Use Of Small Molecule Inhibitors Research Articles

The PERK-Dependent Molecular Mechanisms as a Novel Therapeutic Target for Neurodegenerative Diseases.

Higher prevalence of neurodegenerative diseases is strictly connected with progressive aging of the world population. Interestingly, a broad range of age-related, neurodegenerative diseases is characterized by a common pathological mechanism—accumulation of misfolded and unfolded proteins within the cells. Under certain circumstances, such protein aggregates may evoke endoplasmic reticulum (ER) stress conditions and subsequent activation of the unfolded protein response (UPR) signaling pathways via the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent manner. Under mild to moderate ER stress, UPR has a pro-adaptive role. However, severe or long-termed ER stress conditions directly evoke shift of the UPR toward its pro-apoptotic branch, which is considered to be a possible cause of neurodegeneration. To this day, there is no effective cure for Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), or prion disease. Currently available treatment approaches for these diseases are only symptomatic and cannot affect the disease progression. Treatment strategies, currently under detailed research, include inhibition of the PERK-dependent UPR signaling branches. The newest data have reported that the use of small-molecule inhibitors of the PERK-mediated signaling branches may contribute to the development of a novel, ground-breaking therapeutic approach for neurodegeneration. In this review, we critically describe all the aspects associated with such targeted therapy against neurodegenerative proteopathies.

Progress in acute myeloid leukaemia: small molecular inhibitors with small benefits

The use of small molecule inhibitors in acute myeloid leukaemia (AML) has become ubiquitous with the US Food and Drug Administration approval of multiple agents between 2017 and 2018. Despite the promise, some of these indications are based on early efficacy data (phase I/II), and single-arm studies, and have not been tested in randomised trials. Furthermore, there are important limitations in the evidence that exists in randomised trials. This perspective aims to summarise the data that formed the basis for approval of gilteritinib, glasdegib, ivosidenib, enasidenib and venetoclax. It also aims to shed a light on some of the limitations in the evidence. Clinicians should exercise caution when using drugs that largely have yet to show an improvement in survival over the standard of care in AML.

Novel cell-permeable p38-MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function.

During islet transplantation, mitogen-activated protein kinase (MAPK) p38 is preferentially activated in response to the isolation of islets and the associated inflammation. Although therapeutic effects of p38 inhibitors are expected, the clinical application of small-molecule inhibitors of p38 is not recommended because of their serious adverse effects on the liver and central nervous system. Here we designed peptides to inhibit p38, which were derived from the sites on p38 that mediate binding to proteins such as MAPK kinases. Peptide 11R-p38I110 significantly inhibited the activation of p38. To evaluate the effects of 11R-p38I110 , porcine islets were incubated with 10µmol/L 11R-p38I110 or a mutant form designated 11R-mp38I110 . After islet transplantation, blood glucose levels reached the normoglycemic range in 58.3% and 0% of diabetic mice treated with 11R-p38I110 or 11R-mp38I110 , respectively. These data suggest that 11R-p38I110 inhibited islet apoptosis and improved islet function. Peptide p38I110 is a noncompetitive inhibitor of ATP and targets a unique docking site. Therefore, 11R-p38I110 specifically inhibits p38 activation, which may avoid the adverse effects that have discouraged the clinical use of small-molecule inhibitors of p38. Moreover, our methodology to design "peptide inhibitors" could be used to design other inhibitors derived from the binding sites of proteins.

Current perspectives on targeting PIM kinases to overcome mechanisms of drug resistance and immune evasion in cancer.

PIM kinases are a class of serine/threonine kinases that play a role in several of the hallmarks of cancer including cell cycle progression, metabolism, inflammation and immune evasion. Their constitutively active nature and unique catalytic structure has led them to be an attractive anticancer target through the use of small molecule inhibitors. This review highlights the enhanced activity of PIM kinases in cancer that can be driven by hypoxia in the tumour microenvironment and the important role that aberrant PIM kinase activity plays in resistance mechanisms to chemotherapy, radiotherapy, anti-angiogenic therapies and targeted therapies. We highlight an interaction of PIM kinases with numerous major oncogenic players, including but not limited to, stabilisation of p53, synergism with c-Myc, and notable parallel signalling with PI3K/Akt. We provide a comprehensive overview of PIM kinase's role as an escape mechanism to targeted therapies including PI3K/mTOR inhibitors, MET inhibitors, anti-HER2/EGFR treatments and the immunosuppressant rapamycin, providing a rationale for co-targeting treatment strategies for a more durable patient response. The current status of PIM kinase inhibitors and their use as a combination therapy with other targeted agents, in addition to the development of novel multi-molecularly targeted single therapeutic agents containing a PIM kinase targeting moiety are discussed.

