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In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF-1. The RGG domain can undergo liquid-liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme-RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme-RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes-a kinase, a phosphorylase, and an ATP-dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co-purification of these two enzymes by co-phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis.
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BACKGROUND: ATTR (ATTRv) amyloidosis neuropathy is characterized by progressive sensorimotor and autonomic nerve degeneration secondary to amyloid deposition caused by a misfolded transthyretin protein (TTR). Small nerve fiber neuropathy is an early clinical manifestation of this disease resulting from the dysfunction of the Aδ and C small nerve fibers. Tafamidis, a selective TTR stabilizer, has proven its efficacy in the earlier stages of hATTR. OBJECTIVES: To evaluate the clinical course and utility of cutaneous pathological biomarkers in patients with ATTR amyloidosis treated with tafamidis compared to control patients. METHODS: Forty patients diagnosed with early stages of ATTRv amyloidosis (polyneuropathy disability [PND] scores 0-II) underwent small and large nerve fiber neurological evaluations, and annual skin biopsies for intraepidermal nerve fiber density (IENFD) and amyloid deposition index (ADI) estimation. Thirty patients were allocated to receive tafamidis, and 10 patients served as controls. Tafamidis pharmacokinetics analysis was performed in patients who received the treatment. RESULTS: At baseline, 12% of patients in stage PND 0 and 28% in PND I displayed small nerve fiber denervation in the distal thigh, whereas 23% and 38%, respectively, in the distal leg. Similarly, 72% and 84% had amyloid deposition in the distal thigh and 56% and 69% in the distal leg. Following 1 year of treatment, the tafamidis group showed significant clinical improvement compared to the control group, revealed by the following mean differences (1) -9.3 versus -4 points (p = <.00) in the patient's neuropathy total symptom score 6 (NTSS-6) questionnaire, (2) -2.5 versus +2.8 points (p = <.00) in the Utah Early Neuropathy Score (UENS), and (3) +1.2°C versus -0.6 (p = .01) in cold detection thresholds. Among the patients who received tafamidis, 65% had stable or increased IENFD in their distal thigh and 27% in the distal leg. In contrast, all patients in the control group underwent denervation. The ADI either decreased or remained constant in 31% of the biopsies in the distal thigh and in 24% of the biopsies in the distal leg of the tafamidis-treated patients, whereas it rose across all the biopsies in the control group. At the 4-year follow-up, the tafamidis group continued to display less denervation in the distal thigh (mean difference [MD] of -3.0 vs. -9.3 fibers/mm) and the distal leg (mean difference [MD] -4.9 vs. -8.6 fibers/mm). ADI in tafamidis-treated patients was also lower in the distal thigh (10 vs. 30 amyloid/mm2) and the distal leg (23 vs. 40 amyloid/mm2) compared to control patients. Plasma tafamidis concentrations were higher in patients with IENFD improvement and in patients with reduced amyloid deposition. Patients without amyloid deposition in the distal leg at baseline displayed delayed disease progression at 4 years. CONCLUSIONS: Cutaneous IENFD and amyloid deposition assessments in the skin of the distal thigh and distal leg are valuable biomarkers for early diagnosis of ATTR amyloidosis and for measuring the progression of small nerve fiber neuropathy. Early treatment with tafamidis slows the clinical progression of the disease, skin denervation, and amyloid deposition in the skin. Higher plasma concentrations of tafamidis are associated with better disease outcomes, suggesting that increasing the drug dose could achieve better plasma concentrations and response rates. This study describes the longest small nerve fiber neuropathy therapeutic trial with tafamidis and is the first to report small fiber symptoms, function, and structural assessments as outcomes.
