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Metalloenzymes are central to a wide range of essential biological activities, including nucleic acid modification, protein degradation, and many others. The role of metalloenzymes in these processes also makes them central for the progression of many diseases and, as such, makes metalloenzymes attractive targets for therapeutic intervention. Increasing awareness of the role metalloenzymes play in disease and their importance as a class of targets has amplified interest in the development of new strategies to develop inhibitors and ultimately useful drugs. In this Review, we provide a broad overview of several drug discovery efforts focused on metalloenzymes and attempt to map out the current landscape of high-value metalloenzyme targets.
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Inhibidores Enzimáticos/uso terapéutico , Metaloproteínas/metabolismo , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Dominio Catalítico , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/metabolismo , Metaloproteínas/antagonistas & inhibidores , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Transferasas/antagonistas & inhibidores , Transferasas/metabolismo , Virosis/tratamiento farmacológicoRESUMEN
Protonated states of the nitrogenase active site are mechanistically significant since substrate reduction is invariably accompanied by proton uptake. We report the low pH characterization by X-ray crystallography and EPR spectroscopy of the nitrogenase molybdenum iron (MoFe) proteins from two phylogenetically distinct nitrogenases (Azotobacter vinelandii, Av, and Clostridium pasteurianum, Cp) at pHs between 4.5 and 8. X-ray data at pHs of 4.5-6 reveal the repositioning of side chains along one side of the FeMo-cofactor, and the corresponding EPR data shows a new S = 3/2 spin system with spectral features similar to a state previously observed during catalytic turnover. The structural changes suggest that FeMo-cofactor belt sulfurs S3A or S5A are potential protonation sites. Notably, the observed structural and electronic low pH changes are correlated and reversible. The detailed structural rearrangements differ between the two MoFe proteins, which may reflect differences in potential protonation sites at the active site among nitrogenase species. These observations emphasize the benefits of investigating multiple nitrogenase species. Our experimental data suggest that reversible protonation of the resting state is likely occurring, and we term this state "E0H+", following the Lowe-Thorneley naming scheme.
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Nitrogenasa/metabolismo , Protones , Dominio Catalítico , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Nitrogenasa/químicaRESUMEN
Modulation of the amyloid-ß (Aß) trafficking pathway heralds a new therapeutic frontier for Alzheimer's disease (AD). As CD74 binds to the amyloid-ß precursor protein (APP) and can suppresses Aß processing, we investigated whether recombinant adeno-associated virus (AAV) delivery of CD74 could reduce Aß production and affect disease outcomes. This idea was tested in a mouse AD model. Cotransduction of AAV-tetracycline-controlled transactivator (tTA) and AAV-tet-response element (TRE)-CD74 resulted in CD74 expression, reduced Aß production in mouse neurons containing the human APP with familial AD-linked mutations. Stereotaxic injection of AAV-TRE-GFP or CD74 into the hippocampi of an AD mouse, defined as a TgCRND8 × calmodulin-dependent protein kinase II derived promoter-tTA double-transgenic, reduced Aß loads and pyramidal neuronal Aß accumulation in the hippocampus. Immunofluorescent studies showed that APP colocalization with Lamp1 was increased in CD74-expressing neurons. Moreover, Morris water maze tasks demonstrated that mice treated with AAV-TRE-CD74 showed improved learning and memory compared to AAV-TRE-GFP control animals. These results support the idea that CD74-induced alteration of Aß processing could improve AD-associated memory deficits as shown in mouse models of human disease.
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Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Histocompatibilidad Clase II/genética , Neuronas/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/psicología , Precursor de Proteína beta-Amiloide/fisiología , Amiloidosis/metabolismo , Animales , Células Cultivadas , Dependovirus , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Vectores Genéticos/uso terapéutico , Hipocampo/metabolismo , Hipocampo/cirugía , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Masculino , Aprendizaje por Laberinto , Memoria , Ratones , Ratones Transgénicos , Microinyecciones/métodos , Técnicas EstereotáxicasRESUMEN
In the nitrogenase molybdenum-iron (MoFe) protein, we have identified five potential substrate access pathways from the protein surface to the FeMo-cofactor (the active site) or the P-cluster using experimental structures of Xe pressurized into MoFe protein crystals from Azotobacter vinelandii and Clostridium pasteurianum. Additionally, all published structures of the MoFe protein, including those from Klebsiella pneumoniae, were analyzed for the presence of nonwater, small molecules bound to the protein interior. Each pathway is based on identification of plausible routes from buried small molecule binding sites to both the protein surface and a metallocluster. Of these five pathways, two have been previously suggested as substrate access pathways. While the small molecule binding sites are not conserved among the three species of MoFe protein, residues lining the pathways are generally conserved, indicating that the proposed pathways may be accessible in all three species. These observations imply that there is unlikely a unique pathway utilized for substrate access from the protein surface to the active site; however, there may be preferred pathways such as those described here.
