RESUMEN
A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1-3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.
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Actomiosina , Condensados Biomoleculares , Caenorhabditis elegans , Oocitos , Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Animales , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Emulsiones/química , Emulsiones/metabolismo , Oocitos/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Epigenetic proteins containing YEATS domains (YD) are an emerging target class in drug discovery. Described herein are the discovery and characterization efforts associated with PFI-6, a new chemical probe for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). For hit identification, fragment-like mimetics of endogenous YD ligands (crotonylated histone-containing proteins), were synthesized via parallel medicinal chemistry (PMC) and screened for MLLT1 binding. Subsequent SAR studies led to iterative MLLT1/3 binding and selectivity improvements, culminating in the discovery of PFI-6. PFI-6 demonstrates good affinity and selectivity for MLLT1/3 vs. other human YD proteins (YEATS2/4) and engages MLLT3 in cells. Small-molecule X-ray co-crystal structures of two molecules, including PFI-6, bound to the YD of MLLT1/3 are also described. PFI-6 may be a useful tool molecule to better understand the biological effects associated with modulation of MLLT1/3.
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Histonas , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , Histonas/metabolismo , Dominios Proteicos , Descubrimiento de Drogas , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismoRESUMEN
We present a deep learning model to automatically generate computer models of the human heart from patient imaging data with an emphasis on its capability to generate thin-walled cardiac structures. Our method works by deforming a template mesh to fit the cardiac structures to the given image. Compared with prior deep learning methods that adopted this approach, our framework is designed to minimize mesh self-penetration, which typically arises when deforming surface meshes separated by small distances. We achieve this by using a two-stage diffeomorphic deformation process along with a novel loss function derived from the kinematics of motion that penalizes surface contact and interpenetration. Our model demonstrates comparable accuracy with state-of-the-art methods while additionally producing meshes free of self-intersections. The resultant meshes are readily usable in physics based simulation, minimizing the need for postprocessing and cleanup.
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Aprendizaje Profundo , Humanos , Simulación por Computador , Corazón , Análisis de Elementos Finitos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Force fields form the basis for classical molecular simulations, and their accuracy is crucial for the quality of, for instance, protein-ligand binding simulations in drug discovery. The huge diversity of small-molecule chemistry makes it a challenge to build and parameterize a suitable force field. The Open Force Field Initiative is a combined industry and academic consortium developing a state-of-the-art small-molecule force field. In this report, industry members of the consortium worked together to objectively evaluate the performance of the force fields (referred to here as OpenFF) produced by the initiative on a combined public and proprietary dataset of 19,653 relevant molecules selected from their internal research and compound collections. This evaluation was important because it was completely blind; at most partners, none of the molecules or data were used in force field development or testing prior to this work. We compare the Open Force Field "Sage" version 2.0.0 and "Parsley" version 1.3.0 with GAFF-2.11-AM1BCC, OPLS4, and SMIRNOFF99Frosst. We analyzed force-field-optimized geometries and conformer energies compared to reference quantum mechanical data. We show that OPLS4 performs best, and the latest Open Force Field release shows a clear improvement compared to its predecessors. The performance of established force fields such as GAFF-2.11 was generally worse. While OpenFF researchers were involved in building the benchmarking infrastructure used in this work, benchmarking was done entirely in-house within industrial organizations and the resulting assessment is reported here. This work assesses the force field performance using separate benchmarking steps, external datasets, and involving external research groups. This effort may also be unique in terms of the number of different industrial partners involved, with 10 different companies participating in the benchmark efforts.
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Proteínas , Termodinámica , Ligandos , Proteínas/química , Fenómenos FísicosRESUMEN
Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70-Bcl-2-associated athanogene 3 (Hsp70-Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70-Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70-Bag3-LATS1/2 signaling regulates protein aggregation. We established that the regulated step was the emergence and growth of abnormal protein oligomers containing only a few molecules, indicating that aggregation is regulated at very early stages. The Hsp70-Bag3 complex therefore functions as an important signaling node that senses proteotoxicity and triggers multiple pathways that control cell physiology, including activation of protein aggregation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Complejos Multiproteicos/metabolismo , Agregación Patológica de Proteínas/metabolismo , Deficiencias en la Proteostasis/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Humanos , Complejos Multiproteicos/genética , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patologíaRESUMEN
A direct, stereocontrolled synthesis of acyclic α-chloroenamides is presented. Our methodology showed good yields and substrate scope. Mechanistic insights are provided that account for the high levels of stereoselectivity reported. Subsequent synthetic manipulation of the α-chloroenamides provides direct entry to polyfunctionalized acyclic enamides, compounds of wide use in organic chemistry and the pharmaceutical industry.