16 Improvement of porcine cloned embryo developmental competence via KDM4A overexpression and H3K9me3 methyltransferase inhibitor treatment

Aberrant epigenetic reprogramming is a major reason for the developmental failure of somatic cell nuclear transfer (SCNT) embryos. Histone H3 lysine 9 trimethylation (H3K9me3), a histone marker for transcriptional repression, is considered a key barrier to the development of cloned embryos. In the present study, we found that H3K9me3 was much higher in SCNT embryos than in IVF embryos at the 4-cell and 2-cell stages; H3K9me3 demethylase KDM4A mRNA was injected into cloned embryos 5h after activation. The intensity of H3K9me3 modification decreased obviously after microinjection of KDM4A mRNA, and the developmental efficiency of porcine cloned embryos was enhanced significantly compared with control (32.2±4.2 vs. 21.0±3.5; P<0.05). Moreover, we evaluated the effect of chaetocin, an inhibitor of histone methyltransferase suv39h1/2, on SCNT embryo development. The results showed that 10 nM chaetocin not only suppressed the H3K9me3 modification in porcine embryonic fibroblast but also downregulated the expression of SUV39H1, SUV39H2, and KDM4D. However, treatment of cloned embryos with 10 nM chaetocin efficiently decreased the H3K9me3 level. Importantly, chaetocin treatment at the 4-cell stage for 6h significantly increased the blastocyst rate (57.8±4.3 vs. 43.5±1.8; P<0.05) compared with the control group. Furthermore, because a recent study showed that a high level of H3K9me3 appears in the reprogramming resistance region of nuclear transfer embryos during zygogene activation (ZGA), we examined the expression levels of ZGA-related genes. The qPCR results showed that the expression of ZGA-related genes increased significantly in SCNT embryos with chaetocin treatment compared with the control. These results suggested that chaetocin treatment can improve the efficiency of SCNT reprogramming during ZGA. In summary, our results suggested that H3K9me3 acts as an epigenetic barrier in porcine SCNT reprogramming and that a suv39h1/2 inhibitor can effectively reduce the H3K9me3 level in the early reprogramming phase and further improve the invitro developmental competence. Due to the existence of a variety of abnormal epigenetic mechanisms during somatic cell reprogramming, the combined use of small-molecule inhibitors is required in future studies. Data analyses were performed using SPSS software. Significance was set at P<0.05 unless otherwise specified. The results are expressed as the mean±standard deviation. This work was supported by the National Natural Science Foundation of China (grant number 31601942); the Postdoctoral Science Foundation of Heilongjiang Province (grant number LBH-Z17010); and the Fund for the National Key Research and Development program of China-Stem Cell and Translational Research (2016YFA0100200).

Systematic Characterization of Genes Representing Preferential Molecular Vulnerabilities for Myeloma Cells Compared to Other Neoplasias - Implications for the Biology and Therapeutic Targeting of Myeloma

In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while >80% and >75% of these genes have detectable transcript levels (RPKM>1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Abstract 1897: Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3

