Current Medicinal Chemistry (v.19, #28)

Ligands for Fluorescence Correlation Spectroscopy on G Protein-Coupled Receptors by D. Jakobs, T. Sorkalla, H. Haberlein (4722-4730).
G protein-coupled receptors (GPCRs) comprise a large protein family of transmembrane receptors involved in many physiological processes. They are engaged in various transduction processes of extracellular signals into intracellular responses. Due to their involvement in numerous diseases they represent an important pharmacological target. Fluorescence correlation spectroscopy (FCS) poses a very sensitive analytical technique well-suited for the investigation of GPCRs. It is minimally invasive and operates on a single molecular level. It further provides detailed pharmacological information on receptor kinetics and quantities of activated receptors on the cell membrane. In addition, FCS allows distinguishing between different receptor states based on different diffusion time constants. In order to be applicable for FCS, the molecule of interest has to be fluorescently labeled. This review focuses on the physical requirements for dyes intended for FCS, their influence on the binding characteristics of coupled ligands and strategies to generate dye labeled ligands, exemplified on GPCR ligands.

Activatable Fluorescent Probes: A New Concept in Optical Molecular Imaging by E. Lacivita, M. Leopoldo, F. Berardi, N. A. Colabufo, R. Perrone (4731-4741).
Activatable fluorescent probes share the unique feature of being turned on only under specific conditions: they are "silent" when not interacting with a specific target protein, microenvironment, or reactive species. Several activatable fluorescence probes have demonstrated their potential in cell biology study, disease study and diagnosis, and even in the rapidly expanding field of image-guided surgery. In this review, we will summarize progress in the design of activatable probes and their application in studying cell biology or in optical imaging. Some of the most effective examples of activatable fluorescent probes will be presented and their application will be discussed.

Receptor-targeted optical imaging of cancer is emerging as an attractive strategy for early cancer diagnosis and surgical guidance. The success of such strategy depends largely upon the development of receptor-targeted fluorescent probes with high specificity and binding affinity to the target receptors. Recently, a host of such probes have been reported to target cancer-specific receptors, such as somatostatin receptors (SSTRs), integrin receptors, cholecystokinin-2 (CCK2) receptor, gastrin-releasing peptide (GRP) receptor, endothelin A (ETA) receptor, translocator protein (TSPO) receptor, epidermal growth factor (EGF) receptor, human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF) receptor, folate receptor (FR), transferrin receptor (TFR), low-density lipoprotein (LDL) receptors, type I insulin-like growth factor receptor (IGF1R), vasoactive intestinal peptide (VIP) receptors, urokinase plasminogen activator (uPA) and estrogen receptor (ER). This review will describe the recent advances in synthetic targeting optical imaging probes and demonstrate their in vivo imaging potentials. Moreover, current status of near infrared (NIR) fluorescent dyes, targeting moieties and coupling reactions, as well as strategies for designing targeted probes, will also be discussed.

Recent Advances in Optical Cancer Imaging of EGF Receptors by G. Kramer-Marek, M. R. Longmire, P. L. Choyke, H. Kobayashi (4759-4766).
Epidermal growth factor (EGF) receptors are commonly expressed on the cell membrane of cancer cells and activity of these receptors results in accelerated cell growth and carcinogenesis. A variety of targeted molecules have been developed to block ligand binding and/or inhibit the function of these receptor tyrosine kinases, and several have proven therapeutic benefits. Along with the advent of new therapeutic agents comes a need for non-invasive tools to diagnose, characterize, and monitor tumor responsiveness to therapy. Imaging EGF receptors with radionuclides has been performed for decades. However, recently this area has advanced considerably with the development of EGF receptor-targeted optical imaging probes. Herein, we review recent advances in molecular imaging of the EGF receptor family, focusing specifically on optical imaging. Such agents provide the opportunity for earlier diagnosis, improved tumor characterization, and the ability to measure and monitor tumor responsiveness to anti-EGF receptor treatment strategies.

