RESUMEN
Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.
Asunto(s)
Glicoproteínas de Membrana , Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Glicoproteínas de Membrana/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ubiquitina/metabolismo , Citoplasma/metabolismo , Factores de TranscripciónRESUMEN
Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4-pyridyl of the prazole expands the prazole site on the UEV domain toward the ß-hairpin in the Ub-binding site and is coupled to increased inhibition of virus-like particle production in HIV-1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4-pyridyl substituent to form an irreversible species, with implications for increasing the half-life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS-COV-2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.
Asunto(s)
Antivirales , VIH-1 , Antivirales/farmacología , Antivirales/química , Humanos , VIH-1/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Relación Estructura-Actividad , Tratamiento Farmacológico de COVID-19 , Replicación Viral/efectos de los fármacosRESUMEN
PIM kinases have important pro-tumorigenic roles and mediate several oncogenic traits, including cell proliferation, survival, and chemotherapeutic resistance. As a result, multiple PIM inhibitors have been pursued as investigational new drugs in cancer; however, response to PIM inhibitors in solid tumors has fallen short of expectations. We found that inhibition of PIM kinase activity stabilizes protein levels of all three PIM isoforms (PIM1/2/3), and this can promote resistance to PIM inhibitors and chemotherapy. To overcome this effect, we designed PIM proteolysis targeting chimeras (PROTACs) to target PIM for degradation. PIM PROTACs effectively downmodulated PIM levels through the ubiquitin-proteasome pathway. Importantly, degradation of PIM kinases was more potent than inhibition of catalytic activity at inducing apoptosis in prostate cancer cell line models. In conclusion, we provide evidence of the advantages of degrading PIM kinases versus inhibiting their catalytic activity to target the oncogenic functions of PIM kinases.
Asunto(s)
Resistencia a Antineoplásicos , Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Fosforilación , Apoptosis , Proliferación Celular , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-pim-1RESUMEN
The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.
Asunto(s)
Histona Desacetilasas , Péptidos y Proteínas de Señalización Intracelular , FN-kappa B , Arginina Deiminasa Proteína-Tipo 4 , Factores de Transcripción , Humanos , Epigénesis Genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Proteómica , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Línea Celular TumoralRESUMEN
Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3ß-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3ß-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.
Asunto(s)
Colesterol , Peróxido de Hidrógeno , Proteínas de la Membrana , Metionina Sulfóxido Reductasas , Colesterol/análogos & derivados , Colesterol/metabolismo , Peróxido de Hidrógeno/metabolismo , Metionina/metabolismo , Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/metabolismo , Oxidación-Reducción , Sulfóxidos/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismoRESUMEN
Pentosinane is a structurally complex nonenzymatic post-translational modification of proteins believed to be present in all living things. It falls into the category of advanced glycation end products (AGEs) and is structurally related to the other AGEs pentosidine and glucosepane. Although pentosidine and glucosepane have been widely studied for their role in wide-ranging conditions (e.g., diabetes mellitus, Alzheimer's disease, and human aging), relatively little is known about pentosinane. Interestingly, previous reports have suggested that pentosidine may derive from pentosinane. The (patho)physiological significance of pentosinane in humans is largely unexplored. As a first step to address this knowledge gap, we report herein the first total synthesis of pentosinane. Our synthesis is high yielding (1.7% over seven steps), concise, and enantioselective, and it leverages a strategy for synthesizing 2,5-diaminoimidazoles previously developed by our lab. Access to synthetic pentosinane has allowed us to perform additional studies showing that its oxidation to pentosidine is both pH and oxygen dependent and is substantially slower under physiological conditions than previously believed. Additionally, pentosinane rapidly decomposes under harshly acidic conditions typically employed for pentosidine isolation. Taken together, these results suggest that pentosinane is likely to be more abundant in vivo than previously appreciated. We believe these results represent a critical step toward illuminating the role(s) of pentosinane in human biology.