Abstract Introduction: Tumors are complex systems where cell-cell communication between intra-tumoral and extra-tumoral cells play a crucial role. Extracellular vesicles (EVs), membrane-limited vesicles secreted by normal and malignant cells, have been proposed to act as mediators of intercellular communication in physiological and pathological scenarios. It is now well established that integrins are constantly endocytosed and recycled back to the plasma membrane through multiple routes. Accumulating evidence clearly indicates that endocytosis and recycling of integrins plays a crucial role during cancer progression, invasion, and metastasis. Here we describe a fundamental link between endocytosis mediated integrin trafficking and the integrin-mediated uptake of exosomes. Objectives: As the integrins are crucial for the role of exosomes in cancer progression and particularly in metastasis we were aiming at delineating the role of ITGB3 in exosome-mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231. Methodology: Using stable shRNA of ITGB3 (shITGB3) in the triple-negative breast cancer cell line MDA.MB.231, EVs were isolated by ultracentrifugation and characterized by Western blot analysis to determine the protein composition and by NTA and CryoEM to measure the amount of EVs. An LC-MS/MS-based survey was performed, to determine the differences in the protein composition of EVs after knock-down of ITGB3. Uptake of fluorescently labeled exosomes into recipient cells was measured by flow cytometry. The colony forming capacity induced by conditioned medium was used to assess the functional impact on the role of ITGB3 in intercellular communication. Results: Analysis of the EVs fraction of shITGB3 cell revealed a striking decrease of classical markers of exosomes as judged by western blot analysis of proteins such as TSG101, CD81, but not others like Flotillin-1. LC-MS/MS analysis of shITGB3-MDA.MB.231 derived-exosomes furthermore revealed clear defects in protein complexes required for exosome formation, namely the ESCRT complex and the Syndecan-syntenin-ALIX complex. Both complexes are required for the formation of endosomal intraluminal vesicles that get released as exosomes. On the other hand, NTA analysis and CryoEM showed the overall amount of EVs was increased. In line with this, the uptake of fluorescently labeled EVs was strongly impaired in shITGB3 cells. By the use of small molecule inhibitors, we can furthermore demonstrate that this defect linked to endocytosis of EVs. Conclusions: Here we demonstrate that ITGB3 plays a crucial role in controlling the composition of different vesicle populations in the extracellular space, linked to the endocytosis of integrins and interference with this mechanism results in defects in the uptake of EVs and impedes the generation of exosomes. The functional effects are discussed Citation Format: Pedro Fuentes Varela, Marta Sesé, Pedro Jesús Guijarro, Marta Emperador, Héctor Peinado, Stefan Hümmer, Santiago Ramon y Cajal. Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1897.

Antitumor effects of a covalent cyclin-dependent kinase 7 inhibitor in colorectal cancer.

Although both antiepidermal growth factor receptor and vascular endothelial growth factor therapies have been shown to be effective against colorectal cancer (CRC), their beneficial effects are limited to a small proportion of patients and are not sustainable. Cyclin-dependent kinase 7 (CDK7) is an important regulator of the transcriptional machinery. Use of small-molecule inhibitors of the transcriptional machinery has shown promising selectivity for cancer cells and potent antiproliferative effects. In this study, the effects of a covalent CDK7 inhibitor THZ1 as a potent anti-CRC compound were evaluated in vitro and in vivo. THZ1 significantly inhibited cell growth and induced apoptosis of CRC cells in vitro. In addition, it also decreased xenograft tumor growth in vivo. RNA-Seq showed that THZ1 induced inhibition of a number of oncogenic transcripts. Taken together, our results indicate that pharmacological modulation of CDK7 kinase activity by THZ1 may represent a potential strategy in the treatment of CRC.

Use of small molecule inhibitors of metabolism to enhance T cell proliferation and phenotype for adoptive T cell therapies

Abstract Adoptive T cell therapies including CAR T therapy are limited by poor in-vivo persistence and moderate anti-tumor efficacy. According to literature, metabolism plays a critical role in the phenotypic state and fate of T cells post-expansion. For instance, glycolytic T cells are more active and exhibit a terminal effector phenotype, while quiescent T cells (naïve and memory T cells) rely more on oxidative phosphorylation with better proliferative capacity. Therefore, this study aims to determine whether metabolic inhibitors used during ex vivo T cell expansion promote a more quiescent phenotype leading to T cells with increased in vivo persistence and efficacy. A screening was conducted using inhibitors of T cell metabolism to alter the balance between T cell activation and senescence, thereby enhancing T cells proliferative capacity. The two inhibitors screened were: idelalisib, an inhibitor of PI3K δ and ibrutinib, an inhibitor of Bruton’s tyrosine kinase. The study involved culturing human T cells with the above-mentioned compounds for 15 days with anti- CD3/CD28 beads and 30 U/mL of IL-2. T cells cultured with idelalisib and ibrutinib had increased expansion (50- fold: idelalisib and 21-fold ibrutinib) when compared to control (cells with beads alone) with only 6-fold expansion. Phenotypic analysis performed using flow cytometry showed an increased percentage of CD27+ CD28+ CD8+ T cells in the idelalisib treated group and decreased number of senescent T cells that are double negative for CD27 and CD28. Future experiments focus on other modulators of metabolism that may benefit T cell expansion. They will also focus on metabolic analysis of T cells using the Seahorse assay and in-depth analysis of the PI3K pathway using western blot.

