Tsaokoic Acid: A New Bicyclic Nonene from the Fruits of Amomum tsao-ko with Acetylcholinesterase Inhibitory Activity.

A new bicyclic nonene, tsaokoic acid (1), was isolated from the fruits of Amomum tsao-ko, together with three known compounds (2-4). The structure of 1 was elucidated by analyzing spectroscopic data including 1D and 2D NMR spectra and compounds 2-4 were identified as tsaokoin, vanillin, and tsaokoarylone, respectively, by comparing their NMR spectra with previously reported data. Compounds 1-4 showed possible inhibitory activity against acetylcholinesterase (AChE) in silico molecular docking simulations. They were submitted to in vitro assay system and exhibited moderate inhibitory activity with IC50 values of 32.78, 41.70, 39.25, and 31.13 µM, respectively.

Triphenylphosphonium conjugation to a TRAP1 inhibitor, 2-amino-6-chloro-7,9-dihydro-8H-purin-8-one increases antiproliferative activity.

Tumor-necrosis-factor-receptor associated protein 1 (TRAP1), a mitochondrial paralog of heat shock protein 90 family proteins, is overexpressed in many cancer cells and supports tumorigenesis by rewiring vital metabolic and cell death pathways. The triphenylphosphonium moiety is used to deliver therapeutic cargo to increase drug uptake into mitochondria. Various aryl- or alkyl-substituted phosphonium analogs were conjugated with TRAP1-selective inhibitors 4a-c to optimize anticancer activity. Among these various phosphonium-conjugated compounds, (6-(2-amino-9-(4-bromo-2-fluorobenzyl)-6-chloro-8-oxo-8,9-dihydro-7H-purin-7-yl)hexyl)triphenylphosphornium (6a) was identified as a potential anticancer agent. Compound 6a had IC50 values of 0.30-3.24 µM in seven different cancer cell lines and potently suppressed tumor growth without any noticeable in vivo toxicity in a nude mouse model xenografted with PC3 prostate cancer cells.

Mitoquinone Inactivates Mitochondrial Chaperone TRAP1 by Blocking the Client Binding Site.

Heat shock protein 90 (Hsp90) family proteins are molecular chaperones that modulate the functions of various substrate proteins (clients) implicated in pro-tumorigenic pathways. In this study, the mitochondria-targeted antioxidant mitoquinone (MitoQ) was identified as a potent inhibitor of mitochondrial Hsp90, known as a tumor necrosis factor receptor-associated protein 1 (TRAP1). Structural analyses revealed an asymmetric bipartite interaction between MitoQ and the previously unrecognized drug binding sites located in the middle domain of TRAP1, believed to be a client binding region. MitoQ effectively competed with TRAP1 clients, and MitoQ treatment facilitated the identification of 103 TRAP1-interacting mitochondrial proteins in cancer cells. MitoQ and its redox-crippled SB-U014/SB-U015 exhibited more potent anticancer activity in vitro and in vivo than previously reported mitochondria-targeted TRAP1 inhibitors. The findings indicate that targeting the client binding site of Hsp90 family proteins offers a novel strategy for the development of potent anticancer drugs.

Physiological involvement of presynaptic L-type voltage-dependent calcium channels in GABA release of cerebellar molecular layer interneurons.

While high threshold voltage-dependent Ca2+ channels (VDCCs) of the N and P/Q families are crucial for evoked neurotransmitter release in the mammalian CNS, it remains unclear to what extent L-type Ca2+ channels (LTCCs), which have been mainly considered as acting at postsynaptic sites, participate in the control of transmitter release. Here, we investigate the possible role of LTCCs in regulating GABA release by cerebellar molecular layer interneurons (MLIs) from rats. We found that BayK8644 (BayK) markedly increases mIPSC frequency in MLIs and Purkinje cells (PCs), suggesting that LTCCs are expressed presynaptically. Furthermore, we observed (1) a potentiation of evoked IPSCs in the presence of BayK, (2) an inhibition of evoked IPSCs in the presence of the LTCC-specific inhibitor Compound 8 (Cp8), and (3) a strong reduction of mIPSC frequency by Cp8. BayK effects are reduced by dantrolene, suggesting that ryanodine receptors act in synergy with LTCCs. Finally, BayK enhances presynaptic AP-evoked Ca2+ transients and increases the frequency of spontaneous axonal Ca2+ transients observed in TTX. Taken together, our data demonstrate that LTCCs are of primary importance in regulating GABA release by MLIs.

