Recent Patents on Anti-Cancer Drug Discovery - Volume 6, Issue 3, 2011

The Plasma Membrane: A Catalyst in the Decision to Die or not to Die? DEATH RECEPTOR-MEDIATED CELL DEATH Induction of cell death plays a crucial role in morphogenesis, homeostasis, immune tolerance and surveillance and chemotherapy. Supernumerary, damaged, transformed, or infected cells can be eliminated through intrinsic or extrinsic cell death programs. The induction of the extrinsic signal occurs when cytokines (i.e., CD95L, TRAIL, TNF-α) present in serum or anchored in immune cells (e.g., T-lymphocytes, natural killer cells) bind to their respective death receptors, namely CD95 (also called APO-1 or Fas), DR4 and DR5 or TNF-R1. Interaction with the ligand orchestrates aggregation and conformational alteration of the death receptors, whose intracellular domains recruit adaptor proteins (i.e., TRADD, FADD), which in turn drive, through protein/protein interactions, the induction of caspases evoking the death program. PLASMA MEMBRANE AND “DEATH RECEPTOR” SIGNALING PATHWAYS The extensive characterization of the protein/protein interactions promoting the ignition of the apoptotic signal, led to underestimate the role played by the lipid bilayer plasma membrane in the signaling induced by death receptors. In this regard, death receptors are anchored into a 3-D support, the plasma membrane, which exhibits a complex structure and exerts constraints affecting receptor motility, aggregation, conformation and consequently signaling. Recent evidences established that not only the lipid composition of the membrane bilayer, but also the partition of the death receptors into subdomains designated lipid rafts, detergent-resistant membranes (DRMs), or merely microdomains may affect the transmission of the apoptotic pathway. For instance, distribution of CD95 into lipid rafts dramatically enhances the induction of the signal [1-6]. More strikingly, not only the ligand fixation is able to redistribute CD95 into lipid rafts, but also different anti-tumoral agents have been reported to achieve the partition of CD95 into large platforms constituted of aggregated lipid rafts [2, 7-12]. It is noteworthy that the reorganization of CD95 into DRMs can occur independently of its ligand upon addition of certain chemotherapeutic drugs (e.g., rituximab [13], resveratrol [9, 14], edelfosine [7, 11], aplidin [15], perifosine [16], cisplatin [12]). The molecular mechanisms that underlie this process remain to be elucidated. Nevertheless, the current evidence let us to envision that intracellular signal(s) or modulation of the plasma membrane biophysical properties mimic the death cytokinedriven initial events [17]. It is noteworthy that designation of these receptors as “death receptors” originated from initial studies that were seeking for apoptotic inducers [18, 19]. However, this appellation leads to a misunderstanding since all death receptors are able to induce non-apoptotic signals that, in certain context, promote carcinogenesis [20-23]. While theoretically speaking, decrease in the apoptotic threshold has been reported to switch the CD95 signal from a non-apoptotic to an apoptotic signaling pathway [24- 26], it remains to identify the molecular mechanism(s) underlying this phenomenon. Endocytosis of CD95 may reduce the apoptotic threshold and thereby, may discriminate between apoptosis and non-apoptosis signaling. Indeed, cells harboring a mutation in the AP-2-binding motif of CD95 (e.g., Y291F) were not only unable to internalize the death receptor and to transmit the apoptotic signal, but they also continued to induce non-apoptotic signals in the presence of CD95L [27]. In this topic issue, Milosavljenic and colleagues reported that endocytosis relies on the forces applied on the membrane and thus, on the composition of the membrane itself. Consequently, we may envision that the plasma membrane composition, which diverges between normal and tumor cells, controls the fate of the CD95 signal, even if the role of endocytosis in death receptor signaling remains controversial [28, 29]. Likewise, the partition of CD95 into aggregated lipid rafts, whose micrometer-sized structure accumulates and/or excludes critical death modulators, may alternatively contribute to modulating the apoptotic threshold and thus, the cell fate. The partition of death receptors together with downstream apoptotic signaling molecules in aggregated DRMs [15, 16, 30-32] has led to the emerging concept of “liquid-ordered” plasma membrane platform designated as “cluster of apoptotic signaling molecule-enriched rafts” (CASMER) [33]. These CASMERs may reduce the apoptotic signal threshold by stabilizing protein/protein interactions and thereby, catalyze the transmission of the apoptotic signal [33]. BIOPHYSICAL PROPERTIES OF THE PLASMA MEMBRANE AND “DEATH RECEPTOR” SIGNALING PATHWAYS Another parameter regulating the biophysical properties of plasma membrane and its composition is the intracellular pH, which in turn alters the induction of the apoptotic signal induced upon death receptor engagement. Indeed, reduction of the intracellular pH not only modulates the plasma membrane composition by activating acidic sphingomyelinase, which in turn generates ceramides, but also promotes protonation of the lipid polar heads present in the inner leaflet. This latter effect reduces membrane packing and tension, which both influence drug permeation and membrane endocytosis, affecting the amounts of chemotherapeutic drugs retained in tumor cells and the death receptor signaling, respectively. In addition, numerous pollutants alter the biophysical properties of the plasma membrane through the production of reactive oxygen species (ROS), which in turn enhance lipid peroxidation [34], activate acid sphingomyelinase [35], and modulate gene expression involved in lipid metabolism, and thus modify plasma membrane composition......