The success of detecting cancer at early stages relies greatly on the sensitivity and specificity of in vivo molecular imaging. Optical imaging with near infrared (NIR) luminescent molecular nanoprobes currently attracts much attention because of many advantages of this imaging modality. It provides real time imaging with relatively inexpensive cost, produces images with high sensitivity and spatial resolution, and avoids exposure to ionizing irradiation. Raman spectroscopy/microscopy imaging with surface enhanced Raman scattering (SERS) nanoparticles allows scientists to detect biological events in living cells or organisms in real time and with high sensitivity. The photoacoustic imaging has emerged as a hybrid of optical and ultrasound imaging for sensitive and quantitative tumor detection. Given the recent advances in nanoscience and biomedicine, receptor-targeted NIR nanoprobes promise to improve the cancer early detection with relatively high sensitivity and specificity. We summarize various targeted NIR nanoprobes and their potential applications in cancer targeting and in vivo imaging and discuss the potential of multimodality imaging of NIR nanoprobes. With ongoing efforts to enhance their targeting ability and endow more functions, NIR nanoprobes hold great promise for clinical translation.

Noninvasive Fluorescence Imaging in Animal Models of Stroke by N. Stemmer, J. Mehnert, J. Steinbrink, A. Wunder (4786-4793).
Noninvasive fluorescence imaging (NFI) is a powerful tool to study physiology and pathophysiology in animal disease models. NFI has been successfully applied in a number of animal disease models including cancer, arthritis, and stroke. Furthermore, several applications in humans have been described. NFI is widely available in research laboratories because it has a number of advantages: It uses non-ionizing radiation and requires comparably simple, inexpensive instrumentation, and easy to handle. Fluorochromes can be detected with high sensitivity, and image acquisition time is relatively short. Furthermore, a plethora of fluorescent imaging agents is available including unspecific, target-specific, and activatable imaging probes. With these probes, biological processes such as inflammation, cell death or enzyme activity, and many others can be visualized in living animals. This review offers an overview of current approaches in NFI of stroke pathophysiology in animal models of cerebral ischemia. First, the instrumentation and the different types of imaging agents for NFI are described. Second, a short introduction to animal models of stroke is provided. Third, examples for NFI in animal models of stroke are given. Finally, the use of NFI in human stroke is critically discussed.

The Bioisosteric Concept Applied to Cannabinoid Ligands by C. Mugnaini, S. Pasquini, F. Corelli (4794-4815).
Bioisosterism is widely used in medicinal chemistry as an approach aimed at either rationally modifying a hit compound into a more potent and/or selective molecule or a lead compound into a more drug-like one. Two different cannabinoid receptors have been cloned from mammalian tissues, the CB1 receptor, mostly expressed in brain, and the CB2 receptor, mostly expressed in the immune system, both regulating a variety of physiological functions. Synthetic cannabinoids have been developed that act as highly selective agonists or antagonists/inverse agonists at one or other of these receptor types with the ultimate goal of modulating the endocannabinoid system. This review takes into account the use of the bioisosteric substitution in the field of cannabinoid ligands as a tool for improving both their pharmacodynamic and pharmacokinetic properties.

