Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Protective Potential of a Botanical-Based Supplement Ingredient against the Impact of Environmental Pollution on Cutaneous and Cardiopulmonary Systems: Preclinical Study.

Air pollution is a growing threat to human health. Airborne pollution effects on respiratory, cardiovascular and skin health are well-established. The main mechanisms of air-pollution-induced health effects involve oxidative stress and inflammation. The present study evaluates the potential of a polyphenol-enriched food supplement ingredient comprising Lippia citriodora, Olea europaea, Rosmarinus officinalis, and Sophora japonica extracts in mitigating the adverse effects of environmental pollution on skin and cardiopulmonary systems. Both in vitro and ex vivo studies were used to assess the blend's effects against pollution-induced damage. In these studies, the botanical blend was found to reduce lipid peroxidation, inflammation (by reducing IL-1α), and metabolic alterations (by regulating MT-1H, AhR, and Nrf2 expression) in human skin explants exposed to a mixture of pollutants. Similar results were also observed in keratinocytes exposed to urban dust. Moreover, the ingredient significantly reduced pollutant-induced ROS production in human endothelial cells and lung fibroblasts, while downregulating the expression of apoptotic genes (bcl-2 and bax) in lung fibroblasts. Additionally, the blend counteracted the effect of urban dust on the heart rate in zebrafish embryos. These results support the potential use of this supplement as an adjuvant method to reduce the impact of environmental pollution on the skin, lungs, and cardiovascular tissues.

Telomerase Reverse Transcriptase-Promoter Mutation in Young Patients with Bladder Tumors.

The TERT (Telomerase Reverse Transcriptase) gene promoter mutation is one of the most prevalent mutations in urothelial bladder tumors and this mutation is related to bladder tumor progression. Our purpose was to evaluate the presence of this mutation in a population of patients who were first diagnosed at age ≤ 40 years and to examine its relationship with tumor characteristics and progression. A molecular study was performed to detect the two most prevalent mutations in the TERT promoter (C228T and C250T). The study included 29 patients, with a mean follow-up of 152 months. There were no statistically significant differences in the clinical or tumor characteristics according to the presence or absence of the mutation. Although the mutation group showed poorer recurrence-free survival (RFS), there was no statistically significant difference and there was no difference in progression-free survival by group (p > 0.05). The pTERT mutations in bladder tumor cells occurred less frequently in younger patients than in older patients, a finding that could indicate different mechanisms of carcinogenesis. The trend towards lower RFS in patients with mutated pTERT needs to be confirmed by further studies, given the small number of patients included in these studies due to the low incidence of bladder tumors in this age group.

Human fecal alpha-glucosidase activity and its relationship with gut microbiota profiles and early stages of intestinal mucosa damage.

OBJECTIVES: We investigated potential relationships among initial lesions of the intestinal mucosa, fecal enzymatic activities and microbiota profiles. METHODS: Fecal samples from 54 volunteers were collected after recruitment among individuals participating in a colorectal cancer (CRC) screening program in our region (Northern Spain) or attending for consultation due to clinical symptoms; intestinal mucosa samples were resected during colonoscopy. Enzymatic activities were determined in fecal supernatants by a semi-quantitative method. The fecal microbiota composition was determined by 16S rRNA gene-based sequencing. The results were compared between samples from clinical diagnosis groups (controls and polyps), according with the type of polyp (hyperplastic polyps or conventional adenomas) and considering the grade of dysplasia for conventional adenomas (low and high grade dysplasia). RESULTS: High levels of α-glucosidase activity were more frequent among samples from individuals diagnosed with intestinal polyps, reaching statistical significance for conventional adenomas and for low grade dysplasia adenomas when compared to controls. Regarding the microbiota profiles, higher abundance of Christensenellaceae_R-7 group and Oscillospiraceae_UCG-002 were found in fecal samples displaying low α-glucosidase activity as compared with those with higher activity as well as in controls with respect to conventional adenomas. A relationship was evidenced among intestinal mucosal lesions, gut glucosidase activities and intestinal microbiota profiles. CONCLUSIONS: Our findings suggest a relationship among altered fecal α-glucosidase levels, the presence of intestinal mucosal lesions, which can be precursors of CRC, and shifts in defined microbial groups of the fecal microbiota.

Randomized phase II study of weekly carfilzomib 70 mg/m2 and dexamethasone with or without cyclophosphamide in relapsed and/or refractory multiple myeloma patients.

