Prominent Therapeutic Target Research Articles

Natural Compound-Rhein and PROTACs Unleash Potent VEGFR-2 Degraders.

VEGFR-2 is a prominent therapeutic target in antitumor drug research to block tumor angiogenesis. This study focused on the synthesis and optimization of PROTACs based on the natural product rhein, resulting in the successful synthesis of 15 distinct molecules. In A549 cells, D9 exhibited remarkable antitumor efficacy with an IC50 of 5.88±0.50 μM, which was 15-fold higher compared to rhein (IC50=88.45±2.77 μM). An in-depth study of the effect of D9 on the degradation of VEGFR-2 revealed that D9 was able to induce the degradation of VEGFR-2 in A549 cells in a time-dependent manner. The observed effect was reversible, contingent upon the proteasome and ubiquitination system, and demonstrably linked to CRBN. Further experiments revealed that D9 induced apoptosis in A549 cells and led to cell cycle arrest in the G1 phase. Molecular docking simulations validated the binding mode of D9 to VEGFR, establishing the potential of D9 to bind to VEGFR-2 in its natural state. In summary, this study confirms the feasibility of natural product-bound PROTAC technology for the development of a new generation of VEGFR-2 degraders, offering a novel trajectory for the future development of pharmacological agents targeting VEGFR-2.

From Deep Learning to the Discovery of Promising VEGFR-2 Inhibitors.

Vascular endothelial growth factor receptor 2 (VEGFR-2) stands as a prominent therapeutic target in oncology, playing a critical role in angiogenesis, tumor growth, and metastasis. FDA-approved VEGFR-2 inhibitors are associated with diverse side effects. Thus, finding novel and more effective inhibitors is of utmost importance. In this study, a deep learning (DL) classification model was first developed and then employed to select putative active VEGFR-2 inhibitors from an in-house chemical library including 187 druglike compounds. A pool of 18 promising candidates was shortlisted and screened against VEGFR-2 by using molecular docking. Finally, two compounds, RHE-334 and EA-11, were prioritized as promising VEGFR-2 inhibitors by employing PLATO, our target fishing and bioactivity prediction platform. Based on this rationale, we prepared RHE-334 and EA-11 and successfully tested their anti-proliferative potential against MCF-7 human breast cancer cells with IC50 values of 26.78±4.02 and 38.73±3.84 μM, respectively. Their toxicities were instead challenged against the WI-38. Interestingly, expression studies indicated that, in the presence of RHE-334, VEGFR-2 was equal to 0.52±0.03, thus comparable to imatinib equal to 0.63±0.03. In conclusion, this workflow based on theoretical and experimental approaches demonstrates effective in identifying VEGFR-2 inhibitors and can be easily adapted to other medicinal chemistry goals.

Post-transcriptional Regulation of Gene Expression via Unproductive Splicing.

Unproductive splicing is a mechanism of post-transcriptional gene expression control in which premature stop codons are inserted into protein-coding transcripts as a result of regulated alternative splicing, leading to their degradation via the nonsense-mediated decay pathway. This mechanism is especially characteristic of RNA-binding proteins, which regulate each other's expression levels and those of other genes in multiple auto- and cross-regulatory loops. Deregulation of unproductive splicing is a cause of serious human diseases, including cancers, and is increasingly being considered as a prominent therapeutic target. This review discusses the types of unproductive splicing events, the mechanisms of auto- and cross-regulation, nonsense-mediated decay escape, and problems in identifying unproductive splice isoforms. It also provides examples of deregulation of unproductive splicing in human diseases and discusses therapeutic strategies for its correction using antisense oligonucleotides and small molecules.

Exploring the Synergistic Effect of Sildenafil and Green Tea Polyphenols on Breast Cancer Stem Cell-like Cells and their Parental Cells: A Potential Novel Therapeutic Approach.

