Discovery of Novel Oleamide Analogues as Brain-Penetrant Positive Allosteric Serotonin 5-HT2C Receptor and Dual 5-HT2C/5-HT2A Receptor Modulators.

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.

Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site.

Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners.

Small-Molecule Inhibitors Targeting the Canonical WNT Signaling Pathway for the Treatment of Cancer.

Canonical WNT signaling is an important developmental pathway that has attracted increased attention for anticancer drug discovery. From the production and secretion of WNT ligands, their binding to membrane receptors, and the ß-catenin destruction complex to the expansive ß-catenin transcriptional complex, multiple components have been investigated as drug targets to modulate WNT signaling. Significant progress in developing WNT inhibitors such as porcupine inhibitors, tankyrase inhibitors, ß-catenin/coactivators, protein-protein interaction inhibitors, casein kinase modulators, DVL inhibitors, and dCTPP1 inhibitors has been made, with several candidates (e.g., LGK-974, PRI-724, and ETC-159) in human clinical trials. Herein we summarize recent progress in the drug discovery and development of small-molecule inhibitors targeting the canonical WNT pathway, focusing on their specific target proteins, in vitro and in vivo activities, physicochemical properties, and therapeutic potential. The relevant opportunities and challenges toward maintaining the balance between efficacy and toxicity in effectively targeting this pathway are also highlighted.

Discovery of a Small Molecule Inhibitor of Human Adenovirus Capable of Preventing Escape from the Endosome.

Human adenoviruses (HAdVs) display a wide range of tissue tropism and can cause an array of symptoms from mild respiratory illnesses to disseminated and life-threatening infections in immunocompromised individuals. However, no antiviral drug has been approved specifically for the treatment of HAdV infections. Herein, we report our continued efforts to optimize salicylamide derivatives and discover compound 16 (JMX0493) as a potent inhibitor of HAdV infection. Compound 16 displays submicromolar IC50 values, a higher selectivity index (SI > 100) and 2.5-fold virus yield reduction compared to our hit compound niclosamide. Moreover, unlike niclosamide, our mechanistic studies suggest that the antiviral activity of compound 16 against HAdV is achieved through the inhibition of viral particle escape from the endosome, which bars subsequent uncoating and the presentation of lytic protein VI.

Small Molecules Selectively Targeting Sigma-1 Receptor for the Treatment of Neurological Diseases.

The sigma-1 (σ1) receptor, an enigmatic protein originally classified as an opioid receptor subtype, is now understood to possess unique structural and functional features of its own and play critical roles to widely impact signaling transduction by interacting with receptors, ion channels, lipids, and kinases. The σ1 receptor is implicated in modulating learning, memory, emotion, sensory systems, neuronal development, and cognition and accordingly is now an actively pursued drug target for various neurological and neuropsychiatric disorders. Evaluation of the five selective σ1 receptor drug candidates (pridopidine, ANAVEX2-73, SA4503, S1RA, and T-817MA) that have entered clinical trials has shown that reaching clinical approval remains an evasive and important goal. This review provides up-to-date information on the selective targeting of σ1 receptors, including their history, function, reported crystal structures, and roles in neurological diseases, as well as a useful collation of new chemical entities as σ1 selective orthosteric ligands or allosteric modulators.

Discovery of 4-Phenylpiperidine-2-Carboxamide Analogues as Serotonin 5-HT2C Receptor-Positive Allosteric Modulators with Enhanced Drug-like Properties.

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.

Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological Evaluation.

Bromodomain-containing protein 4 (BRD4) represents a promising drug target for anti-inflammatory therapeutics. Herein, we report the design, synthesis, and pharmacological evaluation of novel chromone derivatives via scaffold hopping to discover a new class of orally bioavailable BRD4-selective inhibitors. Two potent BRD4 bromodomain 1 (BD1)-selective inhibitors 44 (ZL0513) and 45 (ZL0516) have been discovered with high binding affinity (IC50 values of 67-84 nM) and good selectivity over other BRD family proteins and distant BD-containing proteins. Both compounds significantly inhibited the expression of Toll-like receptor-induced inflammatory genes in vitro and airway inflammation in murine models. The cocrystal structure of 45 in complex with human BRD4 BD1 at a high resolution of 2.0 Å has been solved, offering a solid structural basis for its binding validation and further structure-based optimization. These BRD4 BD1 inhibitors demonstrated impressive in vivo efficacy and overall promising pharmacokinetic properties, indicating their therapeutic potential for the treatment of inflammatory diseases.

