Optical Control of Insect Behavior and Receptors with Azobenzene-Bridged Fipronil and Imidacloprid.

Photopharmacology can be implemented in a way of regulating drug activities by light-controlling the molecular configuations. Three photochromic ligands (PCLs) that bind on one or two sites of GABARs and nAChRs were reported here. These multiphoton PCLs, including FIP-AB-FIP, IMI-AB-FIP, and IMI-AB-IMI, are constructed with an azobenzene (AB) bridge that covalently connects two fipronil (FIP) and imidacloprid (IMI) molecules. Interestingly, the three PCLs as well as FIP and IMI showed great insecticidal activities against Aedes albopictus larvae and Aphis craccivora. IMI-AB-FIP in both trans/cis isomers can be reversibly interconverted depending on light, accompanied by insecticidal activity decrease or increase by 1.5-2.3 folds. In addition, IMI-AB-FIP displayed synergistic effects against A. craccivora (LC50, IMI-AB-FIP = 14.84-22.10 µM, LC50, IMI-AB-IMI = 210.52-266.63 µM, LC50, and FIP-AB-FIP = 36.25-51.04 µM), mainly resulting from a conceivable reason for simultaneous targeting on both GABARs and nAChRs. Furthermore, modulations of wiggler-swimming behaviors and cockroach neuron function were conducted and the results indirectly demonstrated the ligand-receptor interactions. In other words, real-time regulations of receptors and insect behaviors can be spatiotemporally achieved by our two-photon PCLs using light.

Structure-based drug design for TSPO: Challenges and opportunities.

The translocator protein 18 kDa (TSPO) is an evolutionarily conserved mitochondrial transmembrane protein implicated in various neuropathologies and inflammatory conditions, making it a longstanding diagnostic and therapeutic target of interest. Despite the development of various classes of TSPO ligand chemotypes, and the elucidation of bacterial and non-human mammalian experimental structures, many unknowns exist surrounding its differential structural and functional features in health and disease. There are several limitations associated with currently used computational methodologies for modelling the native structure and ligand-binding behaviour of this enigmatic protein. In this perspective, we provide a critical analysis of the developments in the uses of these methods, outlining their uses, inherent limitations, and continuing challenges. We offer suggestions of unexplored opportunities that exist in the use of computational methodologies which offer promise for enhancing our understanding of the TSPO.

Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles.

TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein. We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, w:w), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased. Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: Dmax ∼95 Å in DPC alone versus ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (Kd ∼70 µM in DPC alone versus ∼0.91 µM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST). In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.

The mutual and dynamic role of TSPO and ligands in their binding process: An example with PK-11195.

Translocator protein (TSPO) is an 18 kDa transmembrane protein, localized primarily on the outer mitochondrial membrane. It has been found to be involved in various physiological processes and pathophysiological conditions. Though studies on its structure have been performed only recently, there is little information on the nature of dynamics and doubts about some structures referenced in the literature, especially the NMR structure of mouse TSPO. In the present work, we thoroughly study the dynamics of mouse TSPO protein by means of atomistic molecular dynamics simulations, in presence as well as in absence of the diagnostic ligand PKA. We considered two starting structures: the NMR structure and a homology model (HM) generated on the basis of X-ray structures from bacterial TSPO. We examine the conformational landscape in both the modes for both starting points, in presence and absence of the ligand, in order to measure its impact for both structures. The analysis highlights high flexibility of the protein globally, but NMR simulations show a surprisingly flexibility even in the presence of the ligand. Interestingly, this is not the case for HM calculations, to the point that the ligand seems not so stable as in the NMR system and an unbinding event process is partially sampled. All those results tend to show that the NMR structure of mTSPO seems not deficient but is just in another portion of the global conformation space of TSPO.

Shisa reduces the sensitivity of homomeric RDL channel to GABA in the two-spotted spider mite, Tetranychus urticae Koch.

