Crystal structure of active CDK4-cyclin D and mechanistic basis for abemaciclib efficacy.

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.

Normalization of DNA encoded library affinity selection results driven by high throughput sequencing and HPLC purification.

The output of an affinity selection screening results in a huge amount of valuable data that, after conducting the appropriate analysis, lead to the correct identification of the compounds enriched in the target of interest. The approach chosen to perform these analyses has become a key step in the development of a successful DNA Encoded Library platform. In this paper, we describe the combination of High Performance Liquid Chromatography purification during the library production with the Next Generation Sequencing analysis of the libraries to assess the yield of the chemical reactions prior to the affinity selection. This process allows us, apart from achieving higher quality libraries, to enable a normalization analysis of the affinity selection output, thus minimizing the bias induced by the chemical yield of each reaction as a misleading factor within the analysis and subsequent compound short-listing for off-DNA synthesis.

The Angiopoietin-Like Protein 3 and 8 Complex Interacts with Lipoprotein Lipase and Induces LPL Cleavage.

Lipoprotein lipase (LPL) is the key enzyme that hydrolyzes triglycerides from triglyceride-rich lipoproteins. Angiopoietin-like proteins (ANGPTL) 3, 4, and 8 are well-characterized protein inhibitors of LPL. ANGPTL8 forms a complex with ANGPTL3, and the complex is a potent endogenous inhibitor of LPL. However, the nature of the structural interaction between ANGPTL3/8 and LPL is unknown. To probe the conformational changes in LPL induced by ANGPTL3/8, we found that HDX-MS detected significantly altered deuteration in the lid region, ApoC2 binding site, and furin cleavage region of LPL in the presence of ANGPTL3/8. Supporting this HDX structural evidence, we found that ANGPTL3/8 inhibits LPL enzymatic activities and increases LPL cleavage. ANGPTL3/8-induced effects on LPL activity and LPL cleavage are much stronger than those of ANGPTL3 or ANGPTL8 alone. ANGPTL3/8-mediated LPL cleavage is blocked by both an ANGPTL3 antibody and a furin inhibitor. Knock-down of furin expression by siRNA significantly reduced ANGPT3/8-induced cleavage of LPL. Our data suggest ANGPTL3/8 promotes furin-mediated LPL cleavage.

A Decoupled Automation Platform for Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein-ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a "real-time" serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.

Median filter algorithm for estimating the threshold of detection on custom protein arrays.

We constructed protein arrays according to a titration design to estimate the assay sensitivities over varying concentrations of flu vaccine and human immunoglobulin G (IgG). After imaging, we considered the problem of appropriately distinguishing background noise from foreground signal. We applied the median filter smoothing technique and estimated the differences of the observed signal compared to the smoothed signal. If the absolute value of the difference was large, the feature was easily detectable, indicating that the spot did not blend with its surrounding neighbors. After estimating the residuals, we applied thresholding algorithms to estimate the limits of detection for each assay. At sufficiently large smoothing spans, our median filter approach performed as well or better than visual inspection and two other competing analysis methods. This suggests that a median filter approach has utility in high-throughput arrays where visual inspection is impractical.

Solution structure of the C-terminal domain of Ku80 suggests important sites for protein-protein interactions.

The solution structure of Ku80 CTD from residue 566 to 732 has been solved in order to gain insights into the mechanisms of its interactions with other proteins. The structure reveals a topology similar to several common scaffolds for protein-protein interactions, in the absence of significant sequence similarity to these proteins. Conserved surface amino acid residues are clustered on two main surface areas, which are likely involved in mediating interactions between Ku80 and other proteins. The Ku70/Ku80 heterodimer has been shown to be involved in at least three processes, nonhomologous end joining, transcription, and telomere maintenance, and thus it needs to interact with different proteins involved in these different processes. The three-dimensional structure of the Ku80 C-terminal domain and the availability of NMR chemical shift assignments provide a basis for further investigation of the interactions between Ku80 and other proteins in these Ku-dependent cellular functions.

Synthesis, anticholinesterase activity and structure-activity relationships of m-Aminobenzoic acid derivatives.