RESUMEN
A variety of long-lived carbocations containing the p-(pentafluorosulfanyl)phenyl and m-(pentafluorosulfanyl)phenyl groups have been characterized by low-temperature NMR spectroscopy. In the case of potential nonclassical carbocations substituted with the p-(pentafluorosulfanyl)phenyl substituent, deviations from linearity when the Hammett parameter (σC+) is plotted versus 13C NMR shifts of the carbocationic center were observed. Plotting the experimentally derived 13C NMR shifts versus σC+ or σ+ of classical 4-phenyl-X substituted carbocations also provides a means to accurately back-calculate the σ+ and σC+ parameters of the -SF5 substituent.
RESUMEN
Nine propargylic acetates, bearing a stereogenic center (-C*HXR(2)) adjacent to the electrophilic carbon atom, were prepared and subjected to SN1-type substitution reactions with various silyl nucleophiles employing bismuth trifluoromethanesulfonate [Bi(OTf)3] as the Lewis acid. The diastereoselectivity of the reactions was high when the alkyl group R(2) was tertiary (tert-butyl), irrespective of the substituent X. Products were formed consistently with a diastereomeric ratio larger than 95:5 in favor of the anti-diastereoisomer. If the alkyl substitutent R(2) was secondary, the diastereoselectivity decreased to 80:20. The reaction was shown to proceed stereoconvergently, and the relative product configuration was elucidated. The reaction outcome is explained by invoking a chiral propargylic cation as an intermediate, which is preferentially attacked by the nucleophile from one of its two diastereotopic faces. Density functional theory (DFT) calculations suggest a preferred conformation in which the group R(2) is almost perpendicular to the plane defined by the three substituents at the cationic center, with the nucleophile approaching the electrophilic center opposite to R(2). Transition states calculated for the reaction of allyltrimethylsilane with two representative cations support this hypothesis. Tertiary propargylic cations with a stereogenic center (-C*HXR(2)) in the α position were generated by ionization of the respective alcohol precursors with FSO3H in SO2ClF at -80 °C. Nuclear magnetic resonance (NMR) spectra were obtained for five cations, and the chemical shifts could be unambiguously assigned. The preferred conformation of the cations as extracted from nuclear Overhauser experiments is in line with the preferred conformation responsible for the reaction of the secondary propargylic cations.
RESUMEN
We present simultaneous measurements of angular-dependent magnetoresistance and thermopower along all three crystal axes in (TMTSF)2PF6 for pressures to 7.4 kbar and magnetic fields to 35 T. (TMTSF)2PF6 under pressure shows the coexistence of spin density wave and metal-superconducting orders. We suggest that this coexistence results neither in microscopic coexistence nor in a new soliton wall phase, contrary to previous suggestions, but in phase separation into domains of the high-pressure metal and the low-pressure spin density wave phases. Simultaneous measurement of transport along all crystal axes allows us to unambiguously describe the domain structure, whereas the superconducting transition temperature and four independent Fermi surface-sensitive magnetoresistance signatures allow us to unambiguously characterize the coexisting metallic domains.
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Biomolecular condensation has emerged as a key organizing principle governing the formation of membraneless cellular assemblies. Revealing the mechanism of formation of biomolecular condensates requires the quantitative examination of their growth kinetics. Here, we introduce mass balance imaging (MBI) as a general method to study compositional growth dynamics based on fluorescent images of multicomponent clusters. MBI allows the visualization and measurement of composition-dependent growth rates of biomolecular condensates and other assemblies. We provide a computational pipeline and demonstrate the applicability of our method by investigating cortical assemblies containing N-WASP (WSP-1) and F-actin that appear during oocyte cortex activation in C. elegans. In general, the method can be broadly implemented to identify interactions that underlie growth kinetics of multicomponent assemblies in vivo and in vitro.
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Actinas , Orgánulos , Animales , Condensados Biomoleculares , Caenorhabditis elegans , CinéticaRESUMEN
A series of small-molecule YEATS4 binders have been discovered as part of an ongoing research effort to generate high-quality probe molecules for emerging and/or challenging epigenetic targets. Analogues such as 4d and 4e demonstrate excellent potency and selectivity for YEATS4 binding versus YEATS1,2,3 and exhibit good physical properties and in vitro safety profiles. A new X-ray crystal structure confirms direct binding of this chemical series to YEATS4 at the lysine acetylation recognition site of the YEATS domain. Multiple analogues engage YEATS4 with nanomolar potency in a whole-cell nanoluciferase bioluminescent resonance energy transfer assay. Rodent pharmacokinetic studies demonstrate the competency of several analogues as in vivo-capable binders.
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Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , Dominios Proteicos , Acetilación , Epigénesis GenéticaRESUMEN
Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state.
