RESUMO
Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured in vitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated in vitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Interleucina-17/metabolismo , Proteínas Culina/metabolismo , Linfócitos T CD4-Positivos , Camundongos Endogâmicos C57BLRESUMO
Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.
Assuntos
Ligantes , Ubiquitina-Proteína Ligases , Ubiquitinação , Animais , Humanos , Camundongos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Culina/metabolismo , Suramina/farmacologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteína NEDD8/metabolismoRESUMO
Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1α (casein kinase 1α) and ß-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4CRBN and CRL1ßTrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new "apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated ß-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated ß-catenin and CRL1ßTrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified ß-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1ßTrCP. These findings support a role for conjugated Ub in enhancing interactions with E3.
Assuntos
Ubiquitina , Ubiquitinação , beta Catenina , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Antineoplásicos/química , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Inibidores Enzimáticos/química , Feminino , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ubiquitina/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short-chain enoyl-CoA hydratase (ECHS1 or crotonase) deficiency, 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA), and methylmalonic aciduria (MMA). Isobutyryl-CoA dehydrogenase (ACAD8) and short/branched-chain acyl-CoA dehydrogenase (SBCAD, ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl-CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2-methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase, but not ACAD8. MCPA treatment of wild-type and PA HEK-293 cells caused a pronounced decrease in C3-carnitine. Furthermore, deletion of ACADSB in HEK-293 cells led to an equally strong decrease in C3-carnitine when compared to wild-type cells. Deletion of ECHS1 in HEK-293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued by ACAD8 deletion. MCPA was able to rescue lipoylation in ECHS1 KO cells, but only in cells with prior ACAD8 deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK-293 cells for the isobutyryl-CoA substrate. Substrate promiscuity appeared less prominent for 2-methylbutyryl-CoA at least in HEK-293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further.
Assuntos
Ácido 2-Metil-4-clorofenoxiacético , Acidemia Propiônica , Humanos , Valina/genética , Valina/metabolismo , Acil-CoA Desidrogenase/metabolismo , Isoleucina/metabolismo , Células HEK293 , CarnitinaRESUMO
BACKGROUND: Patients with incidentally found musculoskeletal lesions are regularly referred to orthopaedic oncology. Most orthopaedic oncologists understand that many incidental findings are nonaggressive and can be managed nonoperatively. However, the prevalence of clinically important lesions (defined as those indicated for biopsy or treatment, and those found to be malignant) remains unknown. Missing clinically important lesions can result in harm to patients, but needless surveillance may exacerbate patient anxiety about their diagnosis and accrue low-value costs to the payor. QUESTIONS/PURPOSES: (1) What percentage of patients with incidentally discovered osseous lesions referred to orthopaedic oncology had lesions that were clinically important, defined as those receiving biopsy or treatment or those found to be malignant? (2) Using standardized Medicare reimbursements as a surrogate for payor expense, what is the value of reimbursements accruing to the hospital system for the imaging of incidentally found osseous lesions performed during the initial workup period and during the surveillance period, if indicated? METHODS: This was a retrospective study of patients referred to orthopaedic oncology for incidentally found osseous lesions at two large academic hospital systems. Medical records were queried for the word "incidental," and matches were confirmed by manual review. Patients evaluated at Indiana University Health between January 1, 2014, and December 31, 2020, and those evaluated at University Hospitals between January 1, 2017, and December 31, 2020, were included. All patients were evaluated and treated by the two senior authors of this study and no others were included. Our search identified 625 patients. Sixteen percent (97 of 625) of patients were excluded because their lesions were not incidentally found, and 12% (78 of 625) were excluded because the incidental findings were not bone lesions. Another 4% (24 of 625) were excluded because they had received workup or treatment by an outside orthopaedic oncologist, and 2% (10 of 625) were excluded for missing information. A total of 416 patients were available for preliminary analysis. Among these patients, 33% (136 of 416) were indicated for surveillance. The primary indication for surveillance included lesions with a benign appearance on imaging and low clinical suspicion of malignancy or fracture. A total of 33% (45 of 136) of these patients had less than 12 months of