The γ-aminobutyric acid receptors (GABARs) mediate fast inhibitory transmission in central nervous system of insects and are important targets of insecticides. An auxiliary subunit, Shisa7, was identified in mammals as a single-passing transmembrane protein. However, the homology gene(s) of Shisa in invertebrates has not been reported to date. In the present study, a homolog Shisa gene was identified from the two-spotted spider mite, Tetranychus urticae Koch. Its open reading frame had 927 base pairs and encoded 308 amino acid residues, which has a typical Shisa domain at 13th-181st amino acid residues. According to the phylogenetic tree, the invertebrate Shisa was categorized apart with those of vertebrate, and TuShisa showed closest relationship with the Shisa9 of velvet mite, Dinothrombium tinctorium (L.). In the electrophysiological assay with two-electrode voltage clamp, the GABA-activated TuRDL channel was functionally formed in the Africa clawed frog Xenopus laevis (Daudin) oocytes (EC50 = 53.34 µM). No GABA-activated current could be observed in TuShisa-expressed oocytes, whereas TuShisa could reduce the sensitivity of TuRDL/TuShisa (mass ratio of 1: 4) channel to GABA. The homology structural models of TuRDL and TuShisa were built by the SWISS-MODEL server, their interaction was predicted using Z-DOCK and three predicted hydrogen bonds and interface residues were analysed by PyMOL. Meanwhile, the key interface residues of TuShisa affected the stability of complex were calculated by Discovery Studio 2019. In conclusion, the TuShisa, as the first reported invertebrate Shisa, was explored and functionally examined as the GABARs auxiliary subunit. Our findings provide a basis for research of invertebrate Shisa.

The Haemonchus contortus LGC-39 subunit is a novel subtype of an acetylcholine-gated chloride channel.

The nematode genome exhibits a vast array of Cys-loop receptors that are activated by a diverse set of neurotransmitters and anthelmintic drugs such as ivermectin and levamisole. While many Cys-loop receptors have been functionally and pharmacologically characterized, there remains a large subset of orphan receptors where the agonist remains unknown. We have identified an orphan Cys-loop receptor, LGC-39, from the parasitic nematode Haemonchus contortus that is a novel type of cholinergic-sensitive ligand-gated chloride channel. This receptor groups outside of the acetylcholine-gated chloride channel family, in the previously named GGR-1 (GABA/Glycine Receptor-1) group of Cys-loop receptors. We found that LGC-39 forms a functional homomeric receptor when expressed in Xenopus laevis oocytes and is activated by several cholinergic ligands including acetylcholine, methacholine and surprisingly, atropine with an EC50 for atropine on the low µM range. A homology model was generated which revealed some key features of the LGC-39 ligand-binding pocket that may explain some of the elements important for atropine recognition of the LGC-39 receptor. Overall these results suggest that the GGR-1 family (now called LGC-57) of Cys-loop receptors includes novel acetylcholine-gated chloride channel subtypes and may represent important future drug targets.

Effect of amphiphilic environment on the solution structure of mouse TSPO translocator protein.

The translocator protein (TSPO) is a ubiquitous transmembrane protein of great pharmacological interest thanks to its high affinity to many drug ligands. The only high-resolution 3D-structure known for mammalian TSPO was obtained by NMR for the mouse mTSPO in DPC detergent only in presence of the high-affinity PK 11195 ligand. An atomic structure of free-ligand mTSPO is still missing to better understand the interaction of ligands with mTSPO and their effects on the protein conformation. Here, we decipher the solution structures of the recombinant mTSPO without ligand both in (i) SDS, the detergent used to extract and purify the protein from E. coli inclusion bodies, and (ii) DPC, the detergent used to solve the PK 11195-binding mTSPO NMR structure. We report partially refolded and less flexible mTSPO helices in DPC compared to SDS. Besides, DPC stabilizes the tertiary structure of mTSPO, as shown by a higher intrinsic Trp fluorescence and changes in indole environment. We evaluate by SEC-MALLS that ∼135 SDS and ∼100 DPC molecules are bound to mTSPO. SEC-small-angle X-ray (SAXS) and neutron (SANS) scattering confirm a larger mTSPO-detergent complex in SDS than in DPC. Using the contrast-matching technique in SEC-SANS, we demonstrate that mTSPO conformation is more compact and less flexible in DPC than in SDS. Combining ab initio modeling with SANS, we confirm that mTSPO conformation is less elongated in DPC than in SDS. However, the free-ligand mTSPO envelope in DPC is not as compact as the PK 11195-binding protein NMR structure, the ligand stiffening the protein.

Solid-state NMR study of structural heterogeneity of the apo WT mouse TSPO reconstituted in liposomes.