Ca2+ is a highly versatile intracellular second messenger in the central nervous system, and regulates many complicated cellular processes, including excitation, plasticity and apoptosis. Influx of Ca2+ from the extracellular fluid is required for sustained elevation of the cytosolic Ca2+ concentration and full activation of Ca2+-dependent processes. Voltage-dependent Ca2+ channels (VDCCs) serve as the principal routes of Ca2+ entry into electrically excitable cells such as neurons. The nervous system expresses VDCCs with unique cellular and subcellular distribution and specific functions. L-type voltage-dependent Ca2+ channels (L-VDCCs) are distributed at neuronal cell bodies, dendrites and spines, and the postsynaptic L-VDCCs regulate neuronal excitability and gene expression. Presynaptic P/Qand N-type VDCCs trigger neurotransmitter release, and T-type channels support neuronal rhythmic burst firing. Evidence from natural mutants, knockout mice, and human genetic disorders indicates a fundamental role of some VDCCs in a wide variety of neurological disorders, including vascular dementia (VaD), Alzheimer's disease (AD), Parkinson's disease (PD) and Prion disease. Amyloid β peptides, causative factors for AD, potentiate the influx of Ca2+ into neurons via L-VDCCs. L-VDCCs blockers prevent neurons from undergoing amyloid β-induced apoptosis. The present review highlights some recent findings on biochemical characterizations, physiological functions, pathological roles and pharmacological applications of the L-VDCCs and their implication in neurologic diseases.

Privileged structures bind to multiple receptors with high affinity, thus aiding the development of novel biologically active compounds. Indoles are classed as privileged structures, and as a result of the indole nucleus being present in a broad range of biologically active molecules, it has been suggested that indoles probably represent the most important of all structural classes in drug discovery. Amongst the indole class of compounds is a particular subset – 2-arylindoles – which appear to be a most promising lead for drug development. This review summarises the wide-ranging activities of 2-arylindoles and some of their important biological activities reported in the literature over the past two decades.

Modulation of Poly(A)-specific Ribonuclease (PARN): Current Knowledge and Perspectives by N. A.A. Balatsos, P. Maragozidis, D. Anastasakis, C. Stathopoulos (4838-4849).
Deadenylation is the exoribonucleolytic shortening of eukaryotic poly(A) tails. It is often the first and rate-limiting step for mRNA decay and translational silencing. The process is catalysed by a diversity of deadenylases, which provide robust and flexible means to control mRNA levels and gene expression. Poly(A)-specific ribonuclease (PARN) is a major mammalian deadenylase and the only known to concurrently bind the 5' cap-structure and the 3' poly(A), thus enhancing the degradation rate and amplifying its processivity. PARN is important during oocyte maturation, embryogenesis, early development, DNA damage, and in cell-cycle progression, but also in processes beyond mRNA metabolism, such as the maturation of snoRNAs. The enzyme also participates in nonsense-mediated mRNA decay and in the regulation of cytoplasmic polyadenylation. Importantly, PARN is involved in the degradation of several cancerrelated genes, while its expression is altered in cancer. Apart from the direct interaction with the cap structure, several strategies regulate PARN activity, such as phosphorylation, interaction with RNA-binding proteins (RBPs), and natural nucleotides. Recent studies have focused on the regulation of its activity by synthetic nucleoside analogues with therapeutic potential. In this context, the wide repertoire of RBPs and molecules that regulate PARN activity, together with the established role of deadenylases in miRNA-mediated regulation of mRNA expression, suggest that mRNA turnover is more complex than it was previously thought and PARN holds a key role in this process. In this review, we highlight the importance of PARN during RNA’s lifecycle and discuss clinical perspectives of modulating its activity.

Role of Oxidative Stress and Molecular Changes in Liver Fibrosis: A Review by V. Sanchez-Valle, N. C. Chavez-Tapia, M. Uribe, N. Mendez-Sanchez (4850-4860).
Liver fibrosis represents a health problem with significant morbidity and mortality that affects 100 million people worldwide. It is a final pathway to several chronic liver diseases and is characterized by excess collagen and accumulation of extracellular matrix in response to chronic hepatocellular damage. Clinical and experimental data suggest that oxidative stress (OS) mediates the progression of fibrosis, and that OS-related molecules may act as mediators of molecular and cellular events implicated in liver fibrosis. The generation of reactive oxygen species (ROS) plays an important role in producing liver damage and initiating hepatic fibrogenesis. OS disrupts lipids, proteins and DNA, induces necrosis and apoptosis of hepatocytes and amplifies the inflammatory response. ROS also stimulate the production of profibrogenic mediators from Kupffer cells and circulating inflammatory cells and directly activate hepatic stellate cells, resulting in the initiation of fibrosis. Advances in understanding the mechanisms involved in fibrosis have identified new molecular targets with therapeutic potential for more targeted and personalized control of this disease. This review will highlight recent concepts in OS, antioxidants and the molecular pathways involved in hepatic fibrosis.

