Chapter 1: Quest for Magic Bullets to Solve the Endocannabinoid Puzzle Free

Marijuana is the most widely used recreational drug in the Western world, and is consumed by ∼3% of the world's population (∼185 million individuals).¹  The legal cannabis turnover was ∼$7 billion in 2016 in the United States alone, and it is expected that it will grow to $22 billion by 2021.²  The advent of legalized cannabis in multiple regions of the United States, with currently 28 and 8 States having accepted medical and recreational marijuana, respectively, raises concerns about its potential hazard to health. Nevertheless, research on the therapeutic potential of cannabis extracts-based drugs suggests them to be clinically useful in a wide range of pathological conditions, including neurological³  and psychiatric disorders.⁴  Conversely, repeated cannabis use has been associated with short-term and long-term side effects, including cognitive alterations, psychosis, schizophrenia and mood disorders,⁴  as well as respiratory and cardiovascular diseases.⁵  In this context, the existence of different species and cultivars of cannabis must be taken into account when evaluating the impact on health outcomes. Cannabis sativa and Cannabis indica are the most widespread and best characterized species of cannabis, and their extracts contain phytocannabinoids of therapeutic interest, such as Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD), both shown in Table 1.1. The effect of cannabis extracts depends on the amount of THC and CBD, as well as on the presence and concentration of >110 additional phytocannabinoids, and >440 non-phytocannabinoid compounds like terpenoids, flavonoids and sterols.⁶  Thus, different cannabis extracts may be different “chemovars” with a different chemical profile, which means that they may contain different components and/or different amounts of them. In addition, the modes of cultivation, harvest, extraction of active principles and administration may further affect the final chemical composition, clearly suggesting that there is no “one cannabis” but several mixtures even from the same plant.³  As yet, there is little understanding of the pharmacological efficacy of cannabis extracts, and these uncertainties represent a warning for the clinical applications of these natural compounds.³  The complexity of the plant extracts is even exceeded by the complexity of the molecular targets that they can hit in our body, as described in the following section.

It is well established that THC binds to and activates specific G protein-coupled receptors, known as type-1 (CB₁) and type-2 (CB₂) cannabinoid receptors, that endogenously are triggered by ligands that were identified in the 1990s as anandamide (N-arachidonoylethanolamine, AEA)⁷  and 2-arachidonoylglycerol (2-AG).^8,9  These two compounds, an amide and an ester of arachidonic acid, respectively (Table 1.1), are the most active and best studied endocannabinoids (eCBs).^5,10  Both molecules are metabolized by a complex array of biosynthetic enzymes, hydrolases and oxygenases, and are transported through the plasma membrane and intracellularly by distinct carriers. Altogether receptors, enzymes and transporters of eCBs form the “eCB system”, that has been recently discussed in a comprehensive review.¹¹ 

The various components of the eCB system support and control the manifold actions of eCBs, both in the central nervous system^10,12  and at the periphery.⁵  In particular, the number of receptors activated by eCBs in the same cell, both on the plasma membrane and in the nucleus, appears striking. Indeed, the most relevant eCB-binding receptors include: i) CB₁ and CB₂,¹³  as well as G protein-coupled orphan receptors (GPR) 55¹⁴  and 119¹⁵  (all on the plasma membrane and with an extracellular binding site); ii) transient receptor potential vanilloid 1 (TRPV1)¹⁶  and additional transient receptor potential (TRP) channels TRPV2, TRPV3, TRPV4, TRPA1 and TRPM8 (all on the plasma membrane, but with an intracellular binding site);¹⁷  and iii) nuclear peroxisome proliferator-activated receptors (PPARs) α,¹⁸  γ¹⁹  and δ,²⁰  that are all transcription factors that regulate gene expression. It is of paramount importance that receptor-mediated activities of eCBs are subjected to a stringent “metabolic control”, which means that their cellular concentration (and hence biological activity) depends on a balance between synthesis and degradation by different biosynthetic and hydrolytic enzymes.¹¹  Among the latter, N-acyltransferase (NAT),²¹  N-acyl phosphatidylethanolamines-specific phospholipase D (NAPE-PLD),²²  and α/β hydrolase domain 4 (ABHD4)²³  catalyze parallel routes for the release of AEA from phospholipid precursors; instead, fatty acid amide hydrolase (FAAH)²⁴  and N-acylethanolamine acid amidase (NAAA)²⁵  cleave AEA and other eCBs, terminating their signalling activity. Much like AEA, diacylglycerol lipases (DAGL) α and β synthesize 2-AG,²⁶  that instead is cleaved through different routes by monoacylglycerol lipase (MAGL),²⁷  ABHD2,²⁸  ABHD6²⁹  or ABHD12.³⁰  In addition to synthesis and degradation, a further level of complexity in eCB metabolism is represented by the addition of oxygen to the fatty acid moiety by cyclooxygenase-2 (COX-2), lipoxygenases (LOXs) like 5-LOX, 12-LOX and 15-LOX, and cytochrome P450.³¹  Interestingly, the oxidative products of eCBs are endowed with their own biological activities, distinct from those of eCBs (ref. 32, and references therein). The stringent metabolic control of eCB tone is further modulated by distinct transporters that facilitate the movement of eCBs both across the plasma membrane (via a purported eCB membrane transporter, EMT),³³  and intracellularly,³⁴  as well as by storage of eCBs in cytosolic organelles like adiposomes.³⁵  Among the intracellular transporters of eCBs are heat shock protein (HSP) 70 and albumin,³⁶  and fatty acid binding proteins (FABP) 5 and 7.³⁷ 

To date, 3D structures of 23 out of the 34 major components of the eCB system have been resolved, as shown in Figure 1.1. The remaining 3D structures of 11 eCB system components are still elusive, thus preventing our understanding of their regulation, cross-talk with other eCB system components, and ultimately their impact on eCB signalling.¹¹  This information gap appears particularly troubling for enzymes involved in 2-AG metabolism: out of 6 most important enzymes discovered so far (DAGLα, DAGLβ, MAGL, ABHD2, ABHD6 and ABHD12), only MAGL has a known 3D structure (Figure 1.1).