follow-up and were excluded from further analysis. No minimum follow-up criteria were applied to patients not indicated for surveillance because this would artificially inflate our estimated rate of clinically important findings. A total of 371 patients were included in the final study group. Notes from all clinical encounters with orthopaedic and nonorthopaedic providers were screened for our endpoints (biopsy, treatment, or malignancy). Indications for biopsy included lesions with aggressive features, lesions with nonspecific imaging characteristics and a clinical picture concerning for malignancy, and lesion changes seen on imaging during the surveillance period. Indications for treatment included lesions with increased risk of fracture or deformity, certain malignancies, and pathologic fracture. Diagnoses were determined using biopsy results if available or the documented opinion of the consulting orthopaedic oncologist. Imaging reimbursements were obtained from the Medicare Physician Fee Schedule for 2022. Because imaging charges vary across institutions and reimbursements vary across payors, this method was chosen to enhance the comparability of our findings across multiple health systems and studies. RESULTS: Seven percent (26 of 371) of incidental findings were determined to be clinically important, as previously defined. Five percent (20 of 371) of lesions underwent tissue biopsy, and 2% (eight of 371) received surgical intervention. Fewer than 2% (six of 371) of lesions were malignant. Serial imaging changed the treatment of 1% (two of 136) of the patients, corresponding to a rate of one in 47 person-years. Median reimbursements to work up the incidental findings analyzed was USD 219 (interquartile range USD 0 to 404), with a range of USD 0 to 890. Among patients indicated for surveillance, the median annual reimbursement was USD 78 (IQR USD 0 to 389), with a range of USD 0 to 2706. CONCLUSION: The prevalence of clinically important findings among patients referred to orthopaedic oncology for incidentally found osseous lesions is modest. The likelihood of surveillance resulting in a change of management was low, but the median reimbursements associated with following these lesions was also low. We conclude that after appropriate risk stratification by orthopaedic oncology, incidental lesions are rarely clinically important, and judicious follow-up with serial imaging can be performed without incurring high costs. LEVEL OF EVIDENCE: Level III, therapeutic study.
Assuntos
Medicare , Neoplasias , Humanos , Idoso , Estados Unidos/epidemiologia , Estudos Retrospectivos , Prevalência , Osso e OssosRESUMO
Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid.
Assuntos
Acil Coenzima A/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Encefalopatias Metabólicas/genética , Glutaril-CoA Desidrogenase/deficiência , Complexo Cetoglutarato Desidrogenase/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/patologia , Células Cultivadas , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Células HEK293 , Humanos , Cetona Oxirredutases/genética , Especificidade por Substrato/genéticaRESUMO
Studies performed in healthy smokers have documented a diminished responsiveness to tussive challenges, and several lines of experimental evidence implicate nicotine as an antitussive component in both cigarette smoke and the vapors generated by electronic cigarettes (eCigs). We set out to identify the nicotinic receptor subtype involved in the antitussive actions of nicotine and to further evaluate the potential of nicotinic receptor-selective agonists as cough-suppressing therapeutics. We confirmed an antitussive effect of nicotine in guinea pigs. We additionally observed that the alpha-4 beta-2 (α 4 ß 2)-selective agonist Tc-6683 was without effect on evoked cough responses in guinea pigs, while the α 7-selective agonist PHA 543613 dose-dependently inhibited evoked coughing. We subsequently describe the preclinical evidence in support of ATA-101, a potent and highly selective (α 7) selective nicotinic receptor agonist, as a potential candidate for antitussive therapy in humans. ATA-101, formerly known as Tc-5619, was orally bioavailable and moderately central nervous system (CNS) penetrant and dose-dependently inhibited coughing in guinea pigs evoked by citric acid and bradykinin. Comparing the effects of airway targeted administration versus systemic dosing and the effects of repeated dosing at various times prior to tussive challenge, our data suggest that the antitussive actions of ATA-101 require continued engagement of α 7 nicotinic receptors, likely in the CNS. Collectively, the data provide the preclinical rationale for α 7 nicotinic receptor engagement as a novel therapeutic strategy for cough suppression. The data also suggest that α 7 nicotinic acetylcholine receptor (nAChR) activation by nicotine may be permissive to nicotine delivery in a way that may promote addiction. SIGNIFICANCE STATEMENT: This study documents the antitussive actions of nicotine and identifies the α7 nicotinic receptor subtype as the target for nicotine during cough suppression described in humans. We additionally present evidence suggesting that ATA-101 and other α7 nicotinic receptor-selective agonists may be promising candidates for the treatment of chronic refractory cough.