In the last decades, ligand binding to human TSPO has been largely used in clinical neuroimaging, but little is known about the interaction mechanism. Protein conformational mobility plays a key role in the ligand recognition and both, ligand-free and ligand-bound structures, are mandatory for characterizing the molecular binding mechanism. In the absence of crystals for mammalian TSPO, we have exploited solid-state nuclear magnetic resonance (ssNMR) spectroscopy under magic-angle spinning (MAS) to study the apo form of recombinant mouse TSPO (mTSPO) reconstituted in lipids. This environment has been previously described to permit binding of its high-affinity drug ligand PK11195 and appears therefore favourable for the study of molecular dynamics. We have optimized the physical conditions to get the best resolution for MAS ssNMR spectra of the ligand-free mTSPO. We have compared and combined various ssNMR spectra to get dynamical information either for the lipids or for the mTSPO. Partial assignment of residue types suggests few agreements with the published solution NMR assignment of the PK11195-bound mTSPO in DPC detergent. Moreover, we were able to observe some lateral chains of aromatic residues that were not assigned in solution. 13C double-quantum NMR spectroscopy shows remarkable dynamics for ligand-free mTSPO in lipids which may have significant implications on the recognition of the ligand and/or other protein partners.

Characterization of adjacent charged residues near the agonist binding site of the nematode UNC-49 GABA receptor.

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features. Here we investigated three residues in the UNC-49 receptor from the parasitic nematode Haemonchus contortus: K181, E183, and T230. Analysis of these residues was conducted via site-directed mutagenesis, electrophysiology, MD simulations, and mutant cycling analysis. In the UNC-49 receptor, E183 lies in close proximity to K181 where together they appear to play a role in GABA sensitivity and pharmacology, possibly interacting via an ionic bond. While the introduction of single alanine residues at each position separately had a negative impact on GABA EC50, the double alanine mutant (K181A/E183A) exhibited wildtype-level GABA EC50 and some differences in pharmacology. Overall, this study has revealed a potentially novel role for these two residues in nematode UNC-49 GABA receptors that could aid in understanding their function.

Identification of the probable structure of the sAPPα-GABABR1a complex and theoretical solutions for such cases.

Amyloid precursor protein (APP) is the core of the pathogenesis of Alzheimer's disease (AD). Existing studies have shown that the soluble secreted APP (sAPPα) fragment obtained from the hydrolysis of APP by α-secretase has a synaptic function. Thereinto, a nine-residue fragment (APP9mer) of the extension domain region of sAPPα can bind directly and selectively to the N-terminal sushi1 domain (SD1) of the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a) protein, which can influence synaptic transmission and plasticity by changing the GABABR1a conformation. APP9mer is a highly flexible, disordered region, and as such it is difficult to experimentally determine the optimal APPmer-SD1 binding complex. In this study we constructed two types of APP9mer-SD1 complexes through molecular docking and molecular dynamics simulation, aiming to explore the recognition function and mechanism of the specific binding of APP9mer with SD1, from which the most probable APPmer-SD1 model conformation is predicted. All the data from the analyses of RMSD, RMSF, PCA, DCCM and MM/PBSA binding energy as well as comparison with the experimental dissociation constant Kd suggest that 2NC is the most likely conformation to restore the crystal structure of the experimental APP9mer-SD1 complex. Of note, the key recognition residues of APP9mer are D24, D25, D27, W29 and W30, which mainly act on the 9-45 residue domain of SD1 (consisting of two loops and three short ß-chains at the N-terminus of SD1). The mini-model with key residues identified establishes the molecular basis with deep insight into the interaction between APP and GABABR1a and provides a target for the development of therapeutic strategies for modulating GABABR1a-specific signaling in neurological and psychiatric disorders. More importantly, the study offers a theoretical solution for how to determine a biomolecular structure with a highly flexible, disordered fragment embedded within. The flexible fragment involved in a protein structure has to be deserted usually during the structural determination with experimental methods (e.g. X-ray crystallography, etc.).

The Repertoire of Small-Molecule PET Probes for Neuroinflammation Imaging: Challenges and Opportunities beyond TSPO.

Neuroinflammation is an adaptive response of the central nervous system to diverse potentially injurious stimuli, which is closely associated with neurodegeneration and typically characterized by activation of microglia and astrocytes. As a noninvasive and translational molecular imaging tool, positron emission tomography (PET) could provide a better understanding of neuroinflammation and its role in neurodegenerative diseases. Ligands to translator protein (TSPO), a putative marker of neuroinflammation, have been the most commonly studied in this context, but they suffer from serious limitations. Herein we present a repertoire of different structural chemotypes and novel PET ligand design for classical and emerging neuroinflammatory targets beyond TSPO. We believe that this Perspective will support multidisciplinary collaborations in academic and industrial institutions working on neuroinflammation and facilitate the progress of neuroinflammation PET probe development for clinical use.