Against this background, it is apparent that the need for more structural data is urgent, especially if one aims at developing selective drugs of therapeutic relevance that are able to modulate a distinct eCB system element, without affecting the others. In particular, it should be appreciated that eCBs are quite short lived substances, and that metabolic enzymes and transporters are the main drivers of their timely delivery (and in the right concentration) to the right target among many alternatives in the same cell. Thus, ensuring the right eCB tone at the right time and in the right place has a major impact on signal-transduction pathways, and hence on the overall cell functioning. Notably, a detailed knowledge of the 3D structures of the proteins of the eCB system is mandatory also to interrogate and understand the mode of action of exogenous (plant-derived) and endogenous signals responsible for their regulation, turning these proteins into novel targets of effective drugs.

From the previous sections, it appears that the complexity of cannabis extracts is mirrored (and even exceeded) by the complexity of the endogenous array of proteins that altogether support eCB signalling in vivo. Unfortunately, the functional properties of only a few elements of the eCB system have been thoroughly investigated, and natural compounds have been tested as potential modulators of them. Also, new synthetic compounds have been designed, often based on natural scaffolds, in search for magic bullets able to hit a selected element without affecting the others. During many years of intensive efforts, selective drugs have been indeed developed to modulate CB₁, CB₂, non-CB₁/non-CB₂ receptors, TRP receptors, as well as NAPE-PLD, FAAH and MAGL enzymes, along with eCB transporters. Instead, chemical tools to interrogate the other members of the eCB system are still scarce (if any). Available eCB-oriented drugs will be presented in the different chapters of this book, written by widely recognized leaders in the field, who will also discuss recent advances and future perspectives of drug design. It will be shown that not only single-target inhibitors, but also dual- and multi-target compounds have been developed and tested. In fact, the multi-target directed ligand approach has been applied in the last 20 years, stemming from the concept of “network medicine”, that takes into account mutual effects of biological and medical occurrences.³⁸  On this basis, a disease is considered the consequence of the systemic breakdown of physiological networks, due to the inhibition or activation of certain stages (variation of input–output). Consequently, the aim of a therapy is to restore the perturbed disease networks by simultaneously targeting key components or checkpoints.³⁹  Indeed, restoration of the networks through a single node of intervention (i.e., one protein target or one signalling pathway) is very difficult, because usually any complex biological network is controlled by several mechanisms. Instead, modulation of several targets at once through a well-concerted polypharmacological approach may be more effective to achieve the desired therapeutic effect.³⁹  Overall, the network models suggest that (at least partial) inhibition of a small number of targets can be more efficient than complete inhibition of a single target. Such a concept holds true also for eCB signalling,⁴⁰  and for instance it has recently led to development of novel dual-class inhibitors of FAAH.⁴¹ 

In this book, Chapter 2 will introduce the differences between phytocannabinoids and eCBs, along with a modern view of the eCB system.⁴²  Then, Chapter 3 will describe natural compounds and synthetic drugs to target CB₁ receptor,⁴³  and Chapters 4, 5 and 6 will deliver the same information for CB₂,⁴⁴  non-cannabinoid⁴⁵  and TRP receptors,⁴⁶  respectively. After eCB-binding receptors, the focus will be on metabolic enzymes of eCBs. Indeed, Chapter 7 will discuss natural compounds and synthetic drugs to target NAPE-PLD,⁴⁷  and Chapters 8 and 9 will present those able to hit FAAH⁴⁸  and MAGL,⁴⁹  respectively, whereas not much is known about DAGL inhibitors.⁵⁰  Last but not least, Chapter 10 will address eCB transporters and drugs able to modulate them,⁵¹  a highly debated hot topic within the (endo)cannabinoid community for many years. Needless to say, only when we become able to dissect the distinct contribution of each element of the eCB system to eCB signalling and its fine regulation, will we be able to better appreciate the effects of exogenous, plant-derived cannabinoids, and possibly to exploit them to cure, or at least slow down, peripheral and central human diseases. Until then, the potential benefits of therapeutic cannabis will be counterbalanced by potential threats due to unwanted side-effects. Unfortunately, this has been the case with the anorexigenic drug Acomplia^® (Rimonabant^®), a CB₁ antagonist/inverse agonist that was withdrawn shortly after its first use in human subjects, because of serious neuropsychiatric effects.⁵²  Even worse was the case of BIA 10-2474, a purportedly specific FAAH inhibitor that led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others.^53,54  A subsequent careful profiling of BIA 10-2474 showed that its neurotoxicity was not due to FAAH inhibition but rather to substantial alterations of lipid networks in human cortical neurons, hence causing metabolic dysregulation and death.⁵⁵ 

Overall, it seems more urgent now than ever before to solve 3D structures of the components of the eCB system, and to develop reliable methods to assess their functional activity. Only then can magic bullets be designed and tested, expanding present knowledge and advancing the state-of-the-art reported in this book.

I thank Dr Daniel Tortolani (Campus Bio-Medico University of Rome) for his support in preparing the artwork.