Assuntos
Antitussígenos/uso terapêutico , Benzofuranos/uso terapêutico , Tosse/tratamento farmacológico , Agonistas Nicotínicos/uso terapêutico , Quinuclidinas/uso terapêutico , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Antitussígenos/farmacologia , Benzofuranos/farmacologia , Tosse/metabolismo , Cobaias , Masculino , Nicotina/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Quinuclidinas/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistasRESUMO
Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by acute encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. We investigated the efficacy of substrate reduction through inhibition of 2-aminoadipic semialdehyde synthase (AASS), an enzyme upstream of the defective glutaryl-CoA dehydrogenase (GCDH), in a cell line and mouse model of GA1. We show that loss of AASS function in GCDH-deficient HEK-293 cells leads to an approximately fivefold reduction in the established GA1 clinical biomarker glutarylcarnitine. In the GA1 mouse model, deletion of Aass leads to a 4.3-, 3.8-, and 3.2-fold decrease in the glutaric acid levels in urine, brain, and liver, respectively. Parallel decreases were observed in urine and brain 3-hydroxyglutaric acid levels, and plasma, urine, and brain glutarylcarnitine levels. These in vivo data demonstrate that the saccharopine pathway is the main source of glutaric acid production in the brain and periphery of a mouse model for GA1, and support the notion that pharmacological inhibition of AASS may represent an attractive strategy to treat GA1.
Assuntos
Ácido 2-Aminoadípico/análogos & derivados , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Encefalopatias Metabólicas/metabolismo , Encéfalo/metabolismo , Glutaratos/metabolismo , Glutaril-CoA Desidrogenase/deficiência , Fígado/metabolismo , Ácido 2-Aminoadípico/genética , Ácido 2-Aminoadípico/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Animais , Encéfalo/patologia , Encefalopatias Metabólicas/genética , Encefalopatias Metabólicas/terapia , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Feminino , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Células HEK293 , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
OBJECTIVE: The purpose of this article is to provide a primer for radiologists outlining the modern systemic therapies used in melanoma brain metastases, including tyrosine kinase inhibitors and immune checkpoint inhibitors. The role of radiologic treatment response evaluation will be discussed from the standpoint of both modern systemic therapies and more traditional treatments. CONCLUSION: Understanding the role of systemic treatments in melanoma brain metastases is critical for oncologic imaging interpretation in this unique patient population.
Assuntos
Neoplasias Encefálicas , Melanoma/patologia , Neoplasias Cutâneas/patologia , Idoso , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/secundário , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Radioterapia , Tomografia Computadorizada por Raios XRESUMO
Recently, we have shown that harmine induces ß-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. We explore structure-activity relationships of the 7-position of harmine for both DYRK1A kinase inhibition and ß-cell proliferation based on our related previous structure-activity relationship studies of harmine in the context of diabetes and ß-cell specific targeting strategies. 33 harmine analogs of the 7-position substituent were synthesized and evaluated for biological activity. Two novel inhibitors were identified which showed DYRK1A inhibition and human ß-cell proliferation capability. The DYRK1A inhibitor, compound 1-2b, induced ß-cell proliferation half that of harmine at three times higher concentration. From these studies we can draw the inference that 7-position modification is limited for further harmine optimization focused on ß-cell proliferation and cell-specific targeting approach for diabetes therapeutics.