Targeting the 5-HT2C Receptor in Biological Context and the Current State of 5-HT2C Receptor Ligand Development.

Serotonin (5-HT) 5-HT2C receptor (5-HT2CR) is recognized as a critical mediator of diseaserelated pathways and behaviors based upon actions in the central nervous system (CNS). Since 5-HT2CR is a class A G protein-coupled receptor (GPCR), drug discovery efforts have traditionally pursued the activation of the receptor through synthetic ligands with agonists proposed for the treatment of obesity, substance use disorders and impulse control disorders while antagonists may add value for the treatment of anxiety, depression and schizophrenia. The most significant agonist discovery to date is the FDAapproved anti-obesity medication lorcaserin. In recent years, efforts towards developing other mechanisms to enhance receptor function have resulted in the discovery of Positive Allosteric Modulators (PAMs) for the 5-HT2CR, with several molecule series now reported. The biological significance and context for signaling and function of the 5-HT2CR, and the current status of 5-HT2CR agonists and PAMs are discussed in this review.

Structure-activity relationship studies on Bax activator SMBA1 for the treatment of ER-positive and triple-negative breast cancer.

In an effort to develop novel Bax activators for breast cancer treatment, a series of diverse analogues have been designed and synthesized based on lead compound SMBA1 through several strategies, including introducing various alkylamino side chains to have a deeper access to S184 pocket, replacing carbon atoms with nitrogen, and reducing the nitro group of 9H-fluorene scaffold. Compounds 14 (CYD-2-11) and 49 (CYD-4-61) have been identified to exhibit significantly improved antiproliferative activity compared to SMBA1, with IC50 values of 3.22 µM and 0.07 µM against triple-negative breast cancer MDA-MB-231 and 3.81 µM and 0.06 µM against ER-positive breast cancer MCF-7 cell lines, respectively. Mechanism of action studies of compound 49 indicated that it can activate Bax protein to induce cytochrome c release and regulate apoptotic biomarkers, leading to cancer cell apoptosis. Further in vivo efficacy studies of compounds 14 and 49 in nude mice bearing MDA-MB-231 tumor xenografts demonstrated that these drug candidates can significantly suppress tumor growth, indicating their therapeutic potential for the treatment of breast cancer.

Applications of Bioorthogonal Chemistry in Tumor-Targeted Drug Discovery.

Chemical reactions that can proceed in living systems while not interfering with native biochemical processes are collectively referred to as bioorthogonal chemistry. Selectivity, efficiency, and aqueous compatibility are three significant characteristics of an ideal bioorthogonal reaction. To date, the specialized bioorthogonal reactions that have been reported include: Cu (I)-catalyzed [3 + 2] azido- alkyne cycloadditions (CuAAC), strain-promoted [3 + 2] azide-alkyne cycloadditions (SPAAC), Staudinger ligation, photo-click 1,3-dipolar cycloadditions, strain-promoted alkyne-nitrone cycloadditions (SPANC), transition metal catalysis (TMC), and inverse electron demand Diels-Alder (IEDDA). These reactions are divided into two subtypes, 1) bond-formation reactions (e.g. CuAAC, SPAAC, photo-click cycloadditions, SPANC), which can be conventionally applied in the chemical biology field for target identification, protein-specific modifications and others; and 2) bond-release reactions (e.g. Staudinger ligation, TMC, and IEDDA), which are emerging as powerful approaches for the study of protein activation and drug discovery. Over the past decade, bioorthogonal chemistry has enabled important compound design features in targeted drug discovery and has expanded biological knowledge on intractable targets. Research groups have also focused on the discovery of reactions with improved biocompatibility and increased reaction rates, which will undoubtably prove essential for future therapeutic development. Herein, we highlight two significant applications of bioorthogonal chemistry to drug discovery, which are tumor-targeted prodrug delivery and activation, and self-assembly of bifunctional molecules. The relevant challenges and opportunities are also discussed.

