A concise review on advances in development of small molecule anti-inflammatory therapeutics emphasising AMPK: An emerging target

Abstract

Inflammatory diseases are complex, multi-factorial outcomes of evolutionarily conserved tissue repair processes. For decades, non-steroidal anti-inflammatory drugs and cyclooxygenase inhibitors, the primary drugs of choice for the management of inflammatory diseases, addressed individual targets in the arachidonic acid pathway. Unsatisfactory safety and efficacy profiles of the above have necessitated the development of multi-target agents to treat complex inflammatory diseases. Current anti-inflammatory therapies still fall short of clinical needs and the clinical trial results of multi-target therapeutics are anticipated. Additionally, new drug targets are emerging with improved understanding of molecular mechanisms controlling the pathophysiology of inflammation. This review presents an outline of small molecules and drug targets in anti-inflammatory therapeutics with a summary of a newly identified target AMP-activated protein kinase, which constitutes a novel therapeutic pathway in inflammatory pathology.

Small molecules in drug discovery

‘Small molecules are at the heart of chemical biology.’ ¹

There is a growing interest in the discovery of small molecules to deal with the emergent healthcare crisis resulting from the mounting incidence of chronic illnesses worldwide. Since the second half of the 20th century, researchers have often focused on biologically active small molecules that act quickly (within minutes to hours) and reversibly, allowing both inhibition and activation of a protein function. Small molecules (molecular mass <1000 Da) have been at the centre stage of drug discovery in both academia and the pharmaceutical industry. The US Food and Drug Administration (FDA) approved 113 first-in-class drugs from 1999 to 2013, among which the majority (n = 45) were small molecules. Phenotypic screening identified 28 and 17 were discovered based on a target-based approach. Therefore, small molecule drugs are of paramount interest in drug discovery.^2,3

An overview of inflammation

Inflammation, the hallmark of the body’s immediate response to injury is a complex biological process triggered by tissue/cellular damage or a variety of harmful stimuli such as pathogens, irritants (chemicals) or infection. A multifactorial network of biochemical mediators (reactive oxygen species, nitric oxide, bradykinin, prostaglandins, prostacyclins, pro-inflammatory cytokines and chemokines) are released locally after tissue injury, stimulating and propagating the inflammatory response that may be broadly divided into acute and chronic inflammation. Acute inflammation is a short-term adaptive response for repair while chronic inflammation is a prolonged, dysregulated and maladaptive response. Uncontrolled and persistent inflammation may develop into a chronic malady, becoming the fundamental basis for the pathogenesis of many chronic diseases including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), metabolic disorders (diabetes and obesity), cardiovascular disorders (ischaemic heart disease, atherosclerosis), neurodegenerative diseases (Parkinson’s and Alzheimer’s) and cancer, many of which are life-threatening. Inflammatory diseases are among the commonest cause of chronic ill-health globally. The mounting burden of healthcare costs may be dominantly attributed to the severity and complexity of inflammatory disorders.^4,5

Drug targets and small molecules for the treatment of inflammation

Historically, anti-inflammatory agents had their origin in the serendipitous discovery (chiefly from plant extracts), during the 19th and 20th centuries. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase inhibitors (COXIBs) were primary drugs of choice for treating pain and inflammation. Later, advances in chemical, molecular, cellular biological and technological developments led to identification of numerous drug targets for anti-inflammatory drug discovery. ⁶ Here, we shall first discuss NSAIDs and COXIBs followed by multi-target therapeutics and recent advances in AMP-activated protein kinase (AMPK) as an emerging drug target in inflammation.

NSAIDs and COXIBs

NSAIDs and COXIBs mainly act by modulating the arachidonic acid (AA) cascade (Figure 1). Traditionally, NSAIDs (Figure 2) are the most widely prescribed therapeutic agents for treating pain, inflammation (acute and chronic) and fever. In 1990s, COXIBs (Figure 3) were developed to overcome the side effects of traditional NSAIDs. ⁷

Figure 1.

The AA cascade. Prostaglandin (PG) D₂/E₂/F_2αa/G₂/H₂; thromboxane A₂ (TXA₂); epoxyeicosatrienoic acids (EETs); dihydroxyeicosatrienoic acids (DHETs); hydroxyeicosatetraenoic acids (HETEs); hydroperoxy-6,8-trans-11,14-cis-eicosatetraenoic acid (HPETE).

Figure 2.

Chemical structures of traditional NSAIDs.

Figure 3.

Chemical structures of COXIBs: (a) first-generation selective COX-2 inhibitors; (b) second-generation selective COX-2 inhibitors.