Many cancer studies have intensely focused on the role of diet, among other factors involved in cancer establishment. The positive effect of green tea polyphenols (GTP) on controlling breast cancer cells has been reported in several studies. Cancer stem cell-like cells (CSC-LCs) possessing self-renewal, metastatic, and drug-resistant capacities are considered prominent therapeutic targets. In many tumors, inducible nitric oxide synthase (iNOS) expression levels are high; however, they have a dual effect on breast cancer pathogenesis. This study aimed to investigate the cytotoxicity of the iNOS agonist (Sildenafil) and antagonist (LNAME), both alone and in combination with GTP, on MDA-MB-231, CD44+/CD24- CSC-LCs, and their parental cells (MCF-7). The cell viability assay has been studied using the MTT assay. To analyze drug-drug combinations, CompuSyn and Combenefit software were used. The cytotoxicity mechanism was determined using flow cytometric analysis. L-NAME and GTP showed a synergistic effect on MDA-MB-231 and CSC-LCs. Such an effect was not observed on MCF-7. Sildenafil and GTP, on the other hand, showed synergistic cytotoxicity in all the cells mentioned above. Flow cytometric tests resulted in more than 70% apoptosis in MDA-MB-231 and MCF-7. Also, sub-G1 arrest among MCF-7 cells and a considerable decrease in ROS production by MDA-MB-231 cells following treatment with Sildenafil and GTP were observed. Sildenafil, in combination with flavonoids, may be considered a novel strategy for cancer treatment.

A Novel Time-Resolved Fluorescence Resonance Energy Transfer Assay for the Discovery of Small-Molecule Inhibitors of HIV-1 Tat-Regulated Transcription.

Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.

Development of machine learning models for the screening of potential HSP90 inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone playing a significant role in the folding of client proteins. This cellular protein is linked to the progression of several cancer types, including breast cancer, lung cancer, and gastrointestinal stromal tumors. Several oncogenic kinases are Hsp90 clients and their activity depends on this molecular chaperone. This makes HSP90 a prominent therapeutic target for cancer treatment. Studies have confirmed the inhibition of HSP90 as a striking therapeutic treatment for cancer management. In this study, we have utilized machine learning and different in silico approaches to screen the KCB database to identify the potential HSP90 inhibitors. Further evaluation of these inhibitors on various cancer cell lines showed favorable inhibitory activity. These inhibitors could serve as a basis for future development of effective HSP90 inhibitors.

Sympathetic activation in heart failure.

Sympathetic activation has been long appreciated exclusively as a fundamental compensatory mechanism of the failing heart and, thus, welcome and to be supported. In the initial clinical phases of heart failure (HF), the sympathetic nervous system overdrive plays a compensatory function aimed at maintaining an adequate cardiac output despite the inotropic dysfunction affecting the myocardium. However, when the sympathetic reflex response is exaggerated it triggers a sequence of unfavourable remodelling processes causing a further contractile deterioration that unleashes major adverse cardiovascular consequences, favouring the HF progression and the occurrence of fatal events. Eventually, the sympathetic nervous system in HF was demonstrated to be a ‘lethality factor’ and thus became a prominent therapeutic target. The existence of an effective highly specialized intracardiac neuronal network immediately rules out the old concept that sympathetic activation in HF is merely the consequence of a drop in cardiac output. When a cardiac damage occurs, such as myocardial ischaemia or a primary myocardial disorder, the adaptive capability of the system may be overcame, leading to excessive sympatho-excitation coupled with attenuation till to abolishment of central parasympathetic drive. Myocardial infarction causes, within a very short time, both a functional and anatomical remodelling with a diffuse up-regulation of nerve growth factor (NGF). The subsequent nerve sprouting signal, facilitated by a rise in the levels of NGF in the left stellate ganglion and in the serum, triggers an increase in cardiac nerve density in both peri-infarct and non-infarcted areas. Finally, NFG production decreases over time, supposedly as an adaptative response to the prolonged exposure to sympathetic overactivity, leading in the end to a reduction in sympathetic nerve density. Accordingly, NGF levels were markedly reduced in patients with severe congestive heart failure. The kidney is the other key player of the sympathetic response to HF as it indeed reacts to under-perfusion and to loop diuretics to preserve filtration at the cost of many pathological consequences on its physiology. This vicious loop ultimately participates to the chronic and disruptive sympathetic overdrive.In conclusion, sympathetic activation is the natural physiological consequence to life stressors but also to any condition that may harm our body. It is the first system of reaction to any potential life-threatening event. However, in any aspect of life over reaction is never effective but, in many instances, is, actually, life threatening. One for all is the case of ischaemia-related ventricular fibrillation which is, strongly facilitated by sympathetic hyperactivity. The take home message? When, in a condition of harm, everybody is yelling failure is just around the corner.