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Azotobacter vinelandii/enzimología , Proteínas Bacterianas/metabolismo , Clostridium/enzimología , Klebsiella pneumoniae/enzimología , Modelos Moleculares , Molibdoferredoxina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Ligandos , Molibdoferredoxina/química , Presión , Conformación Proteica , Propiedades de Superficie , Xenón/químicaRESUMEN
The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08â Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the ß-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.
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Azotobacter vinelandii/química , Proteínas Bacterianas/química , Clostridium/química , Molibdoferredoxina/química , Sitios de Unión , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína/químicaRESUMEN
The two-dimensional assembly of multicarboxylated arenes is explored at the liquid-graphite interface using scanning tunneling microscopy. Symmetry variations were introduced via phenylene spacer addition and the influence of these perturbations on the formation of hydrogen-bonded motifs from an alkanoic acid solvent is observed. This work demonstrates the importance of symmetry in 2D crystal formation and draws possible links of this behavior to prediction of coordination modes in three-dimensional coordination polymers.
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Aberrations in hippocampal neurogenesis are associated with learning and memory, synaptic plasticity and neurodegeneration in Alzheimer's disease (AD). However, the linkage between them, ß-amyloidosis and neuroinflammation is not well understood. To this end, we generated a mouse overexpressing familial AD (FAD) mutant human presenilin-1 (PS1) crossed with a knockout (KO) of the CC-chemokine ligand 2 (CCL2) gene. The PS1/CCL2KO mice developed robust age-dependent deficits in hippocampal neurogenesis associated with impairments in learning and memory, synaptic plasticity and long-term potentiation. Neurogliogenesis gene profiling supported ß-amyloid independent pathways for FAD-associated deficits in hippocampal neurogenesis. We conclude that these PS1/CCL2KO mice are suitable for studies linking host genetics, immunity and hippocampal function.
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Enfermedad de Alzheimer/fisiopatología , Quimiocina CCL2/genética , Modelos Animales de Enfermedad , Hipocampo/fisiopatología , Neurogénesis , Presenilina-1/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/psicología , Animales , Quimiocina CCL2/metabolismo , Hipocampo/metabolismo , Humanos , Potenciación a Largo Plazo/genética , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Mutación , Neuronas/metabolismo , Neuronas/fisiología , Presenilina-1/metabolismoRESUMEN
BACKGROUND & AIMS: The risk of gastrointestinal (GI) bleeding (GIB) and thromboembolic events may increase with continuous-flow left ventricular assist devices (CF-LVADs). We aimed to characterize GIB and thromboembolic events that occurred in patients with CF-LVADs and compare them with patients receiving anticoagulation therapy. METHODS: We performed a retrospective analysis of 159 patients who underwent CF-LVAD placement at 2 large academic medical centers (mean age, 55 ± 13 y). We identified and characterized episodes of GIB and thromboembolic events through chart review; data were collected from a time period of 292 ± 281 days. We compared the rates of GIB and thromboembolic events between patients who underwent CF-LVAD placement and a control group of 159 patients (mean age, 64 ± 15 y) who received a cardiac valve replacement and were discharged with anticoagulation therapy. RESULTS: Bleeding events occurred in 29 patients on CF-LVAD support (18%; 45 events total). Sixteen rebleeding events were identified among 10 patients (range, 1-3 rebleeding episodes/patient). There were 34 thrombotic events among 27 patients (17%). The most common source of bleeding was GI angiodysplastic lesions (n = 20; 44%). GIB and thromboembolic events were more common in patients on CF-LVAD support than controls; these included initial GIB (18% vs 4%, P < .001), rebleeding (6% vs none, P = .001), and thromboembolic events (17% vs 8%, P = .01). CONCLUSIONS: Patients with CF-LVADS receiving anticoagulants have a significantly higher risk of GIB and thromboembolic events than patients receiving anticoagulants after cardiac valve replacement surgery. GI angiodysplastic lesions are the most common source of bleeding.
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Hemorragia Gastrointestinal/epidemiología , Corazón Auxiliar/efectos adversos , Tromboembolia/epidemiología , Adulto , Anciano , Animales , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Ratas , Estudios RetrospectivosRESUMEN
The synthesis of well-defined cyclic polymers is crucial to exploring applications spanning engineering, energy, and biomedicine. These materials lack chain-ends and are therefore imbued with unique bulk properties. Despite recent advancements, the general methodology for controlled cyclic polymer synthesis via ring-expansion metathesis polymerization (REMP) remains challenging. Low initiator activity leads to high molar mass polymers at short reaction times that subsequently "evolve" to smaller polymeric products. In this work, we demonstrate that in situ addition of pyridine to the tethered ruthenium-benzylidene REMP initiator CB6 increases ancillary ligand lability to synthesize controlled and low dispersity cyclic poly(norbornene) on a short time scale without relying on molar mass evolution events.
