Role of AMPK signaling in mediating the anticancer effects of silibinin in esophageal squamous cell carcinoma

Objective: Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK) may suppress cancer growth. Identification of novel AMPK activators is therefore crucial to exploit AMPK as a potential target for cancer prevention and treatment.

Research design and methods: We determined the expression status and role of AMPK in esophageal squamous cell carcinoma (ESCC) and investigated whether silibinin, a nontoxic natural product, could activate AMPK to inhibit ESCC development.

Results: Our results from 49 pairs of human ESCC and normal tissues showed that AMPK was constitutively inactive in the majority (69.4%) of ESCC. We found that silibinin induced apoptosis, and inhibited ESCC cell proliferation in vitro and tumorigenicity in vivo without any adverse effects. Silibinin also markedly suppressed the invasive potential of ESCC cells in vitro and their ability to form lung metastasis in nude mice. The anticancer effects of silibinin were abrogated by the presence of compound C or shRNA against AMPK. More importantly, silibinin enhanced the sensitivity of ESCC cells and tumors to the chemotherapeutic drugs, 5-fluorouracil and cisplatin.

Conclusions: This preclinical study supports that AMPK is a valid therapeutic target and suggests that silibinin may be a potentially useful therapeutic agent and chemosensitizer for esophageal cancer.     

The AMPK enzyme-complex: from the regulation of cellular energy homeostasis to a possible new molecular target in the management of chronic inflammatory disorders

Introduction: Adenosine monophosphate-activated protein kinase (AMPK), known as an enzymatic complex that regulates the energetic metabolism, is emerging as a pivotal enzyme and enzymatic pathway involved in the regulation of immune homeostatic networks. It is also involved in the molecular mechanisms underlying the pathophysiology of chronic inflammatory diseases.

Areas covered: AMPK is expressed in several immune cell types including macrophages, lymphocytes, neutrophils and dendritic cells, and governs a broad array of cell functions, which include cytokine production, chemotaxis, cytotoxicity, apoptosis and proliferation. Based on its wide variety of immunoregulatory actions, the AMPK system has been targeted to reveal its impact on the course of immune-related diseases, such as atherosclerosis, psoriasis, joint inflammation and inflammatory bowel diseases.

Expert opinion: The identification of AMPK subunits responsible for specific anti-inflammatory actions and the understanding of the underlying molecular mechanisms will promote the generation of novel AMPK activators, endowed with improved pharmacodynamic and pharmacokinetic profiles. These new tools will aid us to utilize AMPK pathway activation in the management of acute and chronic inflammatory diseases, while minimizing potential adverse reactions related to the effects of AMPK on metabolic energy.     

Exploiting the anti-inflammatory effects of AMP-activated protein kinase activation

Introduction: AMP-activated protein kinase (AMPK) is the downstream component of a serine/threonine protein kinase cascade involved in the regulation of metabolism. Many studies have also revealed that AMPK activation can exert significant anti-inflammatory and immunosuppressive effects in a variety of cell types and models of inflammatory/autoimmune disease. Because metformin, an AMPK activator that is a favored first-line therapeutic option for type 2 diabetes, may confer benefits in chronic inflammatory diseases and cancers independent of its ability to normalize blood glucose, there is now considerable interest in identifying and exploiting AMPK's anti-inflammatory effects.

Areas covered: The authors provide a background to AMPK signaling and describe the pro-inflammatory signaling pathways and processes shown to be regulated by AMPK activation.

Expert opinion: Identification of AMPK subunits responsible for specific anti-inflammatory effects, and a molecular understanding of the mechanisms involved, will be necessary to exploit AMPK pathway activation in acute and chronic inflammatory disease settings while minimizing adverse reactions due to deregulation of AMPK's wide-ranging effects on metabolism.     

Targeting the 5′-AMP-activated protein kinase and related metabolic pathways for the treatment of prostate cancer

Introduction: Increasing evidence suggests that prostate cancer cells undergo unique metabolic reprogramming during transformation. A master regulator of cellular homeostasis, 5′-AMP-activated protein kinase (AMPK), directs metabolic adaptation that supports the growth demands of rapidly dividing cancer cells. The utilization of AMPK as a therapeutic target may therefore provide an effective strategy in the treatment of prostate cancer.

