The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR localize to the brain and share overlapping signal transduction facets that contribute to their roles in cognition, mood, learning, and memory. Achieving selective targeting of these receptors is challenged by the similarity in their 5-HT orthosteric binding pockets. A fragment-based discovery approach was employed to design and synthesize novel oleamide analogues as selective 5-HT2CR or dual 5-HT2CR/5-HT2AR positive allosteric modulators (PAMs). Compound 13 (JPC0323) exhibited on-target properties, acceptable plasma exposure and brain penetration, as well as negligible displacement to orthosteric sites of ∼50 GPCRs and transporters. Furthermore, compound 13 suppressed novelty-induced locomotor activity in a 5-HT2CR-dependent manner, suggesting 5-HT2CR PAM, but not 5-HT2AR, activity at the level of the whole organism at the employed doses of 13. We discovered new selective 5-HT2CR PAMs and first-in-class 5-HT2CR/5-HT2AR dual PAMs that broaden the pharmacological toolbox to explore the biology of these vital receptors.

Abstract The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a class of severe and chronic diseases of the gastrointestinal (GI) tract with recurrent symptoms and significant morbidity. Long-term persistence of chronic inflammation in IBD is a major contributing factor to neoplastic transformation and the development of colitis-associated colorectal cancer. Conversely, persistence of transmural inflammation in CD is associated with formation of fibrosing strictures, resulting in substantial morbidity. The recent introduction of biological response modifiers as IBD therapies, such as antibodies neutralizing tumor necrosis factor (TNF)-α, have replaced nonselective anti-inflammatory corticosteroids in disease management. However, a large proportion (~40%) of patients with the treatment of anti-TNF-α antibodies are discontinued or withdrawn from therapy because of (1) primary nonresponse, (2) secondary loss of response, (3) opportunistic infection, or (4) onset of cancer. Therefore, the development of novel and effective therapeutics targeting specific signaling pathways in the pathogenesis of IBD is urgently needed. In this comprehensive review, we summarize the recent advances in drug discovery of new small molecules in preclinical or clinical development for treating IBD that target biologically relevant pathways in mucosal inflammation. These include intracellular enzymes (Janus kinases, receptor interacting protein, phosphodiesterase 4, IκB kinase), integrins, G protein-coupled receptors (S1P, CCR9, CXCR4, CB2) and inflammasome mediators (NLRP3), etc. We will also discuss emerging evidence of a distinct mechanism of action, bromodomain-containing protein 4, an epigenetic regulator of pathways involved in the activation, communication, and trafficking of immune cells. We highlight their chemotypes, mode of actions, structure-activity relationships, characterizations, and their in vitro/in vivo activities and therapeutic potential. The perspectives on the relevant challenges, new opportunities, and future directions in this field are also discussed.

The G protein-coupled receptors (GPCRs) control crucial functions in the central nervous system (CNS) and are acknowledged as important medication targets. The serotonin (5-HT) 5-HT2R family (5-HT2AR, 5-HT2BR, 5-HT2CR) mediates a myriad of CNS functions. Of note, 5-HT2CR signaling is implicated in metabolic disorders (e.g., obesity) and a variety of neuropsychiatric disorders (e.g., anxiety, schizophrenia and substance use disorders). Activation of the brain 5-HT2AR mediates psychedelic states and mood and cognitive alterations evoked by hallucinogens, which is attractive for the application of psychedelic compounds in psychiatry. We sought to discover novel modulators to selectively potentiate 5-HT2CR and 5-HT2AR signaling while avoiding the undesired activation of 5-HT2BR, widely known as associated with valvulopathy and pulmonary hypertension adverse effects. The application of positive allosteric modulators (PAMs) is an appealing strategy to modulate 5-HT2CR and 5-HT2AR functional response by binding to a less conserved allosteric site. Our PAM discovery efforts have included a focus on the well-known natural product oleamide, which has been implicated in signaling modulation of 5-HT2AR and 5-HT2CR as well as 5-HT1AR and 5-HT7R, with an overall promiscuous pharmacological profile. Three series of oleamide-derived 5-HT2CR and 5-HT2AR PAMs are designed and synthesized via a fragment-based drug discovery approach. Pharmacological evaluation of novel small molecules was conducted in a cell-based, 5-HT-induced, intracellular calcium (Ca2+) release assay with physiologically relevant 5-HT2CR or 5-HT2AR expression levels. Multiple 5‑HT2CR and 5‑HT2ARPAMs were obtained with a functional increase of 5-HT-induced Ca2+ release by 20% or greater in the in vitro assay. In vitro distribution, metabolism, and pharmacokinetic analyses, off-target radioligand binding displacement studies and physicochemical properties indicate that our 5‑HT2CR and 5‑HT2AR PAMs display promising lead- and drug-like attributes. In silico molecular docking with the 5-HT2CR X-ray crystal structure has identified a potential PAM binding site for the oleamide-derived 5-HT2CR PAMs that is shared with our previously reported PAMs that do not share an oleamide scaffold. In conclusion, we have designed, synthesized, and optimized a novel series of 5‑HT2CR and 5‑HT2AR PAMs with promising in vitropharmacological andphysicochemical profiles as potential neurotherapeutics for impaired serotonergic controlled substance use disorders and other CNS disorders.

