Protac: redirigiendo los sistemas de degradación de proteínas a nuevos sustratos / Protac: redirecting protein degradation systems to new substrates

Tradicionalmente el diseño de fármacos se ha basado en el diseño de moléculas pequeñas hasta la aparición de terapias basadas en ácidos nucleicos, ya sea por la modificación de ciertos genes o por impedir que las proteínas se transcriban de forma efectiva. Empleando estas nuevas aproximaciones se han podido modificar dianas que hasta el momento se consideraban inmodificables o al menos no lo podían ser por moléculas pequeñas.Sin embargo, estas nuevas aproximaciones no están carentes de limitaciones como la baja biodisponibilidad debido a su limitada estabilidad y dificultad para poder atravesar las barreras celulares. Además, en muchas ocasiones las modificaciones que generan son irreversibles con el consiguiente riesgo de padecer efectos adversos de forma crónica.Como alternativa, han surgido con fuerza una serie de compuestos quiméricos heterobifuncionales denominados PROTACs (Protein Targeting Chimeras). Estos PROTACs son capaces de mantener en la proximidad de la ligasa E3 a una proteína de interés, marcándola con ubiquitina y finalmente, promoviendo su degradación mediada por el proteasoma. Esta aproximación permite la generación de diferentes estructuras de PROTAC por diseño racional o basado en la estructura y, además, permite las modificaciones estructurales necesarias para mejorar su perfil de estabilidad y farmacocinético manteniendo su actividad.Esta revisión pretende dar una visión general de qué son los PROTACs, qué ligasas E3 se emplean por el momento, factores relevantes a la hora de desarrollar un PROTAC y otras aproximaciones similares que no emplean el proteasoma como ruta de degradación. (AU)

The small molecules development has dominated the design of new drugs until the rise of nucleic acid-based therapies, either by modifying a gene or by preventing it from being effectively transcribed. Taking advantage of this new approaches, the pharmacological intervention in therapeutic targets that are considered unmodifiable up to now with small molecules were allowed.However, these new approaches are not devoid of defects such as low bioavailability due to their stability and pharmacokinetic problems, in addition to being irreversible DNA modifications in many cases, with the subsequent risk of suffering chronic adverse effects.Alternatively, a series of chimeric heterobifunctional compounds, called PROTACs (Protein Targeting Chimeras), have emerged with force in recent years. These PROTACs are able to bring E3 ligases closer with proteins of interest in space to label them with ubiquitin. Finally, it was degraded by the proteasome. This approach enables the generation of different PROTACs structures by rational design and, also, allows the chemical structure modification to improve their stability and pharmacokinetic profile keeping their activity.This review aims to give a comprehensive approach of what PROTACs are, what E3 ligases recruit, relevant factors in PROTAC development, and other approaches similar to this but that use non-proteasomal degradation pathways. (AU)

Protactinium and Actinium Monohydrides: A Theoretical Study on Their Spectroscopic and Thermodynamic Properties.

Spectroscopic and thermodynamics properties including bond dissociation energies (BDEs), adiabatic electron affinities (AEAs), and ionization energies (IEs) have been predicted for AcH and PaH using the Feller-Peterson-Dixon composite approach. Comparisons with previous studies on ThH and UH were performed to identify possible trends in the actinide series. Multireference CASPT2 calculations were used to predict the spin-orbit effects and obtain potential energy curves for the low-lying Ω states around the equilibrium distance as well as the vertical detachment energies (VDEs) from AcH- and PaH- to excited states of the neutral species. The calculated AEA for AnH (An = Ac, Th, Pa, U) showed that the AEA increases from AcH (0.425 eV) to ThH (0.820 eV) and decreases to PaH (0.781 eV) and to UH (0.457 eV), whereas the IE values are 5.887 eV (AcH), 6.181 eV (ThH), 6.204 eV (PaH), and 6.182 eV (UH). The ground state of AcH, AcH-, PaH, and PaH- are predicted to be1Σ+0,2Π3/2, 3H4, and 4I9/2, respectively. The BDEs for AcH and PaH are 276.4 and 237.2 kJ/mol, and those for AcH- and PaH- are 242.8 and 239.8 kJ/mol, respectively. The natural bond analysis shows a significant ionic character, An+H-, in the bonding of the neutral hydrides.

