Current approaches for the discovery of drugs that deter substance and drug abuse

Abstract

Introduction

Much has been presented and debated on the topic of drug abuse and its multidimensional nature, including the role of society and its customs and laws, economical factors, and the magnitude and nature of the burden. Given the complex nature of the receptors and pathways implicated in regulation of the cognitive and behavioral processes associated with addiction, a large number of molecular targets have been interrogated during recent years to discover starting points for development of small molecule interventions.

Areas covered

This review describes recent developments in the field of early drug discovery for drug abuse interventions, with a special emphasis on advances published during the 2012-2014 period.

Expert Opinion

Technologically, the processes/platforms utilized in drug abuse drug discovery are nearly identical to those used in the other disease areas. A key complicating factor in drug abuse research is the enormous biological complexity surrounding the brain processes involved and the associated difficulty in finding “good” targets and achieving exquisite selectivity of treatment agents. While tremendous progress has been made during recent years to use the power of high-throughput technologies to discover proof-of-principle molecules for many new targets, next-generation models will be especially important in this field; examples include seeking advantageous drug-drug combinations, use of automated whole-animal behavioral screening systems, advancing our understanding of the role of epigenetics in drug addiction, and the employment of organoid-level 3D test platforms (also referred to as tissue-chip or organs-on-chip).

1. Introduction

The use of xenobiotics as remedies for illness has enabled society to combat diseases that for centuries were thought to be not curable or treatable. Unfortunately, not all medicines are benevolent, as the ability to cure often comes with some serious side-, or off-target effects, one of them being addiction or abuse. Substance abuse[1], also known as drug abuse (other terms being used in this context are drugs of abuse, addiction, substance abuse, substance disorders, substance use disorders) is a patterned use of a substance (drug) in which the user consumes the substance in amounts or with methods which are harmful to themselves or others. Drug addiction is defined as the continued compulsive use of drugs despite adverse health or social consequences. These consequences are far from minor: in 2008, The National Institute on Drug Abuse (NIDA) reported that an estimated $559 billion/year is spent on substance abuse and addiction in the form of health care costs, productivity loss, crime, incarceration, and drug enforcement [2,3].

Substance abuse presents a long-term vexing problem for society and as such the topic is well reviewed in the scientific literature [4-10], with recurring themes being prevention, identification, treatment, and the social and pharmacological ways to diagnose and treat each. Developing agents that treat substance abuse employs a process nearly identical to that used in the traditional-disease therapeutic discovery and development pipeline: initial research into the underlying biology and identification of a suitable molecular target, followed by the associated steps of early discovery and optimization, next testing in animal models, and proceeding to human trials. After an introduction of a treatment modality, the final step of post-market surveillance aids in understanding the public's response and the effects of the new intervention [11-14]. This has typically been a reactionary process, with researchers and clinicians on the defensive. One of the most common treatments for substance/drug abuse has been, ironically, drugs, due to their ability for quick onset of action in eliminating the users' desire for said substance. But with typical drugs taking well over a decade to be developed, finding a quick solution, especially in response to a new emerging substance of abuse, has been very difficult[15]. With its ability to speed up the discovery of drug candidates and lower overall costs, high-throughput screening (HTS) plays an important role in the process [16].

The literature is rich in reports of molecular targets, pathways, and mechanisms thought be associated with the action of drugs of abuse [6,17-21]. Typically, the substances of abuse exert their action by modulating the production and transmission of chemicals (for example, neurotransmitters such as serotonin) through the receptors they bind, which in turn leads to a dysregulation of the cellular signaling[6]. Modulating such receptors with new drugs offers a potential therapy at curbing abuse [22,23] and high-throughput screening (HTS)[24], with its ability to facilely test thousands-to-millions of compounds, while also recently incorporating dose-response format [25], provides a welcome avenue for early drug discovery. Herein, we highlight the variety of assay platforms employed to interrogate targets implicated in substance abuse. In addition to examples of how HTS has been employed in the discovery of small molecule modulators, we also briefly cover the topics of biologics [26] and vaccines[27] as potential interventions for substance abuse.