Discovery of 2-((4-resorcinolyl)-5-aryl-1,2,3-triazol-1-yl)acetates as potent Hsp90 inhibitors with selectivity over TRAP1.

As the most abundant heat shock protein (HSP), Hsp90 is actively involved in tumor cell growth and various responses to anti-carcinogenic stress. Hsp90 has thus emerged as a potential drug target. A structure-based drug design approach was applied to develop novel resorcinolyltriazole derivatives as Hsp90 inhibitors. Structure-activity relationships (SARs) and molecular docking were investigated to provide a rationale for binding affinity and paralog selectivity. Click chemistry between iodoethynylresorcinol and an azido derivative was used to synthesize a new family of 2-((4-resorcinolyl)-5-aryl-1,2,3-triazol-1-yl) acetates that exhibited Hsp90 binding affinities of 40-100 nM (IC50). Among the synthesized molecules, the triazole alkyl acetates displayed the highest Hsp90 binding affinities. Their potency against Hsp90 was over 100-fold stronger than against TRAP1 and 1-3-fold stronger than against Grp94. In particular, compounds 18, 19, and 30 had Hsp90 inhibitory activities of ~45 nM (IC50) and they displayed over 350-fold selectivity for Hsp90 over TRAP1.

Practical direct synthesis of N-aryl-substituted azacycles from N-alkyl protected arylamines using TiCl4 and DBU.

A novel transformation of N-alkyl protected arylamines and cyclic ethers into N-aryl substituted azacycles is described. Alkyl groups have been used for the protection of amines in organic syntheses. In this synthesis, N-alkyl protected arylamines were reacted with cyclic ethers in the presence of TiCl4 and DBU, crucial reagents affording five- and six-membered azacycles. In particular, utilization of the novel TiCl4/DBU-mediated reaction allows various N-alkyl protected arylamines such as N-methyl-, N-ethyl-, N-isopropyl, and N-tert-butyl arylamines to be readily converted into N-aryl substituted azacycles in high yields. This practical approach using various N-alkyl arylamines leads to the efficient preparation of azacycles.

Development of pyrazolo[3,4-d]pyrimidine-6-amine-based TRAP1 inhibitors that demonstrate in vivo anticancer activity in mouse xenograft models.

TNF Receptor Associated Protein 1 (TRAP1) is a mitochondrial paralog of Hsp90 related to the promotion of tumorigenesis in various cancers via maintaining mitochondrial integrity, reducing the production of reactive oxygen species, and reprogramming cellular metabolism. Consequently, Hsp90 and TRAP1 have been targeted to develop cancer therapeutics. Herein, we report a series of pyrazolo[3,4-d]pyrimidine derivatives that are mitochondria-permeable TRAP1 inhibitors. Structure-based drug design guided the optimization of potency, leading to the identification of compounds 47 and 48 as potent TRAP1 and Hsp90 inhibitors with good metabolic and plasma stability as well as acceptable CYP and hERG inhibition. X-ray co-crystallization studies confirmed both 47 and 48 interact with the ATP binding pocket in the TRAP1 protein. Compounds 47 and 48 demonstrated excellent anticancer efficiency in various cancer cells, with limited toxicity over normal hepatocyte and prostate cells. Mouse PC3 xenograft studies showed 47 and 48 significantly reduced tumor growth.

Metal-Free Synthesis of N-Aryl-Substituted Azacycles from Cyclic Ethers Using POCl3.