Verteporfin-Loaded Polymeric Microparticles for Intratumoral Treatment of Brain Cancer.

Glioblastoma (GBMs) is the most common and aggressive type of primary brain tumor in adults with dismal prognosis despite radical surgical resection coupled with chemo- and radiotherapy. Recent studies have proposed the use of small-molecule inhibitors, including verteporfin (VP), to target oncogenic networks in cancers. Here we report efficient encapsulation of water-insoluble VP in poly(lactic- co-glycolic acid) microparticles (PLGA MP) of ∼1.5 μm in diameter that allows tunable, sustained release. Treatment with naked VP and released VP from PLGA MP decreased cell viability of patient-derived primary GBM cells in vitro by ∼70%. Moreover, naked VP treatment significantly increased radiosensitivity of GBM cells, thereby enhancing overall tumor cell killing ability by nearly 85%. Our in vivo study demonstrated that two intratumoral administrations of sustained slow-releasing VP-loaded PLGA MPs separated by two weeks significantly attenuated tumor growth by ∼67% in tumor volume in a subcutaneous patient-derived GBM xenograft model over 26 d. Additionally, our in vitro data indicate broader utility of VP for treatment for other solid cancers, including chordoma, malignant meningioma, and various noncentral nervous system-derived carcinomas. Collectively, our work suggests that the use of VP-loaded PLGA MP may be an effective local therapeutic strategy for a variety of solid cancers, including unresectable and orphan tumors, which may decrease tumor burden and ultimately improve patient prognosis.

Targeting metabolic vulnerabilities of cancer: Small molecule inhibitors in clinic.

Altered cell metabolism is an established hallmark of cancer. Advancement in our understanding of dysregulated cellular metabolism has aided drastically in identifying metabolic vulnerabilities that can be exploited therapeutically. Indeed, this knowledge has led to the development of a multitude of agents targeting various aspects of tumor metabolism. The intent of this review is to provide insight into small molecule inhibitors that target tumor metabolism and that are currently being explored in active clinical trials as either preventive, stand-alone, or adjuvant therapies for various malignancies. For each inhibitor, we outline the mechanism (s) of action, preclinical/clinical findings, and limitations. Sections are divided into three aspects based on the primary target of the small molecule inhibitor (s): those that impact (1) cancer cells directly, (2) immune cells present in the tumor microenvironment, or (3) both cancer cells and immune cells. We highlight small molecule targeting of metabolic pathways including de novo fatty acid synthesis, NAD+ biosynthesis, 2-hydroxyglutarate biosynthesis, polyamine metabolism, the kynurenine pathway, as well as glutamine and arginine metabolism. Use of small molecule inhibitors aimed at exploiting tumor metabolic vulnerabilities continues to be an active area of research. Identifying metabolic dependencies specific to cancer cells and/or constituents of the tumor microenvironment is a viable area of therapeutic intervention that holds considerable clinical potential.

Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs.

There are several challenges towards the development and clinical use of small molecule inhibitors, which are currently the main type of targeted therapies towards intracellular proteins. PROteolysis-TArgeting Chimeras (PROTACs) exploit the intracellular ubiquitin-proteasome system to selectively degrade target proteins. Recently, small-molecule PROTACs with high potency have been frequently reported. In this review, we summarize the emerging characteristics of small-molecule PROTACs, such as inducing a rapid, profound and sustained degradation, inducing a robust inhibition of downstream signals, displaying enhanced target selectivity, and overcoming resistance to small molecule inhibitors. In tumor xenografts, small-molecule PROTACs can significantly attenuate tumor progression. In addition, we also introduce recent developments of the PROTAC technology such as homo-PROTACs. The outstanding advantages over traditional small-molecule drugs and the promising preclinical data suggest that small-molecule PROTAC technology has the potential to greatly promote the development of targeted therapy drugs.

Frontal Bone Healing Is Sensitive to Wnt Signaling Inhibition via Lentiviral-Encoded Beta-Catenin Short Hairpin RNA.