Cholesterol- and sphingolipid-rich membrane domains, termed lipid rafts, have been recently involved in the triggering of death receptor-mediated apoptosis. The alkyl-lysophospholipid analogue edelfosine was the first antitumor drug reported to induce apoptosis in cancer cells through co-clustering of lipid rafts and Fas/CD95 death receptor. Recruitment and aggregation of Fas/CD95 in lipid raft clusters was independent of its cognate ligand FasL/CD95L, and could be pharmacologically modulated. The adaptor molecule Fas-associated death domain protein (FADD) and procaspase- 8 were also recruited into lipid rafts following edelfosine treatment, forming the death-inducing signaling complex (DISC), and hence these membrane microdomains can act as scaffolds for Fas/CD95 death signaling. Edelfosine accumulated in lipid rafts of cancer cells, altering raft protein and lipid composition. Subsequently, an increasing number of antitumor drugs have been found to induce apoptosis through recruitment of Fas/CD95 into membrane rafts, and some of these compounds accumulated in raft membrane domains. Additional downstream apoptotic signaling molecules have also been reported to be recruited into rafts following treatment of cancer cells with antitumor agents, thus facilitating proteinprotein interactions and conveying apoptotic signals. On these grounds, lipid rafts have become an appealing and promising target for therapeutic intervention in cancer chemotherapy. Co-clustering of lipid rafts and Fas/CD95 signaling provides a new insight in the regulation of death receptor-mediated apoptosis, opening a new avenue in cancer therapy. In this regard, an increasing number of patents are dealing with the above insights in order to improve cancer treatment.

It is well known that tumor formation arises from the imbalance between cell death and proliferation. For many years, cancer research has engaged an important part of its efforts to find new therapeutic strategies based on cell death induction. One of the predominant ways to kill tumor cells is to trigger apoptosis by chemotherapy. However tumor responsiveness to chemotherapy is dependent on different biological factors including cancer types, genetics and pharmacogenetics. Although, molecular mechanisms involved in chemotherapy-induced apoptosis are diverse and depend on celltype and drugs used, a common pathway leading to tumor cell death has been shown to implicate the generation of a simple cellular sphingolipid, ceramide. Ceramide is released by the activity of neutral or acidic sphingomyelinases or de novo synthesis during treatment with chemotherapy. This review in particular focuses on enzymes involved in chemotherapyinduced cell death such as neutral or acidic sphingomyelinases and ceramide synthases, the role of ceramide in cellular effects of chemotherapy at the plasma membrane or the mitochondria and the induction of cell death by ceramide. It also includes recent advances on novel patented sphingolipid compounds and cancer therapeutic strategies based on ceramide release.