Assuntos
Harmina/química , Harmina/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Harmina/análogos & derivados , Células Secretoras de Insulina/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The derivatization of pharmaceuticals is a core activity in the discovery and development of new medicines. Late-stage functionalization via modern CH functionalization chemistry has emerged as a powerful technique with which to diversify advanced pharmaceutical intermediates. We report herein a case study in late-stage functionalization towards the development of a new class of indazole-based mineralocorticoid receptor antagonists (MRA). An effort to modify the electronics of the core indazole heterocycle inspired the use of modern CH borylation chemistry. New reactivity patterns were revealed and studied computationally. Ultimately, a de novo synthesis delivered a key 6-fluoroindazole compound 26, a potent MRA with excellent metabolic stability.
Assuntos
Desenvolvimento de Medicamentos/métodos , Indazóis/química , Antagonistas de Receptores de Mineralocorticoides/química , Estrutura MolecularRESUMO
Previously disclosed benzimidazole-based DGAT1 inhibitors containing a cyclohexane carboxylic acid moiety suffer from isomerization at the alpha position of the carboxylic acid group, generating active metabolites which exhibit DGAT1 inhibition comparable to the corresponding parent compounds. In this report, we describe the design, synthesis and profiling of benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety. Our results show that single isomer 3A maintains in vitro and in vivo inhibition against DGAT1. In contrast to previous lead compounds, 3A does not undergo isomerization during in vitro hepatocyte incubation study or in vivo mouse study.
Assuntos
Benzimidazóis/química , Ácidos Carboxílicos/química , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Animais , Benzimidazóis/metabolismo , Ácidos Carboxílicos/metabolismo , Cromatografia Líquida de Alta Pressão , Cicloexanonas/química , Diacilglicerol O-Aciltransferase/metabolismo , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Hepatócitos/química , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Isomerismo , Espectrometria de Massas , Camundongos , RatosRESUMO
OBJECTIVES: To evaluate the effect of precompression on power Doppler visualization of blood flow in breast masses. METHODS: This Institutional Review Board-approved and Health Insurance Portability and Accountability Act-compliant study evaluated 30 patients with breast masses (16 benign and 14 malignant) undergoing ultrasound-guided breast biopsy. A computational mathematics program was used to calculate the number of color pixels in a region of interest at various degrees of compression of the breast by the transducer. The amount of precompression was calculated as previously described. The percentage of color pixels compared to minimal compression was plotted against the percentage of precompression. The amount of precompression needed to decrease the number of color pixels by 50% and 100% was calculated. The differences between benign and malignant lesions were compared. RESULTS: The mean percentages of precompression ± SD needed to decrease the number of color voxels by 50% in were 15.9% ± 6.43% (range, 8%-30%) for benign lesions and 14.0% ± 4.17% (range, 8%-20%) for malignant lesions (P = .35). The percentages of precompression needed to decrease the number of color pixels by 100% in were 34.7% ± 12.33% (range, 23%-62%) for benign lesions and for malignant lesions 26.7% ± 3.89% (range, 18%-31%), which were statistically significant (P = .027). CONCLUSIONS: The amount of precompression normally used when obtaining B-mode images can substantially decrease the number of color voxels on power Doppler sonography. When performing quantitative work on Doppler evaluation of breast lesions, precompression needs to be controlled.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/diagnóstico por imagem , Neovascularização Patológica/patologia , Ultrassonografia Doppler em Cores , Ultrassonografia Mamária , Adulto , Idoso , Idoso de 80 Anos ou mais , Mama/irrigação sanguínea , Mama/diagnóstico por imagem , Mama/patologia , Neoplasias da Mama/patologia , Diagnóstico Diferencial , Feminino , Humanos , Biópsia Guiada por Imagem , Pessoa de Meia-Idade , Neovascularização Patológica/diagnóstico por imagem , Sensibilidade e Especificidade , Ultrassonografia de IntervençãoRESUMO