Muscimol Directly Activates the TREK-2 Channel Expressed in GABAergic Neurons through Its N-Terminus.

The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.

Neuroactive Type-A γ-Aminobutyric Acid Receptor Allosteric Modulator Steroids from the Hypobranchial Gland of Marine Mollusk, Conus geographus.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E (1-5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1-5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1ß1γ2 and α4ß3γ2 receptors (IC50 1.5 and 1.0 µM, respectively). Although the structures of 1-6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.

Mechanistic evaluation of a novel cyclohexenone derivative's functionality against nociception and inflammation: An in-vitro, in-vivo and in-silico approach.

The synthesis of a novel cyclohexanone derivative (CHD; Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-enecarboxylate) was described and the subsequent aim was to perform an in vitro, in vivo and in silico pharmacological evaluation as a putative anti-nociceptive and anti-inflammatory agent in mice. Initial in vitro studies revealed that CHD inhibited both cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes and it also reduced mRNA expression of COX-2 and the pro-inflammatory cytokines TNF-α and IL-1ß. It was then shown that CHD dose dependently inhibited chemically induced tonic nociception in the abdominal constriction assay and also phasic thermal nociception (i.e. anti-nociception) in the hot plate and tail immersion tests in comparison with aspirin and tramadol respectively. The thermal test outcomes indicated a possible moderate centrally mediated anti-nociception which, in the case of the hot plate test, was pentylenetetrazole (PTZ) and naloxone reversible, implicating GABAergic and opioidergic mechanisms. CHD was also effective against both the neurogenic and inflammatory mediator phases induced in the formalin test and it also disclosed anti-inflammatory activity against the phlogistic agents, carrageenan, serotonin, histamine and xylene compared with standard drugs in edema volume tests. In silico studies indicated that CHD possessed preferential affinity for GABAA, opioid and COX-2 target sites and this was supported by molecular dynamic simulations where computation of free energy of binding also favored the formation of stable complexes with these sites. These findings suggest that CHD has prospective anti-nociceptive and anti-inflammatory properties, probably mediated through GABAergic and opioidergic interactions supplemented by COX-2 and 5-LOX enzyme inhibition in addition to reducing pro-inflammatory cytokine expression. CHD may therefore possess potentially beneficial therapeutic effectiveness in the management of inflammation and pain.

5-O-methylcneorumchromone K Exerts Antinociceptive Effects in Mice via Interaction with GABAA Receptors.

The proper pharmacological control of pain is a continuous challenge for patients and health care providers. Even the most widely used medications for pain treatment are still ineffective or unsafe for some patients, especially for those who suffer from chronic pain. Substances containing the chromone scaffold have shown a variety of biological activities, including analgesic effects. This work presents for the first time the centrally mediated antinociceptive activity of 5-O-methylcneorumchromone K (5-CK). Cold plate and tail flick tests in mice showed that the 5-CK-induced antinociception was dose-dependent, longer-lasting, and more efficacious than that induced by morphine. The 5-CK-induced antinociception was not reversed by the opioid antagonist naloxone. Topological descriptors (fingerprints) were employed to narrow the antagonist selection to further investigate 5-CK's mechanism of action. Next, based on the results of fingerprints analysis, functional antagonist assays were conducted on nociceptive tests. The effect of 5-CK was completely reversed in both cold plate and tail-flick tests by GABAA receptor antagonist bicuculline, but not by atropine or glibenclamide. Molecular docking studies suggest that 5-CK binds to the orthosteric binding site, with a similar binding profile to that observed for bicuculline and GABA. These results evidence that 5-CK has a centrally mediated antinociceptive effect, probably involving the activation of GABAergic pathways.

Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick cholesterol analogue.

The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive cholesterol analogue. PhotoClick cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s).

The Interplay of Cholesterol and Ligand Binding in hTSPO from Classical Molecular Dynamics Simulations.

