Current Pharmaceutical Design - Volume 19, Issue 30, 2013

Normal cell cycle progression is controlled by the sequential action of cyclin-dependent kinases (CDKs), the activity of which depends on their binding to regulatory partners (cyclins). Deregulation of cell cycle is one of the first steps that transform normal cells into tumor cells. Indeed, most cancer cells bear mutations in members of the pathways that control the CDK activity. For this reason, this kinase family is a crucial target for the development of new drugs for cancer therapy. Recently, both ATP-competitive CDK inhibitors and the last generation of non-ATP-competitive inhibitors are emerging as promising agents for targeted therapies. Many clinical trials are in progress, using CDK inhibitors both as single agents and in combination with traditional cytotoxic agents. In this review, we will discuss new therapeutic strategies based on the use of CDK inhibitors in cancer.

Lung cancer is the leading cause of mortality world-wide. Non Small Cell Lung Cancer (NSCLC) is a particularly aggressive cancer, the optimum management of which is still being determined. In the next years modest survival improvement can be expected by chemotherapy. Advances in understanding of the molecular pathogenesis of lung cancer have led to the identification of several specific targets for therapeutic agents. Targeting the epidermal growth factor receptor (EGFR) has played a central role in advancing NSCLC research, treatment, and patient outcome over the last several years. In lung cancer, 10-15% of NSCLC contain activating mutations in the EGFR kinase conferring hypersensitivity to the oral TKIs gefitinib and erlotinib, have been demonstrated to be important predictive factors when selecting patients to be treated with these two agents. More recently, another molecular abnormality, the translocation of the anaplastic lymphoma kinase (ALK) gene that drives NSCLC in a different group of patients has been found in 4 to 5% of NSCLC. The rearrangement results in an EML4 – AKL fusion gene, which increases ALK activity. Inhibitors of ALK kinase have been developed and investigated. Crizotinib, an orally ALK and met proto-oncogene (MET) inhibitor, was very well tolerated and produced dramatic antitumor activity in early-stage trials which facilitated a faster than normal move into late-stage trials for EML4-ALK –positive NSCLC patients treatment. In a phase III randomized that showed progression free survival benefit as compared to chemotherapy in second-line setting. Several novel selective inhibitors of ALK kinase are currently in preclinical or early clinical testing. Since the discovery that Met pathway is one of the most frequently dysregulated pathways in human cancer, Met inhibitors, with varying kinase selectivity profiles ranging from highly selective to multi-targeted have been studied in the clinic and good progress has been achieved. A number of studies suggest that the PI3K/Akt signaling pathway is central to NSCLC growth and survival. Given the importance of activated PI3K signaling in cancer, several PI3K inhibitors are currently one of the most recent drug targets in oncology, with several small molecules in early stages of clinical development. This review will focus on the role of EGFR, ALK, MET, and PI3K inhibitors in the treatment of NSCLC.

Drug “repurposing” is the process of finding new therapeutic indications for existing drugs, and can be considered as a more efficient and realistic strategy for the design of therapies against rare diseases than the current efforts to develop targeted-drugs. In this review, we explore the difficulties related to the identification and development of tailored therapies for individual patients with sarcomas, which are relatively rare diseases characterized by an extreme genetic and histologic variability. Overall, sarcomas comprise about 1% of all adult tumors and 10% of pediatric cancers. They are conventionally divided in bone and soft-tissue sarcomas, considering their site of origin. However, each group is highly heterogeneous and recent global characterization of their genetic alterations has clearly identified the existence of peculiarities that render these group of tumors even more “orphan” for pharmaceutical companies to develop and market specific- targeted drugs. The present review highlights key examples of molecular targets identification in bone sarcomas, reexamining the history of insulin-like growth factor receptor (IGF-IR) and its role in physiology and in cancer as well as developments regarding phase I to II clinical trials of agents directed against this receptor. The IGF system is quite complex, with many players in the field. Insulin receptor function in cancer cells has certainly been underestimated, but also little attention was paid to the type of ligands that are mainly involved in each tumor type. Strategies considering the system in its complex are encouraged and, in this context, drugs aimed at reducing circulating insulin levels, such as metformin, should receive attention as potential anti-cancer agents.

Myelodysplastic Syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis that often develop into acute myeloid leukemia (AML). MDSs are predominant in the elderly with an incidence of 20/100000 at 70 years of age. To date, the only curative treatment is allogeneic stem cell transplantation; however, a majority of patients are not eligible for this therapy. Azacitidine (AZA), a hypomethylating agent, remains the primary treatment for MDS patients, which leads to a significant increase in overall survival (OS), although it is not curative. Although it is well known that the impairment of apoptosis and differentiation are important features of this complex disease, the implication of autophagy in the pathogenesis of MDS is an emerging concept. Another significant advance in MDS pathogenesis research is the recent identification of mutations in genes encoding transcription factors implicated in hematopoiesis and proteins involved in splicing (SF3B1), methylation (DNMT3A), regulation of methylation (TET2 and IDH), DNA conformation (EZH2 and ASXL1) and differentiation (N- and K-RAS). Additionally, BCL2 family member expression and regulation may also affect the physiopathology of this disease. We have recently reported that targeting autophagy may be an interesting option for the treatment of AZA-resistant patients. Thus, targeting the products of the above-mentioned genes or the signaling pathways affected by the corresponding proteins may be of great interest for the development of a new arsenal of molecules to fight MDS. In this review, we discuss the new aspects of MDS physiopathology and how recent advances in MDS pathogenesis research may impact future treatments to improve the outcome of MDS patients.