Areas covered: Our review describes the regulation of AMPK by androgens and upstream kinases including the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in prostate cancer. Oncogenic, AMPK-regulated pathways that direct various metabolic processes are also addressed. Furthermore, we discuss the role of AMPK in growth arrest and autophagy as a potential survival pathway for cancer cells. In addition, by regulating non-metabolic pathways, AMPK may stimulate migration and mitosis. Finally, this review summarizes efforts to treat prostate cancer with pharmacological agents capable of modulating AMPK signaling.

Expert opinion: Current research is primarily focused on developing drugs that activate AMPK as a treatment for prostate cancer. However, oncogenic aspects of AMPK signaling calls for caution about employing such therapies. We think that inhibitors of CaMKK2 or AMPK, or perhaps the modulation of downstream targets of AMPK, will gain importance in the clinical management of prostate cancer.     

Novel strategies for anti-aging drug discovery

Introduction: Scientific achievements in the last few decades, leading to effective therapeutic interventions, have dramatically improved human life expectancy. Consequently, aging has become a significant problem and represents the major risk factor for most human pathologies including diabetes, cardiovascular diseases, neurological disorders, and cancer. Scientific discoveries over the past two decades have been instrumental in dissecting molecular mechanism(s) which play important roles in determining longevity. The same understanding has also led to the acknowledgement of the plurality of ‘causes’ which act either alone or in combination to create the condition which can be defined as ‘aging’.

Areas covered: Over the years, several concepts have been put forward for the development of a viable anti-aging regimen. In this review, the authors extensively review anti aging interventions based on caloric restriction, activation of telomerase, autophagy inducers, senolytic therapeutics, plasma membrane redox system (PMRS) activators, epigenetic modulators, and stem cell therapies.

Expert opinion: Based upon our current understanding, one of the most promising approaches for a successful anti-aging strategy includes the activation of adenosine monophosphate dependent protein kinase (AMPK). Another strategy may involve activation of PMRS. Future research efforts are likely to focus on nutrient and energy sensing molecular pathways which include mTOR, IGF-1, AMPK and the sirtuins.     

Screening methods for AMP-activated protein kinase modulators: a patent review

Introduction: AMP-activated protein kinase (AMPK) functions as a cellular energy gauge that maintains cellular homeostasis and has been suggested to play important roles in tumorigenesis, lifespan and autophagy. Accordingly, AMPK is a potential target of drugs for controlling a growing number of human diseases ranging from metabolic disorders to cancer, highlighting the need for rational and robust screening systems for identifying compounds that modulate AMPK.

Areas covered: The relevant screening methods in the patent and scientific literature were analyzed, and key features of direct AMPK modulators are discussed in the context of their physiological relevance and the three-dimensional structure of the AMPK complex.

Expert opinion: The mechanism of action of modulators is important in designing drugs with enhanced efficacy, specificity and stability. Most patented assay formats for identifying AMPK modulators are based on classical enzyme assays that monitor AMPK activity or changes in AMPK-dependent cellular physiology. However, these systems do not provide information about underlying mechanisms. Two patented assay systems use a specific domain or the three-dimensional structure of AMPK to identify AMPK modulators. The recent identification of two AMPK modulators, A-769662 and C-2 (or its prodrug, C-13), suggests the promise of structure-based assays in discovering more potent and specific modulators of AMPK.     

Focal adhesion kinase inhibitors in the treatment of metastatic cancer: a patent review

Introduction: Focal adhesion kinase (FAK) plays a prominent role in integrin signaling. FAK activation increases phosphorylation of Tyr397 and other sites of the protein. FAK-dependent activation of signaling pathways implicated in controlling essential cellular functions including growth, proliferation, survival and migration. FERM (F for the 4.1 protein, ezrin, radixin and moesin) domain-enhanced p53 degradation plays a critical role in proliferation and survival. FAK, overexpressed in metastatic tumors, has emerged as an important therapeutic target for the development of selective inhibitors. FAK inhibitors achieved tumor growth inhibition and induced apoptosis. Strategies targeting FAK inhibition using novel compounds have created an exciting opportunity for anticancer therapy.