The Wnt/β-catenin signaling pathway plays an integral role in skeletal biology, spanning from embryonic skeletal patterning through bone maintenance and bone repair. Most experimental methods to antagonize Wnt signaling in vivo are either systemic or transient, including genetic approaches, use of small-molecule inhibitors, or neutralizing antibodies. We sought to develop a novel, localized model of prolonged Wnt/β-catenin signaling blockade by the application and validation of a lentivirus encoding β-catenin short hairpin RNA (shRNA). Efficacy of lentiviral-encoded β-catenin shRNA was first confirmed in vitro using bone marrow mesenchymal stromal cells, and in vivo using an intramedullary long bone injection model in NOD SCID mice. Next, the effects of β-catenin knockdown were assessed in a calvarial bone defect model, in which the frontal bone demonstrates enhanced bone healing associated with heightened Wnt/β-catenin signaling. Lentivirus encoding either β-catenin shRNA or random sequence shRNA with enhanced green fluorescent protein (control) was injected overlying the calvaria of NOD SCID mice and bone defects were created in either the frontal or parietal bones. Among mice treated with lentivirus encoding β-catenin shRNA, frontal bone defect healing was significantly reduced by all radiographic and histologic metrics. In contrast, parietal bone healing was minimally impacted by β-catenin shRNA. In aggregate, our data document the application and validation of a lentivirus encoding β-catenin shRNA model that represents an easily replicable tool for examining the importance of locoregional Wnt/β-catenin signaling in bone biology and regeneration.

Antileukemic Efficacy of BET Inhibitor in a Preclinical Mouse Model of MLL-AF4+ Infant ALL.

MLL-rearranged acute lymphoblastic leukemia (ALL) occurring in infants is a rare but very aggressive leukemia, typically associated with a dismal prognosis. Despite the development of specific therapeutic protocols, infant patients with MLL-rearranged ALL still suffer from a low cure rate. At present, novel therapeutic approaches are urgently needed. Recently, the use of small molecule inhibitors targeting the epigenetic regulators of the MLL complex emerged as a promising strategy for the development of a targeted therapy. Herein, we have investigated the effects of bromodomain and extra-terminal (BET) function abrogation in a preclinical mouse model of MLL-AF4+ infant ALL using the BET inhibitor I-BET151. We reported that I-BET151 is able to arrest the growth of MLL-AF4+ leukemic cells in vitro, by blocking cell division and rapidly inducing apoptosis. Treatment with I-BET151 in vivo impairs the leukemic engraftment of patient-derived primary samples and lower the disease burden in mice. I-BET151 affects the transcriptional profile of MLL-rearranged ALL through the deregulation of BRD4, HOXA7/HOXA9, and RUNX1 gene networks. Moreover, I-BET151 treatment sensitizes glucocorticoid-resistant MLL-rearranged cells to prednisolone in vitro and is more efficient when used in combination with HDAC inhibitors, both in vitro and in vivo Given the aggressiveness of the disease, the failure of the current therapies and the lack of an ultimate cure, this study paves the way for the use of BET inhibitors to treat MLL-rearranged infant ALL for future clinical applications. Mol Cancer Ther; 17(8); 1705-16. ©2018 AACR.

The importance of 15-lipoxygenase inhibitors in cancer treatment.

Cancer-targeted therapy is an expanding and successful approach in treatment of many types of cancers. One of the main categories of targeted therapy is use of small molecule inhibitors. 15-Lipoxygenase (15-LOX) is an enzyme which reacts with polyunsaturated fatty acids and produces metabolites that are implicated in many important human diseases, such as cancer. Considering the role of 15-LOX (mainly 15-LOX-1) in the progression of some cancers, the discovery of 15-LOX inhibitors could potentially lead to development of novel cancer therapeutics and it can be claimed that 15-LOX inhibitors might be suitable as chemotherapy agents in the near future. This article reviews relevant publications on 15-LOX inhibitors with focus on their anticancer activities in vitro and in vivo. Many 15-LOX inhibitors have been reported for which separate studies have shown their anticancer activities. This review paves the way to further explore the mechanism of their antiproliferative effects via 15-LOX inhibition.

Recent Advances in Next Generation Snakebite Antivenoms

With the inclusion of snakebite envenoming on the World Health Organization’s list of Neglected Tropical Diseases, an incentive has been established to promote research and development effort in novel snakebite antivenom therapies. Various technological approaches are being pursued by different research groups, including the use of small molecule inhibitors against enzymatic toxins as well as peptide- and oligonucleotide-based aptamers and antibody-based biotherapeutics against both enzymatic and non-enzymatic toxins. In this article, the most recent advances in these fields are presented, and the advantages, disadvantages, and feasibility of using different toxin-neutralizing molecules are reviewed. Particular focus within small molecules is directed towards the inhibitors varespladib, batimastat, and marimastat, while in the field of antibody-based therapies, novel recombinant polyclonal plantivenom technology is discussed.

