic importance. As such, the design of inhibitors of this class of interaction is of significant interest. We present methodology to modify N-alkylated aromatic oligoamide α-helix mimetics using 'click' chemistry. The effect is shown to modulate the binding properties of a series of selective p53/hDM2 inhibitors.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/química , Amidas/química , Biomimética , Química Click , Humanos , Concentração Inibidora 50 , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteômica/métodos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Solventes/química , Propriedades de Superfície , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína bcl-X/químicaRESUMO
Piperazines are found widely in commercially-available compounds and bioactive molecules (including many drugs). However, in the vast majority of these molecules, the piperazine ring is isolated (i.e. not fused to another ring) and is not substituted on any of its carbon atoms. A modular synthetic approach is described in which combinations of cyclic sulfamidate and hydroxy sulfonamide building blocks may be converted into piperazines and related 1,4-diazepine and 1,5-diazocane scaffolds. By variation of the combinations of building blocks used, it was possible to vary the ring size, substitution and configuration of the resulting heterocyclic scaffolds. The approach was exemplified in the synthesis of a range of heterocyclic scaffolds that, on decoration, would target lead-like chemical space. It was demonstrated that lead-like small molecules based on these scaffolds would likely complement those found in large compound collections.
Assuntos
Azepinas/síntese química , Compostos Heterocíclicos com 1 Anel/síntese química , Piperazinas/síntese química , Azepinas/química , Compostos Heterocíclicos com 1 Anel/química , Estrutura Molecular , Piperazina , Piperazinas/químicaRESUMO
Ubiquitination is a key post-translational modification on protein lysine sidechains known to impact protein stability, signal transduction cascades, protein-protein interactions, and beyond. Great strides have been made towards developing new methods to generate discrete chains of polyubiquitin and conjugate them onto proteins site-specifically, with methods ranging from chemical synthetic approaches, to enzymatic approaches and many in between. Previous work has demonstrated the utility of engineered variants of the bacterial transpeptidase enzyme sortase (SrtA) for conjugation of ubiquitin site-specifically onto target proteins. In this manuscript, we've combined the classical E1/E2-mediated polyubiquitin chain extension approach with sortase-mediated ligation and click chemistry to enable the generation of mono, di, and triubiquitinated proteins sfGFP and PCNA. We demonstrate the utility of this strategy to generate both K48-linked and K63-linked polyubiquitins and attach them both N-terminally and site-specifically to the proteins of interest. Further, we highlight differential activity between two commonly employed sortase variants, SrtA 5M and 7M, and demonstrate that while SrtA 7M can be used to conjugate these ubiquitins to substrates, SrtA 5M can be employed to release the ubiquitin from the substrates as well as to cleave C-terminal tags from the ubiquitin variants used. Overall, we envision that this approach is broadly applicable to readily generate discrete polyubiquitin chains of any linkage type that is accessible via E1/E2 systems and conjugate site-specifically onto proteins of interest, thus granting access to bespoke ubiquitinated proteins that are not currently possible.
RESUMO
PRMT1 plays many important roles in both normal and disease biology, thus understanding it's regulation is crucial. Herein, we report the role of p300-mediated acetylation at K228 in triggering PRMT1 degradation through FBXL17-mediated ubiquitination. Utilizing mass-spectrometry, cellular biochemistry, and genetic code-expansion technologies, we elucidate a crucial mechanism independent of PRMT1 transcript levels. These results underscore the significance of acetylation in governing protein stability and expand our understanding of PRMT1 homeostasis. By detailing the molecular interplay between acetylation and ubiquitination involved in PRMT1 degradation, this work contributes to broader efforts in deciphering post-translational mechanisms that influence protein homeostasis.
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PURPOSE: The goal of our study was to characterize the dynamics of intracellular oxygen during application of radiation at conventional (CONV) and FLASH dose rates and obtain evidence for or against the oxygen depletion hypothesis as a mechanism of the FLASH effect. METHODS AND MATERIALS: The measurements were performed by the phosphorescence quenching method using probe Oxyphor PtG4, which was delivered into the cellular cytosol by electroporation. RESULTS: Intracellular radiochemical oxygen depletion (ROD) g-value for a dose rate of 100 Gy/s in the normoxic range was found to be 0.58 ± 0.03 µM/Gy. Intracellular ROD g-values for FLASH and CONV dose rates in the normoxic range were found to be nearly equal. As in solution-based studies, intracellular ROD was found to exhibit strong dependence on oxygen concentration in the range of 0 to â¼40 µM [O2]. CONCLUSIONS: Depletion of oxygen in cells in vitro by a clinical dose of proton radiation delivered as FLASH is unable to produce a transient state of hypoxia and, therefore, unable to induce radioprotection. The difference between ROD g-values for FLASH and CONV dose rates, detected previously in solutions-based experiments, disappears when measurements are conducted inside cells. Understanding this phenomenon should provide additional insight into the role of oxygen in FLASH radiation therapy and help to decipher the mechanism of the FLASH effect.