Determination of Picogram-per-Gram Concentrations of 231Pa and 230Th in Sediments by Melt Quenching and Laser Ablation Mass Spectrometry.

Uranium, thorium, and protactinium radionuclides in marine sediments are important proxies for understanding the earth's environmental evolution. Conventional solution-based methods, which typically involve isotope spike preparation, concentrated acid sample digestion, column chemistry, and mass spectrometry, allow precise but time-consuming and costly measurements of these nuclide concentrations (i.e., 230Th and 231Pa). In this work, we have established an efficient method for 230Th and 231Pa measurement of marine sediments down to the picogram-per-gram level without purification and enrichment. Our method first transforms a small amount of thermally decomposed sediments (∼0.1-0.2 g) to homogeneous silicate glass using a melt quenching technique and then analyzes the glass with laser ablation multicollector inductively coupled plasma-mass spectrometry. Standard sample bracketing with isotope-spike-calibrated glass standards prepared in this study was used to correct for instrumental fractionation during measurement. It is demonstrated that our method can accurately determine the U-Th-Pa concentrations of typical marine sediments in the late Pleistocene with precision of a few percent. Compared with the conventional solution-based methods, the turnover time of sample preparation and measurement with our established protocol is greatly reduced, facilitating future application of U-series radionuclides in reconstructing oceanic processes at high temporal and spatial resolution.

Glacial episodes of a freshwater Arctic Ocean covered by a thick ice shelf.

Following early hypotheses about the possible existence of Arctic ice shelves in the past1-3, the observation of specific erosional features as deep as 1,000 metres below the current sea level confirmed the presence of a thick layer of ice on the Lomonosov Ridge in the central Arctic Ocean and elsewhere4-6. Recent modelling studies have addressed how an ice shelf may have built up in glacial periods, covering most of the Arctic Ocean7,8. So far, however, there is no irrefutable marine-sediment characterization of such an extensive ice shelf in the Arctic, raising doubt about the impact of glacial conditions on the Arctic Ocean. Here we provide evidence for at least two episodes during which the Arctic Ocean and the adjacent Nordic seas were not only covered by an extensive ice shelf, but also filled entirely with fresh water, causing a widespread absence of thorium-230 in marine sediments. We propose that these Arctic freshwater intervals occurred 70,000-62,000 years before present and approximately 150,000-131,000 years before present, corresponding to portions of marine isotope stages 4 and 6. Alternative interpretations of the first occurrence of the calcareous nannofossil Emiliania huxleyi in Arctic sedimentary records would suggest younger ages for the older interval. Our approach explains the unexpected minima in Arctic thorium-230 records9 that have led to divergent interpretations of sedimentation rates10,11 and hampered their use for dating purposes. About nine million cubic kilometres of fresh water is required to explain our isotopic interpretation, a calculation that we support with estimates of hydrological fluxes and altered boundary conditions. A freshwater mass of this size-stored in oceans, rather than land-suggests that a revision of sea-level reconstructions based on freshwater-sensitive stable oxygen isotopes may be required, and that large masses of fresh water could be delivered to the north Atlantic Ocean on very short timescales.

Production of 230Pa by proton irradiation of 232Th at the LANL isotope production facility: Precursor of 230U for targeted alpha therapy.

Uranium-230 (t1/2 = 20.8 d) is an alpha-emitting radionuclide that has potential application in targeted alpha therapy (TAT) of cancer. Its parent isotope 230Pa (t1/2 = 17.4 d), can be produced by proton irradiation of thorium metal targets. Preliminary 230Pa production runs were performed at the Los Alamos National Laboratory Isotope Production Facility (LANL-IPF) using thin thorium metal targets and a proton beam energy of 15-30 MeV, followed by radiochemical separation of 230Pa from the irradiated target matrix. The measured 230Pa production yields were found to exceed the predicted values in most of the experiments that were performed. This data will inform further production efforts for providing 230Pa/230U for clinical trials.