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[This corrects the article DOI: 10.1039/C9SC05586J.].
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BACKGROUND: Since the start of the pandemic, text-based communication with patients has become increasingly common. Leicester Medical School introduced experiential teaching in this field in 2014 but identified a need to develop teaching on the key skills required for effective consultations. Shout 85258 offers a text messaging support service for anyone who is struggling with their mental health. They have developed an evidence-based training programme enabling volunteers to deliver quality crisis support via the medium of text messaging. APPROACH: Leicester Medical School and Shout 85258 collaborated, developing a small group teaching session for delivery to second-year medical students. The training programme and framework developed by Shout 85258 was used as the basis for a 1.5-hour session with didactic teaching and experiential role play. It was hoped that the collaboration would raise the profile of Shout 85258 in the student body. EVALUATION: The students enjoyed the mixture of didactic teaching and role play. They saw the relevance of the topic and recognised the key difficulties of text-based consultations such as in rapport building. Tutors valued the structure of the session and the framework provided. The session raised the profile of Shout 85258 amongst students and tutors. Further evaluation is needed to measure changes in the use of the service or volunteering by students following the session. IMPLICATIONS: Text-based consultation is an increasingly important area of communication in health care. Collaborating with a charitable organisation allowed sharing of established best practice in this area and raised the profile of the charity within the student body.
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Estudiantes de Medicina , Envío de Mensajes de Texto , Comunicación , Humanos , Facultades de Medicina , Estudiantes de Medicina/psicología , EnseñanzaRESUMEN
In this work, we provide the first in vitro characterization of two essential proteins from Staphylococcus aureus (S. aureus) involved in iron-sulfur (Fe-S) cluster biogenesis: the cysteine desulfurase SufS and the sulfurtransferase SufU. Together, these proteins form the transient SufSU complex and execute the first stage of Fe-S cluster biogenesis in the SUF-like pathway in Gram-positive bacteria. The proteins involved in the SUF-like pathway, such as SufS and SufU, are essential in Gram-positive bacteria since these bacteria tend to lack redundant Fe-S cluster biogenesis pathways. Most previous work characterizing the SUF-like pathway has focused on Bacillus subtilis (B. subtilis). We focus on the SUF-like pathway in S. aureus because of its potential to serve as a therapeutic target to treat S. aureus infections. Herein, we characterize S. aureus SufS (SaSufS) by X-ray crystallography and UV-vis spectroscopy, and we characterize S. aureus SufU (SaSufU) by a zinc binding fluorescence assay and small-angle X-ray scattering. We show that SaSufS is a type II cysteine desulfurase and that SaSufU is a Zn2+-containing sulfurtransferase. Additionally, we evaluated the cysteine desulfurase activity of the SaSufSU complex and compared its activity to that of B. subtilis SufSU. Subsequent cross-species activity analysis reveals a surprising result: SaSufS is significantly less stimulated by SufU than BsSufS. Our results set a basis for further characterization of SaSufSU as well as the development of new therapeutic strategies for treating infections caused by S. aureus by inhibiting the SUF-like pathway.
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[This corrects the article DOI: 10.1039/C9SC05586J.].
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Various carboxylic acid substitution patterns on the 1,3,5-triphenylbenzene nucleus were explored, and their influence on the symmetry of the resulting two-dimensional (2D) crystal structures was assessed. The symmetry of 1,3,5-benzenetribenzoic acid (H(3)BTB) was reduced by modifying the substitution pattern of the arene and/or adding an additional carboxylic acid. Four analogues belonging to various point groups were studied. Comparison of the monolayers of the analogues to that of H(3)BTB shows that plane group symmetry and molecular symmetry are not correlated: H(3)BTB and its analogues exhibit the same plane group p2 at the heptanoic acid/graphite interface. The 2D crystal structure of the H(3)BTB analogues is more strongly controlled by the geometry of hydrogen-bonding interactions rather than molecular symmetry. Other significant observations in this study include porosity, uncommon hydrogen-bonding motifs, and an unusually high number of inquivalent molecules (Z' = 3) present in the 2D crystal of the lowest symmetry analogue. This research demonstrates that reduction of molecular symmetry based on geometric modification of noncovalent interactions allows for control over porosity of the 2D crystals (close-packed structures to nanoporous networks) without changing the core shape of the molecule.