In this randomized phase II study (GEM-KyCyDex, clinicaltrials gov. Identifier: NCT03336073), the combination of weekly carfilzomib 70 mg/m2, cyclophosphamide and dexamethasone (KCd) was compared to carfilzomib and dexamethasone (Kd) in relapsed/refractory multiple myeloma (RRMM) after 1-3 prior lines (PL). One hundred and ninety-seven patients were included and randomized 1:1 to receive KCd (97 patients) or Kd (100 patients) in 28-day cycles until progressive disease or unacceptable toxicity occurred. Patient median age was 70 years, and the median number of PL was one (range, 1-3). More than 90% of patients had previously been exposed to proteasome inhibitors, approximetely 70% to immunomodulators, and approximetely 50% were refractory to their last line (mainly lenalidomide) in both groups. After a median follow-up of 37 months, median progression-free survival (PFS) was 19.1 and 16.6 months in KCd and Kd, respectively (P=0.577). Of note, in the post hoc analysis of the lenalidomide-refractory population, the addition of cyclophosphamide to Kd resulted in a significant benefit in terms of PFS: 18.4 versus 11.3 months (hazard ratio =1.7, 95% confidence interval: 1.1-2.7; P=0.043). The overall response rate and the percentage of patients who achieved complete response was around 70% and 20% in both groups. The addition of cyclophosphamide to Kd did not result in any safety signal, except for severe infections (7% vs. 2%). In conclusion, the combination of cyclophosphamide with Kd 70 mg/m2 weekly does not improve outcomes as compared with Kd alone in RRMM after 1-3 PL, but a significant benefit in PFS was observed with the triplet combination in the lenalidomide-refractory population. The administration of weekly carfilzomib 70 mg/m2 was safe and convenient, and, overall, the toxicity was manageable in both arms.

Small Molecule Pytren-4QMn Metal Complex Slows down Huntington's Disease Progression in Male zQ175 Transgenic Mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder considered a rare disease with a prevalence of 5.7 per 100,000 people. It is caused by an autosomal dominant mutation consisting of expansions of trinucleotide repeats that translate into poly-glutamine enlarged mutant huntingtin proteins (mHTT), which are particularly deleterious in brain tissues. Since there is no cure for this progressive fatal disease, searches for new therapeutic approaches are much needed. The small molecule pytren-4QMn (4QMn), a highly water-soluble mimic of the enzyme superoxide dismutase, has shown in vivo beneficial anti-inflammatory activity in mice and was able to remove mHTT deposits in a C. elegans model of HD. In this study, we assessed 4QMn therapeutic potential in zQ175 neo-deleted knock-in mice, a model of HD that closely mimics the heterozygosity, genetic injury, and progressive nature of the human disease. We provide evidence that 4QMn has good acute and chronic tolerability, and can cross the blood-brain barrier, and in male, but not female, zQ175 mice moderately ameliorate HD-altered gene expression, mHtt aggregation, and HD disease phenotype. Our data highlight the importance of considering sex-specific differences when testing new therapies using animal models and postulate 4QMn as a potential novel type of small water-soluble metal complex that could be worth further investigating for its therapeutic potential in HD, as well as in other polyglutamine diseases.

Immunometabolic Profile Associated with Progressive Damage of the Intestinal Mucosa in Adults Screened for Colorectal Cancer: Association with Diet.

Environmental factors such as diet and lifestyle have been shown to influence the development of some intestinal mucosal lesions that may be precursors of colorectal cancer (CRC). The presence of these alterations seems to be associated with misbalanced immunological parameter levels. However, it is still unclear as to which immunological parameters are altered in each phase of CRC development. In this work, we aimed to study the potential relationships of immunological and metabolic parameters with diet in a CRC-related lesion context. Dietary information was obtained using an annual semi-quantitative food-frequency questionnaire (FFQ) from 93 volunteers classified via colonoscopy examination according to the presence of intestinal polyps or adenocarcinoma. Cytokines, chemokines, and adipokines were determined from serum samples. We observed a reduction in adiponectin according to the damage to the mucosa, accompanied by an increase and decrease in C-X-C motif chemokine ligand 10 (CXCL10) and resistin, respectively, in CRC cases. The presence of aberrant crypt foci (ACF) in the polyp group was associated with higher tumor necrosis factor-alpha (TNF-α) concentrations. Vegetables were directly correlated with adiponectin and resistin levels, while the opposite occurred with red meat. A bioactive compound, soluble pectin, showed a negative association with TNF-α. Future dietary strategies could be developed to modulate specific immunological parameters in the context of CRC.