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Neuropatías Amiloides Familiares , Benzoxazoles , Piel , Humanos , Masculino , Femenino , Persona de Mediana Edad , Neuropatías Amiloides Familiares/tratamiento farmacológico , Benzoxazoles/farmacología , Benzoxazoles/administración & dosificación , Anciano , Piel/patología , Piel/inervación , Piel/efectos de los fármacos , Biomarcadores/metabolismo , Prealbúmina , Adulto , Resultado del Tratamiento , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/patologíaRESUMEN
In recent studies, we have established the unique adapter chimeric antigen receptor (CAR) platform RevCAR which uses, as an extracellular CAR domain, a peptide epitope instead of an antibody domain. RevCAR adapters (termed RevCAR target modules, RevTMs) are bispecific antibodies that enable the reversible ON/OFF switch of the RevCAR system, improving the safety compared to conventional CARs. Here, we describe for the first time its use for retargeting of both T and NK-92 cells. In addition, we describe the development and preclinical validation of a novel RevTM for targeting of the fibroblast growth factor-inducible 14 (Fn14) surface receptor which is overexpressed on Glioblastoma (GBM) cells, and therefore serves as a promising target for the treatment of GBM. The novel RevTM efficiently redirects RevCAR modified T and NK-92 cells and leads to the killing of GBM cells both in vitro and in vivo. Tumor cell killing is associated with increased IL-2, TNF-α and/or IFN-γ secretion. Hence, these findings give an insight into the complementary potential of both RevCAR T and NK-92 systems as a safe and specific immunotherapeutic approach against GBM.
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Glioblastoma , Receptores Quiméricos de Antígenos , Receptor de TWEAK , Glioblastoma/inmunología , Glioblastoma/terapia , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patología , Humanos , Animales , Ratones , Receptor de TWEAK/metabolismo , Receptor de TWEAK/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/genética , Línea Celular Tumoral , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Inmunoterapia Adoptiva/métodos , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19RESUMEN
Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.
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Glioblastoma , Linfocitos T , Humanos , Glioblastoma/patología , Línea Celular Tumoral , Receptores ErbB/metabolismo , Epítopos/metabolismoRESUMEN
BACKGROUND: One-third of the risk for Alzheimer's disease is explained by environment and lifestyle, but Alzheimer's disease pathology might also affect lifestyle and thereby impair the individual potential for health behavior and prevention. METHODS: We examined in mice how the AppNL-F/NL-F (NL-F) knockin mutation affects the presymptomatic response to environmental enrichment (ENR) as an experimental paradigm addressing nongenetic factors. We assessed the emergence of interindividual phenotypic variation under the condition that both the genetic background and the shared environment were held constant, thereby isolating the contribution of individual behavior (nonshared environment). RESULTS: After 4 months of ENR, the mean and variability of plasma ApoE were increased in NL-F mice, suggesting a presymptomatic variation in pathogenic processes. Roaming entropy as a measure of behavioral activity was continuously assessed with radiofrequency identification (RFID) technology and revealed reduced habituation and variance in NL-F mice compared with control animals, which do not carry a Beyreuther/Iberian mutation. Intraindividual variation decreased, while behavioral stability was reduced in NL-F mice. Seven months after discontinuation of ENR, we found no difference in plaque size and number, but ENR increased variance in hippocampal plaque counts in NL-F mice. A reactive increase in adult hippocampal neurogenesis in NL-F mice, known from other models, was normalized by ENR. CONCLUSIONS: Our data suggest that while NL-F has early effects on individual behavioral patterns in response to ENR, there are lasting effects on cellular plasticity even after the discontinuation of ENR. Hence, early behavior matters for maintaining individual behavioral trajectories and brain plasticity even under maximally constrained conditions.
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Enfermedad de Alzheimer , Individualidad , Animales , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Modelos Animales de Enfermedad , Ratones TransgénicosRESUMEN
Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as "on-target, off-tumor" toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on hea9lthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA+ and EpCAM+ cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.
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Antígeno Carcinoembrionario , Neoplasias Colorrectales , Humanos , Linfocitos T , Molécula de Adhesión Celular Epitelial , Antígenos de NeoplasiasRESUMEN
Bulk and surface trap-states in the Sb2S3films are considered one of the crucial energy loss mechanisms for achieving high photovoltaic performance in planar Sb2S3solar cells. Because ionic liquid additives offer interesting physicochemical properties to control the synthesis of inorganic material, in this work we propose the addition of 1-Butyl-3-methylimidazolium hydrogen sulfate (BMIMHS) into a Sb2S3hydrothermal precursor solution as a facile way to fabricate low-defect Sb2S3solar cells. Lower presence of small particles on the surface, as well as higher crystallinity are demonstrated in the BMIMHS-assisted Sb2S3films. Moreover, analyses of dark current density-voltageJ-Vcurves, surface photovoltage transient and intensity-modulated photocurrent spectroscopy have suggested that adding BMIMHS results in high-quality Sb2S3films and a successful defect passivation. Consequently, the best-performing BMIMHS-assisted device exhibits a 15.4% power conversion efficiency enhancement compared to that of control device. These findings show that ionic liquid BMIMHS can effectively be used to obtain high-quality Sb2S3films with low-defects and improved optoelectronic properties.