Polarization of Microglia and Its Therapeutic Potential in Sepsis.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection, leaving the inflammation process without a proper resolution, leading to tissue damage and possibly sequelae. The central nervous system (CNS) is one of the first regions affected by the peripheral inflammation caused by sepsis, exposing the neurons to an environment of oxidative stress, triggering neuronal dysfunction and apoptosis. Sepsis-associated encephalopathy (SAE) is the most frequent sepsis-associated organ dysfunction, with symptoms such as deliriums, seizures, and coma, linked to increased mortality, morbidity, and cognitive disability. However, the current therapy does not avoid those patients’ symptoms, evidencing the search for a more optimal approach. Herein we focus on microglia as a prominent therapeutic target due to its multiple functions maintaining CNS homeostasis and its polarizing capabilities, stimulating and resolving neuroinflammation depending on the stimuli. Microglia polarization is a target of multiple studies involving nerve cell preservation in diseases caused or aggravated by neuroinflammation, but in sepsis, its therapeutic potential is overlooked. We highlight the peroxisome proliferator-activated receptor gamma (PPARγ) neuroprotective properties, its role in microglia polarization and inflammation resolution, and the interaction with nuclear factor-κB (NF-κB) and mitogen-activated kinases (MAPK), making PPARγ a molecular target for sepsis-related studies to come.

The Fra-1/AP-1 Oncoprotein: From the “Undruggable” Transcription Factor to Therapeutic Targeting

Simple SummaryCancer-associated mortality largely depends on metastatic dissemination. Metastasizing cancer cells exhibit drastic phenotypic changes, including the ability to migrate, invade surrounding tissues, survive in the bloodstream, adapt to different microenvironments, and resist to therapeutic treatments. These changes depend on the genetic reprogramming orchestrated by relatively few players (transcription factors). Among the components of the dimeric transcription factor AP-1, the nuclear oncoprotein Fra-1 is strongly implicated in metastasis mechanisms. Therefore, Fra-1, along with other proinvasive transcription factors, represents an ideal therapeutic target. However, as for other DNA-binding proteins, the design of inhibitory drugs is hampered by the structural features of Fra-1. In this Review, after summarizing the Fra-1 functions and mechanisms of accumulation in invasive tumors, we survey the possible application of multiple strategies and emerging technologies aimed at the inhibition of Fra-1 expression and activity, to prevent metastatic dissemination and therapeutic resistance. The genetic and epigenetic changes affecting transcription factors, coactivators, and chromatin modifiers are key determinants of the hallmarks of cancer. The acquired dependence on oncogenic transcriptional regulators, representing a major determinant of cancer cell vulnerability, points to transcription factors as ideal therapeutic targets. However, given the unavailability of catalytic activities or binding pockets for small-molecule inhibitors, transcription factors are generally regarded as undruggable proteins. Among components of the AP-1 complex, the FOS-family transcription factor Fra-1, encoded by FOSL1, has emerged as a prominent therapeutic target. Fra-1 is overexpressed in most solid tumors, in response to the BRAF-MAPK, Wnt-beta-catenin, Hippo-YAP, IL-6-Stat3, and other major oncogenic pathways. In vitro functional analyses, validated in onco-mouse models and corroborated by prognostic correlations, show that Fra-1-containing dimers control tumor growth and disease progression. Fra-1 participates in key mechanisms of cancer cell invasion, Epithelial-to-Mesenchymal Transition, and metastatic spreading, by driving the expression of EMT-inducing transcription factors, cytokines, and microRNAs. Here we survey various strategies aimed at inhibiting tumor growth, metastatic dissemination, and drug resistance by interfering with Fra-1 expression, stability, and transcriptional activity. We summarize several tools aimed at the design and tumor-specific delivery of Fra-1/AP-1-specific drugs. Along with RNA-based therapeutics targeting the FOSL1 gene, its mRNA, or cognate regulatory circRNAs, we will examine the exploitation of blocking peptides, small molecule inhibitors, and innovative Fra-1 protein degraders. We also consider the possible caveats concerning Fra-1 inhibition in specific therapeutic contexts. Finally, we discuss a recent suicide gene therapy-based approach, aimed at selectively killing the Fra-1-overexpressing neoplastic cells.