The Culture-Repopulating Ability (CRA) assays is a method to measure in vitro the bone marrow-repopulating potential of haematopoietic cells. The method was developed in our laboratory in the course of studies based on the use of growth factorsupplemented liquid cultures to study haematopoietic stem/progenitor cell resistance to, and selection at, low oxygen tensions in the incubation atmosphere. These studies led us to put forward the first hypothesis of the existence in vivo of haematopoietic stem cell niches where oxygen tension is physiologically lower than in other bone marrow areas. The CRA assays and incubation in low oxygen were later adapted to the study of leukaemias. Stabilized leukaemia cell lines, ensuring genetically homogeneous cells and enhancing repeatability of results, were found nevertheless phenotypically heterogeneous, comprising cell subsets exhibiting functional phenotypes of stem or progenitor cells. These subsets can be assayed separately, provided an experimental system capable to select one from another (such as different criteria for incubation in low oxygen) is established. On this basis, a two-step procedure was designed, including a primary culture of leukaemia cells in low oxygen for different times, where drug treatment is applied, followed by the transfer of residual cell population (CRA assay) to a drug-free secondary culture incubated at standard oxygen tension, where the expansion of population is allowed. The CRA assays, applied to cell lines first and then to primary cells, represent a simple and relatively rapid, yet accurate and reliable, method for the pre-screening of drugs potentially active on leukaemias which in our opinion could be adopted systematically before they are tested in vivo.

Plants produce many low molecular mass natural compounds endowed with biological activity. Among them, resveratrol (3,5,4’-trihydroxystilbene) has been demonstrated to be able to affect a plethora of pivotal cellular molecular processes, including transduction pathways and gene expression. These activities result, in turn, in several different cell phenotypes. Particularly, frequent effects of resveratrol treatment appear to be the reduction of growth and the activation of programmed cell death. Accordingly, a number of trials are currently under development to evaluate the possibility of using resveratrol in cancer therapy, both as single agent or in association with other anticancer compounds. However, some reports suggest that, at low concentrations, not only resveratrol does not inhibit the proliferation and/or the survival of cells but, conversely, it induces proliferation and/or protects cells against toxic agents. On the basis of these biphasic effects, it has been proposed that resveratrol belongs to the so-called hormetic compounds. Hormesis is an expression employed by toxicologists to describe a U-shaped (or J-shaped) dose response characterized by a beneficial effect at low doses and a toxic (or inhibitory) activity at high dose. In this review, we will reappraise data that might suggest or disprove that resveratrol is endowed with clear hormetic properties.

Inorganic phosphate (Pi) is an essential nutrient to living organisms. It plays a key role in diverse biological processes, including osteoblast differentiation and skeletal mineralization. Maintenance of proper Pi homeostasis is a critical event, as any deviation from that state can lead to several acute and chronic disease states and influence the ageing process and lifespan. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, renal tubular reabsorption and depends mainly on the activity of Na/Pi cotransporters. Pi is abundant in the diet and intestinal absorption of Pi is efficient and minimally regulated. The kidney is a major regulator of Pi homeostasis and can increase or decrease its Pi reabsorptive capacity to accommodate Pi need. Relevantly, Pi is emerging as an important signalling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression and protein abundance in many cell types. However, little is known about the initial events involving the detection of changes in serum or local Pi concentrations and the subsequent downstream regulation cascade. Previously, we provided evidence that Pi inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. More recently, we demonstrated that Pi is capable also of inducing sensitization of osteosarcoma cells to doxorubicin in a p53-dependent manner and through a mechanism involving ERK1/2 down-regulation. This review summarizes the current knowledge regarding inorganic phosphate as a novel specific signaling molecule in bone and other cell types in mammals and discuss how targeting Pi levels at local sites might represent a potential strategy for improving osteosarcoma therapy.

Over the past few years, the SNAP-tag technology has become a methodology with great potential in a variety of applications, e.g. the (specific) visualization of individual proteins and studies of protein interaction in living cells. Furthermore, the tag can be used for immunopurification and detection of recombinant proteins or site-specific coupling of recombinant proteins to surfaces. Next to the in vitro applications, it also enables detection of tagged proteins in vivo. This review gives an overview of the SNAP-tag technology in different fields of research and its potential for future developments.