Design, Synthesis, and Characterization of 4-Undecylpiperidine-2-carboxamides as Positive Allosteric Modulators of the Serotonin (5-HT) 5-HT2C Receptor.

An impaired signaling capacity of the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) has been implicated in the neurobehavioral processes that promote relapse vulnerability in cocaine use disorder (CUD). Restoration of the diminished 5-HT2CR signaling through positive allosteric modulation presents a novel therapeutic approach. Several new molecules with the 4-alkylpiperidine-2-carboxamide scaffold were designed, synthesized, and pharmacologically evaluated, leading to the discovery of selective 5-HT2CR positive allosteric modulators (PAMs). Compound 16 (CYD-1-79) potentiated 5-HT-evoked intracellular calcium release in cells stably expressing the human 5-HT2CR but not the 5-HT2AR cells. A topographically distinct allosteric site was identified based on the newly solved 5-HT2CR structure. Compound 16 modulated 5-HT2CR-mediated spontaneous ambulation, partially substituted for the training dose of the 5-HT2CR agonist WAY163909, synergized with a low dose of WAY163909 to substitute fully for the stimulus effects of WAY163909, and attenuated relapse vulnerability as assessed in a rodent self-administration model, indicating its therapeutic promise for CUD.

Allosteric Modulation of Class A GPCRs: Targets, Agents, and Emerging Concepts.

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.

Orphan Receptor GPR88 as an Emerging Neurotherapeutic Target.

Although G protein-coupled receptors (GPCRs) are recognized as pivotal drug targets involved in multiple physiological and pathological processes, the majority of GPCRs including orphan GPCRs (oGPCRs) are unexploited. GPR88, a brain-specific oGPCR with particularly robust expression in the striatum, regulates diverse brain and behavioral functions, including cognition, mood, movement control, and reward-based learning, and is thus emerging as a novel drug target for central nervous system disorders including schizophrenia, Parkinson's disease, anxiety, and addiction. Nevertheless, no effective GPR88 synthetic ligands have yet entered into clinical trials, and GPR88 endogenous ligands remain unknown. Despite the recent discovery and early stage study of several GPR88 agonists, such as 2-PCCA, RTI-13951-33, and phenylglycinol derivatives, further research into GPR88 pharmacology, medicinal chemistry, and chemical biology is urgently needed to yield structurally diversified GPR88-specific ligands. Drug-like pharmacological tool function and relevant signaling elucidation will also accelerate the evaluation of this receptor as a viable neurotherapeutic target.

Regio- and Stereospecific Synthesis of Oridonin D-Ring Aziridinated Analogues for the Treatment of Triple-Negative Breast Cancer via Mediated Irreversible Covalent Warheads.

Covalent drug discovery has undergone a resurgence in recent years due to comprehensive optimization of the structure-activity relationship (SAR) and the structure-reactivity relationship (SRR) for covalent drug candidates. The natural product oridonin maintains an impressive pharmacological profile through its covalent enone warhead on the D-ring and has attracted substantial SAR studies to characterize its potential in the development of new molecular entities for the treatment of various human cancers and inflammation. Herein, for the first time, we report the excessive reactivity of this covalent warhead and mediation of the covalent binding capability through a Rh2(esp)2-catalyzed mild and concise regio- and stereospecific aziridination approach. Importantly, aziridonin 44 (YD0514), with a more-druglike irreversible covalent warhead, has been identified to significantly induce apoptosis and inhibit colony formation against triple-negative breast cancer with enhanced antitumor effects in vitro and in vivo while displaying lower toxicity to normal human mammary epithelial cells in comparison to oridonin.

Therapeutic Potential of Oridonin and Its Analogs: From Anticancer and Antiinflammation to Neuroprotection.