Density Functional Theory Investigations on the Mechanism of Formation of Pa(V) Ion in Hydrous Solutions.

Due to the enormous threat of protactinium to the environment and human health, its disposal and chemistry have long been important topics in nuclear science. [PaO(H2O)6]3+ is proposed as the predominant species in hydrous and acidic solutions, but little is known about its formation mechanism. In this study, density functional theory (DFT) calculations demonstrate a water coordination-proton transfer-water dissociation mechanism for the formation of PaO3+ in hydrous solutions. First, Pa(V) ion preferentially forms hydrated complexes with a coordination number of 10. Through hydrogen bonding, water molecules in the second coordination sphere easily capture two protons on the same coordinated H2O ligand to form [PaO(H2O)9]3+. Water dissociation then occurs to generate the final [PaO(H2O)6]3+, which is the thermodynamic product of Pa(V) in hydrous solutions.

Separation of Protactinium Employing Sulfur-Based Extraction Chromatographic Resins.

Protactinium-230 ( t1/2 = 17.4 d) is the parent isotope of 230U ( t1/2 = 20.8 d), a radionuclide of interest for targeted alpha therapy (TAT). Column chromatographic methods have been developed to separate no-carrier-added 230Pa from proton irradiated thorium targets and accompanying fission products. Results reported within demonstrate the use of novel sulfur bearing chromatographic extraction resins for the selective separation of protactinium. The recovery yield of 230Pa was 93 ± 4% employing a R3P═S type commercially available resin and 88 ± 4% employing a DGTA (diglycothioamide) containing custom synthesized extraction chromatographic resin. The radiochemical purity of the recovered 230Pa was measured via high purity germanium γ-ray spectroscopy to be >99.5% with the remaining radioactive contaminant being 95Nb due to its similar chemistry to protactinium. Measured equilibrium distribution coefficients for protactinium, thorium, uranium, niobium, radium, and actinium on both the R3P═S type and the DGTA resin in hydrochloric acid media are reported, to the best of our knowledge, for the first time.

Saline-induced Pd/Pa ratio predicts functional significance of coronary stenosis assessed using fractional flow reserve.

AIMS: Fractional flow reserve (FFR), assessed using distal coronary pressure/aortic pressure (Pd)/(Pa) ratio, functionally evaluates coronary stenosis. An assessment method without vasodilators would be helpful. A single intracoronary bolus of saline decreases Pd because of the speculated low-viscosity effect. We hypothesised that saline-induced Pd/Pa ratio (SPR) could functionally evaluate coronary stenosis. This study aimed to test the accuracy and utility of SPR for predicting FFR ≤0.80. METHODS AND RESULTS: In 137 coronary lesions with over 50% angiographic diameter stenosis, SPR was assessed using an intracoronary bolus of saline (2 mL/s) for five heartbeats (SPR-5) and three heartbeats (SPR-3). FFR was obtained after intravenous adenosine infusion (140 µg/kg/min). There was a strong correlation between FFR and SPR-5 or SPR-3 (R=0.941 and R=0.933, respectively). Receiver operating characteristic (ROC) curve analysis demonstrated good accuracy (86.3%) for SPR-5, with a cut-off of ≤0.84 for predicting FFR ≤0.80 (area under ROC curve 0.96, specificity 94.3, sensitivity 79.9). Thirty-three lesions (24%) were located in the "grey zone" (SPR 0.83-0.88). No complications were observed in 673 SPR measurements. CONCLUSIONS: SPR may accurately predict FFR and can limit adenosine use to one in four lesions. Further studies are needed to confirm the validity of SPR.

Nuclear Data Evaluation for Mass Chain A=217:Odd-Proton Nuclei.