A facile method for the synthesis of N-aryl-substituted azacycles from arylamines and cyclic ethers has been developed. In this study, arylamines were treated with cyclic ethers in the presence of POCl3 and DBU to provide five- and six-membered azacycles. Using this method, various azacycloalkanes, isoindolines, and tetrahydroisoquinolines were prepared in high yields. This synthetic method offers an efficient approach to the production of azacycles from cyclic ethers.

Synthesis and evaluation of novel potent TSPO PET ligands with 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide.

Translocator protein (TSPO) expression is closely related with neuroinflammation and neuronal damage which might cause several central nervous system diseases. Herein, a series of TSPO ligands (11a-c and 13a-d) with a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide structure were prepared and evaluated via an in vitro binding assay. Most of the novel ligands exhibited a nano-molar affinity for TSPO, which was better than that of DPA-714. Particularly, 11a exhibited a subnano-molar TSPO binding affinity with suitable lipophilicity for in vivo brain studies. After radiolabeling with fluorine-18, [18F]11a was used for a dynamic positron emission tomography (PET) study in a rat LPS-induced neuroinflammation model; the inflammatory lesion was clearly visualized with a superior target-to-background ratio compared to [18F]DPA-714. An immunohistochemical examination of the dissected brains confirmed that the uptake location of [18F]11a in the PET study was consistent with a positively activated microglia region. This study proved that [18F]11a could be employed as a potential PET tracer for detecting neuroinflammation and could give possibility for diagnosis of other diseases, such as cancers related with TSPO expression.

Evolution of In Silico Strategies for Protein-Protein Interaction Drug Discovery.

The advent of advanced molecular modeling software, big data analytics, and high-speed processing units has led to the exponential evolution of modern drug discovery and better insights into complex biological processes and disease networks. This has progressively steered current research interests to understanding protein-protein interaction (PPI) systems that are related to a number of relevant diseases, such as cancer, neurological illnesses, metabolic disorders, etc. However, targeting PPIs are challenging due to their "undruggable" binding interfaces. In this review, we focus on the current obstacles that impede PPI drug discovery, and how recent discoveries and advances in in silico approaches can alleviate these barriers to expedite the search for potential leads, as shown in several exemplary studies. We will also discuss about currently available information on PPI compounds and systems, along with their usefulness in molecular modeling. Finally, we conclude by presenting the limits of in silico application in drug discovery and offer a perspective in the field of computer-aided PPI drug discovery.

CF3-Substituted Mollugin 2-(4-Morpholinyl)-ethyl ester as a Potential Anti-inflammatory Agent with Improved Aqueous Solubility and Metabolic Stability.

Although mollugin, the main ingredient of the oriental medicinal herb Rubia cordifolia, has considerable anti-inflammatory effects, it has poor aqueous solubility as well as poor metabolic and plasma stability. To overcome these shortfalls, various mollugin derivatives have been synthesized and evaluated for their ability to inhibit U937 monocyte cell adhesion to HT-29 colonic epithelial cells in TNF-α- or IL-6-induced models of colon inflammation. The 2-(4-morpholinyl)-ethyl ester of CF3-substituted mollugin (compound 15c) showed good water solubility, improved metabolic and plasma stability, and greater inhibitory activity than mesalazine in both the TNF-α- and IL-6-induced colonic epithelial cell adhesion assays, suggesting that 15c is a potential anti-inflammatory agent.

Electrostatic Control of Isoform Selective Inhibitor Binding in Nitric Oxide Synthase.

Development of potent and isoform selective nitric oxide synthase (NOS) inhibitors is challenging because of the structural similarity in the heme active sites. One amino acid difference between NOS isoforms, Asp597 in rat neuronal NOS (nNOS) versus Asn368 in bovine endothelial NOS (eNOS), has been identified as the structural basis for why some dipeptide amide inhibitors bind more tightly to nNOS than to eNOS. We now have found that the same amino acid variation is responsible for substantially different binding modes and affinity for a new class of aminopyridine-based inhibitors.

Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease.

Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.

Structural and biological studies on bacterial nitric oxide synthase inhibitors.

Nitric oxide (NO) produced by bacterial NOS functions as a cytoprotective agent against oxidative stress in Staphylococcus aureus, Bacillus anthracis, and Bacillus subtilis. The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds impede the growth of B. subtilis under oxidative stress, and crystal structures show that each compound exhibits a unique binding mode. Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

Inhibitor Bound Crystal Structures of Bacterial Nitric Oxide Synthase.

Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors.

Development of nitric oxide synthase inhibitors for neurodegeneration and neuropathic pain.

Nitric oxide (NO) is an important signaling molecule in the human body, playing a crucial role in cell and neuronal communication, regulation of blood pressure, and in immune activation. However, overproduction of NO by the neuronal isoform of nitric oxide synthase (nNOS) is one of the fundamental causes underlying neurodegenerative disorders and neuropathic pain. Therefore, developing small molecules for selective inhibition of nNOS over related isoforms (eNOS and iNOS) is therapeutically desirable. The aims of this review focus on the regulation and dysregulation of NO signaling, the role of NO in neurodegeneration and pain, the structure and mechanism of nNOS, and the use of this information to design selective inhibitors of this enzyme. Structure-based drug design, the bioavailability and pharmacokinetics of these inhibitors, and extensive target validation through animal studies are addressed.

The mobility of a conserved tyrosine residue controls isoform-dependent enzyme-inhibitor interactions in nitric oxide synthases.

Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS-inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity.

Microwave-assisted protection of primary amines as 2,5-dimethylpyrroles and their orthogonal deprotection.

Primary amines can be readily doubly protected as N-substituted 2,5-dimethylpyrroles. Although this protecting group is stable toward strong bases and nucleophiles, long reaction times are required for both the protection and deprotection steps, generally resulting in low deprotection yields. By employing microwave irradiation, protection and deprotection reaction times are dramatically reduced. Furthermore, deprotection with dilute hydrochloric acid in ethanol increases reaction yields. Diverse deprotection conditions have been developed in conjunction with microwave irradiation, so that protection as an N-substituted 2,5-dimethylpyrrole can be orthogonal to other standard amine protecting groups, such as tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), and 9-fluorenylmethyloxycarbonyl (Fmoc).

Antagonism of L-type Ca2+ channels CaV1.3 and CaV1.2 by 1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine mimics.

The L-type calcium channel (LTCC) CaV1.3 is regarded as a new potential therapeutic target for Parkinson's disease. Calcium influx through CaV1.3 LTCC during autonomous pacemaking in adult dopaminergic neurons of the substantia nigra pars compacta is related to the generation of mitochondrial oxidative stress in animal models. Development of a CaV1.3 antagonist selective over CaV1.2 is essential because CaV1.2 pore-forming subunits are the predominant form of LTCCs and are abundant in the central nervous and cardiovascular systems. We have explored 1,4-dihydropyrimidines and 4H-pyrans to identify potent and selective antagonists of CaV1.3 relative to CaV1.2 LTCCs. A library of 36 dihydropyridine (DHP)-mimic 1,4-dihydropyrimidines and 4H-pyrans was synthesized, and promising chiral compounds were resolved. The antagonism studies of CaV1.3 and CaV1.2 LTCCs using DHP mimic compounds showed that dihydropyrimidines and 4H-pyrans are effective antagonists of DHPs for CaV1.3 LTCCs. Some 1,4-dihydropyrimidines are more selective than isradipine for CaV1.3 over CaV1.2, shown here by both calcium flux and patch-clamp electrophysiology experiments, where the ratio of antagonism is around 2-3. These results support the hypothesis that the modified hydrogen bonding donor/acceptors in DHP-mimic dihydropyrimidines and 4H-pyrans can interact differently with DHP binding sites, but, in addition, the data suggest that the binding sites of DHP in CaV1.3 and CaV1.2 LTCCs are very similar.