Identification of novel leads as potent inhibitors of HDAC3 using ligand-based pharmacophore modeling and MD simulation

In the landscape of epigenetic regulation, histone deacetylase 3 (HDAC3) has emerged as a prominent therapeutic target for the design and development of candidate drugs against various types of cancers and other human disorders. Herein, we have performed ligand-based pharmacophore modeling, virtual screening, molecular docking, and MD simulations to design potent and selective inhibitors against HDAC3. The predicted best pharmacophore model ‘Hypo 1’ showed excellent correlation (R2 = 0.994), lowest RMSD (0.373), lowest total cost value (102.519), and highest cost difference (124.08). Hypo 1 consists of four salient pharmacophore features viz. one hydrogen bond acceptor (HBA), one ring aromatic (RA), and two hydrophobic (HYP). Hypo 1 was validated by Fischer's randomization with a 95% of confidence level and the external test set of 60 compounds with a good correlation coefficient (R2 = 0.970). The virtual screening of chemical databases, drug-like properties calculations followed by molecular docking resulted in identifying 22 representative hit compounds. Performed 50 ns of MD simulations on top three hits were retained the salient π-stacking, Zn2+ coordination, hydrogen bonding, and hydrophobic interactions with catalytic residues from the active site pocket of HDAC3. Total binding energy calculated by MM-PBSA showed that the Hit 1 and Hit 2 formed stable complexes with HDAC3 as compared to reference TSA. Further, the PLIP analysis showed a close resemblance between the salient pharmacophore features of Hypo 1 and the presence of molecular interactions in co-crystallized FDA-approved drugs. We conclude that the screened hit compounds may act as potent inhibitors of HDAC3 and further preclinical and clinical studies may pave the way for developing them as effective therapeutic agents for the treatment of different cancers and neurodegenerative disorders.

In vitro enzyme inhibition and in vivo anti-hyperuricemic potential of eugenol: An experimental approach

: Xanthine oxidase (XO) was accountable for the uric acid synthesis in the body and is considered as a prominent therapeutic target in urate lowering treatment. Eugenol is a natural compound commonly found in the clove, cinnamon etc. and have various biological activities. This study was designed to examine the anti-hyperuricemic effect of eugenol by in vitro and in vivo studies. Potassium oxonate (PO) was used to induce hyperuricemia in Wistar rats. Different doses of eugenol (1.25, 2.5 and 5mg/kg bwt orally) were used for the treatment and various biological function markers (renal, hepatic and hematological) were analyzed. The IC50 value obtained for eugenol was 3.51 ± 0.002 μM. The kinetic studies revealed that the eugenol exhibited a mixed type of inhibition. Abnormality in the levels of various biological function markers were observed in the PO treated rats. Upon the eugenol treatment, those biological function markers were retained near to its normal values. The study proved the anti-hyperuricemic potential of eugenol against the PO induced hyperuricemia model.

