RESUMEN
Opioid withdrawal is a liability of chronic opioid use and misuse, impacting people who use prescription or illicit opioids. Hyperactive autonomic output underlies many of the aversive withdrawal symptoms that make it difficult to discontinue chronic opioid use. The locus coeruleus (LC) is an important autonomic centre within the brain with a poorly defined role in opioid withdrawal. We show here that pannexin-1 (Panx1) channels expressed on microglia critically modulate LC activity during opioid withdrawal. Within the LC, we found that spinally projecting tyrosine hydroxylase (TH)-positive neurons (LCspinal) are hyperexcitable during morphine withdrawal, elevating cerebrospinal fluid (CSF) levels of norepinephrine. Pharmacological and chemogenetic silencing of LCspinal neurons or genetic ablation of Panx1 in microglia blunted CSF NE release, reduced LC neuron hyperexcitability, and concomitantly decreased opioid withdrawal behaviours in mice. Using probenecid as an initial lead compound, we designed a compound (EG-2184) with greater potency in blocking Panx1. Treatment with EG-2184 significantly reduced both the physical signs and conditioned place aversion caused by opioid withdrawal in mice, as well as suppressed cue-induced reinstatement of opioid seeking in rats. Together, these findings demonstrate that microglial Panx1 channels modulate LC noradrenergic circuitry during opioid withdrawal and reinstatement. Blocking Panx1 to dampen LC hyperexcitability may therefore provide a therapeutic strategy for alleviating the physical and aversive components of opioid withdrawal.
Asunto(s)
Conexinas , Locus Coeruleus , Proteínas del Tejido Nervioso , Probenecid , Médula Espinal , Síndrome de Abstinencia a Sustancias , Animales , Locus Coeruleus/metabolismo , Locus Coeruleus/efectos de los fármacos , Conexinas/metabolismo , Conexinas/genética , Conexinas/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Síndrome de Abstinencia a Sustancias/metabolismo , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Ratones , Masculino , Ratas , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Probenecid/farmacología , Morfina/farmacología , Microglía/efectos de los fármacos , Microglía/metabolismo , Analgésicos Opioides/farmacología , Norepinefrina/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Ratones Endogámicos C57BL , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismo , Ratones NoqueadosRESUMEN
Targeted protein degradation with monovalent molecular glue degraders is a powerful therapeutic modality for eliminating disease causing proteins. However, rational design of molecular glue degraders remains challenging. In this study, we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation-an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein-targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2/4, and BCR-ABL/c-ABL. Our study reveals a DCAF16-based covalent degradative and linker-less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.
RESUMEN
[This corrects the article DOI: 10.1021/acscentsci.2c01317.].
RESUMEN
Targeted protein degradation with molecular glue degraders has arisen as a powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify a transposable chemical handle that would convert protein-targeting ligands into molecular degraders of their corresponding targets. Using the CDK4/6 inhibitor ribociclib as a prototype, we identified a covalent handle that, when appended to the exit vector of ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Further modification of our initial covalent scaffold led to an improved CDK4 degrader with the development of a but-2-ene-1,4-dione ("fumarate") handle that showed improved interactions with RNF126. Subsequent chemoproteomic profiling revealed interactions of the CDK4 degrader and the optimized fumarate handle with RNF126 as well as additional RING-family E3 ligases. We then transplanted this covalent handle onto a diverse set of protein-targeting ligands to induce the degradation of BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, HDAC1/3, and SMARCA2/4. Our study undercovers a design strategy for converting protein-targeting ligands into covalent molecular glue degraders.
RESUMEN
To develop new degrader molecules from an existing protein ligand a linkage vector must be identified and then joined with a suitable E3 ligase without disrupting binding to the respective targets. This is typically achieved through empirically evaluating the degradation efficacy of a series of synthetic degraders. Our strategy for determining optimal linkage sites utilises biotinylated protein ligands, linked via potential conjugation sites of an inhibitor to confirm whether target protein is maintained after forming a conjugate. This method provides low-cost, qualitative evidence that the addition of a linker moiety at a specific position can be tolerated, guiding further optimisation. We demonstrate the application of this method through the exploration of linkage vectors on A-485, a known ligand of p300/CBP, and found a conjugation site through a urea moiety. Pomalidomide was then conjugated through this site with several different linkers and cell viability and degradation were assessed for this library using a myeloma cell line, MM1.S. Compound 18i, with a PEG4 linker, was found to be the most effective p300 degrader and linker length greater than 10 atoms afforded enhanced degradation.
RESUMEN
The reactions of bicyclic divinyl ketones display wavelength-dependent changes in product formation. UV irradiation results in the formation of competitive [6,3,5] and [7,3,5] tricyclic unsaturated ketones that subsequently undergo ring expansion and reaction with a range of nucleophiles. DFT calculations and transient absorption experiments were completed that are consistent with a vinylogous Type II Norrish pathway.
RESUMEN
Gamma delta T cells (γδTc) have tremendous anti-tumoral activity, thus γδTc immunotherapy is currently under development for various malignancies. We targeted breast cancer stem-like cells (BCSC), a rare cell population responsible for patient mortality. BCSC were mostly susceptible to γδTc immunotherapy, yet some escaped. The BCSC secretome rendered γδTc hypo-responsive, and resistant BCSC expressed more PD-L1 and anti-apoptotic protein MCL-1 than non-stem-like cells (NSC). BCSC resistance was partially overcome by dMCL1-2, an MCL-1 degrader, or more fully by blocking PD-1 on γδTc. Increased MICA shedding was prevented by the ADAM inhibitor GW280264X, rendering BCSC as sensitive to γδTc cytotoxicity as NSC. Our data show promising potential for γδTc immunotherapy against BCSC while unraveling immune evasion mechanisms exploited by BCSC, which likely also enable their resistance to cytotoxic T and NK cells. Overcoming this resistance, as we have done here, will improve cancer immunotherapy, leading to better cancer patient outcomes.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inmunoterapia , Linfocitos Intraepiteliales/inmunología , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Humanos , Linfocitos Intraepiteliales/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein-protein interactions (PPIs) have emerged as significant targets for therapeutic development, owing to their critical nature in diverse biological processes. An ideal PPI-based target is the protein myeloid cell leukemia 1 (MCL1), a critical prosurvival factor in cancers such as multiple myeloma where MCL1 levels directly correlate to disease progression. Current strategies for halting the antiapoptotic properties of MCL1 revolve around inhibiting its sequestration of proapoptotic factors. Existing inhibitors disrupt endogenous regulatory proteins; however, this strategy actually leads to an increase of MCL1 protein levels. Here, we show the development of hetero-bifunctional small molecules capable of selectively targeting MCL1 using a proteolysis targeting chimera (PROTAC) methodology leading to successful degradation. We have confirmed the involvement of the E3 ligase CUL4A-DDB1 cereblon ubiquitination pathway, making these PROTACs a first step toward a new class of antiapoptotic B-cell lymphoma 2 family protein degraders.