Thallium (81(217)Tl, Bismuth (83(217)Bi), Astatine (85(217)At), Francium (87(217)Fr), Actinium (89(217)Ac) and Protactinium (91(217)Pa) are of odd-proton numbers among the mass chain A = 217. In the present work, the half-lives and gamma transitions for the six nuclei have been studied and adopted based on the recently published interactions or unevaluated nuclear data sets XUNDL. The Q (α) has been updated based on the recent published work of the Atomic Mass Evaluation AME2012 as well. Moreover, the total conversion electrons as well as the K-Shell to L-Shell, L-Shell to M-Shell and L-Shell to N-Shell Conversion Electron Ratios have been calculated using BrIcc code v2.3. An updated skeleton decay scheme for each of the above nuclei has been presented here. The decay hindrance factors (HF) calculated using the ALPHAD program, which is available from Brookhaven National Laboratory's website, have been calculated for the α- decay data sets for (221)Fr-, (221)Ac- and (221)Pa-α-decays.

A simple-rapid method to separate uranium, thorium, and protactinium for U-series age-dating of materials.

Uranium-series dating techniques require the isolation of radionuclides in high yields and in fractions free of impurities. Within this context, we describe a novel-rapid method for the separation and purification of U, Th, and Pa. The method takes advantage of differences in the chemistry of U, Th, and Pa, utilizing a commercially-available extraction chromatographic resin (TEVA) and standard reagents. The elution behavior of U, Th, and Pa were optimized using liquid scintillation counting techniques and fractional purity was evaluated by alpha-spectrometry. The overall method was further assessed by isotope dilution alpha-spectrometry for the preliminary age determination of an ancient carbonate sample obtained from the Lake Bonneville site in western Utah (United States). Preliminary evaluations of the method produced elemental purity of greater than 99.99% and radiochemical recoveries exceeding 90% for U and Th and 85% for Pa. Excellent purity and yields (76% for U, 96% for Th and 55% for Pa) were also obtained for the analysis of the carbonate samples and the preliminary Pa and Th ages of about 39,000 years before present are consistent with (14)C-derived age of the material.

Relativistic small-core pseudopotentials for actinium, thorium, and protactinium.

Small-core pseudopotentials for actinium, thorium, and protactinium have been energy-adjusted to multiconfiguration Dirac-Hartree-Fock reference data based on the Dirac-Coulomb-Breit Hamiltonian and the Fermi nucleus model. Corresponding optimized valence basis sets of polarized valence quadruple-ζ quality are presented. Atomic test calculations for the first four ionization potentials show satisfactory results at both the Hartree-Fock and the multireference averaged coupled-pair functional level. Highly correlated Fock-space coupled cluster calculations demonstrate that the new pseudopotentials yield ionization potentials, which are in excellent agreement with corresponding all-electron results and experimental data. The pseudopotentials and basis sets supplement a similar set previously published for uranium.

(232)Th(d,4n)(230)Pa cross-section measurements at ARRONAX facility for the production of (230)U.

INTRODUCTION: (226)Th (T1/2=31 min) is a promising therapeutic radionuclide since results, published in 2009, showed that it induces leukemia cells death and activates apoptosis pathways with higher efficiencies than (213)Bi. (226)Th can be obtained via the (230)U α decay. This study focuses on the (230)U production using the (232)Th(d,4n)(230)Pa(ß-)(230)U reaction. METHODS: Experimental cross sections for deuteron-induced reactions on (232)Th were measured from 30 down to 19 MeV using the stacked-foil technique with beams provided by the ARRONAX cyclotron. After irradiation, all foils (targets as well as monitors) were measured using a high-purity germanium detector. RESULTS: Our new (230)Pa cross-section values, as well as those of (232)Pa and (233)Pa contaminants created during the irradiation, were compared with previous measurements and with results given by the TALYS code. Experimentally, same trends were observed with slight differences in orders of magnitude mainly due to the nuclear data change. Improvements are ongoing about the TALYS code to better reproduce the data for deuteron-induced reactions on (232)Th. CONCLUSIONS: Using our cross-section data points from the (232)Th(d,4n)(230)Pa reaction, we have calculated the thick-target yield of (230)U, in Bq/µA·h. This value allows now to a full comparison between the different production routes, showing that the proton routes must be preferred.

Persistent export of 231Pa from the deep central Arctic Ocean over the past 35,000 years.