Novel Mechanisms of Acalabrutinib Resistance in Patients with Chronic Lymphocytic Leukemia By Whole Genome Methylome Sequencing

Novel Mechanisms of Acalabrutinib Resistance in Patients with Chronic Lymphocytic Leukemia By Whole Genome Methylome Sequencing

Design, synthesis and biological evaluation of tyrosinase-targeting PROTACs

The human tyrosinase is the most prominent therapeutic target for pigmentary skin disorders. However, the overwhelming majority efforts have been devoted to search mushroom tyrosinase inhibitors, which show poor inhibitory activity on human tyrosinase and certain side effects that cause skin damage in practical application. Herein, a series of degraders that directly targeted human tyrosinase was firstly designed and synthesized based on newly developed PROTAC technology. The best PROTAC TD9 induced human tyrosinase degradation obviously in dose and time-dependent manner, and its mechanism of inducing tyrosinase degradation has also been clearly demonstrated. Besides, encouraging results that low-toxicity PROTAC TD9 was applied to reduce zebrafish melanin synthesis have been obtained, highlighting the potential to treatment of tyrosinase-related disorders. Moreover, this work has innovatively expanded the application scope of PROTAC technology and laid a solid foundation for further development of novel drugs treating pigmentary skin disorders.

Abstract P325: The Role Of Runx1 In Cardiomyocyte Cell Cycle And Ploidy

Adult mammalian cardiomyocytes (CMs) are thought to be post-mitotic and therefore unable to regenerate the myocardium after injury. In recent years various studies have shown that the adult mammalian CM is capable of a small amount of proliferation, potentially restricted to a subset of CMs. One such study demonstrated that having greater percentages of the rare mononuclear diploid cardiomyocyte (MNDCM) is associated with improved outcomes after myocardial infarction (MI). An accompanying genome-wide association analysis identified genetic loci associated with the frequency of the MNDCM population. One candidate to come out of this screen was Runx1. Concurrently, RUNX1 captured the attention of cardiac regeneration researchers due to its increased presence in disease states, with some suggesting it may be a marker for dedifferentiation (fetal gene induction). One recent study demonstrated improved calcium handling and decreased eccentric hypertrophy following RUNX1 ablation after injury, perhaps corroborating the idea that RUNX1 is involved with CM dedifferentiation. We hypothesize that Runx1 influences dedifferentiation in CMs, impacting ploidy, as well as CM cell cycle activity and post-MI outcomes. We found that CM-specific overexpression (OE) of Runx1 results in a doubling of the MNDCM population, thereby validating its influence on the population. Via multiple contexts including postnatal development and adult injury, knocking out Runx1 decreases DNA synthesis while Runx1 OE increases DNA synthesis. Furthermore, an initial analysis of RNAseq data demonstrates that RUNX1 OE in a neonatal mouse hearts demonstrated differential expression in genes related to cardiac conduction, contraction, heart development, regeneration, and regulation of cell differentiation . After MI in the adult mouse heart, the effects of Runx1 OE resulted in transient benefits which included increased cell cycle activity and preservation of function. These data suggest that Runx1 is not simply a marker of CM dedifferentiation, but also a regulator of the process including cell cycle activation. Ongoing work will tease apart this role in more detail and could establish RUNX1 as a prominent therapeutic target for mitigating effects of cardiac injury.

Pain as Defining Feature of Health Status and Prominent Therapeutic Target in Patients with Hidradenitis Suppurativa.

Background. Pain is one of the main aspects of hidradenitis suppurativa that strongly affects the quality of life of patients. We explored the relationship between pain and clinical severity as well as its role in defining the health status in patients with HS. Methods. Pain was defined by three measures: (a) question 1 (“my skin hurts”) of the Skindex-17; (b) Bodily Pain (BP) scale of the SF-36; and (c) Visual Analog Scale (VAS). Clinical severity of HS was assessed by the Hurley staging, the Sartorius HS Score, and the International HS Severity Score System. Results. The study population included 341 HS patients with complete data for the VAS pain, 316 for question 1 of the Skindex-17, and 294 for BP. Clinical severity was positively associated with pain. This result was observed for all three severity scores and all three pain evaluation methods. In addition, the number of fistulae, abscesses, and nodules were significantly associated with the three severity measures of pain, while the association with scars was not observed for question 1 of the Skindex-17 and BP. Conclusions. Pain may be a good proxy of clinical severity and efficacy of a treatment in HS and therefore a crucial hallmark of patients’ health status.