2. High-throughput screening

HTS is made possible by the use of robotic equipment and automated liquid handlers that operate autonomously, combined with the ability to track samples and process the data accurately [28-30]. A key enabling factor is the density of samples that can be assayed, with 384-, 1,536-, and 3,456-well plate formats now a commonplace, allowing millions of samples to be tested in a week [28]. While often being characterized as a still-emerging approaching, recent analyses have shown that utilization of HTS in the discovery process has resulted in benefits, as judged by the generation of new molecular entities [16,31], highlighting the immediate impact of HTS on all therapeutic areas including the field of substance abuse [32] (Table 1).

Table 1.

Selected Neurotransmitter-Receptor HTS Assays.

Neurotransmitters	Receptors	HTS Assays	Reference
Acetylcholine	Muscarinic; Nicotinic	Phenotypic	[48], [49]
Norepinephrine	Adrenergic	Phenotypic	[40]
Dopamine	Dopamine; Neurotensin	Phenotypic	[40],[42,43]; [54]
Serotonin	Serotonin	Phenotypic and Target	[39,40]
Glutamate	NMDA; Metabotropic glutamate	Phenotypic	[51,52]
Opioids	delta, kappa, and mu	Phenotypic	[36,37]
Neuropeptides	y2; galanin; S; orexin; ΔFosB	Phenotypic and Target	[62]; [64]; [67]; [69]; [77]

2.1. Drug abuse target categories addressed via cell-based HTS

A factor that significantly complicates substance abuse research is the number of biological components involved: for example, a recent chemogenomics knowledgebase publication listed over 85 G-protein coupled receptors (GPCRs) alone thought to be involved in substance abuse, a daunting number [33]. Below, we provide limited examples on the recent utilization of high-throughput assays to discover cell-active modulators of several key target classes implicated in drug abuse/addiction (also see Table 2).

Table 2. Example Tool Compounds from Receptor cell-based HTS Assays.

Name	Structure	Receptor	Plate Density	Reporter	References
ML326		Muscarinic acetylcholine	384	Ca2+ (Fluo-4)	[129]
ML375		Muscarinic acetylcholine	384	Ca2+ (Fluo-4)	[49]
ML381		Muscarinic acetylcholine (M5)	1,536	Ca2+ (Fluo-4)	[48]
VU0463841		Metabotropic glutamate receptor subtype 5 (mGlu5)	384	Ca2+ (Fluo-4)	[51]
ML138		Kappa Opioid Receptor	1,536	β-arrestin w/Luciferase	[130]
ML139		Kappa Opioid Receptor	1,536	β-arrestin w/Luciferase	[130]
ML140		Kappa Opioid Receptor	1,536	β-arrestin w/Luciferase	[130]
ML190		Kappa Opioid Receptor	1,536	β-arrestin w/Luciferase	[130]
ML191		GPR55	384	GFP-labeled β-arrestin	[58]
ML192		GPR55	384	GFP-labeled β-arrestin	[58]
ML193		GPR55	384	GFP-labeled β-arrestin	[58]
ML321		Dopamine (D2)	1,536	Ca2+ (Fluo-8)	[42]
ML297		GIRK Potassium Channel	384	Thallium Flux (Thallos-AM)	[57]
ML154		Neuropeptide S	1,536	Ca2+ (Fluo-4)	[67]
72		Orexin 1	96	calcium 5	[69]
32c		Sigma 1	96	[3H] Radiolabeled	[131]
ML314		Neurotensin NTR1	1,536	GFP-labeled β-arrestin	[54]

2.1.1. Opioids/Opiates

The κ-opioid receptor (KOR)-dynorphin system has been implicated in the control of affect, cognition, and motivation, and is thought to be dysregulated in mood and psychotic disorders, as well as in various phases of opioid dependence [34,35]. Using miniaturized assays based on a genetically engineered firefly luciferase cyclic AMP (cAMP) biosensor, the Tango™ and bioluminescence resonance energy transfer (BRET) technologies, several KOR-selective ligand scaffolds were recently identified, displaying a range of signaling biases in vitro [36]. In another recent study, two classes of biased KOR agonists that potently activate G-protein coupling but weakly recruit arrestin 2 were discovered through a 96-well radiolabeled [³⁵S]GTPγS and a 384-well GFP-labeled U2OS-hKOR-βarrestin2 cell methods, respectively[37].

2.1.2. Serotonin and Dopamine

During the past few decades evidence has steadily accumulated that 5-HT_2C receptor agonists alter various behaviors and underlying neurobiological systems relevant to drug abuse and addiction, supporting the possibility for use of selective 5-HT_2C agonists to treat nicotine and psychostimulant dependence [38]. Using a 96-well serotonin radiolabel assay, and a 384-well Cy3b-labeled fluorescence polarization (FP) binding assay, the small molecule 5a was identified as a high-affinity tracer ligand for assessing the binding affinity of novel ligands for 5HT_2C [39].

