Meclizine, a piperazine-derivative antihistamine, binds to dimerized translationally controlled tumor protein and attenuates allergic reactions in a mouse model.

Translationally controlled tumor protein (TCTP), a highly conserved protein present in most eukaryotes, is involved in numerous biological processes. Only the dimeric form of TCTP (dTCTP) formed during inflammatory conditions exhibits cytokine-like activity. Therefore, dTCTP is considered as a therapeutic target for allergic diseases. Because monomeric TCTP (mTCTP) and dTCTP share a high topological similarity, we hypothesized that small molecules interacting with mTCTP would also bind to dTCTP and interfere with dTCTP-based cellular processes. In this study, nine compounds listed in the literature as interacting with mTCTP were investigated for their ability to suppress the activity of extracellular dTCTP in bronchial epithelial cells. It was found that one of the nine, meclizine, a piperazine-derivative antihistamine, significantly reduced IL-8 release and suppressed the NF-κB pathway. The direct interaction of meclizine with dTCTP was confirmed by surface plasmon resonance (SPR). Also, we found that meclizine can attenuate ovalbumin (OVA)-induced airway inflammation in mice. Therefore, meclizine might be a potential anti-allergic drug as an inhibitor for dTCTP.

Structure, Function, and Inhibitors of the Mitochondrial Chaperone TRAP1.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone modulating cellular metabolism and signaling pathways by altering the conformation, activity, and stability of numerous substrate proteins called clients. It exerts its chaperone function as an adaptive response to counter cellular stresses instead of maintaining housekeeping protein homeostasis. However, the stress-adaptive machinery becomes dysregulated to support the progression and maintenance of human diseases, such as cancers; therefore, TRAP1 has been proposed as a promising target protein for anticancer drug development. In this review, by collating recent reports on high-resolution TRAP1 structures and structure-activity relationships of inhibitors, we aimed to provide better insights into the chaperoning mechanism of the emerging drug target and to suggest an efficient strategy for the development of potent TRAP1 inhibitors.

2-Amino-1,3,4-thiadiazoles as Glutaminyl Cyclases Inhibitors Increase Phagocytosis through Modification of CD47-SIRPα Checkpoint.

Glutaminyl cyclases (QC, isoQC) convert N-terminal glutamine or glutamate into pyroglutamate (pGlu) on substrates. IsoQC has recently been demonstrated to promote pGlu formation on the N-terminus of CD47, the SIRPα binding site, contributing to the "don't eat me" cancer immune signaling of CD47-SIRPα. We developed new QC inhibitors by applying a structure-based optimization approach starting from fragments identified through library screening. Screening of metal binding fragments identified 5-(1H-benzimidazol-5-yl)-1,3,4-thiadiazol-2-amine (9) as a potent fragment, and further modification provided 5-(1-(3-methoxy-4-(3-(piperidin-1-yl)propoxy)benzyl)-1H-benzo[d]imidazol-5-yl)-1,3,4-thiadiazol-2-amine (22b) as a potent QC inhibitor. Treatment with 22b in A549 and H1975 lung cancer cells decreased the CD47/αhCD47-CC2C6 interaction, indicative of the CD47/SIRPα interaction, and enhanced the increased phagocytic activity of both THP-1 and U937 macrophages.

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.

Design and Synthesis of TRAP1 Selective Inhibitors: H-Bonding with Asn171 Residue in TRAP1 Increases Paralog Selectivity.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is overexpressed in the mitochondria of various cancer cells, reprograms cellular metabolism to enable cancer cells to adapt to harsh tumor environments. As inactivation of TRAP1 induces massive apoptosis in cancer cells in vitro and in vivo, the development of TRAP1-selective inhibitors has become an attractive approach. A series of purine-8-one and pyrrolo[2,3-d]pyrimidine derivatives was developed based on TRAP1 structure and identified to be highly selective in vitro for TRAP1 over the paralogous enzymes, Hsp90α and Grp94. The TRAP1-selective inhibition strategy via utilization of the Asn171 residue of the ATP-lid was investigated using X-ray crystallography and molecular dynamics simulation studies. Among various synthesized potent TRAP1 inhibitors, 5f possessed a 65-fold selectivity over Hsp90α and a 13-fold selectivity over Grp94. Additionally, 6f had a half-maximal inhibitory concentration (IC50) of 63.5 nM for TRAP1, with a 78-fold and 30-fold selectivity over Hsp90α and Grp94, respectively.

Deciphering Fatty Acid Synthase Inhibition-Triggered Metabolic Flexibility in Prostate Cancer Cells through Untargeted Metabolomics.

Fatty acid synthase (FAS) is a key enzyme involved in de novo lipogenesis that produces lipids that are necessary for cell growth and signal transduction, and it is known to be overexpressed, especially in cancer cells. Although lipid metabolism alteration is an important metabolic phenotype in cancer cells, the development of drugs targeting FAS to block lipid synthesis is hampered by the characteristics of cancer cells with metabolic flexibility leading to rapid adaptation and resistance. Therefore, to confirm the metabolic alterations at the cellular level during FAS inhibition, we treated LNCaP-LN3 prostate cancer cells with FAS inhibitors (Fasnall, GSK2194069, and TVB-3166). With untargeted metabolomics, we observed significant changes in a total of 56 metabolites in the drug-treated groups. Among the altered metabolites, 28 metabolites were significantly changed in all of the drug-treated groups. To our surprise, despite the inhibition of FAS, which is involved in palmitate production, the cells increase their fatty acids and glycerophospholipids contents endogenously. Also, some of the notable changes in the metabolic pathways include polyamine metabolism and energy metabolism. This is the first study to compare and elucidate the effect of FAS inhibition on cellular metabolic flexibility using three different FAS inhibitors through metabolomics. We believe that our results may provide key data for the development of future FAS-targeting drugs.