New Clinical Perspectives of Hypolipidemic Drug Therapy in Severe Hypercholesterolemia by C. Stefanutti, C. Morozzi, S. Di Giacomo (4861-4868).
Patients with homozygous familial hypercholesterolemia (HoFH) represent the most severe patients within the spectrum of dyslipidemias. Untreated Low-Density Lipoprotein Cholesterol (LDL-C) levels in these patients are usually in the range 500 to 1200 mg/dL. Moreover, these patients exhibit a scarce responsiveness or even non responsiveness to oral lipid lowering agents. Patients with heterozygous familial hypercholesterolemia (HetFH) tend to have untreated LDL-C levels of 250-500 mg/dL. Many of these patients are responsive to 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA-reductase) inhibitors (statins) and/or other specific drugs. Unfortunately, a significant subset of these patients (5-10%) have a severe and/or refractory form of HetFH and after current maximal oral therapy, they remain significantly far from treatment goals (The National Cholesterol Education Program (NCEP) ATPIII guidelines). This would be defined as LDL-C levels of ≥ 190 mg/dL - prior Coronary Heart Disease (CHD) or CHD equivalent - or ≥ 250 mg/dL (no prior CHD or CHD risk-equivalent). The only current therapy option for these patients is Low Density Lipoprotein-apheresis (LDL_a). While LDL_a is very effective in reducing LDL-C, many patients do not receive this extracorporeal therapy because of costs and limited availability of LDL_a centers. Recently, new potent lipid-lowering drugs have been developed and are currently under investigation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role controlling the levels of LDL-C. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of the Low-Density Lipoprotein receptor (LDLR) protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels. Since the loss of a functional PCSK9 in human is not associated with apparent deleterious effects, this protease is becoming an attractive target for lowering plasma LDL-C levels either alone or in combination with statins. Mipomersen, an apolipoprotein B (ApoB) synthesis inhibitor, for lowering of LDL-C showed to be an effective therapy to reduce LDL-C concentrations in patients with HoFH who are already receiving lipid-lowering drugs, including high-dose statins. Lomitapide is a potent inhibitor of microsomal triglyceride transfer protein and is highly efficacious in reducing LDL-C and triglycerides (TG). Lomitapide is currently being developed for patients with HoFH at doses up to 60 mg/d. These new powerful lipid-lowering drugs might be possibly superior than available hypolipidemic agents. Their mechanisms of action, effectiveness, safety, and indication in severe, genetically determined dyslipidemias, are reviewed.

Steryl glycosides are derivatives of sterols where the 3β-hydroxy group is glycosylated. Some of them are further converted to steryl O-acyl glycosides. Steryl glycosides and their derivatives are widely distributed in plants, algae, and fungi, but are relatively rarely distributed in bacteria and animals. Accumulating evidence suggests that glycosylation of sterols not only modifies physicochemical properties of cell membranes but also alters immunogenicity of the cells. Helicobacter pylori, that colonizes the stomach and causes gastric diseases, is auxotrophic for cholesterol, so that it extracts this lipid from plasma membranes of epithelial cells of the host stomach. Since incorporation of cholesterol promotes immune responses of the host, Helicobacter pylori converts cholesterol to cholesteryl glucoside (ChG) and then to cholesteryl 6'-O-acyl glucoside (ChAcG) to evade the immune surveillance. We have found that ChAcG thus produced is specifically recognized by invariant Vα14-Jα18 TCR+ (Vα14) NKT cells in a CD1-dependent manner. We have also found that activation of Vα14 NKT cells by administration of ChAcG retains homeostasis of immunity upon exposure to allergens and reduces the incidence of allergy. In this article, overview of immunological functions of steryl glycosides with an emphasis on the immunoregulatory functions of ChAcG, is demonstrated.