The synthesis, acetylcholinesterase inhibitory capacity and structure-activity relationships of simple-structured m-Aminobenzoic acid derivatives are reported. Compound 1b was found to be more potent than galanthamine and tacrine in inhibiting acetylcholinesterase.

Utilidad de la carga bucal con calcio en la litiasis renal / Usefulness of the oral calcium test in renal lithiasis

Se describen los resultados de la prueba bucal con calcio efectuada en 317 pacientes con litiasis renal, única o múltiple. Se midió el umbral de reabsorción tubular máxima de fosfatos (TmP04/DCr, umbral de fosfatos), en vez de cuantificar PTH o AMPc, para simplificar la prueba y reducir los costos del estudio. La prueba consistió en colectar la orina de los pacientes de 7 a 9 y de 9 a 13h y proporcionar a las 9h 1g de calcio mezclado con los alimentos. Se cuantificaron las concentraciones de calcio, fósforo y creatinina en sangre y en orina y se calcularon los índices: calciuria en ayunas, calciurina postcarga de calcio, calcemia y TmP04/DCr. Se identificaron 97 pacientes sin trastorno en el metabolismo del calcio como causa de litiasis. De estos, 183 (57 por ciento) cursaron con hipercalciuria idiopática y se clasificaron en cuatro subgrupos: hipercalciuria absortiva normofosfatémica, hipercalciuria absortiva hipofosfatémica (por fuga renal de fosfatos), hipercalciuria renal normofosfatémica e hipercalciuria renal hipofosfatémica. Por último se identificaron 37 sujetos con hiperatiroidismo primario

Efectos del salvado de trigo sobre el metabolismo de calcio y fósforo / Wheat bran effects on calcium and phosphorus metabolism

Con objeto de investigar si la administración de salvado de trigo altera la absorción intestinal de calcio, fósforo, sodio y potasio, se estudiaron cinco individuos con diabetes no complicada. Recibieron una dieta fija durante dos periodos consecutivos de ocho días de duración; el primero fue el periodo testigo y en el periodo siguiente se agregaron a los alimentos 90 g de salvado de trigo diariamente. Se determinaron todos los días las cifras séricas y la eliminación urinaria de los electrólitos y la creatinina. Con la administración de salvado se observó un aumento no significativo en la eliminación urinaria de calcio; la FeCa fue significativamente mayor, aumentaron la eliminarión urinaria de fósforo y de sodio la FeP y la FeNa, mientras que la eliminación urinaria de potasio y la FeK disminuyeron (P < 0.05), y los niveles séricos no se modificaron. Los resultados se atribuyeron a un mayor aporte de fósforo y calcio contenidos en el salvado y a disminución en la absorción de potasio. A diferencia de lo informado por otros autores, no se comprobó que la administración de salvado de trigo disminuya la absorción intestinal de calcio

Participación de factores mecánicos y renohumorales en la elevación de la tensión arterial en pacientes con coartación de la aorta / Mechanical and renohumoral factors participating in arterial pressure rising in patients with aortic coarctation

Se estudian siete pacientes con coartación aórta bajo cuatro situaciones diferentes para valorar la participación de los factores mecánico y renohumoral en la hipertensión de la coartación: 1) control, 2) tratamiento con propranolol, 3) tratamiento con captopril y 4) periodo postoperatorio. La tensión arterial humoral media disminuyó de 108 + 11 mmHg en el período de control a 95 ñ 13 mmHg con propranolol, 91 ñ 11 mmHg con captopril y 85 ñ 10 en periodo postoperatorio. La disminución fue significativamente mayor con captopril que con propranolol (p < 0.01), y también con la correción quirúrgica de la coartación que con cualesquiera de los dos fármacos (p < 0.01). La actividad de renina plasmática y la frecuencia cardiaca aumentaron en el periodo postoperatorio con respecto al período de control de 5.23 ñ 3.5 a 12.000 ñ 3.24 ng/ml/h (p < 0.05) y, 84 ñ 9 a 93 ñ 10 lpm (p < 0.001), respectivamente. Estos datos pueden explicarse por resituación de los barorreceptores. El sistema de renina y angiotensina participa en la patogénesis de la hipertensión de la coartación aórtica, aunque no es el único factor