The bulk of the approved drugs for inflammatory conditions (e.g. arthritis, asthma, pain relief and fever) target cyclooxygenases (both COX-1 and COX-2) and lipoxygenases (5-LOX, 12-LOX, 15-LOX), key players in the AA cascade. Despite the popularity of these drugs, long-term use at high doses result in myocardial infarctions, stroke, increased blood pressure, deaths (COXIBs), gastrointestinal ulcers, bleeding and platelet dysfunction (NSAIDs).^8,9 Moreover, chronic inflammatory diseases fail to respond to conventional therapy. Discovery of more effective molecules with improved safety profiles is of supreme relevance today.

Multi-target anti-inflammatory therapeutics

Therapeutic agents acting on single targets often show suboptimal efficacy and undesirable safety profiles. Many reports suggest that balanced modulation of several but relevant and interconnected targets can be more efficient than the selective action upon individual/single targets. To enhance therapeutic efficacy and improve safety profile, anti-inflammatory research is currently focusing on multi-target ligands for treating complex, multi-factorial inflammation.^10–13 Some of the reported multi-targets include: the combination of COX and LOX; phospholipase A₂ and leukotriene A₄ hydrolase (PLA₂ and LTA₄H); microsomal prostaglandin E₂ synthase-1 and 5-lipoxygenase (mPGES-1 and 5-LOX); 5-lipoxygenase and thromboxane A₂ synthetase (5-LOX and TXA₂); cyclooxygenases and leukotriene A₄ hydrolyase (COX-2/COX-1 and LTA₄H); and fatty acid amide hydrolase and endocannabinoid substrate-specific cyclooxygenase-2 (FAAH/CB₂ and COX-2). A multi-target approach is an important deviation from the classical strategy. Although results of clinical trials of dual inhibitors are limited, their success is bound to elevate this strategy to the helm of the emerging paradigm of drug discovery.

Emerging therapeutic targets for inflammation

Target-based approaches began to dominate drug discovery research following revolutionary advances in molecular biology and genomics that provided valuable insight into cellular targets and signalling pathways. ¹⁴ Likewise, the anti-inflammatory research focus also shifted towards signalling pathways, such as p38 mitogen activated protein (MAP) kinase; tumour necrosis factor (TNF-α) inhibitors; nuclear transcription factor (NF-kB); glycogen synthase kinase 3 (GSK-3); Janus kinases and signal transducers and activators of transcription (JAK/STAT); matrix degrading enzymes like matrix metalloproteinases (MMPs); peroxisome proliferator-activated receptor (PPARγ-agonists); and 5’-Adenosine monophosphate-activated protein kinase/AMPK. ¹⁵ Employing these alternate targets constitute an important strategy in addressing the underlying cause of chronic inflammation. In the section below, we discuss recent advances of one of the very rapidly emerging anti-inflammatory targets: AMPK.

AMPK as a potent new anti-inflammatory target

AMPK is an evolutionarily conserved heterotrimeric (αβγ) serine/threonine protein kinase (~145 kDa) and a critical signaling molecule that regulates energy homeostasis.^16,17

Structural biology of AMPK and its regulation

AMPK, which was originally discovered for its ability to inhibit two key enzymes (acetyl-CoA carboxylase and HMG-CoA reductase) that regulate lipid biosynthesis, is present in all eukaryotic cells. AMPK is composed of a catalytic α subunit (AMPK-α) and regulatory β and γ subunits (AMPK-β and AMPK-γ). In mammals, all three subunits occur as multiple isoforms (α1, α2; β1, β2; γ1, γ2, γ3) encoded by distinct genes leading to 12 hetero-trimeric combinations. The roles of many isoforms remain unclear. Stressors that increase intracellular AMP:ATP ratio (hypoxia, exercise, muscle contraction and long-term starvation) promote AMPK activation. A rise in AMP levels results in the binding of the γ subunit of AMPK to AMP, inducing a conformational change and allosteric activation of the phosphorylation state of Thr172 in the α-subunit. Phosphorylation of Thr172 by upstream kinases (LKB1, CaMKKβ and TAK1) results in AMPK activation. The β subunit acts as a scaffold for the binding of other two subunits (α and γ) bridging the heterotrimeric complex assembly of AMPK. The glycogen-binding domain (GBD) might play a role in glycogen metabolism. The γ subunit contains four tandem cystathionine-β-synthase (CBS) motifs for the binding of AMP and other adenine nucleotides (ADP and ATP).^5,18,19

Structure of full length human AMPK

Xiao et al. recently resolved the X-ray crystal structure of full length human α2β1γ1 AMPK bound to small molecule activator Compound 991, a cyclic benzimidazole derivative (3.02 Å resolution; PDB ID - 4CFE) and A-769662, thienopyridone derivative (3.92 Å resolution; PDB ID - 4CFF). Later, Calabrese et al. determined the full length structure of the α1β1γ1 AMPK complexed with A-769662 with better resolution (3.35 Å). Additionally, researchers reported AMPK binding with salicylate, although with low (3.9 Å) resolution (anomalous diffraction data supports that salicylate binds at the CBM-KD interface like A-769662). These crystal structures offer new opportunities for better design and discovery of small molecules to treat human diseases.^16,20