The Arctic Ocean has an important role in Earth's climate, both through surface processes such as sea-ice formation and transport, and through the production and export of waters at depth that contribute to the global thermohaline circulation. Deciphering the deep Arctic Ocean's palaeo-oceanographic history is a crucial part of understanding its role in climatic change. Here we show that sedimentary ratios of the radionuclides thorium-230 ((230)Th) and protactinium-231 ((231)Pa), which are produced in sea water and removed by particle scavenging on timescales of decades to centuries, respectively, record consistent evidence for the export of (231)Pa from the deep Arctic and may indicate continuous deep-water exchange between the Arctic and Atlantic oceans throughout the past 35,000 years. Seven well-dated box-core records provide a comprehensive overview of (231)Pa and (230)Th burial in Arctic sediments during glacial, deglacial and interglacial conditions. Sedimentary (231)Pa/(230)Th ratios decrease nearly linearly with increasing water depth above the core sites, indicating efficient particle scavenging in the upper water column and greater influence of removal by lateral transport at depth. Although the measured (230)Th burial is in balance with its production in Arctic sea water, integrated depth profiles for all time intervals reveal a deficit in (231)Pa burial that can be balanced only by lateral export in the water column. Because no enhanced sink for (231)Pa has yet been found in the Arctic, our records suggest that deep-water exchange through the Fram strait may export (231)Pa. Such export may have continued for the past 35,000 years, suggesting a century-scale replacement time for deep waters in the Arctic Ocean since the most recent glaciation and a persistent contribution of Arctic waters to the global ocean circulation.

Determination of weight distribution ratios of Pa(V) and Np(V) with some extraction chromatography resins and the AG1-X8 resin.

Literature data on distribution ratios (Dw) of Np(V) and Pa(V) for the AG1-X8 resin are scarce whereas those related on resin capacity factors (k') values for TEVA, TRU and U/TEVA resins are absent. Therefore, batch extraction experiments for Pa(V) and Np(V) from HCl and HNO3 media were realized, at tracer scale, with AG1-X8 and EIChroM resins (TEVA, TRU and U/TEVA). Based on the new Dw and k' values obtained in this study, a new protocol for Pa/Np separation has been developed leading to a better separation factor of 10(5) and a chemical yield of 97±3% and 99±1% for Pa and Np, respectively. A separation of (231)Pa from uranium matrix was successfully tested.

LAFARA: a new underground laboratory in the French Pyrénées for ultra low-level gamma-ray spectrometry.

We describe a new underground laboratory, namely LAFARA (for "LAboratoire de mesure des FAibles RAdioactivités"), that was recently created in the French Pyrénées. This laboratory is primarily designed to analyze environmental samples that display low radioactivity levels using gamma-ray spectrometry. Two high-purity germanium detectors were placed under 85 m of rock (ca. 215 m water equivalent) in the tunnel of Ferrières (Ariège, France). The background is thus reduced by a factor of ∼20 in comparison to above-ground laboratories. Both detectors are fully equipped so that the samples can be analyzed in an automatic mode without requiring permanent presence of a technician in the laboratory. Auto-samplers (twenty positions) and systems to fill liquid nitrogen automatically provide one month of autonomy to the spectrometers. The LAFARA facility allows us to develop new applications in the field of environmental sciences based on the use of natural radionuclides present at low levels in the environment. As an illustration, we present two of these applications: i) dating of marine sediments using the decay of (226)Ra in sedimentary barite (BaSO(4)), ii) determination of (227)Ac ((231)Pa) activities in marine sediment cores.

Actinide sulfides in the gas phase: experimental and theoretical studies of the thermochemistry of AnS (An = Ac, Th, Pa, U, Np, Pu, Am and Cm).