Autoregulatory control of microtubule binding in doublecortin-like kinase 1.

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

Ruxolitinib in Combination with Vorinostat Suppresses Tumor Growth and Alters the Metabolic Phenotype in Haematological Diseases

Background and Objectives Jak-2 dysregulation has a significative role as an oncogenic driver and as a prominent therapeutic target in hematological malignancies. Ruxolitinib is pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2 and minor activity against JAK3. Vorinostat is an HDAC inhibitor able to reduce Jak-2 expression affecting Jak-2 mRNA expression and increasing Jak-2 proteasomal deterioration. In this study we hypothesized that Ruxolitinib and Vorinostat combination could have a synergistic effects on haematological disease. Methods. We tested Ruxolitinib and Vorinostat alone and in combination for 24 and 48 hrs in 12 lymphoma and myeloma cell lines. The synergism was assessed by Chou-Talalay method. The cell cycle, the apoptosis, the caspase activation, the autophagy, the interleukin expression, the ROS generation, the ATP levels, the lactate, the phosphorylation status of protein kinases were evaluated in all 12 cell lines after 24 hrs. Co-coltures with bone marrow stromal cells were also performed after 24 to 120 hrs of treatments. Results. Treatment of 12 cell lines with low doses of Ruxolitinib and Vorinostat as single agents for 24h was rather ineffective at blocking cell growth if compared with the efficacy of the combination of the two drugs. The combination was additive in two lines of mantle cell lymphoma, in one line of Hodgkin lymphoma, in one line of myeloma, in one line of T cell lymphoma and in on line of B cell lymphoma. In the remaining cell lines the combination of the two drugs was highly synergistic. This synergistic cytotoxic action was maintained over time up to 120 hrs also in the presence of stromal cells. Ruxolitinib and Vorinostat combination influenced the cell cycle distribution increasing G2-M phase arrest in all 12 cell lines after 24 hrs. The levels of p21 and p27 resulted up-regulated while the levels of Cyclin D and Aurora A were downregulated in all 12 cell line after combined treatment. The apoptotic AnnexinV-positive cells after 24 hrs of treatment with Ruxolitinib or Vorinostat alone was less than 10%. However, the percentage of apoptotic cells grew up to 50 % after treatment of the cells with Ruxolitinib in combination with Vorinostat. After 24 of combined treatment, we detected a cleavage of caspase 8 and caspase 3 in all 12 cell line, suggesting activation of the extrinsic apoptotic pathway. Quantification of caspase 9 in all 12 cell lines has resulted in observation of an increase in cleaved form only in the six most sensitive cell lines, suggesting a caspase activation mitochondrial events regulating the intrinsic apoptosis. Bax, Bid and BAD levels increased in all 12 cell lines treated with combined drugs, but apoptotic inhibitors BLC-2 and MCL-1 were down regulated only in the 6 more sensitive cell lines. Our experiment showed that Ruxolitinib in combination with Vorinostat reduced IL-10 and IL-6 secretion in all 12 cell lines in a comparable way. The expression of IL-17A after combined treatment was significantly decreased in only the 6 most sensitive cell lines. Ruxolitinib and Vorinostat alone and in combination induced a decrease of the amount of autophagy-related protein p62 in cancer cells, indicating the activation of autophagic system. Autophagy and apoptosis increased the oxidative stress leading to ROS production and in this work, we showed that Ruxolitinib and Vorinostat synergistically induce apoptosis in 6 more sensitive cell lines, associated with a marked increase in ROS generation. Interestingly, our data have shown that combined therapy alters tumor cell metabolism by reducing the glycolic state by causing greater sensitivity of the six cell lines to the combined treatment. Conclusions. In conclusions, the combination of Ruxolitinib and Vorinostat is able to decrease glucose metabolism and this partial reversion of Warburg effect is associated to ROS production, apoptosis and cell growth inhibition. The drugs tested under different cell culture conditions provide informations on drug efficacy and could help predict pharmacological effects that may be difficult to translate from in vitro and ex vivo conditions to the clinic. This study gives evidence of synergistic interaction between Ruxolitinib and Vorinostat in lymphoid cells and provides the rationale for clinical studies with the combination of both agents in patients. DisclosuresNo relevant conflicts of interest to declare.