Areas covered: This review summarizes the current research with available data from early phase clinical trials and discusses the available small-molecule inhibitors of FAK from patents. The importance of inhibiting FAK activity in cancer patients is discussed.

Expert opinion: Emerging data from clinical trials with orally available small-molecule inhibitors of FAK are promising. Although this approach is appropriate as a targeted therapeutic approach against several metastatic cancer types, several compounds in research are yet to prove their preclinical efficacy. This report lays special emphasis on the available patent data of FAK inhibitors for such targeted molecular therapies. This review summarizes current knowledge about FAK inhibition in cancer therapy.     

Targeting the liver kinase B1/AMP-activated protein kinase pathway as a therapeutic strategy for hematological malignancies

Introduction: Despite considerable advances, several hematological malignancies remain incurable with standard treatments. Therefore, there is a need for novel targeted and less toxic therapies, particularly for patients who develop resistance to traditional chemotherapeutic drugs. The liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling pathway has recently emerged as a tumor suppressor axis. A critical point is that the LKB1/AMPK network remains functional in a wide range of cancers and could be stimulated by drugs, such as N,N-dimethylimidodicarbonimidic diamide (metformin) or 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR).

Areas covered: The literature data show that drugs activating LKB1/AMPK signaling induced cell cycle arrest, caspase-dependent apoptosis or autophagy in hematopoietic tumors. Moreover, metformin effectively inhibited mammalian target of rapamycin complex 1 (mTORC1)-controlled oncogenetic protein translation, which does not occur with allosteric mTORC1 inhibitors, such as rapamycin and its derivatives. Metformin was also capable of targeting leukemic stem cells, the most relevant target for leukemia eradication.

Expert opinion: Data emerging from preclinical settings suggest that the LKB1/AMPK pathway is critically involved in regulating proliferation and survival of malignant hematopoietic cells. Thus, it is proposed that drugs activating the LKB1/AMPK axis may offer a novel and less toxic treatment option for some types of hematological malignancies.     

5-Lipoxygenase inhibitors: a review of recent patents (2010 – 2012)

Introduction: 5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) with the help of FLAP, the 5-LO-activating protein. LTs are inflammatory mediators playing a pathophysiological role in different diseases like asthma, allergic rhinitis as well as cardiovascular diseases and certain types of cancer. With the rising number of indications for anti-LT therapy, 5-LO inhibitor drug development becomes increasingly important.

Areas covered: Here, both recent findings regarding the pathophysiological role of 5-LO and the patents claimed for 5-LO inhibitors are discussed. Focusing on direct inhibitors, several patents disclosing FLAP antagonists are also subject of this review. Novel compounds include 1,5-diarylpyrazoles, indolizines and indoles and several natural product extracts.

Expert opinion: Evaluation of the patent activities revealed only quite moderate action. Nevertheless, several auspicious drug-like molecules were disclosed. It seems that in the near future, FLAP inhibitors can be expected to enter the market for the treatment of asthma. With the resolved structure of 5-LO, structure-based drug design is now applicable. Together with the identification of downstream enzyme inhibitors and dual-targeting drugs within the AA cascade, several tools are at hand to cope with 5-LOs increasing pathophysiological roles.     

A patent review of sirtuin activators: an update

Introduction : Reversible acetylation is a key post-translational modification of target proteins. Sirtuin deacetylases represent the homolog of the yeast silent information regulator (SIR2). Although seven sirtuins have been found in mammals, all sirtuin activators described to date act through SIRT1.

Areas covered : Areas covered in this paper include a review of the patent literature associated with SIRT1 activators, with a focus on therapeutic applications, primarily related to the use of pharmaceuticals and nutraceuticals containing resveratrol (RSV), and the development of second-generation activators unrelated to RSV. Also discussed is the current controversy over whether or not these molecules are actual SIRT1 activators.