The SNAP-tag labeling technology provides a simple, robust, and versatile approach to the imaging of fusion proteins for a wide range of experimental applications. Owing to the specific and covalent nature of the labeling reaction, SNAP-tag is well suited for the analysis and quantification of fused target protein using fluorescence microscopy techniques. In this report, we present our most recent findings on the labeling of SNAP-tag fusion proteins both in vitro and in cell culture with SNAP-tag substrates derived from single regioisomers of carboxyrhodamine dyes. Carboxyrhodamines are invaluable fluorescent dyes for biotechnology applications including DNA sequencing, detection on microarrays, and fluorescence in situ hybridization. We found that SNAP-tag reacts preferentially with the 6-positional regioisomer of carboxyrhodamine fluorescent dyes, whereas the 5-regioisomer predominantly contributes to background fluorescence. Our experimental study also indicates that benzylchloropyrimidine (CP) conjugates of 6-carboxyrhodamines exhibit a dramatic increase in the signal-to-noise ratio of fluorescently labeled cellular proteins compared to the benzylguanine (BG) conjugates, presumably due to higher cell permeability. These new SNAP-tag substrates based on pure 6-regioisomers can significantly improve fluorescence labeling in live cells and should become powerful tools for bioimaging applications.

Display technologies are powerful tools that are widely used for selecting protein binders against different targets by mimicking natural selection. Such in vitro display methods have been pivotal in the isolation and evolution of antibodies with affinities greater than those produced by the natural immune response. Here we describe SNAP display, which covalently links genotype with phenotype by the action of the SNAP-tag in an emulsion microdroplet. Successful model selections suggest that SNAP display, has potential as a robust display platform suitable for selecting protein binders. A multivalent format of SNAP display using dendrimer-like DNA has also been established to probe avidity effects in multivalent protein display selections. This review describes the principles of SNAP display and extrapolates its scope for in vitro selections.

Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O6-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface® Alexa Fluor® 488 or SNAP-surface® Alexa Fluor® 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development.

In the past two decades, immense advances have been achieved in the engineering, production and purifying of recombinant proteins. These proteins are being widely utilized in many fields of biology, biotechnology and medicine, including diagnostic and therapeutic applications. These applications often require the modification or conjugation of these proteins with other molecules. Researchers are spending many efforts to develop and improve the methods of protein modifications. A main challenge they face is derivatizing proteins without affecting their structure and biological function. The conjugation methods available today include random and specific chemical modifications on endogenous amino acids or carbohydrate of the protein of interest. Other methods utilize self-labeling tags as fusion partners to the original protein enabling site-specific conjugation. SNAP-tag is one of the most promising self-labeling tags, which reacts specifically, rapidly and covalently with benzylguanine (BG) derivatives. SNAP-tag fusion proteins have been successfully used for imaging living cells. Recently, several studies have utilized the SNAP technology for generating antibody-based diagnostic and therapeutic tools. We here review these approaches and their possible impact on improving cancer targeting.

SNAP-tag technology has been an important tool for protein study for more than a decade and in the meanwhile has found a number of applications beyond the field of molecular biology and protein purification. Based on covalent interaction of SNAP-tag, 20 kDA mutant of DNA repair protein and benzylguanine, it enables irreversible and controllable protein modification. In this mini review, recent developments in the use of SNAP-tag for the design of protein arrays and nanoparticle biofunctionalization are presented and discussed. A short overview of other applications that paved the way to surface modifications is also given with emphasis on fluorescent labeling through the use of SNAP-tag fusion proteins. Finally, the future of the SNAP-tag methodology for surface patterning and 3D structural scaffolding is addressed.

Biosensors are used for a variety of applications in medicine and biology. A critical step during the development of such devices is the coordination of biological and technical requirements. The design of the device, as well as of the sample chamber and its functionalized surface is of great importance. Depending on the surface, the method of coupling of the desired receptor has to be adapted to guarantee functionality and biological activity during the measuring process. By using the SNAP-tag technology, a site-specific coupling of molecules with unaltered activity to a variety of O6-benzylguanine functionalized surfaces is possible, making it a versatile tool for the setup of biomedical devices.

Galectins,β-galactoside binding proteins, function in several physiological and pathological processes. The further evaluation of these processes as well as possible applications of galectins in diagnosis and therapy has raised high scientific interest. Therefore, easy and reliable test systems are necessary. Here we present the simple and cost-efficient production of recombinant human galectins as fusion proteins with SNAP-tag and fluorescent proteins. These constructs show binding specificities and oligomerisation properties generally comparable to recombinant galectins. Their direct fluorescence signal was utilised by ELISA-type assay and flow cytometry analysis with human and ovine mesenchymal stem cells (MSC). Flow cytometry demonstrated glycan mediated binding of His6-SNAP-YFP-Gal- 3 to both MSC types, which was specifically inhibited by lactose. Moreover, directed immobilisation by SNAP-tag technology onto benzylguanine- activated sepharose was utilised to prepare galectin affinity columns for glycoprotein analysis and purification. The SNAPtag directed coupling yielded up to three-fold higher binding capacities for the glycoprotein standard asialofetuin compared to nondirected coupled galectin suggesting improved functionality following directed coupling.