The death receptors CD95, TRAILR1 and TRAILR2 induce cell death in many types of tumor cells. Activation of these receptors has received considerable interest due to its potential use in cancer therapy. In particular the observation that most primary cells are not or only barely TRAIL-sensitive resulted in the development of targeted therapy concepts that base on activation of the TRAIL death receptors by recombinant TRAIL or agonistic antibodies. Indeed, a variety of preclinical studies and several phase I and II clinical trials show that activation of TRAIL death receptors effectively induces apoptosis in cancer cells in vivo without therapy-limiting toxicity on normal cells. Primary tumor cells are often sparsely sensitive for TRAIL death receptor-mediated apoptosis or acquire resistance during therapy. Sensitization/ resensitization of tumor cells by chemotherapeutic drugs or radiation can therefore be necessary for TRAIL-based therapies, but this involves the danger of triggering side effects related to the breakage of apoptosis resistance of nontransformed cells. Thus, there is a foreseeable need to develop optimized combination therapies or to locally restrict TRAIL receptor activation to fully exploit the antitumoral potential of TRAIL death receptors in the clinic. Although the high sensitivity of hepatocytes for CD95-mediated apoptosis prohibits therapies resulting in systemic activation of CD95, several studies have shown that this limitation can be overcome by ex vivo treatment regimes or by CD95 activating agonists with cell type-specific activity. This patent review is focused on the death receptor agonists currently under consideration in clinical trials, but also addresses the hurdles that have to be cleared to broaden and to improve the applicability of the currently used clinical concepts related to death receptor activation.

The use of TRAIL/APO2L and monoclonal antibodies targeting TRAIL receptors for cancer therapy holds great promise, due to their ability to restore cancer cell sensitivity to apoptosis in association with conventional chemotherapeutic drugs in a large variety of tumors. TRAIL-induced cell death is tightly regulated right from the membrane and at the DISC (Death-Inducing Signaling Complex) level. The following patent and literature review aims to present and highlight recent findings of the deadly discussion that determines tumor cell fate upon TRAIL engagement.

Resistance to death receptor ligands (such as FasL and TRAIL) and anticancer treatments is a hallmark of cancer cells. Ceramide, a biologically active sphingolipid, antagonizes cell growth and promotes apoptosis and non-apoptotic forms of cell death. The intracellular levels of ceramide are highly regulated via complex metabolic pathways. Sphingomyelin synthases (SMS) 1 and 2 convert ceramide to sphingomyelin (SM), a ubiquitous phospholipid in mammals. A growing body of evidence in the literature indicates that SMSs likely modulate hematological cell growth and sensitivity to stress-induced apoptosis. On one hand, complete and sustained inhibition of SMS activity is likely to alter membrane composition and properties through membrane SM depletion, perturbing intracellular signaling pathways and leukemia cell growth and conferring partial resistance to death receptor ligands. On the other hand, different patents & reports point to anti-apoptotic functions for SMSs. In patients with chemoresistant leukemia, a decreased intracellular ceramide level was associated with a higher SMS activity. Thus, SMSs and cofactors may constitute original pharmacological targets to treat leukemia.

“How do drugs cross the plasma membrane?” this may seem like a trivial question. This question is often overlooked to focus primarily on the different complex macro-molecular aspects involved in drug delivery or drug resistance. However, recent studies have highlighted the theme that to be fully understood, more knowledge of the underlying biology of the most complex biological processes involved in the delivery and resistance to drugs is needed. After all, why would a drug interact with a transporter then subsequently be excluded from P-glycoprotein (P-gp) expressing drug resistant cells? What are the determinants of this transition in behavior? Full consideration of the physical biology of drug delivery has allowed a better understanding of the reasons why specific membrane proteins are upregulated or overexpressed in drug resistant cells. This, in turn, allows us to identify new targets for drug chemicals. Better yet, it increases the significance of recents patents and underlines their importance in multi drug resistance.

In the last decade, a lot of patents have been filled regarding molecular biology and functions of cellular membranes. The membrane bilayer model has evolved from a static, passive, homogeneous barrier to a highly dynamic, asymmetric, heterogeneous structure composed of distinct domains. Changes in membrane fluidity and composition of microdomains have been proven to be involved in the regulation of many important physiological signaling pathways. Recently, several xenobiotics, including various drugs and environmental pollutants, have been reported to change plasma membrane characteristics, thereby altering cell physiology. Interestingly, it has been suggested that a cross talk between chemical-induced cellular membrane effects and DNA damages may be important for the final mutation outcome of genotoxic chemicals. Thus, effects on plasma membrane remodeling may give additional mechanistic explanations to how certain chemicals exert their carcinogenic effect. With respect to such effects, recent patents suggest to focus on plasma membrane and its components like caveolin-1 for cancer screening and chemotherapy. Here, we review the effects of environmental toxicants on cellular plasma membrane structure and function, and further describe possible implication for health and disease.