Maternal Diet Shapes the Breast Milk Microbiota Composition and Diversity: Impact of Mode of Delivery and Antibiotic Exposure.

BACKGROUND: Breast milk is a complex biofluid that provides nutrients and bioactive agents, including bacteria, for the development of the infant gut microbiota. However, the impact of maternal diet and other factors, such as mode of delivery and antibiotic exposure, on the breast milk microbiota has yet to be understood. OBJECTIVES: This study aimed to examine the association between maternal diet and breast milk microbiota and to ascertain the potential role of mode of delivery and antibiotic exposure. METHODS: In a cross-sectional study of the MAMI cohort, breast milk microbiota profiling was assessed in 120 samples from healthy mothers by 16S rRNA gene sequencing. Maternal dietary information was recorded through an FFQ, and clinical characteristics, including mode of delivery, antibiotic exposure, and exclusive breastfeeding, were collected. RESULTS: Maternal diet was grouped into 2 clusters: Cluster I (high intake of plant protein, fiber, and carbohydrates), and Cluster II (high intake of animal protein and lipids). Breast milk microbiota was shaped by maternal dietary clusters. Staphylococcus and Bifidobacterium were associated with carbohydrate intake whereas the Streptococcus genus was associated with intakes of the n-3 PUFAs [EPA and docosapentaenoic acid (22:5ω-3)]. Mode of delivery and antibiotic exposure influenced breast milk microbiota in a diet cluster-dependent manner. Differences between/among the maternal dietary clusters were found in the milk microbiota of the cesarean-section (C-section)/antibiotic group, whereas no differences were observed in vaginal births. Lower abundances of Lactobacillus, Bacteroides, and Sediminibacterium genera were observed in Cluster II/C-section/antibiotic exposure compared with the other groups. CONCLUSIONS: Maternal diet shapes the composition and diversity of breast milk microbiota, with the most important contributions coming from dietary fiber and both plant and animal protein intakes. The relation between the maternal diet and the milk microbiota needs further research because it has a key impact on infant microbiota development and contributes to infant health outcomes in the short and long term.This trial was registered at clinicaltrials.gov as NCT03552939.

New players in the relationship between diet and microbiota: the role of macromolecular antioxidant polyphenols.

PURPOSE: Solid evidence has emerged supporting the role of polyphenols and fibers as gut microbiota modulators. These studies have been limited to the data available in food composition databases, which did not include the food content of non-extractable polyphenols (NEPP). The main objective of this work is to quantify the intake of the different types of dietary polyphenols including NEPP and to evaluate their impact on the composition and activity of the intestinal microbiota. METHODS: Cross-sectional descriptive study conducted on a sample of 147 adults with no declared pathologies. Dietary intake has been registered by a semi-quantitative Food Frequency Questionnaire (FFQ) and transformed into extractable (EPP) and NEPP, and dietary fibers based on available databases. Major phylogenetic types of the intestinal microbiota were determined by qPCR and fecal SCFA quantification was performed by gas chromatography. RESULTS: NEPP account for two-thirds of the total polyphenols intake. A combined analysis by stepwise regression model including all dietary fiber and (poly)phenols has identified hydrolysable (poly)phenol (HPP) intake, as the best predictor of Bacteroides-Prevotella-Porphyromonas group and Bifidobacterium levels in feces. Also, HPPs were positively associated with butyric acid, while insoluble fiber was identified as a predictor of propionic acid in feces. CONCLUSION: The intake of macromolecular (poly)phenols could contribute to modulate the gut microbiota by increasing the levels of certain intestinal microorganisms with proven health benefits.

Association of Maternal Microbiota and Diet in Cord Blood Cytokine and Immunoglobulin Profiles.