OBJECTIVES: To compare the agreement and interobserver variability of diagnostic handheld ultrasound (US) and a single volume on an automated breast volume scanner (ABVS) and to determine whether there was a significant difference if the ABVS was used by a sonographer or mammographic technologist. METHODS: Ninety patients scheduled for diagnostic US examinations were randomized to either handheld US or the ABVS first. The AVBS was randomized between a sonographer and a mammographic technologist performing the study. The studies were blinded, randomized, and read by 2 radiologists. The lesion with the highest Breast Imaging Reporting and Data System (BI-RADS) score was used in the analysis. Final diagnoses were made by core biopsy or follow-up for 2 years. Lesions included 9 malignant and 81 benign. RESULTS: The 90 patients had a mean age ± SD of 53.1 ± 16.3 years. The κ value for agreement between the ABVS and handheld US was 0.831 (95% confidence interval, 0.744-0.925), whereas the global agreement for a 7-point BI-RADS score was 0.488 (0.372-0.560). The agreement between the ABVS and handheld US was nearly the same when the ABVS was used by a mammographic technologist (κ = 0.858 [0.723-0.963]) or sonographer (κ = 0.803 [0.596-1.000]; P = .47). The areas under the receiver operating characteristic curves for characterization by the ABVS were 0.91 (0.84-0.96) for reader 1 and 0.91 (0.83-0.96) for reader 2; those for handheld US were 0.91 (0.84-0.96) for reader 1 and 0.83 (0.74-0.90) for reader 2, with no statistical difference. The agreement based on pathologic images was κ = 0.831 (0.718-0.944); for handheld US, κ = 0.795 (0.623-0.967); and for the AVBS, κ = 0.869 (0.725-1.000). CONCLUSIONS: Performing a single-view diagnostic ABVS examination has good agreement with a handheld diagnostic US workup. There is no difference if the ABVS is used by a sonographer or mammographic technologist.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Reconhecimento Automatizado de Padrão/métodos , Ultrassonografia Mamária/instrumentação , Ultrassonografia Mamária/métodos , Mama/diagnóstico por imagem , Feminino , Humanos , Pessoa de Meia-Idade , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or non-toxic metabolites. Here, we report a novel target, SUGCT, which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA. We report the structure of SUGCT, the first eukaryotic structure of a type III CoA transferase, develop a high-throughput enzyme assay and a cell-based assay, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme validating the screening approach. These results may form the basis for future development of new pharmacological intervention to treat GA1.
RESUMO
Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or nontoxic metabolites. Here, we report a putative novel target, succinyl-CoA:glutarate-CoA transferase (SUGCT), which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA and the derived 3-hydroxyglutaric acid. SUGCT is a type III CoA transferase that uses succinyl-CoA and glutaric acid as substrates. We report the structure of SUGCT, develop enzyme- and cell-based assays, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme in a high-throughput screen of FDA-approved compounds. The cocrystal structure of SUGCT with losartan carboxylic acid revealed a novel pocket in the active site and further validated the high-throughput screening approach. These results may form the basis for the future development of new pharmacological intervention to treat GA1.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Encefalopatias Metabólicas , Humanos , Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Encefalopatias Metabólicas/tratamento farmacológico , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/enzimologia , Glutaratos/metabolismo , Glutaratos/química , Losartan/farmacologia , Losartan/química , Coenzima A-Transferases/metabolismo , Coenzima A-Transferases/antagonistas & inibidores , Coenzima A-Transferases/genética , Coenzima A-Transferases/química , Valsartana , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Cristalografia por Raios X , Domínio Catalítico , Acil Coenzima A/metabolismo , Acil Coenzima A/química , Modelos Moleculares , Ensaios de Triagem em Larga Escala , Glutaril-CoA Desidrogenase/deficiênciaRESUMO