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Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as a target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic positron emission tomography (PET) imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+, the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.
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Most patients with head and neck squamous cell carcinomas (HNSCC) are diagnosed at a locally advanced stage and show heterogeneous treatment responses. Low SLC3A2 (solute carrier family 3 member 2) mRNA and protein (CD98hc) expression levels are associated with higher locoregional control in HNSCC patients treated with primary radiochemotherapy or postoperative radiochemotherapy, suggesting that CD98hc could be a target for HNSCC radiosensitization. One of the targeted strategies for tumor radiosensitization is precision immunotherapy, e.g., the use of chimeric antigen receptor (CAR) T cells. This study aimed to define the potential clinical value of new treatment approaches combining conventional radiotherapy with CD98hc-targeted immunotherapy. To address this question, we analyzed the antitumor activity of the combination of fractionated irradiation and switchable universal CAR (UniCAR) system against radioresistant HNSCC cells in 3D culture. CD98hc-redirected UniCAR T cells showed the ability to destroy radioresistant HNSCC spheroids. Also, the infiltration rate of the UniCAR T cells was enhanced in the presence of the CD98hc target module. Furthermore, sequential treatment with fractionated irradiation followed by CD98hc-redirected UniCAR T treatment showed a synergistic effect. Taken together, our obtained data underline the improved antitumor effect of the combination of radiotherapy with CD98hc-targeted immunotherapy. Such a combination presents an attractive approach for the treatment of high-risk HNSCC patients.
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Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.
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Chimeric antigen receptor (CAR) T cells show remarkable therapeutic effects in some hematological malignancies. However, CAR T cells can also cause life-threatening side effects. In order to minimize off-target and on-target/off-tumor reactions, improve safety, enable controllability, provide high flexibility, and increase tumor specificity, we established a novel humanized artificial receptor platform termed RevCARs. RevCAR genes encode for small surface receptors lacking any antigen-binding moiety. Steering of RevCAR T cells occurs via bispecific targeting molecules (TMs). The small size of RevCAR-encoding genes allows the construction of polycistronic vectors. Here, we demonstrate that RevCAR T cells efficiently kill tumor cells, can be steered by TMs, flexibly redirected against multiple targets, and used for combinatorial targeting following the "OR" and "AND" gate logic.
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Receptores Quiméricos de Antígenos , Tratamiento Basado en Trasplante de Células y Tejidos , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Linfocitos TAsunto(s)
Neuropatías Amiloides Familiares/diagnóstico , Prealbúmina/genética , Neuropatía de Fibras Pequeñas/diagnóstico , Adulto , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Neuropatía de Fibras Pequeñas/genética , Neuropatía de Fibras Pequeñas/patologíaAsunto(s)
Neuropatías Amiloides Familiares/tratamiento farmacológico , Benzoxazoles/administración & dosificación , Prealbúmina/genética , Adulto , Neuropatías Amiloides Familiares/epidemiología , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/patología , Benzoxazoles/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Calidad de VidaAsunto(s)
Amiloidosis/genética , Prealbúmina/genética , Adolescente , Adulto , Anciano , Femenino , Genotipo , Humanos , Masculino , México , Persona de Mediana Edad , Mutación/genética , Adulto JovenRESUMEN
OBJECTIVE: We report the results of a pilot, enrichment-design, placebo-controlled crossover trial of pregabalin for the treatment of prediabetic small-fiber neuropathic pain. METHODS: Individuals with impaired fasting glucose or impaired glucose tolerance and neuropathic pain were evaluated according to UTAH Early Neuropathy Scale (UENS), Quantitative Sensory Testing, and intraepidermal nerve fiber density (IENFD). Symptoms were graded according to the Numeric Rating Scale (NRS). Individuals who responded to the administration of placebo were not eligible. Pregabalin was initiated at a dose of 75 mg qid and tapered up to 300 mg bid. Only individuals with a reduction of pain scores ≥30% were eligible to continue with the double-blind phase, which consisted of a randomized crossover period of 1 month of pregabalin and 1 month of placebo, with 7 days of washout between periods. RESULTS: Forty-five participants were enrolled in the study. There was 36% reduction in the NRS from baseline after 1 month of single-blind pregabalin (NRS=5.1±2.6). Twenty-six participants were eligible for the double-blind phase. There was further reduction of pain in the double-blind pregabalin and the placebo groups, but the pregabalin group had a statistically significant reduction of pain (NRS=3.2±2.2 vs. 4.0±2; P<0.05). Participants who did not respond showed a lower IENFD than those who responded, suggesting more severe nerve damage. CONCLUSIONS: This pilot study showed improvement of prediabetic neuropathic pain. Participants with higher pain scores at baseline had higher UENS scores and a lower IENFD. Limitations of the study include the small number of participants and the carry-over effect.