Transporters for dopamine, norepinephrine and serotonin (DAT, NET and SERT, respectively) represent established targets for many pharmacological agents that affect brain function, including antidepressants and psychostimulants. Using a novel neurotransmitter transporter uptake assay kit based on a 384-well FLIPR^TETRA cell transporter assay with a neurotransmitter loading dye, the authors demonstrated the ability to monitor the dynamic transport activity of DAT, NET and SERT by employing a fluorescent substrate that mimics the biogenic amine neurotransmitters. The labeled substrate is taken up by the cell through the specific transporters, resulting in increased fluorescence intensity, forming the basis of an assay amenable to high-throughput screening and compound characterization [40].

Dopamine D₂ receptors inhibit the activity of neurons on which they are expressed; thus, a decrease in D₂ receptor activity in indirect pathway neurons would take away a layer of inhibition and would be expected to increase indirect pathway activity, thereby decreasing motivation to take substances of abuse, such as cocaine[41]. A recent effort utilizing a highly-miniaturized 1,536-well assay based on the Quest Fluo-8 calcium detection dye in D₂ and D₃ β-arrestin overexpressing cells resulted in the identification of a novel D₂ DAR antagonist series with excellent D₂ versus D₁, D₃, D₄, and D₅ receptor selectivity that carries the potential for treatment of multiple neuropsychiatric and endocrine disorders. [42,43]

Sensitization of adenylyl cyclase signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Previous approaches to study sensitization have been generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Using two D₂ dopamine receptor cellular models, Conley et al. reported converting a laborious sensitization assay (>20 steps taking 4-5 days) to a five-step, single day assay in 384-well format, along with a demonstration that the assay design could also be readily used for reverse transfection of siRNA for future targeted siRNA library screening [44].

2.1.3. Acetylcholine

The muscarinic acetylcholine receptors (mAChRs) are members of the G Protein-Coupled Receptor (GPCR) family A that mediate the metabotropic actions of the neurotransmitter acetylcholine. To date, five distinct subtypes of mAChRs (M1-M5) have been cloned and sequenced, and blocking of M5 receptors has been shown to reduce reinforcement and withdrawal symptoms of morphine, as well as cocaine, addiction [45-47]. Using HTS in 1,536-well format, combined with medicinal chemistry efforts, Gentry et al. discovered ML381 (also referred to as VU0480131) as the most potent and selective M5-orthosteric antagonist reported to date[48]. Similarly, a functional high-throughput screen and subsequent medicinal chemistry effort identified the first mAChR negative allosteric modulator, ML375, with submicromolar potency and high selectivity for the M5 subtype.[49]

2.1.4. Glutamate

Though certain types of behavioral therapy have proven effective for treatment of cocaine addiction, relapse remains high, and there are currently no approved medications for the treatment of cocaine abuse. Recent evidence suggests a critical role for the metabotropic glutamate receptor subtype 5 (mGlu5) in the modulation of neural circuitry associated with the addictive properties of cocaine [50]. Using a 384-well based calcium mobilization assay, Amato et al. developed a potent and selective small molecule (VU0463841) with good CNS exposure in rats. Its utility was further demonstrated by its ability to attenuate drug seeking behaviors in relevant rat models of cocaine addiction[51].

Vesicular Monoamine Transporter 2 (VMAT2) inhibitors are also of interest for treatment of psychostimulant abuse and addiction. The natural product lobeline and its derivatives inhibit methamphetamine-induced dopamine release, as well as methamphetamine self-administration, via the inhibition of VMAT2. These compounds, structurally distinct from reserpine and tetrabenazine, are pursued as novel therapeutics in preclinical and clinical studies of methamphetamine abuse disorders. Recently, a new fluorescent probe, FFN206, was reported as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy. The probe was used in a cell-based fluorescence assay using VMAT2-transfected HEK cells, with excellent Z′-factors of 0.7 – 0.8 reported[52].