Asunto(s)
Procesamiento Proteico-Postraduccional , HumanosRESUMEN
α-Ketoglutarate is a key biomolecule involved in a number of metabolic pathwaysâmost notably the TCA cycle. Abnormal α-ketoglutarate metabolism has also been linked with cancer. Here, isotopic labeling was employed to synthesize [1-13C,5-12C,D4]α-ketoglutarate with the future goal of utilizing its [1-13C]-hyperpolarized state for real-time metabolic imaging of α-ketoglutarate analytes and its downstream metabolites in vivo. The signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH) hyperpolarization technique was used to create 9.7% [1-13C] polarization in 1 minute in this isotopologue. The efficient 13C hyperpolarization, which utilizes parahydrogen as the source of nuclear spin order, is also supported by favorable relaxation dynamics at 0.4 µT field (the optimal polarization transfer field): the exponential 13C polarization buildup constant Tb is 11.0 ± 0.4 s whereas the 13C polarization decay constant T1 is 18.5 ± 0.7 s. An even higher 13C polarization value of 17.3% was achieved using natural-abundance α-ketoglutarate disodium salt, with overall similar relaxation dynamics at 0.4 µT field, indicating that substrate deuteration leads only to a slight increase (â¼1.2-fold) in the relaxation rates for 13C nuclei separated by three chemical bonds. Instead, the gain in polarization (natural abundance versus [1-13C]-labeled) is rationalized through the smaller heat capacity of the "spin bath" comprising available 13C spins that must be hyperpolarized by the same number of parahydrogen present in each sample, in line with previous 15N SABRE-SHEATH studies. Remarkably, the C-2 carbon was not hyperpolarized in both α-ketoglutarate isotopologues studied; this observation is in sharp contrast with previously reported SABRE-SHEATH pyruvate studies, indicating that the catalyst-binding dynamics of C-2 in α-ketoglutarate differ from that in pyruvate. We also demonstrate that 13C spectroscopic characterization of α-ketoglutarate and pyruvate analytes can be performed at natural 13C abundance with an estimated detection limit of 80 micromolar concentration × *%P13C. All in all, the fundamental studies reported here enable a wide range of research communities with a new hyperpolarized contrast agent potentially useful for metabolic imaging of brain function, cancer, and other metabolically challenging diseases.
Asunto(s)
Ácidos Cetoglutáricos , Teofilina , Catálisis , Medios de Contraste , Ácido PirúvicoRESUMEN
Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru ß-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13Pru-producing cancer types.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mieloma Múltiple/metabolismo , Ubiquitinación , Apoptosis , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Mieloma Múltiple/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Ubiquitina/metabolismoRESUMEN
Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.
Asunto(s)
Receptor de Asialoglicoproteína/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Dinitrofenoles/química , Dinitrofenoles/metabolismo , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Here we describe a general method for the synthesis of 2,5-diaminoimidazoles, which involves a thermal reaction between α-aminoketones and substituted guanylhydrazines without the need for additives. As one of the few known ways to access the 2,5-diaminoimidazole motif, our method greatly expands the number of reported diaminoimidazoles and further supports our previous observations that these compounds spontaneously adopt the non-aromatic 4(H) tautomer. The reaction works successfully on both cyclic and acyclic amino ketone starting materials, as well as a range of substituted guanylhydrazines. Following optimization, the method was applied to the efficient synthesis of the advanced glycation end product (AGE) methylglyoxal-derived imidazolium crosslink (MODIC). We expect that this method will enable rapid access to a variety of biologically important 2,5-diaminoimidazole-containing products.
RESUMEN
A facile thermal cyclization of ynamide-tethered 1,3,8-triynes to form a 3,5,6,7-tetrahydro-1H-pyrano[3,4-c]pyridine skeleton is described. Although the mechanism of this unusual reaction has yet to be defined, the formation of either a strained keteniminium or a biradical intermediate followed by a 1,5-hydride or -hydrogen shift is tentatively proposed as the key elementary steps in the reaction sequence. Appropriate electronic activation at the carbon center donating a hydride or hydrogen is crucial for successful cyclization.