Expert opinion : Developing effective strategies to protect against diet-induced metabolic imbalance is necessary to fight against current obesity rates. The hypothalamus is a candidate for developing drugs that suppress SIRT1 degradation, as a strategy for treating metabolic syndrome. Deciphering the basic mechanism of activators is essential to develop effective strategies to alter sirtuin activity. This is true regardless of the apparent controversy of whether in vitro activation of SIRT1 is direct or not, depending on the experimental design, and whether sirtuins may play a major role in longevity. The numerous studies on their positive effects against age-related diseases, obesity and other metabolic disorders are still valid, promising to positively influence the development of treatments to improve human health.     

The eIF2-alpha kinase HRI: a potential target beyond the red blood cell

Introduction: The eIF2α kinase heme-regulated inhibitor (HRI) is one of four well-described kinases that phosphorylate eIF2α in response to various cell stressors, resulting in reduced ternary complex formation and attenuation of mRNA translation. Although HRI is well known for its role as a heme sensor in erythroid progenitors, pharmacologic activation of HRI has been demonstrated to have anti-cancer activity across a wide range of tumor sub-types. Here, the potential of HRI activators as novel cancer therapeutics is explored.

Areas covered: We provide an introduction to eIF2 signaling pathways in general, and specifically review data on the eIF2α kinase HRI in erythroid and non-erythroid cells. We review aspects of targeting eIF2 signaling in cancer and highlight promising data using HRI activators against tumor cells.

Expert opinion: Pharmacologic activation of HRI inhibits tumor growth as a single agent without appreciable toxicity in vivo. The ability of HRI activators to provide direct and sustained eIF2α phosphorylation without inducing oxidative stress or broad eIF2α kinase activation may be especially advantageous for tolerability. Combination therapy with established therapeutics may further augment anti-cancer activity to overcome disease resistance.     

Protein kinase C inhibitors: a patent review (2008 – 2009)

Introduction: The protein kinase C (PKC) is a family of multifunctional isoenzymes involved in apoptosis, migration, adhesion, tumorgenesis, cardiac hypertrophy, angiogenesis, platelet function and inflammation. It also plays a vital role in the regulation of signal transduction, cell proliferation and differentiation through positive and negative regulation of the cell cycle. In this work, we reviewed the existing PKC inhibitors and several patents linked to PKC inhibitors.

Areas covered: Thorough survey on the PKC inhibitors having clinical importance and patents filed for these inhibitors from 2008 – 2009 is reported.

Expert opinion: PKCs are highly potential therapeutic targets for treating diabetic complications, oncological, inflammatory, immunological and dermatological disorders. The clinical trial candidates of PKCs mainly target the catalytic domain, which is highly conserved throughout the PKC family making it difficult to target a particular isoform selectively. Relatively less chemical space and fewer bisubstrate inhibitors targeting both ATP and regulatory domain are explored for PKCs, more research in these areas will be helpful in overcoming existing problems.     

Update on lymphocyte specific kinase inhibitors: a patent survey

Importance of the field: Lck (p56^lck or lymphocyte specific kinase) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T cell receptor (TCR)-mediated signaling, leading to normal T-cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection.

Areas covered in this review: This review covers the patents, patent applications and associated publications for small molecule kinase inhibitors of Lck since 2005 and attempts to place them in context from a structural point of view.

What the reader will gain: Readers will gain an overview of the structural classes and binding modes of Lck inhibitors, the major players in this area and an insight into the current state of the field.

Take home message: The search for a potent and orally active inhibitor of Lck has been an intense area of research for a number of years. Despite tremendous efforts, the identification of a highly selective and potent Lck inhibitor suitable for use as an immunosuppressive agent remains elusive.     

Aurora kinase inhibitor patents and agents in clinical testing: an update (2011 – 2013)

Introduction: Aurora kinase A, B and C, members of serine/threonine kinase family, are key regulators of mitosis. As Aurora kinases are overexpressed in many of the human cancers, small-molecule inhibitors of Aurora kinase have emerged as a possible treatment option for cancer.