The translocator protein (TSPO) is a 18kDa transmembrane protein, ubiquitously present in human mitochondria. It is overexpressed in tumor cells and at the sites of neuroinflammation, thus representing an important biomarker, as well as a promising drug target. In mammalian TSPO, there are cholesterol-binding motifs, as well as a binding cavity able to accommodate different chemical compounds. Given the lack of structural information for the human protein, we built a model of human (h) TSPO in the apo state and in complex with PK11195, a molecule routinely used in positron emission tomography (PET) for imaging of neuroinflammatory sites. To better understand the interactions of PK11195 and cholesterol with this pharmacologically relevant protein, we ran molecular dynamics simulations of the apo and holo proteins embedded in a model membrane. We found that: (i) PK11195 stabilizes hTSPO structural fold; (ii) PK11195 might enter in the binding site through transmembrane helices I and II of hTSPO; (iii) PK11195 reduces the frequency of cholesterol binding to the lower, N-terminal part of hTSPO in the inner membrane leaflet, while this impact is less pronounced for the upper, C-terminal part in the outer membrane leaflet, where the ligand binding site is located; (iv) very interestingly, cholesterol most frequently binds simultaneously to the so-called CRAC and CARC regions in TM V in the free form (residues L150-X-Y152-X(3)-R156 and R135-X(2)-Y138-X(2)-L141, respectively). However, when the protein is in complex with PK11195, cholesterol binds equally frequently to the CRAC-resembling motif that we observed in TM I (residues L17-X(2)-F20-X(3)-R24) and to CRAC in TM V. We expect that the CRAC-like motif in TM I will be of interest in future experimental investigations. Thus, our MD simulations provide insight into the structural features of hTSPO and the previously unknown interplay between PK11195 and cholesterol interactions with this pharmacologically relevant protein.

Chiral Pd-Catalyzed Enantioselective Syntheses of Various N-C Axially Chiral Compounds and Their Synthetic Applications.

Biaryl atropisomers are key structural components in chiral ligands, chiral functional materials, natural products, and bioactive compounds, and their asymmetric syntheses have been reported by many groups. In contrast, although the scientific community has long been aware of atropisomers due to rotational restriction around N-C bonds, they have attracted scant attention and have remained an unexplored research area. In particular, their catalytic asymmetric synthesis and the synthetic applications were unknown until recently. This Account describes studies conducted by our group on the catalytic enantioselective syntheses of N-C axially chiral compounds and their applications in asymmetric reactions.In the presence of a chiral Pd catalyst, the reactions of achiral secondary ortho-tert-butylanilides with 4-iodonitrobenzene proceeded in a highly enantioselective manner (up to 96% ee), affording N-C axially chiral N-arylated ortho-tert-butylanilides in good yields. The application of the present chiral Pd-catalyzed N-arylation reaction to an intramolecular version gave N-C axially chiral lactams with high optical purity (up to 98% ee). These reactions were the first highly enantioselective syntheses of N-C axially chiral compounds with a chiral catalyst. Since the publication of these reactions, N-C axially chiral compounds have been widely accepted as new target molecules for catalytic asymmetric reactions. Furthermore, chiral-Pd-catalyzed intramolecular N-arylations were applied to the enantioselective syntheses of N-C axially chiral quinoline-4-one and phenanthridin-6-one derivatives. We also succeeded in the enantioselective syntheses of various N-C axially chiral compounds using other chiral Pd-catalyzed reactions. That is, optically active N-C axially chiral N-(2-tert-butylphenyl)indoles, 3-(2-bromophenyl)quinazolin-4-ones, and N-(2-tert-butylphenyl)sulfonamides were obtained through chiral Pd-catalyzed 5-endo-hydroaminocyclization, monohydrodebromination (reductive asymmetric desymmetrization), and Tsuji-Trost N-allylation, respectively. The study of the catalytic asymmetric synthesis of axially chiral indoles has contributed to the development of not only N-C axially chiral chemistry but also the chemistry of axially chiral indoles. Subsequently, the catalytic asymmetric syntheses of various indole derivatives bearing a C-C chiral axis as well as an N-C chiral axis have been reported by many groups. Moreover, axially chiral quinazlolin-4-one derivatives, which were obtained through chiral Pd-catalyzed asymmetric desymmetrization, are pharmaceutically attractive compounds; for example, 2-methyl-3-(2-bromophenyl)quinazolin-4-one product is a mebroqualone possessing GABA agonist activity.Most of the N-C axially chiral products have satisfactory rotational stability for synthetic applications, and their synthetic utility was also demonstrated through application to chiral enolate chemistry. That is, the reaction of various alkyl halides with the enolate prepared from the optically active anilide, lactam, and quinazolinone products proceeded with high diastereoselectivity by asymmetric induction due to the N-C axial chirality.At the present time, N-C axially chiral chemistry has become a popular research area, especially in synthetic organic chemistry, and original papers on the catalytic asymmetric syntheses of various N-C axially chiral compounds and their synthetic applications have been published.