Isoform‐Selective Regulation of Akt1 and Akt2 by Interdomain Interaction

The serine/threonine protein kinase PKB/Akt is a key signal transducer in a wide range of cellular responses and a prominent therapeutic target. The three highly homologous isoforms of Akt regulate critical and distinct physiological functions in cell growth, proliferation, and metabolism. However, the mechanisms that determine isoform-specific Akt signaling remain largely unclear. Here we examined the mechanisms by which Akt1 and Akt2, the most broadly expressed isoforms, are differentially activated upon growth factor stimulation. Using a newly developed FRET-based reporter of Akt2 activation (ReAktion2) along with a previously developed reporter of Akt1 activation (ReAktion), we found that growth factor triggers a conformational change in both Akt isoforms at the plasma membrane, which is critically dependent on activation-loop phosphorylation. Interestingly, the allosteric pan-Akt inhibitor, MK-2206, appears to selectively induce a conformational change in Akt2, but not Akt1 at the plasma membrane. This prompts us to further examine whether inter-domain interactions differ between Akt1 and Akt2. Our preliminary results suggest Akt1 activation enhances the interaction between its PH and linker-catalytic domains, which is dependent on phosphorylation of T308 within the catalytic domain, whereas this induced interdomain interaction is absent in Akt2. Furthermore, the different interdomain interaction of Akt isoforms may lead to different membrane departure, as single cell analysis of plasma membrane translocation revealed that Akt2 accumulates at the plasma membrane to a greater extent than Akt1. Taken together, our results suggest that distinct modes of interactions between the PH and linker-catalytic domains modulate the isoform-specific activation of Akt.

Immune checkpoint inhibitors: review of the existing evidence and challenges in breast cancer.

Cancer initiation and progression are associated with immune system responses. Tumor cells use various tricks to scape of immune system, such as activating immune checkpoint pathways that induce immunosuppressive functions. Among the different immune checkpoint receptors, CTLA-4 and PD-1/PD-L1 are prominent therapeutic targets in different cancers. Although the US FDA has approved some immune checkpoint inhibitors for several cancers, concerning breast cancer still different clinical trials are looking for optimizing efficacy and decreasing immune-related adverse events. This review will discuss the existing body of knowledge with regard to cross-talk between immune system and tumor cells and then explore immune checkpoint-related signaling pathways in the context of breast tumors. Finally, we highlight the application of different immune checkpoint blockers in breast cancer patients.

Diospyros lotus L. fruit: A potential antidiabetic functional food targeting intestinal starch hydrolysis

Post-meal hyperglycaemia is considered a prominent therapeutic target to attenuate the progression of diabetes and its associated complications. The present study identified fruit extract of Diospyros lotus Linnaeus, of the Ebenaceae family, as an inhibitor of starch digestion through the inhibition of both alpha amylase and alpha glucosidase. The extract inhibits porcine and human pancreatic amylase with IC50 values of 82.5±2.0 and 130.4±24 μg/ml respectively. The inhibition of intestinal sucrase and maltase activity was however considerably weaker. In vitro hydrolysis of solubilised potato starch into glucose yielded comparable inhibition kinetics for 100 μg/ml D. lotus L. extract and 3.5 μM acarbose. Screening the major phenolic constituents revealed that quercetin and myricetin were the strongest alpha amylase inhibitors. D. lotus L. extract showed strong antioxidant activity; however, this provided no meaningful protection against 2-deoxy-ribose induced oxidative stress in INS-1 cells. Taken together these findings identify D. lotus L. fruit as a multi-component functional food with potential to dampen the onset and development of diabetes through the inhibition of post meal hyperglycaemia.