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Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Procesamiento Proteico-Postraduccional , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada con la Actina/química , Proteína 2 Relacionada con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/química , Complejo 2-3 Proteico Relacionado con la Actina/genética , Humanos , Simulación de Dinámica Molecular , Mutación , Fosforilación , Polimerizacion , Conformación ProteicaRESUMEN
BACKGROUND: Dysbiotic gut bacteria engage in the development and progression of severe alcoholic hepatitis (SAH). We aimed to characterize bacterial communities associated with clinical events (CE), identify significant bacteria linked to CE, and define bacterial relationships associated with specific CE and outcomes at baseline and after treatment in SAH. METHODS: We performed 16-s rRNA sequencing on stool samples (n=38) collected at admission and the last follow-up within 90 days in SAH patients (n=26; 12 corticosteroids; 14 granulocyte colony-stimulating factor, [G-CSF]). Validated pipelines were used to plot bacterial communities, profile functional metabolism, and identify significant taxa and functional metabolites. Conet/NetworkX® was utilized to identify significant non-random patterns of bacterial co-presence and mutual exclusion for clinical events. RESULTS: All the patients were males with median discriminant function (DF) 64, Child-Turcotte-Pugh (CTP) 12, and model for end-stage liver disease (MELD) score 25.5. At admission, 27%, 42%, and 58% had acute kidney injury (AKI), hepatic encephalopathy (HE), and infections respectively; 38.5% died at end of follow-up. Specific bacterial families were associated with HE, sepsis, disease severity, and death. Lachnobacterium and Catenibacterium were associated with HE, and Pediococcus with death after steroid treatment. Change from Enterococcus (promotes AH) to Barnesiella (inhibits E. faecium) was significant after G-CSF. Phenylpropanoid-biosynthesis (innate-immunity) and glycerophospholipid-metabolism (cellular-integrity) pathways in those without infections and the death, respectively, were upregulated. Mutual interactions between Enterococcus cecorum, Acinetobacter schindleri, and Mitsuokella correlated with admission AKI. CONCLUSIONS: Specific gut microbiota, their interactions, and metabolites are associated with complications of SAH and treatment outcomes. Microbiota-based precision medicine as adjuvant treatment may be a new therapeutic area.
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Lesión Renal Aguda , Enfermedad Hepática en Estado Terminal , Microbioma Gastrointestinal , Hepatitis Alcohólica , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Hepatitis Alcohólica/microbiología , Humanos , Masculino , Índice de Severidad de la EnfermedadRESUMEN
Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu â Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.
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Anemia de Células Falciformes/tratamiento farmacológico , Hemoglobina A/efectos de los fármacos , Hemoglobina Falciforme/efectos de los fármacos , Quinolinas/farmacología , Quinolinas/uso terapéutico , Animales , Eritrocitos/metabolismo , Ratones , Oxígeno/metabolismo , Quinolinas/químicaRESUMEN
The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
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Agregado de Proteínas/genética , Agregación Patológica de Proteínas/genética , Pliegue de Proteína , Línea Celular Tumoral , Humanos , Cinética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Transición de Fase , Agregado de Proteínas/fisiología , Agregación Patológica de Proteínas/patologíaRESUMEN
Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can "capture" a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins.
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Conformación Proteica , Algoritmos , Animales , Ácido Aspártico/química , Biología Computacional , Quinasa 2 Dependiente de la Ciclina/química , Bases de Datos de Proteínas , Ácido Glutámico/química , Humanos , Conformación Molecular , Fosforilación , Procesamiento Proteico-Postraduccional , Estructura Terciaria de ProteínaRESUMEN
Histone deacetylases (HDACs) regulate diverse cellular processes, and are promising targets for a number of diseases. Here we describe the design and utilization of a largazole-based chemical probe to quantitatively measure the intracellular occupancy of HDAC1 and HDAC2 by dacinostat. Surprisingly, the probe was unable to enrich HDAC3 despite its nanomolar potency in a biochemical assay, further proving the necessity of cell-based target occupancy assays to understand compound potency in physiologically-relevant settings. This occupancy assay has the potential to aid the development of novel HDAC1/2 inhibitors in drug discovery.
RESUMEN
The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.
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Diseño de Fármacos , Endorribonucleasas/antagonistas & inhibidores , Atrofia Muscular Espinal/tratamiento farmacológico , Quinazolinas/química , Quinazolinas/uso terapéutico , ARN Mensajero/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Células HEK293 , Humanos , Ratones , Simulación del Acoplamiento Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Quinazolinas/farmacocinética , Quinazolinas/farmacología , ARN Mensajero/genética , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
Sulfonyl fluoride electrophiles have found significant utility as reactive probes in chemical biology and molecular pharmacology. As warheads they possess the right balance of biocompatibility (including aqueous stability) and protein reactivity. Their functionality is privileged in this regard as they are known to modify not only reactive serines (resulting in their common use as protease inhibitors), but also context-specific threonine, lysine, tyrosine, cysteine and histidine residues. This review describes the application of sulfonyl fluoride probes across various areas of research and explores new approaches that could further enhance the chemical biology toolkit. We believe that sulfonyl fluoride probes will find greater utility in areas such as covalent enzyme inhibition, target identification and validation, and the mapping of enzyme binding sites, substrates and protein-protein interactions.