Phosphatases of Regenerating Liver (PRL) as Therapeutic Targets in Cancer

Background: Phosphatases of regenerating liver (PRL) represent a class of protein tyrosine phosphatases with oncogenic activity. PRL overexpression enhances cell proliferation, transformation, and promotes metastasis in many human cancers. Most notably, PRLs interact with a family of magnesium transporters, cyclin M (CNNM), to regulate intracellular Mg2+ levels. Thus, PRLs are attractive therapeutic targets given their role in oncogenic and tumour suppressor signaling pathways by modulating cellular growth. Methods: Academic research articles were obtained by searching key terms in the PubMed database. This review specifically focuses on the articles that provided a comprehensive overview of PRLs, CNNMs, and small molecule inhibitors of PRLs. Summary: This review discusses the role of PRLs in promoting cancer metastasis and explores current strategies for targeting PRL activity through the use of small molecule inhibitors. Although several potent PRL inhibitors have been discovered, improvements must be made prior to clinical applications. Therefore, understanding the molecular basis of PRL inhibition is essential for developing novel therapeutic agents in cancer treatments.

Elucidating molecular events underlying topography mediated cardiomyogenesis of stem cells on 3D nanofibrous scaffolds

Toward engineering a cardiac patch, the objective of this work was to assess stem cell response to a three-dimensional (3D) nanofibrous scaffold and probe the underlying molecular mechanisms including both cell signaling and epigenetic changes. Cardiomyogenesis of human mesenchymal stem cells (hMSCs) in 3D poly(ε-caprolactone) (PCL) nanofibers and macroporous scaffolds was compared with two-dimensional (2D) PCL films. In addition, nanofiber mats of PCL and its blend with gelatin (PCL-Gel) were prepared with fibers of random or unidirectional alignment to assess the roles of topography (fibrous architecture and its alignment) and biochemical cue (cell-adhesive sites) in directing cell functions. Cells on 3D random nanofibers, exhibited elevated expression of known cardiac markers such as cardiac actinin, cardiac troponin and β-myocardial heavy chain compared to cells on 2D films suggesting enhanced differentiation that was further accentuated on the aligned fibers. 3D macroporous scaffolds did not enhance the cardiomyogenic differentiation. However, minimal differences were noted between cells on PCL and PCL-Gel fibers, irrespective of alignment. Co-culture with neonatal rat cardiomyocytes induced beating in the differentiated cells. The use of small molecule inhibitors revealed that cytoskeletal elements F-actin, microtubules and downstream ROCK protein are essential for the cardiomyogenesis of hMSCs on the nanofibers. The activation of ERK, AKT and mTOR was observed during cardiomyogenesis. Interestingly, enhanced differentiation on the aligned nanofibers was associated with increased level of the histone deacytelase SIRT6 and decreased level of the acetylated histone H3K9 suggesting a role for epigenetic regulation. This study demonstrates that aligned nanofibrous scaffolds augment cardiomyogenic differentiation wherein topography plays a critical role in driving stem cell function. In addition, this study offers insight into molecular pathways driving the cellular response.

Integrated Microfluidic Device for Drug Studies of Early C. Elegans Embryogenesis.

Small molecules inhibitors are powerful tools for studying multiple aspects of cell biology and stand at the forefront of drug discovery pipelines. However, in the early Caenorhabditis elegans (C. elegans) embryo, which is a powerful model system for cell and developmental biology, the use of small molecule inhibitors has been limited by the impermeability of the embryonic eggshell, the low‐throughput manual embryo isolation methods, and the lack of well‐controlled drug delivery protocols. This work reports a fully integrated microfluidic approach for studies of C. elegans early embryogenesis, including the possibility of testing small molecule inhibitors with increased throughput and versatility. The setup enables robust on‐chip extraction of embryos from gravid adult worms in a dedicated pillar array chamber by mechanical compression, followed by rapid fluidic transfer of embryos into an adjacent microtrap array. Parallel analysis of ≈100 embryos by high‐resolution time‐lapse imaging from the one‐cell stage zygote until hatching can be performed with this device. The implementation of versatile microfluidic protocols, in particular time‐controlled and reversible drug delivery to on‐chip immobilized embryos, demonstrates the potential of the device for biochemical and pharmacological assays.

Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells

SummaryNatural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion.