Current Drug Targets - Volume 9, Issue 8, 2008

More than half of all human proteins are located in (i.e. receptors and ion channels) or on (i.e. lipid-binding peripheral proteins) cellular membranes. These proteins function in cell signaling and membrane trafficking events to maintain cellular metabolism, growth, differentiation, and homeostasis. Thus, cellular membranes are a platform for intracellular communication where different extracellular signals regulate assembly of lipid-protein and protein-protein complexes. The metabolism of lipids by lipid phosphatases, kinases, phospholipases and more enzymes provides cellular membranes with a vast variety of lipid headgroups and acyl chains for spatial and temporal recruitment of peripheral proteins to specific membrane sites in cells. The interactions between lipids and target proteins are of generally high affinity and specificity and most often mediated by modular lipid-binding domains [1, 2]. These small domains (∼60-220 amino acids) have unique surface characteristics such as charge and hydrophobic moieties to dock onto different lipid membranes. These lipid-binding sites may serve as a ‘hot spot’ for drug development aimed at inhibiting protein function through abrogation of membrane binding. This is in contrast to the common target site of most enzyme inhibitors, the catalytic site, and may provide a more selective means of inhibiting or activating function of the lipid-dependent proteins. Recently, this concept was validated with in silico structure-based virtual ligand screening, which identified several lipid-binding inhibitors of a C2 domain [3]. Along with inhibition of lipid binding the different regions of cellular membranes (i.e. polar and nonpolar) represent target sites of potential therapeutic agents that could specifically disrupt or accentuate lipid-protein interactions within the bilayer [4]. The rapidly expanding field of interdisciplinary research surrounding biomembranes warrants a timely overview of lipid-dependent drug targets as well as strategies of targeting their inhibition and/or activation. Thus, this edition of Current Drug Targets is intended to bring together recent findings and novel concepts about peripheral proteins and their potential as drug targets. In the introductory article, the Stahelin lab provides an overview of the structure and function of lipid-protein interactions highlighting important peripheral protein drug targets and strategies of targeting both the peripheral proteins and membranes. The introductory article is followed by three reviews centering on the lipid second messenger diacylglycerol (DAG). DAG signals act as modulators of cell growth and apoptosis through interactions with a zinc finger DAG recognition motif known as the C1 domain. This is first addressed by Christine Gould and Alexandra Newton as they provide a wonderful overview of the DAG binding protein kinase C (PKC) family of proteins and their regulation by lipids, phosphorylation, scaffolding proteins and ubiquitin. They also describe current and future strategies of targeting PKCs and their regulatory proteins. Next, Fumio Sakane and colleagues highlight the mechanisms of diacylglycerol kinase (DGKs) regulation and their metabolism of DAG to phosphatidic acid. The authors discuss with keen insight the basis for DGKs as drug targets in diseases such as cancer, epilepsy, and autoimmune diseases. In the final installment of DAG signaling Peter Blumberg and colleagues present a seminal article on targeting the DAG binding C1 domain in drug development. Here the authors present the potential of the C1 domain as a drug target and draw attention to current therapeutics in clinical trials aimed at C1 domain targeting. Sphingolipids are a class of bioactive lipids that have received much attention in the last two decades as important regulators of cancer. In fact, enzymes associated with sphingolipid synthesis such as sphingosine kinase 1 (SK) [5] and ceramide kinase (CerK) [6] have been classified as peripheral proteins due to their ability to bind cellular membranes. Thus, three articles are included here to highlight the dynamic field of sphingolipids and some of the important drug targets involved in sphingolipid synthesis and/or degradation. In the first article, Yusuf Hannun, Youssef Zeidan and colleagues discuss the molecular targeting of acid ceramidase in cancer therapy. Acid ceramidase is a lipid hydrolase that is able to regulate ceramide and sphingosine-1- phosphate levels, traditionally two bioactive lipids in cancer among other diseases. Hannun, Zeidan and colleagues outline this enzyme from the gene to the protein in an astute and timely fashion. Next, Sarah Spiegel, Dai Shida and colleagues summarize SK and its role in cancer. The authors carefully and creatively address the properties of SK, its role in different types of cancers, and current inhibitor and clinical trial compounds of SK activity.

Interdisciplinary research focused on biological membranes has revealed them as signaling and trafficking platforms for processes fundamental to life. Biomembranes harbor receptors, ion channels, lipid domains, lipid signals, and scaffolding complexes, which function to maintain cellular growth, metabolism, and homeostasis. Moreover, abnormalities in lipid metabolism attributed to genetic changes among other causes are often associated with diseases such as cancer, arthritis and diabetes. Thus, there is a need to comprehensively understand molecular events occurring within and on membranes as a means of grasping disease etiology and identifying viable targets for drug development. A rapidly expanding field in the last decade has centered on understanding membrane recruitment of peripheral proteins. This class of proteins reversibly interacts with specific lipids in a spatial and temporal fashion in crucial biological processes. Typically, recruitment of peripheral proteins to the different cellular sites is mediated by one or more modular lipid-binding domains through specific lipid recognition. Structural, computational, and experimental studies of these lipid-binding domains have demonstrated how they specifically recognize their cognate lipids and achieve subcellular localization. However, the mechanisms by which these modular domains and their host proteins are recruited to and interact with various cell membranes often vary drastically due to differences in lipid affinity, specificity, penetration as well as protein-protein and intramolecular interactions. As there is still a paucity of predictive data for peripheral protein function, these enzymes are often rigorously studied to characterize their lipid-dependent properties. This review summarizes recent progress in our understanding of how peripheral proteins are recruited to biomembranes and highlights avenues to exploit in drug development targeted at cellular membranes and/or lipid-binding proteins.

Protein kinase C (PKC) is a family of kinases that plays diverse roles in many cellular functions, notably proliferation, differentiation, and cell survival. PKC is processed by phosphorylation and regulated by cofactor binding and subcellular localization. Extensive detail is available on the molecular mechanisms that regulate the maturation, activation, and signaling of PKC. However, less information is available on how signaling is terminated both from a global perspective and isozyme-specific differences. To target PKC therapeutically, various ATP-competitive inhibitors have been developed, but this method has problems with specificity. One possible new approach to developing novel, specific therapeutics for PKC would be to target the signaling termination pathways of the enzyme. This review focuses on the new developments in understanding how PKC signaling is terminated and how current drug therapies as well as information obtained from the recent elucidation of various PKC structures and down-regulation pathways could be used to develop novel and specific therapeutics for PKC.

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating (consuming) DAG to yield PA. Ten mammalian DGK isozymes have been identified to date. In addition to two or three cysteine-rich C1 domains (protein kinase C-like zinc finger structures) commonly conserved in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile α motif domain, a MARCKS (myristoylated alaninerich C kinase substrate) phosphorylation site domain and ankyrin repeats. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of mammalian signal transduction pathways conducting growth factor/ cytokine-dependent cell proliferation and motility, seizure activity, immune responses, cardiovascular responses and insulin receptor-mediated glucose metabolism. It is suggested that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes. Unfortunately, there are no DGK isozyme-specific inhibitors/activators at present. Development of these compounds is eagerly awaited for the development of novel drugs targeting DGKs.

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand - C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand - C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.

Increasingly recognized as bioactive molecules, sphingolipids have been studied in a variety of disease models. The impact of sphingolipids on cancer research facilitated the entry of sphingolipid analogues and enzyme modulators into clinical trials. Owing to its ability to regulate two bioactive sphingolipids, ceramide and sphingosine-1-phosphate, acid ceramidase (AC) emerges as an attractive target for drug development within the sphingolipid metabolic pathway. Indeed, there is extensive evidence supporting a pivotal role for AC in lipid metabolism and cancer biology. In this article, we review the current knowledge of the biochemical properties of AC, its relevance to tumor promotion, and its molecular targeting approaches.

Sphingolipid metabolites have emerged as critical players in a number of fundamental biological processes. Among them, sphingosine-1-phosphate (S1P) promotes cell survival and proliferation, in contrast to ceramide and sphingosine, which induce cell growth arrest and apoptosis. These sphingolipids with opposing functions are interconvertible inside cells, suggesting that a finely tuned balance between them can determine cell fate. Sphingosine kinases (SphKs), which catalyze the phosphorylation of sphingosine to S1P, are critical regulators of this balance. Of the two identified SphKs, sphingosine kinase type 1 (SphK1) has been shown to regulate various processes important for cancer progression and will be the focus of this review, since much less is known of biological functions of SphK2, especially in cancer. SphK1 is overexpressed in various types of cancers and upregulation of SphK1 has been associated with tumor angiogenesis and resistance to radiation and chemotherapy. Many growth factors, through their tyrosine kinase receptors (RTKs), stimulate SphK1 leading to a rapid increase in S1P. This S1P in turn can activate S1P receptors and their downstream signaling. Conversely, activation of S1P receptors can induce transactivation of various RTKs. Thus, SphK1 may play important roles in S1P receptor RTK amplification loops. Here we review the role of SphK1 in tumorigenesis, hormonal therapy, chemotherapy resistance, and as a prognostic marker. We will also review studies on the effects of SphK inhibitors in cells in vitro and in animals in vivo and in some clinical trials and highlight the potential of SphK1 as a new target for cancer therapeutics.

Ceramide kinase (CERK) was discovered more than a decade ago. Since then, numerous reports have been published demonstrating a role for CERK in various signal transduction pathways involved in inflammation, immunity or cancer. In this review, the biosynthesis of ceramide-1-phosphate (C1P) and the various roles of CERK and C1P in biological mechanisms will be overviewed. We will focus on the role of C1P in eicosanoid synthesis, more specifically, in the activation and translocation of cPLA2α. Furthermore, the possible therapeutic relevance of inhibitors of these mechanisms is discussed.

Group VI phospholipase A2 (PLA2) is a family of acyl hydrolases that targets the sn-2 fatty acid on the glycerophospholipid (GPL) backbone. These enzymes are grouped together based on structural homologies and catalytic activities that are independent of calcium and hence are also called the iPLA2s. Although the best characterized of these enzymes, iPLA2β and iPLA2γ, have long been proposed as homeostatic enzymes involved in basal GPL metabolism, recent studies indicate roles for these enzymes in biomedically relevant processes as well. For example, iPLA2 modulates calcium homeostasis by promoting replenishment of intracellular calcium stores. This function is likely of importance in the pathogenesis of Duchenne muscular dystrophy and potentially allergy as well. iPLA2 has a variety of roles in bacterial pathogenesis and the host response against bacterial and fungal infections. These characteristics suggest that the enzyme as a potential target to control infectious diseases. iPLA2 is linked to both proliferation and chemotherapy-induced apoptosis of tumor cells. As such, the enzyme is a potential target for cancer chemotherapy. Recent studies indicate essential roles for iPLA2 in glucose homeostasis, maintenance of energy balance, adipocyte development, and hepatic lipogenesis. Thus, the enzyme is an attractive target for drugs to control type II diabetes, fatty liver disease, and other manifestations of the metabolic syndrome. Several recent studies have associated iPLA2 inactivation with neurodegenerative diseases, suggesting the possibility that products of the iPLA2 reaction as potential treatments for these disorders. Together, these observations suggest iPLA2 as a novel and important target for drug development. However given the ubiquitous expression of the enzyme and its roles in basal GPL metabolism, drug strategies targeting iPLA2 must exhibit exquisite selectivity to avoid undesired side effects. Furthermore, the cell-specific nature of many iPLA2 functions may present another challenge in the design and implementation of drugs targeted to the enzyme.

Lysophosphatidic acids (LPAs) are structurally simple lipid phosphate esters with a widely appreciated role as extracellular signaling molecules. LPA binds to selective cell surface receptors to promote cell growth, survival, motility and differentiation. Studies using LPA receptor knockout mice and experimental therapeutics targeting these receptors identify roles for LPA signaling in processes that include cardiovascular disease and function, angiogenesis, reproduction, cancer progression and neuropathic pain. These studies identify considerable functional redundancy between these receptors and raise the possibility that additional lysophosphatidic acid receptors remain to be identified. LPA is present in the blood and other biological fluids at physiologically relevant concentrations and can likely be rapidly generated and degraded in different locations, for example at sites of inflammation, vascular injury and thrombosis or in the tumor micro environment. Recent work identifies a secreted enzyme, autotaxin (ATX), as the key component of an extracellular pathway for generation of lysophosphatidic acid by lysophospholipase D catalyzed hydrolysis of lysophospholipid substrates. In contrast to the apparently redundant functions of LPA receptors, studies using ATX knock out and transgenic mice indicate that this enzyme is uniquely required for LPA signaling during early development and serves as the primary determinant of circulating LPA levels in adult animals. Accordingly, pharmacological inhibition of ATX may be a viable and potentially effective way to interfere with LPA signaling in the cardiovascular system and possibly other settings such as tumor metastasis for therapeutic benefit. In this review we provide an update on recent advances in defining roles for LPA signaling in major disease processes and discuss recent progress in understanding the regulation and function of autotaxin focusing on strategies for the identification and initial evaluation of small molecule autotaxin inhibitors.

Hypertension is a serious medical problem affecting millions of people worldwide. A key protein regulating blood pressure is the Epithelial Na+ Channel (ENaC). In accord, loss of function mutations in ENaC (PHA1) cause hypotension, whereas gain of function mutations (Liddle syndrome) result in hypertension. The region mutated in Liddle syndrome, called the PY motif (L/PPxY), serves as a binding site for the ubiquitin ligase Nedd4-2, a C2-WW-Hect E3 ubiquitin ligase. Nedd4-2 binds the ENaC-PY motif via it WW domains, ubiquitylates the channel and targets it for endocytosis, a process impaired in Liddle syndrome due to poor binding of the channel to Nedd4-2. This leads to accumulation of active channels at the cell surface and increased Na+ (and fluid) absorption in the distal nephron, resulting in elevated blood volume and blood pressure. Compounds that destabilize cell surface ENaC, or enhance Nedd4-2 activity in the kidney, could potentially serve as drug targets for hypertension. In addition, recent discoveries of regulation of activation of ENaC by proteases such as furin, prostasin and elastase, which cleave the extracellular domain of this channel leading to it activation, as well as the identification of inhibitors that block the activity of these proteases, provide further avenues for drug targeting of ENaC and the control of blood pressure.