2.1.5. Neurotensin (NT) receptors

Methamphetamine addiction remains a substantial public health issue and currently no small molecule therapies are available for its treatment. Neurotensin receptors are expressed on dopaminergic neurological pathways associated with reward and the neurotensin receptor 1 (NTR1) has been proposed as a therapeutic target for the treatment of methamphetamine abuse. NTR1 peptide agonists produce behaviors that are exactly opposite of the psychostimulant effects observed with methamphetamine abuse, such as hyperactivity, neurotoxicity, psychotic episodes, and cognitive deficits, and repeated administrations of NTR1 agonists do not lead to the development of tolerance. [53] A high-throughput screening campaign utilizing a 1,536-well-based calcium flux assay, followed by medicinal chemistry optimization, led to the discovery of ML314, a nonpeptidic β-arrestin biased agonist for NTR1[54].

2.1.6. G-protein coupled receptors and associated targets

The body of G-protein-activated inwardly rectifying potassium channel (GIRK) research implicates GIRK in diverse processes such as heart rhythm control, effects on reward/addiction, and modulation of response to analgesic [55]. GIRK regulation by GPCRs is believed to be linked to the biological effects of a variety of GPCR agonists, including opioids, acetylcholine, and the gamma-aminobutyric acid (GABA_B) receptor agonist baclofen. Representing a significant advancement in our ability to selectively probe GIRK's role in physiology, Kaufmann et al. recently reported the development and characterization of ML297 (VU0456810) as the first potent and selective GIRK activator; the molecule was discovered through a thallium-flux based screen of the NIH Molecular Libraries collection [56],[57]. GPR55 is a class A GPCR with roles in pain, metabolic disorder, bone development, and cancer [33]. In another screen of the Molecular Libraries collection, utilizing fluorescence-based β-arrestin 384-well assay, novel GPR55 antagonist chemotypes with IC₅₀ values in the range of 0.16–2.72 μM were identified, serving as a starting point for further development[58].

Regulator of G-protein signaling (RGS) proteins potently suppress GPCR signal transduction by accelerating GTP hydrolysis on G-protein α [59]. RGS4 is overexpressed in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. An HEK293-FlpIn cell line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Using this 384-well calcium-mediated fluorescence assay, several cell-active RGS4 inhibitors have been identified for use in future biological studies. [60]

2.1.7. cAMP levels

Both tolerance and dependence occur because frequent drug use can suppress components of the brain's reward circuit. The cAMP response element-binding protein (CREB) is a transcription factor with multiple targets within the cell. When drugs of abuse are administered, dopamine concentrations rise, leading to increased production of cAMP, which in turn activates CREB, leading to a multitude of downstream signaling events in part responsible for the development of addiction[22]. To develop tools aimed at further understanding the biology surrounding cAMP/CREB and morphine addiction, the late Marshall Nirenberg used a cellular model system of neuroblastoma-glioma hybrid cells (NG108-15). To identify small molecules that can inhibit morphine-induced cAMP overshoot, a homogeneous time-resolved fluorescence (HTRF) cell-based assay that measures cellular cAMP production after morphine withdrawal was utilized with the above cell line. [61]

2.1.8. Neuropeptides

The role of neuropeptide Y Y2 receptor (Y2R) has been implicated in human diseases such as severe obesity, mood disorders, and alcoholism, but the currently available Y2R antagonists do not permit detailed biological studies to fully delineate the receptor's role. A high-throughput screening approach utilizing the FLIPR technology in 1,536-well format resulted in the identification of 5 selective, brain-penetrant small-molecule Y2R antagonists, with reportedly different mechanism of action from previously-reported agents. [62]

The neuropeptide galanin and the galanin receptor 3 (GalR3) have been implicated in addiction, specifically alcoholism, and mood-related disorders such as anxiety and depression[63]. Recently, the generation of a modified GalR3 that facilitates its cell surface expression, while maintaining wild-type receptor pharmacology, has been reported; it is expected that this platform, utilizing the FLIPR technology in 384-well format, will enable identification of GalR3-selective modulators to facilitate dissection of the biological role(s) that GalR3 plays in normal physiology and dysregulated states [64,65]

Neuropeptide S Receptor (NPSR) is a GPCR first described by Sato et al. in 2004 [66]. Utilizing an HTRF cAMP assay in 1,536-well format in combination with dose-response type screening, ML154, a structurally novel potent small molecule modulator of NPS-NPSR neurocircuitry, was discovered. ML154 is reportedly the most potent in vivo active NPSR-targeting compound to date and has superior microsomal stability compared to other lead NPSR antagonists disclosed in the literature, making it a good tool to use in studying NPS/NPSR pharmacology and the role of NPSR antagonism in sleep, anxiety, food intake, and addiction [17,67,68].