Five hundred thirty-seven million people globally suffer from diabetes. Insulin-producing ß cells are reduced in number in most people with diabetes, but most individuals still have some residual ß cells. However, none of the many diabetes drugs in common use increases human ß cell numbers. Recently, small molecules that inhibit dual tyrosine-regulated kinase 1A (DYRK1A) have been shown to induce immunohistochemical markers of human ß cell replication, and this is enhanced by drugs that stimulate the glucagon-like peptide 1 (GLP1) receptor (GLP1R) on ß cells. However, it remains to be demonstrated whether these immunohistochemical findings translate into an actual increase in human ß cell numbers in vivo. It is also unknown whether DYRK1A inhibitors together with GLP1R agonists (GLP1RAs) affect human ß cell survival. Here, using an optimized immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) protocol in mouse kidneys bearing human islet grafts, we demonstrate that combination of a DYRK1A inhibitor with exendin-4 increases actual human ß cell mass in vivo by a mean of four- to sevenfold in diabetic and nondiabetic mice over 3 months and reverses diabetes, without alteration in human α cell mass. The augmentation in human ß cell mass occurred through mechanisms that included enhanced human ß cell proliferation, function, and survival. The increase in human ß cell survival was mediated, in part, by the islet prohormone VGF. Together, these findings demonstrate the therapeutic potential and favorable preclinical safety profile of the DYRK1A inhibitor-GLP1RA combination for diabetes treatment.
Assuntos
Quinases Dyrk , Exenatida , Harmina , Células Secretoras de Insulina , Peptídeos , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Animais , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Exenatida/farmacologia , Exenatida/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Harmina/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Camundongos , Peptídeos/farmacologia , Peptídeos/metabolismo , Peçonhas/farmacologia , Peçonhas/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Quimioterapia Combinada , Proliferação de Células/efeitos dos fármacos , XenoenxertosRESUMO
The small molecule DYRK1A inhibitor, harmine, induces human beta cell proliferation, expands beta cell mass, enhances expression of beta cell phenotypic genes, and improves human beta cell function i n vitro and in vivo . It is unknown whether the "pro-differentiation effect" is a DYRK1A inhibitor class-wide effect. Here we compare multiple commonly studied DYRK1A inhibitors. Harmine, 2-2c and 5-IT increase expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC2A2, SLC30A8, ENTPD3 in normal and T2D human islets. Unexpectedly, GNF4877, CC-401, INDY, CC-401 and Leucettine fail to induce expression of these essential beta cell molecules. Remarkably, the pro-differentiation effect is independent of DYRK1A inhibition: although silencing DYRK1A induces human beta cell proliferation, it has no effect on differentiation; conversely, harmine treatment enhances beta cell differentiation in DYRK1A-silenced islets. A careful screen of multiple DYRK1A inhibitor kinase candidate targets was unable to identify pro-differentiation pathways. Overall, harmine, 2-2c and 5-IT are unique among DYRK1A inhibitors in their ability to enhance both beta cell proliferation and differentiation. While beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor pharmaceutical development.
RESUMO
Hyperlysinemia is a rare autosomal recessive deficiency of 2-aminoadipic semialdehyde synthase (AASS) affecting the initial step in lysine degradation. It is thought to be a benign biochemical abnormality, but reports on cases remain scarce. The description of additional cases, in particular, those identified without ascertainment bias, may help counseling of new cases in the future. It may also help to establish the risks associated with pharmacological inhibition of AASS, a potential therapeutic strategy that is under investigation for other inborn errors of lysine degradation. We describe the identification of a hyperlysinemia case identified in the Provincial Neonatal Urine Screening Program in Sherbrooke, Quebec. This case presented with a profile of cystinuria but with a very high increase in urinary lysine. A diagnosis of hyperlysinemia was confirmed through biochemical testing and the identification of biallelic variants in AASS. The p.R146W and p.T371I variants are novel and affect the folding of the lysine-2-oxoglutarate domain of AASS. The 11-month-old boy is currently doing well without any therapeutic interventions. The identification of this case through newborn urine screening further establishes that hyperlysinemia is a biochemical abnormality with limited clinical consequences and may not require any intervention.