Acute myeloid leukemia is a heterogeneous group of diseases accounting for 15-20% of all tumors diagnosed in children under 15 years of age. The past few decades have yielded remarkable improvements in long-term outcomes for children with acute myeloid leukemia. A better risk-group stratification of patients based upon clinical and biologic features, a more effective use of anti-leukemic agents and enormous improvements in supportive care have increased the probability of cure by approximately 60%. The increase in our understanding of the biology of this disease has resulted in the development of molecularly targeted therapies that are potentially more effective and less toxic than the standard approaches. We here review novel molecularly targeted drugs for the treatment of childhood acute myeloid leukemia such as monoclonal antibodies, inhibitors of signalling molecules, proteasome inhibitors and epigenetic agents. For these recently patented agents, we also provide a detailed analysis of the published preclinical data and the clinical trials that have been completed.

Since tumors cannot grow or spread without forming new blood vessels, inhibiting angiogenesis is an excellent approach for the treatment of cancer. Further, inhibitors of angiogenesis have mild side effects since they act on endothelial cells, which eliminate the possibility of developing resistance or tolerance in tumor cells, unlike that seen with chemotherapy drugs. The anti-vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab acts by preventing new blood vessel formation in solid tumors and is approved by FDA to treat colorectal, lung, breast, glioblastoma and kidney cancers. The registration of this drug and its ongoing success in the clinic has validated the targeting of angiogenesis as an important approach to the treatment of solid tumors. Apelin is a novel angiogenic factor and recent studies indicate that apelin promotes angiogenesis, lymphangiogenesis and tumor growth in vivo and the angiogenic potential of apelin is similar to that of VEGF. Also, apelin expression is upregulated and has been shown to be associated with clinical outcome in certain human cancers. Thus, inhibition of apelin activity might lead to a new class of anti-angiogenesis drugs which should be more efficacious than those currently on the market due to their ability to be both anti-angiogenic as well as anti-lymphangiogenic. There are very few patents on the angiogenic effects of apelin and this review article focuses on these patented claims related to inhibiting apelin signaling and sheds more light on how blocking apelin signaling might open doors to a new class of angiogenic inhibitors.

Docetaxel has until recently been the only agent with a small survival benefit for metastatic castration-resistant prostate cancer (CRPC). To improve clinical outcome in CRPC, numerous classes of drugs targeting specific pathways involved in hormone action, bone metabolism, angiogenesis, apoptosis and immune response have currently been investigated concerning the efficacies of either single agents or combinations with docetaxel. Noteworthy, current two phase III trials of cabazitaxel and sipuleucel-T have demonstrated significant improvements of overall survival in CRPC. From the viewpoint of complexity of mechanisms implicated in prostate cancer progression, effective therapeutic strategies should be developed by multifaceted approaches, such as the composition of novel agents targeting for key molecules, cytotoxic chemotherapy, and immunotherapy. The recent patented molecules (e.g., hyaluronidase, caveolin, Bag1-L, N-cadherin, AR splicing variants, PCGEM-1) have a strong potential as therapeutic options for CRPC. Here, we review the newest evidence of novel agents and patented compounds and methods for the purpose of the future use in CRPC.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Especially in this disease, qualitative and quantitative aspects render the dysregulated epidermal growth factor receptor (HER1/EGFR) an outstanding therapeutic target. A variety of therapeutic compounds was developed to target HER1/EGFR among which the clinically most advanced agents are small molecule tyrosine kinase (TK) inhibitors. Unfortunately, clinical studies examining their therapeutic efficacy have so far failed to document a major therapeutic break-through in the setting of GBM. Thus, the targeted approach against HER1/EGFR likely requires a synergistic drug combination strategy to ultimately become successful in this disease. This patents review focuses on innovative therapeutic strategies combining HER1/EGFR-targeted TK inhibitors with novel agents which for the most part have not been evaluated for the treatment of GBM yet but which constitute interesting candidates for further evaluation in this setting.

The patents annotated in this section have been selected from various patent databases. These recent patents are relevant to the articles published in this journal issue, categorized by therapeutic areas/targets and therapeutic agents related to anti-cancer drug discovery.....