Bromodomain‐containing protein 4 (BRD4) is a member of the bromodomain and extra‐terminal domain‐containing (BET) family of chromatin “reader” proteins. Small molecule BET inhibitors have been developed within the context of cancer and inflammation pathologies, and more recently are of interest for central nervous system (CNS) disorders, including opioid use disorder (OUD). BRD4 has two bromodomains (BDs) that recognize specific post‐translational histone variants to control downstream transcriptional outcomes. The protein‐protein interaction between BRD4 and lysine residues of histones is driven through the two highly conserved N‐terminal bromodomains BD1 and BD2. The aim of the present study is to pharmacologically characterize BRD4‐BD1‐specific inhibitors to facilitate investigations into the role of BRD4 mechanisms in preclinical models of opioid use disorder.

Targeting the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) allosteric site to potentiate endogenous 5-HT tone may provide novel therapeutics to alleviate the impact of costly, chronic diseases such as obesity and substance use disorders. Expanding upon our recently described 5-HT2CR positive allosteric modulators (PAMs) based on the 4-alkylpiperidine-2-carboxamide scaffold, we optimized the undecyl moiety at the 4-position with variations of cyclohexyl- or phenyl-containing fragments to reduce rotatable bonds and lipophilicity. Compound 12 (CTW0415) was discovered as a 5-HT2CR PAM with improved pharmacokinetics and reduced off-target interactions relative to our previous series of molecules. The in vivo efficacy of compound 12 to potentiate the effects of a selective 5-HT2CR agonist was established in a drug discrimination assay. Thus, 12 is reported as a 5-HT2CR PAM with characteristics suitable for in vivo pharmacological studies to further probe the biological and behavioral mechanisms of allosteric modulation of a receptor important in several chronic diseases.

The transcription factor ΔFosB accumulates in response to chronic insults such as drugs of abuse, L-3,4-dihydroxyphenylalanine (l-DOPA) or stress in specific regions of the brain, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, dyskinesia, and depression. Thus, small molecule chemical probes are urgently needed to investigate biological functions of ΔFosB. Herein we describe the identification of a novel phenanthridine analogue ZL0220 (27) as an active and promising ΔFosB chemical probe with micromolar inhibitory activities against ΔFosB homodimers and ΔFosB/JunD heterodimers.

Signal transduction and the modulation thereof characterize G protein‐coupled receptor (GPCR) function in the central nervous system (CNS) while CNS GPCRs are the therapeutic targets for most neuropsychiatric disorders. Endogenous ligands (e.g., neurotransmitters) and GPCRs are embedded into a dynamic landscape of temporally and spatially controlled signalling events that contribute to physiological brain function, and dysregulation of GPCR systems is implicated in pathophysiological states. Utilizing studies of the serotonin (5‐HT) 5‐HT2C receptor (5‐HT2CR), which has been implicated in CNS disorders such as drug use disorders and depression, we sought to discover novel modulators of 5‐HT2CR function that maintain the temporal and spatial characteristics of CNS signaling. Decreased 5‐HT2CR signaling has been implicated in dysregulated impulse control and drug‐seeking in rodent preclinical models, with selective 5‐HT2CR agonists exhibiting affinity to dampen these behavioral outcomes. The utilization of positive allosteric modulators (PAMs) is an attractive strategy to increase 5‐HT2CR functional response while limiting disruption of its dynamic signaling landscape. Our PAM discovery efforts have focused on two unique molecular scaffolds (i.e., CYD‐1‐79 and oleamide) and the derivatization of each. Synthesized, novel small molecules were screened via a cell‐based, 5‐HT‐induced, intracellular calcium (Ca2+) release assay with physiologically relevant 5‐HT2CR expression levels. Our studies have resulted in the discovery of multiple 5‐HT2CR PAMs, including CYD‐1‐79 and oleamide‐based molecular scaffolds, that increase 5‐HT2CR‐dependent function by 20% or greater. In vitro distribution, metabolism, and pharmacokinetic (DMPK) analyses and physicochemical properties indicate that our 5‐HT2CR PAMs display a range of promising lead‐ and drug‐like attributes. In silico molecular docking with the recently published 5‐HT2CR X‐ray crystal structure has identified a potential PAM binding site common to a diverse collection of small molecule 5‐HT2CR PAM derivatives. In conclusion, we have discovered and further optimized novel series of 5‐HT2CR PAMs with the potential for translation towards neurotherapeutics for drug use disorders and other CNS disorders in which 5‐HT2CR dysfunction is implicated as a mediator.Support or Funding InformationSupported by grants R01 DA038446 (JZ/KAC), K05 DA020087 (KAC), P30 DA028821 (KAC), T32 DA07287 (CTW, EAW), F31 DA038922 (CTW), F31 DA045511 (EAW)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

G-protein-coupled receptors (GPCRs) have been tractable drug targets for decades with over one-third of currently marketed drugs targeting GPCRs. Of these, the class A GPCR superfamily is highly represented, and continued drug discovery for this family of receptors may provide novel therapeutics for a vast range of diseases. GPCR allosteric modulation is an innovative targeting approach that broadens the available small molecule toolbox and is proving to be a viable drug discovery strategy, as evidenced by recent FDA approvals and clinical trials. Numerous class A GPCR allosteric modulators have been discovered recently, and emerging trends such as the availability of GPCR crystal structures, diverse functional assays, and structure-based computational approaches are improving optimization and development. This Perspective provides an update on allosterically targeted class A GPCRs and their disease indications and the medicinal chemistry approaches toward novel allosteric modulators and highlights emerging trends and opportunities in the field.