Orexin A and B, also known as hypocretin 1 and 2, are hypothalamic neuropeptides discovered in the late-1990s. They are the endogenous ligands for two GPCRs, orexin 1 (OX1) and orexin 2 (OX2). Increasing evidence implicates the OX1 receptor in reward processes, suggesting that OX1 antagonism could be used as a therapeutic intervention in drug addiction. Starting from an HTS using calcium fluorescence-based assay, and further optimizing hit series through medicinal chemistry, Perrey et al. reported the development of OX1-selective antagonists based on a tetrahydroisoquinoline scaffold[69].

2.2. Biochemical HTS Assays

While less well represented in the area of drug abuse research, biochemical assay platforms play a very important role in discovery of drug leads, particularly given the fact that they often allow the unambiguous identification of modulators of a discrete protein target; furthermore, biochemical assays often tend to be cheaper to implement on a large scale and may permit the evaluation of binding affinity.

Aldehyde dehydrogenase 2 (ALDH2) is an enzyme responsible for degradation of the metabolite acetaldehyde, the chemical responsible for triggering the red face in individuals sensitive to alcohol; modulation of acetaldehyde levels has also been exploited in the past through the use of disulfiram as an alcohol-abuse deterrent. Thus, highly-specific inhibition of ALDH2 may have a potential application for alcohol aversion therapy, and more recently, in cocaine addiction. Past efforts to discover ALDH2-selective inhibitors include in vitro enzyme activity screens as well as virtual computational screens using the structures of the target enzyme [70][71]. Recently, we developed highly-miniaturized 1,536-well assays to discover inhibitors of the broad family of dehydrogenase enzymes, including aldehyde dehydrogenases: the assay is based on the fluorescent detection of the NADH product resulting from the dehydrogenase-catalyzed reduction of its NAD⁺ cofactor, with detection performed in real-time kinetic format. Application of this assay in large-scale robotic screening has resulted in identification of multiple inhibitory chemotypes whose detailed characterization is ongoing[72].

RGS proteins, introduced earlier, are critical modulators of GPCR signaling given their ability to deactivate G_α subunits via GTPase-accelerating protein (GAP) activity. Several biochemical approaches to RGS targeting have been described recently. A Transcreener® FP immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP has been used to detect the GTPase activity of a G_αi1 mutant interacting with RGS4. [73,74] The assay, initially developed in 384-well format, has recently been further miniaturized to 1,536-well density and used to screen a ∼400,000-compound collection in dose-response format[75]. Furthermore, RGS4 was highlighted in a Malachite Green assay for phosphate release: in a pilot screen using this assay, 13 compounds were identified and subjected to further analysis[76].

The transcriptional regulator ΔFosB accumulates in the striatum in response to chronic administration of drugs of abuse, L-DOPA, or stress, with implications to the neurological changes associated with aspects of drug addiction. To discover chemical probes to study ΔFosB, a high-throughput assay was developed to monitor the interaction of ΔFosB with its target DNA sequence through the use of FP detection. From the resulting screen, two compounds capable of disrupting the interaction with low micromolar activity were identified. The two compounds displayed different activities against ΔFosB homodimers compared to ΔFosB/JunD heterodimers, suggesting that they could be useful in studies of the contribution of different ΔFosB complexes to transcription regulation and to further evaluate ΔFosB as a therapeutic target [77,78].

2.3. Model organism and related systems

The difficulty in translating fundamental biological discoveries into tangible therapies has further highlighted the need for better predictive technologies employed in early discovery. The need for better predictive models is applicable to the area of substance abuse research, as well [32]. Among lower model organisms, zebrafish (Danio rerio) have been the subject of growing interest, with recent reviews addressing their applicability as a model in general, and in particular applicability to substance abuse research [79-84][85-88]. While not immediately relevant to drug abuse screening, recent advances in assay development utilizing zebrafish include target-based fluorescent reporter systems to track the level and distribution of proteins of interest [89], imaging algorithms to detect and quantitate blood vessel formation in zebrafish [90], and use of GFP-expressing transgenes to monitor differentiation into specific lineages [91].