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Analgésicos/uso terapéutico , Intolerancia a la Glucosa/complicaciones , Neuralgia/complicaciones , Neuralgia/tratamiento farmacológico , Pregabalina/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Estudios Cruzados , Método Doble Ciego , Femenino , Intolerancia a la Glucosa/tratamiento farmacológico , Intolerancia a la Glucosa/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Neuralgia/fisiopatología , Dimensión del Dolor , Proyectos Piloto , Método Simple Ciego , Resultado del TratamientoRESUMEN
Protein kinases are implicated in diverse signaling cascades and have been targeted with small molecules that typically bind the conserved ATP-binding active site. These inhibitors are often promiscuous and target multiple protein kinases, which has led to the development of alternate strategies to discover selective ligands. We have recently described a fragment-based selection approach, where a small-molecule warhead can be non-covalently tethered to a phage-displayed library of cyclic peptides. This approach led to the conversion of the promiscuous kinase inhibitor, staurosporine, into a selective bivalent inhibitor.
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Descubrimiento de Drogas , Biblioteca de Péptidos , Péptidos Cíclicos , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/química , Animales , Humanos , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Estaurosporina/químicaRESUMEN
Protein kinases phosphorylate client proteins, while protein phosphatases catalyze their dephosphorylation and thereby in concert exert reversible control over numerous signal transduction pathways. We have recently reported the design and validation of split-protein kinases that can be conditionally activated by an added small molecule chemical inducer of dimerization (CID), rapamycin. Herein, we provide the rational design and validation of three split-tyrosine phosphatases (PTPs) attached to FKBP and FRB, where catalytic activity can be modulated with rapamycin. We further demonstrate that the orthogonal CIDs, abscisic acid and gibberellic acid, can be used to impart control over the activity of split-tyrosine kinases (PTKs). Finally, we demonstrate that designed split-phosphatases and split-kinases can be activated by orthogonal CIDs in mammalian cells. In sum, we provide a methodology that allows for post-translational orthogonal small molecule control over the activity of user defined split-PTKs and split-PTPs. This methodology has the long-term potential for both interrogating and redesigning phosphorylation dependent signaling pathways.
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Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Quinasas/química , Dimerización , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismoRESUMEN
At the PBE-D3/cc-pVDZ level of theory, the hydrogen chemisorption on graphene was analyzed using the reaction force and reaction electronic flux (REF) theories in combination with electron population analysis. It was found that chemisorption energy barrier is mainly dominated by structural work (â¼73%) associated to the substrate reconstruction whereas the electronic work is the greatest contribution of the reverse energy barrier (â¼67%) in the desorption process. Moreover, REF shows that hydrogen chemisorption is driven by charge transfer processes through four electronic events taking place as H approaches the adsorbent surface: (a) intramolecular charge transfer in the adsorbent surface; (b) surface reconstruction; (c) substrate magnetization and adsorbent carbon atom develops a sp(3) hybridization to form the σC-H bond; and (d) spontaneous intermolecular charge transfer to reach the final chemisorbed state.
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The activity of protein kinases are naturally gated by a variety of physiochemical inputs, such as phosphorylation, metal ions, and small molecules. In order to design protein kinases that can be gated by user-defined inputs, we describe a sequence dissimilarity based approach for identifying sites in protein kinases that accommodate 25-residue loop insertion while retaining catalytic activity. We further demonstrate that the successful loop insertion mutants provide guidance for the dissection of protein kinases into two fragments that cannot spontaneously assemble and are thus inactive but can be converted into ligand-gated catalytically active split-protein kinases. We successfully demonstrate the feasibility of this approach with Lyn, Fak, Src, and PKA, which suggests potential generality.