Areas covered: In 2009 and 2011, the literature pertaining to Aurora kinase inhibitors and their patents was reviewed. Here, the aim is to update the information for Aurora kinase inhibitors in clinical trials and the patents filed between the years 2011 and 2013. Pubmed, Scopus®, Scifinder®, USPTO, EPO and www.clinicaltrials.gov databases were used for searching the literature and patents for Aurora kinase inhibitors.

Expert opinion: Even though both Aurora sub-type selective as well as pan-selective inhibitors show preclinical and clinical efficacy, so far no Aurora kinase inhibitor has been approved for clinical use. Particularly, dose-limiting toxicity (neutropenia) is a key issue that needs to be addressed. Preliminary evidence suggests that the use of selective Aurora A inhibitors could avoid Aurora B-mediated neutropenia in clinical settings. Also, use of adjunctive agents such as granulocyte stimulating factor to overcome neutropenia associated with Aurora B inhibition could be an answer to overcome the toxicity and bring Aurora inhibitors to market in the future.     

Antiplatelet effect of AMP-activated protein kinase activator and its potentiation by the phosphodiesterase inhibitor dipyridamole

AMP-activated protein kinase (AMPK) activates endothelial nitric oxide synthase (eNOS) via phosphorylation at the activating site. The eNOS-nitric oxide (NO)/soluble guanylate cyclase (sGC)-cGMP/cGMP-dependent protein kinase (PKG) signaling axis is a major antiaggregatory mechanism residing in platelets. Based on the hypothesis that direct activation of AMPK might be a potential strategy to inhibit platelet aggregation, the antiplatelet effect of AMPK activators was investigated. Treatment of isolated platelets with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in AMPK activation and a decrease in aggregation, which was abolished by pretreatment with the AMPK inhibitors compound C (CC) and ara-A. Such an AMPK-dependent antiaggregatory effect was also observed with other AMPK activators such as A-769662 and PT1. AICAR induced eNOS activation was followed by NO synthesis, cGMP production, and subsequent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a PKG substrate. All these events were blocked by CC or ara-A pretreatment, and each event was inhibited by the eNOS inhibitor L-NAME, the sGC inhibitor ODQ, and the PKG inhibitor Rp-8-pCPT-cGMPS. Simultaneous treatment of dipyridamole, a phosphodiesterase (PDE) inhibitor, with AICAR potentiated the antiaggregatory effect by enhancing the cGMP elevation. Administration of AICAR increased platelet cGMP and prolonged FeCl₃-induced arterial occlusion time in rats, which further increased in combination with dipyridamole. In conclusion, AMPK activators inhibited platelet aggregation by stimulating the eNOS-NO/sGC-cGMP/PKG signaling pathway. The antiplatelet effect of AMPK activators could be potentiated in combination with a PDE inhibitor through the common mechanism of elevating cGMP. Thus, AMPK may serve as a potential target for antiplatelet therapy.     

Targeting of AMP-activated protein kinase: prospects for computer-aided drug design

Introduction: Dysregulation of energy homeostasis has been implicated in a number of human chronic diseases including diabetes, obesity, cancer, and inflammation. Given the functional attributes as a central regulator of energy homeostasis, AMP-activated protein kinase (AMPK) is emerging as a therapeutic target for these diseases, and lines of evidence have highlighted the need for rational and robust screening systems for identifying specific AMPK modulators with a therapeutic potential for preventing and/or curing these diseases.

Areas covered: Here, the authors review the recent advances in the understanding of three-dimensional structures of AMPK in relationship with the regulatory mechanisms, potentials of AMPK as a therapeutic target in human chronic diseases, and prospects of computer-based drug design for AMPK.

Expert opinion: Accumulating information of AMPK structure has provided us with deep insight into the molecular basis underlying the regulatory mechanisms, and further discloses several structural domains, which can be served for a target site for computer-based drug design. Molecular docking and simulations provides useful information about the binding sites between potent drugs and AMPK as well as a rational screening format to discover isoform-specific AMPK modulators. For these reasons, the authors suggest that computer-aided virtual screening methods hold promise as a rational approach for discovering more specific AMPK modulators.     