The bi-aryl urea multi-kinase inhibitor Sorafenib (BAY 43-9006, Nexavar) was initially approved for the treatment of unresectable hepatocellular carcinoma and advanced renal cell carcinoma. Eleven years after its first description in PubMed, the therapeutic potential of Sorafenib has been evaluated in an increasing number of studies, mainly focused on solid tumors. More recently, the potential usefullness of Sorafenib has started to emerge also against hematological malignancies. At the molecular level, besides the RAF kinase pathway, which represents the first therapeutic target of Sorafenib, additional kinases, in particular the vascular endothelial growth factor receptor, have been identified as important targets of Sorafenib. A great interest for the potential use of Sorafenib against acute myeloid leukemia (AML) arose when it was demonstrated that a specific mutation of a kinase gene, called FMS-like tyrosin-kinase-3- internal tandem duplication (FLT-3-ITD) and occurring in more than 30% of AML, represents a molecular target of Sorafenib. However, recent phase I and II clinical studies showed that, in spite of its ability to suppress the activity of this mutated kinase, resistence to Sorafenib rapidly occurs in AML, suggesting that Sorafenib will be more effective in combined therapy than used as single drug. Another critical molecular target of Sorafenib is the anti-apoptotic protein Mcl-1. The ability of Sorafenib to rapidly shut-off Mcl-1 in virtually all the hematological malignancies investigated, including the B-chronic lymphocytic leukemia, represents a key element for its antileukemic activity as well as for therapeutic combinations based on Sorafenib. In this respect, it is of particular interest that many chemotherapeutic drugs or innovative anti-neoplastic compounds, such as recombinant TRAIL or inibitors of MDM2 protein, are either unable to down-regulate Mcl-1 or in some instances promote a paradoxical induction of Mcl-1. In this review, the growing evidences for the role of Mcl-1 in mediating the anti-leukemic activity of Sorafenib will be discussed in relationship with promising therapeutic perspectives.

Protocatechuic Acid Alkyl Esters: Hydrophobicity As a Determinant Factor for Inhibition of NADPH Oxidase by C. M.Q.G. de Faria, A. C. Nazare, M. S. Petronio, L. C. Paracatu, M. L Zeraik, L. O. Regasini, D. H.S. Silva, L. M. da Fonseca, V. F. Ximenes (4885-4893).
This study presents the increased efficiency of NADPH oxidase inhibition produced by esterification of protocatechuic acid (P0). Alkyl esters bearing chain lengths of 4 (P4), 7 (P7) and 10 (P10) carbons were synthesized and their oxidation potential, hydrophobicity, antiradical activity, inhibition of superoxide anion (O2 °-), and the abilities to affect hypochlorous acid (HOCl) production by leukocytes and inhibit myeloperoxidase (MPO) chlorinating activity were studied. The increased hydrophobicity (logP, 0.81-4.82) of the esters was not correlated with a significant alteration in their oxidation potential (0.222-0.298 V). However, except for P10, the esters were ~ 2-fold more effective than the acid precursor for the scavenging of DPPH and peroxyl radicals. The esters were strong inhibitors of O2 °- released by activated neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs). A correlation was found between the carbon chain length and the relative inhibitory potency. P7, the most active ester, was ~ 10-fold more efficient as NADPH oxidase inhibitor than apocynin. The esters strongly inhibited the release of HOCl by PMNs, which was a consequence of the inhibition of NADPH oxidase activity in these cells. In conclusion, as effective inhibitors of NADPH oxidase, the esters of protocatechuic acid are promising drugs for treatment of chronic inflammatory diseases. Moreover, this is the first demonstration that, besides the redox active moiety, the hydrophobicity can also be a determinant factor for the design of NADPH oxidase inhibitors.