Mothers confer natural passive immunization to their infants through the transplacental pathway during the gestation period. The objective of the present study was to establish at birth the maternal and cord plasma concentration and relationship of immunoglobulins (Igs), cytokines (CKs), and adipokines. In addition, the impact of the maternal microbiota and diet was explored. The plasma profile of these components was different between mothers and babies, with the levels of many CKs, IgM, IgG2a, IgE, IgA, and leptin significantly higher in mothers than in the cord sample. Moreover, the total Igs, all IgG subtypes, IgE, and the Th1/Th2 ratio positively correlated in the mother-infant pair. Maternal dietary components such as monounsaturated fatty acids-polyunsaturated fatty acids and fiber were positively associated with some immune factors such as IgA in cord samples. The microbiota composition clustering also influenced the plasma profile of some factors (i.e., many CKs, some Ig, and adiponectin). In conclusion, we have established the concentration of these immunomodulatory factors in the maternal-neonatal pair at birth, some positive associations, and the influence of maternal diet and the microbiota composition, suggesting that the immune status during pregnancy, in terms of CKs and Igs levels, can influence the immune status of the infant at birth.

Microbiome: Effects of Ageing and Diet.

The microbial community inhabiting our intestine, known as 'microbiota', and the ensemble of their genomes (microbiome) regulate important functions of the host, being essential for health maintenance. The recent development of next-generation sequencing (NGS) methods has greatly facilitated the study of the microbiota and has contributed to evidence of the strong influence exerted by age and diet. However, the precise way in which the diet and its components modify the functionality of the intestinal microbiome is far from being completely known. Changes in the intestinal microbiota occur during ageing, frequently accompanied by physiological changes of the digestive tract, modification of dietary patterns and impairment of the immune system. Establishing nutritional strategies aiming to counterbalance the specific alterations taking place in the microbiota during ageing would contribute to improved health status in the elderly. This review will analyse changes appearing in the intestinal microbiota from adulthood to old age and their association with dietary patterns and lifestyle factors.

Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation.

Crimean-Congo haemorrhagic fever (CCHF) virus-specific antibody detection in blood donors, Castile-León, Spain, 2017 and 2018.

BackgroundCrimean-Congo haemorrhagic fever virus (CCHFV) is considered an emerging or even a probable re-emerging pathogen in southern Europe. Presence of this virus had been reported previously in Spain in 2010.AimWe aimed to evaluate the potential circulation of CCHFV in western Spain with a serosurvey in asymptomatic adults (blood donors).MethodsDuring 2017 and 2018, we conducted a CCHFV serosurvey in randomly selected asymptomatic blood donors from western Spain. Three assays using specific IgG antibodies against CCHFV were performed: the VectoCrimea ELISA test, an in-house ELISA and indirect immunofluorescence (EuroImmun) test with glycoprotein and nucleoprotein.ResultsA total of 516 blood donors participated in this cross-sectional study. The majority of the study participants were male (68.4%), and the mean age was 46.3 years. Most of the participants came from rural areas (86.8%) and 68.6% had contact with animals and 20.9% had animal husbandry practices. One in five participants (109/516, 21.1%) were engaged in at-risk professional activities such as agriculture and shepherding, slaughtering, hunting, veterinary and healthcare work (mainly nursing staff and laboratory technicians). A total of 15.3% of the participants were bitten by ticks in the days or months before the date of sampling. We detected anti-CCHFV IgG antibodies with two diagnostic assays in three of the 516 individuals and with one diagnostic assay in six of the 516 individuals.ConclusionSeroprevalence of CCHFV was between 0.58% and 1.16% in Castile-León, Spain. This is the first study in western Spain that showed circulation of CCHFV in healthy people.

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production.

Xenobiotics Formed during Food Processing: Their Relation with the Intestinal Microbiota and Colorectal Cancer.

The colonic epithelium is exposed to a mixture of compounds through diet, among which some are procarcinogens, whereas others have a protective effect. Therefore, the net impact of these compounds on human health depends on the overall balance between all factors involved. Strong scientific evidence has demonstrated the relationship between nitrosamines (NA), heterocyclic amines (HCAs), and polycyclic aromatic hydrocarbons (PAHs), which are the major genotoxins derived from cooking and food processing, and cancer. The mechanisms of the relationship between dietary toxic xenobiotics and cancer risk are not yet well understood, but it has been suggested that differences in dietary habits affect the colonic environment by increasing or decreasing the exposure to mutagens directly and indirectly through changes in the composition and activity of the gut microbiota. Several changes in the proportions of specific microbial groups have been proposed as risk factors for the development of neoplastic lesions and the enrichment of enterotoxigenic microbial strains in stool. In addition, changes in the gut microbiota composition and activity promoted by diet may modify the faecal genotoxicity/cytotoxicity, which can be associated with a higher or lower risk of developing cancer. Therefore, the interaction between dietary components and intestinal bacteria may be a modifiable factor for the development of colorectal cancer in humans and deserves more attention in the near future.