Oridonin, a diterpenoid natural product commonly used in East Asian herbal medicine, is garnering increased attention in the biomedical community due to its extensive biological activities that include antitumor, anti-inflammatory, antimicrobial, hepatic fibrosis prevention, and neurological effects. Over the past decade, significant progress has been made in structure activity relationship and mechanism of action studies of oridonin for the treatment of cancer and other diseases. This review provides a brief summary on oridonin and its analogs in cancer drug discovery and antiinflammation and highlights its emerging therapeutic potential in neuroprotection applications.

Drug Discovery Targeting Bromodomain-Containing Protein 4.

BRD4, the most extensively studied member of the BET family, is an epigenetic regulator that localizes to DNA via binding to acetylated histones and controls the expression of therapeutically important gene regulatory networks through the recruitment of transcription factors to form mediator complexes, phosphorylating RNA polymerase II, and by its intrinsic histone acetyltransferase activity. Disrupting the protein-protein interactions between BRD4 and acetyl-lysine has been shown to effectively block cell proliferation in cancer, cytokine production in acute inflammation, and so forth. To date, significant efforts have been devoted to the development of BRD4 inhibitors, and consequently, a dozen have progressed to human clinical trials. Herein, we summarize the advances in drug discovery and development of BRD4 inhibitors by focusing on their chemotypes, in vitro and in vivo activity, selectivity, relevant mechanisms of action, and therapeutic potential. Opportunities and challenges to achieve selective and efficacious BRD4 inhibitors as a viable therapeutic strategy for human diseases are also highlighted.

Therapeutic Potential of Spirooxindoles as Antiviral Agents.

Antiviral therapeutics with profiles of high potency, low resistance, panserotype, and low toxicity remain challenging, and obtaining such agents continues to be an active area of therapeutic development. Due to their unique three-dimensional structural features, spirooxindoles have been identified as privileged chemotypes for antiviral drug development. Among them, spiro-pyrazolopyridone oxindoles have been recently reported as potent inhibitors of dengue virus NS4B, leading to the discovery of an orally bioavailable preclinical candidate (R)-44 with excellent in vivo efficacy in a dengue viremia mouse model. This review highlights recent advances in the development of biologically active spirooxindoles for their antiviral potential, primarily focusing on the structure-activity relationships (SARs) and modes of action, as well as future directions to achieve more potent analogues toward a viable antiviral therapy.

Discovery and development of natural product oridonin-inspired anticancer agents.

Natural products have historically been, and continue to be, an invaluable source for the discovery of various therapeutic agents. Oridonin, a natural diterpenoid widely applied in traditional Chinese medicines, exhibits a broad range of biological effects including anticancer and anti-inflammatory activities. To further improve its potency, aqueous solubility and bioavailability, the oridonin template serves as an exciting platform for drug discovery to yield better candidates with unique targets and enhanced drug properties. A number of oridonin derivatives (e.g. HAO472) have been designed and synthesized, and have contributed to substantial progress in the identification of new agents and relevant molecular mechanistic studies toward the treatment of human cancers and other diseases. This review summarizes the recent advances in medicinal chemistry on the explorations of novel oridonin analogues as potential anticancer therapeutics, and provides a detailed discussion of future directions for the development and progression of this class of molecules into the clinic.

BH4 domain of Bcl-2 as a novel target for cancer therapy.

Overexpression of B cell lymphoma 2 (Bcl-2) proteins is associated with therapy resistance in various human cancers. Traditional approaches target the Bcl-2 homology (BH)3 domain of Bcl-2; however, the BH4 domain represents a superior therapeutic target in light of its unique structure and crucial involvement in many cellular functions. In this critical review, we focus on the structural and functional basis of targeting the BH4 domain of Bcl-2, and highlight the recent advances in drug discovery efforts toward small-molecule BH4 domain inhibitors (e.g. BDA-366). The proof-of-concept studies support the hypothesis that targeting the BH4 domain of Bcl-2 holds promise to offer a novel anticancer therapy through the induction of apoptosis and an increased potential to overcome therapeutic resistance.