Another lower organism that can be raised cost-effectively to be applied in HTS settings is the vinegar fly Drosophila melanogaster. Flies and vertebrates share many metabolic functions, molecular machinery, and analogous organ systems that control nutrient uptake, storage, and metabolism. In a recent screen for Drosophila larva feeding, a serotonin antagonist, metitepine, was identified as a potential modulator of feeding, with possible implications to eating disorders research[92]. The recently reported designer receptors exclusively activated by designer drugs (DREADDs), based on muscarinic acetylcholine GPCR, are also expected to serve as important tools to build Drosophila models of drug abuse [93]. In addition to zebrafish and the vinegar fly, C. elegans (Caenorhabditis elegans) researchers have made enormous strides to make this model worm HTS-amenable [94]. As methods are sought to predict a compound's efficacy and toxicity in man, having a battery of lower organism models employed in prioritization ahead of the more costly and lower-throughput rodent models will speed up the advancement of potential therapeutics.

Use of higher-level species, such as rodents, in drug abuse early discovery has been limited by cost considerations and, just as importantly, by doubts about the relevance of behavioral models based on rodents to the corresponding human physiology. Nonetheless, recent findings suggest that some behavioral abnormalities elicited by psychedelics in rodents might predict such effects in humans[95]. Further, technological advances in computer vision and machine learning are making it possible to conduct automated, potentially scaled-up, observational experiments to monitor complex animal behavior without the need for laborious human intervention: examples include the SmartCube[96], Pattern Array [97], PhenoMaster/LabMaster [98], INTELLICAGE systems [99], and the Behavioral Spectrometer[100].

3. Additional approaches

3.1. Active and passive immunization

As an alternative approach potentially superior to reacting, researchers have moved toward identifying patients with genetic traits that lead to predisposition to addiction and in turn have directed efforts toward finding ways to prevent such an addiction or its long-term effects. Anti-addiction vaccines, aimed at eliciting antibodies that block the pharmacological effects of drugs leading to addiction, have great potential for treating drug abuse [101-104]. Nicotine vaccines [105], in addition to those for cocaine and methamphetamine [106-108], are currently in various stages of development.

In addition to active immunization strategies, a properly designed passive immunization, also referred to as antidote or anti-serum, carries the potential to serve as a first-line defense in cases of severe drug overdose. An example of this line of research is the work by Janda and Koob to develop catalytic antibody-based treatment for cocaine overdose: a catalytic antibody could in principle dramatically accelerate the estherolytic conversion of cocaine to an inactive derivative and thus help lower the cocaine blood levels in an overdose victim in emergency settings. Earlier research provided proof of concept by showing that the catalytic anti-cocaine monoclonal antibody GNC92H2, derived through an immunization with a cocaine-hydrolysis transition-state hapten and subsequent hybridoma production and clonal isolation, catalyzed cocaine hydrolysis and blocked the psychostymulatory effects of cocaine in animal model[109,110]. However, the reaction acceleration afforded by this antibody proved insufficient for its use in the clinic, necessitating the development of better catalysts. Nevertheless, a recent report has highlighted the potential to use a related phage-display strategy to efficiently deliver to the CNS, while protecting from degradation, either improved catalytic antibodies or enzymes such as butyrylcholinesterase, the major human cocaine-metabolizing enzyme, or bacterial cocaine esterase, the most efficient cocaine degradation enzyme, with the goal of achieving enhanced hydrolytic action on drugs of abuse[111].

3.2. High-throughput discovery of advantageous drug-drug combinations

Combination therapies offer the advantage of an increased positive effect, potentially at lower drug doses, and with associated lower toxicity. While the concept of using drug cocktails is not new, only very recently technological advances have allowed combination screening and data analysis to be performed on a truly large scale HTS [112]: in this study, the authors used acoustic dispense technology, coupled with improved informatics analysis processes, to develop a platform for the high-speed testing of many thousands of drug-drug combinations in a 6×6 concentration matrix, followed by a confirmatory testing of select pairs in a more detailed 10×10 matrix. It can be expected that the public availability of the protocols and software code for this matrix-type screening will spur efforts to re-screen established assays, potentially finding new therapeutic avenues.

In summary, early discovery efforts targeting drug abuse comprise a broad spectrum of platform, including traditional cell-based and biochemical assays, along with emerging technologies such as model-organism screening, automated animal-behavior monitoring, and high-throughput discovery of synergistic drug-drug combinations. Appreciation of the role of epigenetics is growing, as well.