RESUMEN
Substituent- and oxidant-dependent transformations of allenes are described. Given the profound influence of the substituent on the reactivity of allenes, the subtle differences in allene structures are manifested in the formation of diverse products when reacted with different electrophiles/oxidants. In general, reactions of nonsilylated allenes involve an allylic cation intermediate by forming a C-O bond, at the sp-hybridized C2, with either DDQ (2,3-dichloro-5,6-dicyano-p-benzoquinone) or TBHP (tert-butyl hydroperoxide), along with FeCl2â 4 H2O (10â mol %). In contrast, silylated allenes favor the formation of propargylic cation intermediates by transferring the allenic hydride to the oxidant, thus generating 1,3-enynes (E1 product) or propargylic THBP ethers (S(N)1 product). The formation of these different putative cationic intermediates from nonsilylated and silylated allenes is strongly supported by DFT calculations.
RESUMEN
α,ß-Unsaturated ketones generally undergo addition reactions with nucleophiles with a preference for either 1,2- or 1,4-addition, but rarely both. However, the right combination of reagents allows for consecutive 1,4- and 1,2-additions to occur: Cyclic α,ß-unsaturated ketones undergo double additions with lithium(trimethylsilyl)diazomethane, effectively generating various molecular frameworks with complexity and diversity. Owing to the sequential generation of several intermediates of multifaceted reactivity, including diazoalkane derivatives and alkylidene carbenes, it is possible to induce novel Grob-type C-C fragmentations, alkylidene carbene mediated Li-N insertions, and dipolar cycloadditions by controlling the reaction parameters.
RESUMEN
Facile and selective Alder-ene reactions of silylallenes involving the activation of an allenic C(sp(2) )H over an allylic C(sp(3) )H bond is described. In this ene reaction, the presence of a silyl substituent was found to be critical for the observed reactivity and selectivity since the corresponding alkyl-substituted allenes show different reaction profiles. Computational studies show that the origin of this unusual reactivity is the lower bond dissociation energy of the α-C(sp(2) )H bond in silylallenes compared to the corresponding nonsilylated allenes.
RESUMEN
Novel oxidative dimerization of silylallenes is described. Treatment of silylallenes with a catalytic amount of copper(I) chloride, a substoichiometric amount of N-hydroxyphthalamide, and a stoichiometric amount of a terminal oxidant diacetoxyiodobenzene afforded head-to-head dimers as the main products. Silyallenes containing a small ring afforded only dimers, whereas as the ring size increased 1,3-enynes became more favorable products. For silylallenes containing an acyclic substituent, dimer formation is a norm with exceptions where N-hydroxyphthalimide reacts at the propargylic center to generate the corresponding aminoxy ethers.
RESUMEN
An efficient nitration of silyl allenes with nitrogen dioxide radical, generated from NaNO2 and AcOH, to form α-nitro-α,ß-unsaturated silyl oximes has been developed. A similar nitration could be achieved by using Fe(NO3)3·9H2O and FeCl3·6H2O, but different from the regioselective oxime formation, two regioisomeric chloride-trapped products were isolated with varying ratios depending on the steric bulk of the silyl group. A novel ring-closure reaction of α-nitro-α,ß-unsaturated silyl oximes upon treating with TBAF to form isooxazolidinone derivatives was also developed.
RESUMEN
A general and stereoselective rearrangement of allenic alcohols to (E,E)-1,3-dien-2-yl triflates and chlorides was developed under metal-free conditions. Subtle electronic effects of the alkyl and aryl substituents on the carbon bearing the hydroxyl group has a profound impact on the reaction rate and efficiency such that vinyl triflates were obtained from electron-deficient substrates and trimethylsilyl triflate whereas vinyl chlorides were generated with an electron-rich substrate and trimethylsilyl chloride.