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Highly symmetric 2D nanoporous molecular networks containing rhombic voids are demonstrated to be accessible from low symmetry amphiphilic molecules. The amide amphiphiles overcome the barrier to symmetry generation in the two-dimensional crystal through forming an aggregate as a building block. This aggregate consists of three inequivalent amphiphiles that assemble to create 3- and 6-fold rotation axes through hydrogen bonding. In the 6-fold rotation axis, an unusual hydrogen bonding network, supported by high resolution scanning tunneling microscopy (STM) images and computation, is observed. This network formed by amide groups significantly contributes to constructing the rhombic nanoporous network, whereas carboxylic acid amphiphiles do not adopt this nanoporous network due to a geometric difference of hydrogen bonding. This investigation demonstrates that a high symmetry pattern is achievable without correlation with molecular symmetry through the proper combination of noncovalent interactions of simple amphiphilic molecules.
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This case study describes the innovative and unique U.S. Army Wounded Warrior Program (AW2), which provides nonmedical case management to the most severely wounded, injured, and ill soldiers and their families. The study describes the program and identifies the features for a successful nonmedical case management program of an identified population who has complex medical needs. Although the article focuses primarily on the role of the AW2 advocate, key components of the program are discussed, including successful initiatives as well as areas that required adjustment. The lessons learned are identified as well as recommendations for future nonmedical case management initiatives.
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Manejo de Caso , Personas con Discapacidad/rehabilitación , Medicina Militar , Personal Militar , Desarrollo de Programa/métodos , Heridas y Lesiones/rehabilitación , Salud de la Familia , Humanos , Evaluación de Programas y Proyectos de Salud , Apoyo Social , Índices de Gravedad del Trauma , Estados UnidosRESUMEN
HYPOTHESIS: Ears with otosclerotic stapes fixation will exhibit lower-than-normal levels of ossicular mobility as measured by Optical Coherence Tomography Doppler Vibrometry (OCT-DV). BACKGROUND: OCT-DV measures the vibration of middle ear structures in response to sound non-invasively through the intact tympanic membrane. This allows, in particular, direct measurement of the vibration at the lenticular process of the incus which is expected to be lower in patients with otosclerotic stapes fixation. METHODS: OCT-DV was performed on ears presumptively diagnosed with otosclerosis (nâ=â13) and a group of normal control ears (nâ=â42). Displacement was measured at the umbo and the lenticular process of the incus in response to 500 and 1000âHz stimulus tones at 100âdBSPL. MAIN OUTCOME MEASURE: The ability to discriminate between groups was assessed using receiver operator characteristic analysis, with the main outcome measures being the area-under-curve (AUC) and the sensitivity and specificity. RESULTS: For the best condition tested (500âHz at the incus), the AUC was 0.998 and discriminated the otosclerotic from normal ears with a sensitivity/specificity of 1.00/0.98. One anomalous patient with surgically confirmed stapes fixation exhibited hypermobility at the umbo, possibly due to a partial ossicular discontinuity. Despite the high umbo mobility, this patient's stapes fixation was correctly discriminated based on the measured incus vibration levels. CONCLUSIONS: OCT-DV is a promising tool for preoperatively assessing ossicular mobility non-invasively in the clinic. Our results suggest OCT-DV may also be useful in discriminating other ossicular pathologies that result in conductive hearing loss.
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Otosclerosis/diagnóstico , Estribo/patología , Tomografía de Coherencia Óptica/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , VibraciónRESUMEN
Fragment-based drug discovery (FBDD) is a powerful strategy for the identification of new bioactive molecules. FBDD relies on fragment libraries, generally of modest size, but of high chemical diversity. Although good chemical diversity in FBDD libraries has been achieved in many respects, achieving shape diversity - particularly fragments with three-dimensional (3D) structures - has remained challenging. A recent analysis revealed that >75% of all conventional, organic fragments are predominantly 1D or 2D in shape. However, 3D fragments are desired because molecular shape is one of the most important factors in molecular recognition by a biomolecule. To address this challenge, the use of inert metal complexes, so-called 'metallofragments' (mFs), to construct a 3D fragment library is introduced. A modest library of 71 compounds has been prepared with rich shape diversity as gauged by normalized principle moment of inertia (PMI) analysis. PMI analysis shows that these metallofragments occupy an area of fragment space that is unique and highly underrepresented when compared to conventional organic fragment libraries that are comprised of orders of magnitude more molecules. The potential value of this metallofragment library is demonstrated by screening against several different types of proteins, including an antiviral, an antibacterial, and an anticancer target. The suitability of the metallofragments for future hit-to-lead development was validated through the determination of IC50 and thermal shift values for select fragments against several proteins. These findings demonstrate the utility of metallofragment libraries as a means of accessing underutilized 3D fragment space for FBDD against a variety of protein targets.