AMPK as a drug target

AMPK is implicated in many human diseases and currently occupies a pivotal position, particularly in metabolic syndrome (type 2 diabetes and obesity).^21–23 By inhibiting a mammalian target of rapamycin (mTOR) signalling, a key regulator of several tumour suppressors (including S6 kinase 1 (S6K1), liver kinase B1 (LKB1), p53, and tuberous sclerosis complex 2 (TSC2)), AMPK is shown to play a therapeutic role in the treatment and prevention of cancer. ¹⁹ AMPK, having emerged as a potential drug target in inflammatory diseases,^15,24–28 its role in inflammation are discussed below.

AMPK and inflammation

Many AMPK activators have demonstrated beneficial anti-inflammatory and immunosuppressive effects in a variety of cell types and pre-clinical models of inflammatory/autoimmune diseases.^5,15,25 Of late, a few anti-inflammatory agents, particularly the acidic class of NSAIDs, were reported to activate AMPK.^27,28 In mouse and rat models of neuropathic pain, AMPK activators (Metformin and A769662) reduced mechanical hypersensitivity, validating AMPK as a novel therapeutic target in inflammatory and neuropathic pain. ²⁹

Anti-inflammatory effects of AMPK activators

Figure 4 illustrates the chemical structures of various AMPK activators with anti-inflammatory actions. Discussed below is an account of small molecule AMPK activators with potent anti-inflammatory activity.

Figure 4.

Diverse AMPK activators with reported anti-inflammatory activities.

Metformin, the most frequently prescribed anti-diabetic drug, either directly or indirectly activates AMPK. Metformin’s role in controlling hepatic gluconeogenesis and tumour is debated and could be independent of AMPK activation at normal pharmacological doses. However, the ability of metformin to control hepatic lipids is attributed to AMPK activation, which increases fatty acid oxidation. ³⁰ Metformin exerts anti-inflammatory activity in many experimental models by reducing oxidative stress and IL-1β-induced pro-inflammatory cytokine (IL-6 and IL-8) production in macrophages, endothelial cells and vascular smooth muscles. ³¹ AMPK activation by metformin can account for the significant angiogenic response. Metformin also attenuates acetic acid-induced gastric ulcers by promoting ulcer healing and mitigating diabetes-induced peptic ulcers. ³²

Rosiglitazone, a selective PPARγ agonist and AMPK activator in mammals, has demonstrated anti-inflammatory effects in rat cardiac myocytes and in DSS-induced colitis in rats. In both obese and the non-obese diabetic patients, rosiglitazone exerts beneficial anti-inflammatory effect at both the cellular and molecular levels. ³³

5-amino-4-imidazole carboxamide riboside (AICAR), a widely studied pharmacological AMPK activator, was recently shown to have anti-inflammatory properties mediated by AMPK activation. AICAR inhibited lipopolysaccharide (LPS) induced expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) production and pro-inflammatory cytokines such as TNF-α, interleukin-1β (IL-1β) and interleukin-6 (IL-6) in both cultured cells (primary rat astrocytes, microglia and peritoneal macrophages) and animals treated with LPS.^34,35 In several in vivo studies (autoimmune encephalomyelitis, acute lung injury, acute and chronic colitis models in mouse), AICAR attenuates the progression of disease with promising benefit. ³⁶ In addition, AICAR significantly reduced nociceptive response in inflammatory nociception mouse model (acute and neuropathic pain) with fewer adverse effects, mostly mediated by regulation of AMPK-α2. ²⁹ AICAR attenuates ocular inflammation by reducing the expression of pro-inflammatory cytokines and chemokines viz. MCP-1, TNF- α and ICAM-1 and experimental autoimmune uveitis (EAU) by inhibiting T-cell proliferation and Th1 and Th17 cytokine production. Further, AICAR inhibits oxidative stress/H₂O₂-induced caveolin-1 phosphorylation via activation of AMPK.^37,38

A-769662, is the first selective small molecule AMPK activator that binds directly to AMPK, on a site distinct from the nucleotides, inducing conformational changes and inhibiting Thr172 dephosporylation, thereby causing allosteric activation. A-769662 fully reversed neuropathic allodynia in an in vivo mouse model and was more potent than metformin, suggesting that AMPK-β1 may be a viable drug target in the pain pathway. ³⁹

Anti-inflammatory agents which activate AMPK

Figure 5 illustrates the chemical structures of anti-inflammatory agents which activate AMPK. NSAIDs and methotrexate were found to activate AMPK and their actions are discussed below.

Figure 5.

Chemical structures of anti-inflammatory agents reported to activate AMPK.