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.

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.

Casein kinase-1γ1 and 3 stimulate tumor necrosis factor-induced necroptosis through RIPK3.

Upon necroptosis activation, receptor interacting serine/threonine kinase (RIPK)1 and RIPK3 form a necrosome complex with pseudokinase mixed lineage kinase-like (MLKL). Although protein phosphorylation is a key event for RIPK1 and RIPK3 activation in response to a necroptosis signal, relatively little is known about other factors that might regulate the activity of these kinases or necrosome formation. Through a gain-of-function screen with 546 kinases and 127 phosphatases, we identified casein kinase 1 gamma (CK1γ) as a candidate necroptosis-promoting factor. Here, we show that the decreased activity or amounts of CK1γ1 and CK1γ3, either by treatment with a chemical inhibitor or knockdown in cells, reduced TNFα-induced necroptosis. Conversely, ectopic expression of CK1γ1 or CK1γ3 exacerbated necroptosis, but not apoptosis. Similar to RIPK1 and RIPK3, CK1γ1 was also cleaved at Asp343 by caspase-8 during apoptosis. CK1γ1 and CK1γ3 formed a protein complex and were recruited to the necrosome harboring RIPK1, RIPK3 and MLKL. In particular, an autophosphorylated form of CK1γ3 at Ser344/345 was detected in the necrosome and was required to mediate the necroptosis. In addition, in vitro assays with purified proteins showed that CK1γ phosphorylated RIPK3, affecting its activity, and in vivo assays showed that the CK1γ-specific inhibitor Gi prevented abrupt death in mice with hypothermia in a model of TNFα-induced systemic inflammatory response syndrome. Collectively, these data suggest that CK1γ1 and CK1γ3 are required for TNFα-induced necroptosis likely by regulating RIPK3.

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.

Exploring G Protein-Coupled Receptors (GPCRs) Ligand Space via Cheminformatics Approaches: Impact on Rational Drug Design.

The primary goal of rational drug discovery is the identification of selective ligands which act on single or multiple drug targets to achieve the desired clinical outcome through the exploration of total chemical space. To identify such desired compounds, computational approaches are necessary in predicting their drug-like properties. G Protein-Coupled Receptors (GPCRs) represent one of the largest and most important integral membrane protein families. These receptors serve as increasingly attractive drug targets due to their relevance in the treatment of various diseases, such as inflammatory disorders, metabolic imbalances, cardiac disorders, cancer, monogenic disorders, etc. In the last decade, multitudes of three-dimensional (3D) structures were solved for diverse GPCRs, thus referring to this period as the "golden age for GPCR structural biology." Moreover, accumulation of data about the chemical properties of GPCR ligands has garnered much interest toward the exploration of GPCR chemical space. Due to the steady increase in the structural, ligand, and functional data of GPCRs, several cheminformatics approaches have been implemented in its drug discovery pipeline. In this review, we mainly focus on the cheminformatics-based paradigms in GPCR drug discovery. We provide a comprehensive view on the ligand- and structure-based cheminformatics approaches which are best illustrated via GPCR case studies. Furthermore, an appropriate combination of ligand-based knowledge with structure-based ones, i.e., integrated approach, which is emerging as a promising strategy for cheminformatics-based GPCR drug design is also discussed.

Paralog Specificity Determines Subcellular Distribution, Action Mechanism, and Anticancer Activity of TRAP1 Inhibitors.

Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.

Regulation of aldosterone secretion by Cav1.3.

Aldosterone-producing adenomas (APAs) vary in phenotype and genotype. Zona glomerulosa (ZG)-like APAs frequently have mutations of an L-type calcium channel (LTCC) CaV1.3. Using a novel antagonist of CaV1.3, compound 8, we investigated the role of CaV1.3 on steroidogenesis in the human adrenocortical cell line, H295R, and in primary human adrenal cells. This investigational drug was compared with the common antihypertensive drug nifedipine, which has 4.5-fold selectivity for the vascular LTCC, CaV1.2, over CaV1.3. In H295R cells transfected with wild-type or mutant CaV1.3 channels, the latter produced more aldosterone than wild-type, which was ameliorated by 100 µM of compound 8. In primary adrenal and non-transfected H295R cells, compound 8 decreased aldosterone production similar to high concentration of nifedipine (100 µM). Selective CaV1.3 blockade may offer a novel way of treating primary hyperaldosteronism, which avoids the vascular side effects of CaV1.2-blockade, and provides targeted treatment for ZG-like APAs with mutations of CaV1.3.

Phenyl Ether- and Aniline-Containing 2-Aminoquinolines as Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase.

Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.

Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability.

The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogs are not bioavailable. To increase cellular permeability of mevalonate analogs, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogs by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogs in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates.

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.

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.