Bioactive compounds from regular diet and faecal microbial metabolites.

PURPOSE: Short-chain fatty acids (SCFAs) formation by intestinal bacteria is regulated by many different factors, among which dietary fibre is currently receiving most attention. However, since fibre-rich foods are usually good dietary sources of phenolic compounds, which are also known to affect the microbiota, authors hypothesize that the regular intake of these bioactive compounds could be associated with a modulation of faecal SCFA production by the intestinal microbiota. METHODS: In this work, food intake was recorded by means of a validated Food Frequency Questionnaire. Fibres were determined using Marlett food composition tables, and phenolic compounds were obtained from Phenol-Explorer Database. Analysis of SCFA was performed by gas chromatography-flame ionization/mass spectrometry and quantification of microbial populations in faeces by quantitative PCR. RESULTS: Klason lignin and its food contributors, as predictors of faecal butyrate production, were directly associated with Bacteroides and Bifidobacterium levels, as well as lignans with Bacteroides. Also, anthocyanidins, provided by strawberries, were associated with faecal propionate and inversely related to Lactobacillus group. CONCLUSIONS: These results support the hypothesis we put forward regarding the association between some vegetable foods (strawberries, pasta, lentils, lettuce and olive oil) and faecal SCFA. More studies are needed in order to elucidate whether these associations have been mediated by the bacterial modulatory effect of the bioactive compounds, anthocyanins, lignans or Klason lignin, present in foodstuffs.

[Spanish validation of Game Addiction Scale for Adolescents (GASA)]. / Validación española de la Escala de Adicción a Videojuegos para Adolescentes (GASA).

OBJECTIVE: The aim of this study is to adapt and validate the Game Addiction Scale for Adolescents (GASA) to the Spanish youth population. DESIGN: Cultural adaptation and validation study. SETTING: Secondary Education centres. PARTICIPANTS: Two independent studies were conducted on a group of 466 young people with a mean age of 15.27 years (13-18, SD: 1.83) and 48.7% ♀ and on another group of 566, with a mean age of 21.24 years (19-26; SD: 1.86) 44.1% ♀. MEASUREMENTS: Addiction to video games (GASA); Game behavior (Game habits usage questionnaire), Impulsiveness (Plutchik Impulsiveness Scale) and Group Pressure (Ad hoc questionnaire). RESULTS: The Spanish version of GASA has shown good reliability and true to the original scale factor structure. As regards criterion validity, GASA scores are significantly different according to four criteria related to problem gambling: Game intensity and frequency, impulsiveness, and peer pressure. CONCLUSIONS: The results show that the adapted version GASA is adequate and a valid tool for assessing problematic gaming behaviour.

Group Selection and Contribution of Minority Variants during Virus Adaptation Determines Virus Fitness and Phenotype.

Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.

Generation of tricyclic imidazo[1,2-a]pyrazines as novel PI3K inhibitors by application of a conformational restriction strategy.

The involvement of the phosphoinositide 3-kinases (PI3Ks) in several diseases, especially in the oncology area, has singled it as one of the most explored therapeutic targets in the last two decades. Many different inhibitor classes have been developed by the industry and academia with a diverse selectivity profile within the PI3K family. In the present manuscript we report a further exploration of our lead PI3K inhibitor ETP-46321 (Martínez González et al., 2012)1 by the application of a conformational restriction strategy. For that purpose we have successfully synthesized novel tricyclic imidazo[1,2-a]pyrazine derivatives as PI3K inhibitors. This new class of compounds had enable the exploration of the solvent-accessible region within PI3K and resulted in the identification of molecule 8q with the best selectivity PI3Kα/δ isoform profile in vitro, and promising in vivo PK data.

Identification of novel PI3K inhibitors through a scaffold hopping strategy.

A scaffold hopping strategy, including intellectual property availability assessment, was successfully applied for the discovery of novel PI3K inhibitors. Compounds were designed based on the chemical structure of the lead compound ETP-46321, a potent PI3K inhibitor, previously reported by our group. The new generated compounds showed good in vitro potency and selectivity, proved to inhibit potently the phosphorylation of AKTSer473 in cells and demonstrated to be orally bioavailable, thus becoming potential back-up candidates for ETP-46321.