4. Expert Opinion

Technologically, drug abuse drug discovery does not appear to be very different from the approaches employed in other disease types; recent examples from the literature provided within indicate that, with few exceptions, the assay formats and detection platforms used in drug abuse screening are the same as those employed elsewhere. The important difference lies in the enormous biological complexity associated with the control of processes such as mood, sense of satisfaction and reward, cravings, and others, that collectively conspire to drive an individual down the path of addiction. With such complexity comes the associated difficulty in finding “good” targets and achieving exquisite selectivity of potential therapeutic agents. As an added demand, sometimes drugs against drug abuse need to be extremely safe and devoid of side effects as they are likely to be applied for extended periods of the subject's life. On the other hand, there exist possibilities for short term treatments to help break addictions whereby a drug against drug abuse would not be administered over long periods.

Complicating factors such as those described above make it especially important for the field to accelerate utilization of next-generation methods and models, and to seek novel therapeutic targets. To this end, efforts have been growing the areas of drug repurposing [113], as well as to gain a better understanding of the role of epigenetic regulation in the predisposition to and genesis of addiction[114-116], with a growing interest in the role of microbiome also being anticipated. Along with the already-reviewed platforms to discover novel drug-drug combinations and increased use of automated whole-animal behavioral screening systems, the employment of organoid-level three-dimensional test platforms (also referred to as tissue-chip or organs-on-chip), which carry the promise of recapitulating the complexity inherent to whole-organism physiology, will be a crucial next step towards better validating initial discoveries made using the more reductionist traditional cell-culture type screens.

While the negative effects of drugs of abuse have been described in detail, it is only relatively recently that potential benefits of these drugs, for which there exist vast clinical, efficacy, and toxicity data from human use, have begun to be investigated. With repurposing now at the forefront of drug discovery programs, public-access programs such as the Molecular Pharmacology Research Program[117] and the NIMH Psychoactive Drug Screening Program[118,119] are allowing for these bioavailable and efficacious compounds to be tested in order to determine what other diseases they may have the potential to treat. Recent examples of the novel use of the psychedelic psilocybin[120,121] and ecstasy[122] for depression and PTSD, respectively, show the potential of repurposing of compounds with known clinical efficacy, and argues for including them in screening collections[123].

In closing, it is worth pointing out again that substance/drug abuse represents an extreme form of “evolution”: while the above examples show how the leading substances currently being abused are being targeted (through therapeutic interventions and - though not the subject of this review – through law enforcement, regulatory, and other mechanisms), there is a just-as-strong of a trend within the drug abuse community to seek new substances that would be undetectable or are currently unregulated so that the addiction, and associated profits thereof, can continue unabated. Indeed, recent publications highlight the rising tide of synthetic substances that sometimes represent constantly-changing chemical analogues of an initial addictive molecule which traditional management approaches through testing and legislative measures fail to capture rapidly enough [124-128].

Highlights.

• In addition to traditional target- and pathway-based cell-based high-throughput screening to discover modulators of addiction, biochemical and whole-organism assay approaches have recently gained prominence.

• Passive and active immunizations are emerging as key approaches to treating drug and substance abuse.

• Emerging areas of research include next generation technologies and target areas, such as three-dimensional models, automated animal-behavior monitoring, high-throughput discovery of synergistic drug-drug combinations, and increased appreciation of the role of epigenetics.

List of abbreviations

IC₅₀

concentration that produces 50% inhibition

HTS

high-throughput screening

PubChem AID

PubChem Assay Identifier

MOA

mechanism of action

SAR

structure-activity relationship

HTRF

homogeneous time-resolved fluorescence

BRET

bioluminescence resonance energy transfer

cAMP

cyclic adenosine monophosphate

CREB

cAMP response element-binding protein

FP

fluorescence polarization

NIDA

The National Institute on Drug Abuse

KOR

κ-opioid receptor

DAT

dopamine

NET

norepinephrine

SERT

serotonin

AC

adenylyl cyclase

mAChRs

muscarinic acetylcholine receptors

GPCR

G Protein-Coupled Receptor

mGlu5

metabotropic glutamate receptor subtype 5

VMAT2

Vesicular Monoamine Transporter 2

NTR1

neurotensin receptor 1

OX1

orexin 1

OX2

orexin 2

ALDH2

aldehyde dehydrogenase 2

GAP

GTPase-accelerating protein