The gas-phase thermochemistry of actinide monosulfides, AnS, was investigated experimentally and theoretically. Fourier transform ion cyclotron resonance mass spectrometry was employed to study the reactivity of An(+) and AnO(+) (An = Th, Pa, U, Np, Pu, Am and Cm) with CS(2) and COS, as well as the reactivity of the produced AnS(+) with oxidants (COS, CO(2), CH(2)O and NO). From these experiments, An(+)-S bond dissociation energies could be bracketed. Density functional theory studies of the energetics of neutral and monocationic AnS (An = Ac, Th, Pa, U, Np, Pu, Am and Cm) provided values for bond dissociation energies and ionization energies; the computed energetics of neutral and monocationic AnO were also obtained for comparison. The theoretical data, together with comparisons with known An(+)-O bond dissociation energies and M(+)-S and M(+)-O dissociation energies for the early transition metals, allowed for the refining of the An(+)-S bond dissociation energy ranges obtained from experiment. Examination of the reactivity of AnS(+) with dienes, coupled to comparisons with reactivities of the AnO(+) analogues, systematic considerations and the theoretical results, allowed for the estimation of the ionization energies of the AnS; the bond dissociation energies of neutral AnS were consequently derived. Estimates for the case of AcS were also made, based on correlations of the data for the other An and the electronic energetics of neutral and ionic An. The nature of the bonding in the elementary molecular actinide chalcogenides (oxides and sulfides) is discussed, based on both the experimental data and the computed electronic structures. DFT calculations of ionization energies for the actinide atoms and the diatomic sulfides and oxides are relatively reliable, but the calculation of bond dissociation energies is not uniformly satisfactory, either with DFT or CCSD(T). A key conclusion from both the experimental and theoretical results is that the 5f electrons do not substantially participate in actinide-sulfur bonding. We emphasize that actinides form strikingly strong bonds with both oxygen and sulfur.

Thermodynamical and structural study of protactinium(V) oxalate complexes in solution.

The complexation of protactinium(V) by oxalate was studied by X-ray absorption spectroscopy (XAS), density functional theory (DFT) calculations, capillary electrophoresis coupled with inductively coupled plasma mass spectrometry (CE-ICP-MS) and solvent extraction. XAS measurements showed unambiguously the presence of a short single oxo-bond, and the deduced structure agrees with theoretical calculations. CE-ICP-MS results indicated the formation of a highly charged anionic complex. The formation constants of PaO(C(2)O(4))(+), PaO(C(2)O(4))(2)(-), and PaO(C(2)O(4))(3)(3-) were determined from solvent extraction data by using protactinium at tracer scale (C(Pa) < 10(-10) M). Complexation reactions of Pa(V) with oxalate were found to be exothermic with relatively high positive entropic variation.

The determination of absolute intensity of 234mPa's 1001 keV gamma emission using Monte Carlo simulation.

The 1001 keV gamma line of (234m)Pa became important in gamma spectrometric measurements of samples with (238)U content with the advent of development of HpGe detectors of great dimension and high efficiency. In this study the emission probability of the 1001 keV (Y(gamma)) peak of (234m)Pa, was determined by gamma-ray spectrometric measurements performed on glass with Uranium content using Monte Carlo simulation code for efficiency calibration. This method of calculation was not applied for the values quoted in literature so far, at least to our knowledge. The measurements gave an average of 0.836 +/- 0.022%, a value that is in very good agreement to some of the recent results previously presented.

Production of 230U/226Th for targeted alpha therapy via proton irradiation of 231Pa.

(230)U and its daughter nuclide (226)Th are novel therapeutic nuclides for application in targeted alpha-therapy of cancer. We have investigated the feasibility of producing (230)U/(226)Th via proton irradiation of (231)Pa according to the reaction (231)Pa(p,2n)(230)U. The experimental excitation function for this reaction is reported for the first time. Cross sections were measured using thin targets of (231)Pa prepared by electrodeposition and (230)U yields were analyzed using alpha-spectrometry. Beam parameters (energy and intensity) were determined both by calculation using a mathematical model based on measured beam orbits and beam current integrator and by parallel monitor reactions on copper foils using high-resolution gamma-spectrometry and IAEA recommended cross-section data. The measured cross sections are in good agreement with model calculations using the EMPIRE-II code and are sufficiently high for the production of (230)U/(226)Th in clinically relevant amounts. A highly effective separation process was developed to isolate clinical grade (230)U from irradiated protactinium oxide targets. Product purity was assessed using alpha- and gamma-spectrometry as well as ICPMS.