Figure 6.

Schematic overview of downstream anti-inflammatory signalling effects mediated by AMPK activation. The descriptive mechanism of action of direct and indirect small molecule AMPK activators is described in the references.^15,17,19 AMPK, AMP-activated protein kinase; PPAR-γ, peroxisome proliferator-activated receptor gamma; NF-kB, nuclear factor kappa B; MMP-9, matrix metallopeptidase 9; Ccr2, C-C chemokine receptor 2; Nrf2, nuclear factor-erythroid 2-related factor 2; NQO-1, NADPH quinone oxidoreductase-1; HO-1, heme oxygenase-1; GST, glutathione S-transferase; iNOS, inducible nitric oxide synthase; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2.

Salicylate, an active metabolite of aspirin, was reported to activate AMPK by binding at the same site as A-769662. ²⁷ Flufenamic acid increased the phosphorylation of AMPKα at Thr172 and significantly inhibited cytokine-induced expression of iNOS in NRK-52E cell lines similar to AICAR. ⁴⁰ A recent study reported that ibuprofen and diclofenac also activated AMPK in a manner similar to aspirin in murine neuronal cells (Neuro-2), human neuronal cells (SH-SY5Y) and mouse liver. ²⁸ Additionally, methotrexate potentiates the ability of AICAR to activate AMPK by increasing the accumulation of ZMP and decreasing ATP levels in various human cancer cell lines. ⁴¹

AMPK, a nodal centre for regulation of downstream inflammatory signalling mechanisms

Many studies have highlighted an interaction between AMPK and inflammation demonstrating that AMPK activation inhibits inflammatory responses and immune response (by reducing the release of pro-inflammatory cytokines and by impairing Th1 and Th17 cells differentiation) induced by NF-κB, the key regulator of inflammation. AMPK activators such as metformin and AICAR can indirectly inhibit NF-κB signalling and suppress inflammation by several downstream targets of AMPK. AMPK-α1 regulates NF-κB-dependent gene expression mediated by TLR4 and TNF-α stimulation. Also, activated AMPK suppresses MMP-9 expression (important in tissue destruction and inflammation) in mouse fibroblasts and Ccr2 (a key pro-inflammatory marker of M1) expression in macrophages by inhibiting the NF-κB pathway. In addition, AMPK-induced stimulation of nuclear factor erythroid-2-related factor-2 (Nrf2 – downstream anti-oxidant pathway to AMPK) signalling can protect against inflammatory disorders. HO-1, one of the target genes of Nrf2, protects and regenerates mucosal tissues by attenuating inflammatory mediators, namely TNF-α and IL1β. Nrf2 also regulates immune responses by inducing antioxidant and phase II detoxifying enzymes, thereby demonstrating both gastro-protective and anti-inflammatory actions. AICAR and metformin rapidly stimulate the expression of Nrf2 in an AMPK-dependent process. Further, AICAR and metformin exerts anti-inflammatory actions by inhibiting iNOS expression and subsequent NO production mediated through AMPK activation. Additionally, PPAR-γ agonists have demonstrated a role in a novel anti-inflammatory pathway, via AMPK-dependent inhibition of iNOS. Thus, AMPK activation suggests a pivotal point of restrictive control in many inter-connected inflammatory signalling cascades. This is not surprising, because inflammation is an energy demanding repair process, which is antagonized by the energy conserving propensity associated with AMPK activation. As the metabolic control of inflammation constitutes a central principle in repair mechanisms, there exists a rationale for the design and development of novel pharmacological agents to tackle inflammation via AMPK activation, over and above its established value in metabolic syndrome and cancer. By mimicking a cellular process that is fundamental to tissue repair, such strategies might help reduce the adverse effects associated with traditional anti-inflammatory drugs and may represent an emerging class of new therapeutic small molecules for the treatment of inflammatory disease.

Conclusion

To conclude, in the last two decades, several new thoughts are redefining the role of small-molecule-based drugs in inflammation. Chronic inflammation is the common underlying thread that eventually leads to several chronic disease states such as autoimmune diseases, atherosclerosis, diabetes and cancer, the incidence of which has been rising steadily with life style changes. Hence, research in this area is both an opportunity and a challenge that requires a multi-dimensional approach, on account of the involvement of multiple pathways that regulate the complex inflammatory cascade mechanisms.

To sum up, the following points are worth mentioning. First, small-molecule-based therapeutics continues to dominate the pharmaceutical landscape despite the rising market share of biologicals for the treatment of complex, multi-factorial inflammatory conditions. Second, multi-target therapeutics is an attractive alternative strategy that could replace the prevailing ‘one drug one target’ concept. Lastly, novel pathways (e.g. AMPK), guided by multiple signalling switches, have already demonstrated potential for modifying the course of chronic inflammation.