Small-molecule pan-IAP antagonists: a patent review

Importance of the field: The inhibitor of apoptosis (IAP) proteins are critical regulators of cancer cell survival that have become important targets for therapeutic intervention in human malignancies. One strategy for targeting IAP proteins involves agents that mimic the amino terminus of the endogenous IAP protein antagonist second mitochondria-derived activator of caspases (Smac)/direct IAP-binding protein with low pI (DIABLO) and thus block critical IAP protein interactions.

Areas covered in this review: This review of the IAP antagonist patent literature covers the period from 2000 to mid-2009. Over 50 patents and patent applications pertaining to IAP antagonists have been published over the past 10 years. In the case of several filings, only the original source is reviewed in this analysis.

What the reader will gain: Readers will gain an overview of IAP protein antagonist scaffolds, with representative examples including monovalent and bivalent Smac mimetics, and an understanding of their structure–activity relationships.

Take home message: The feasibility of disrupting IAP protein interactions with pro-apoptotic proteins using monovalent and bivalent Smac-derived peptidomimetic compounds has been broadly established. Four such compounds have entered or been approved to enter human clinical trials, which will hopefully allow the utility of this potential therapeutic approach to be evaluated in cancer patients.     

An update on anti-allergic patents granted in China: 2009 – 2011

Introduction: The prevalence of allergic diseases has increased dramatically in recent decades. Therefore, there is a pressing need for the development of effective anti-allergic services worldwide.

Areas covered: In previous studies, the authors had analyzed a total of 789 anti-allergic patents granted in China from 1988 to 2008. Herein, they report a further 151 anti-allergic patents issued in China during 2009 – 2011. The current analysis covers the scientific progress in supporting anti-allergic patent applications and granted patent literature, in China, for the last 3 years.

Expert opinion: The 151 anti-allergic patents granted from 2009 to 2011 mainly focus on seven types of products. They are: traditional Chinese medicines (TCM), plant extracts, biological products, synthetic compounds, pharmaceutical preparations, medical apparatus and new treatment modalities. Although the overall number of anti-allergic patent applications made between 2009 and 2011 in China is less than that of the USA and Europe, patents on TCM have increased. This suggests that there are demands for modernization of TCMs. Recently, studies of interesting new immunomodulators have also been conducted, and some of these are likely to represent clinically useful advances. In the last 3 years, several patents on these novel potential drugs have also been granted in China. The large number of anti-allergic patents issued in China, in recent times, suggests that the Chinese market is relatively competitive one that will help pharmaceutical companies make proper decisions for their research and development strategies.     

Analysis of patents on anti-allergic therapies issued in China from 1988 to 2008

Background: The prevalence of allergic diseases has increased dramatically in recent decades. Therefore, there is a pressing need for the development of effective anti-allergic services worldwide. However, little is known what anti-allergic products have been patented in China and what the potential drug candidates for patents are in China.

Objective: To analyze the patents of anti-allergic products for the last 20 years and help pharmaceutical companies and individuals to understand the potential candidates for anti-allergic patents in China.

Methods: Data were obtained from the People’s Republic of China Country Intellectual Property Rights Bureau website and United States Patent and Trademark Office website.

Results: A total of 789 anti-allergic patents have been granted in China during the past 20 years, which all focused on synthetic compounds, traditional Chinese medicines (TCM), combinations of synthetic compounds and TCM, biological products and medical apparatus. It appears that more and more effective therapeutic components of TCM rather than whole herbs have been patented in recent years and alteration of natural molecules to produce more therapeutically effective molecules has emerged as a novel trend for the modernization of TCM. The patents on synthetic anti-allergic compounds in China mainly focus on well-known targets, such as histamine receptor and leukotrienes, which consist of 93% of patents for validated targets.

Conclusion: The number of anti-allergic patents applied in China is far lower than that in the US. Therefore, there are great opportunities for obtaining anti-allergic patents, particularly patents on active ingredients from TCM in China.