Description: Janus kinase (JAK) 2 plays a pivotal role in the tumorigenesis of signal transducers and activators of transcription (STAT) 3 constitutively activated solid tumors. JAK2 mutations are involved in the pathogenesis of various types of hematopoietic disorders, such as myeloproliferative disorders, polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Thus, small-molecular inhibitors targeting JAK2 are potent for therapy of these diseases. In this study, we screened 1,062,608 drug-like molecules from the ZINC database and 2080 natural product chemicals. We identified a novel JAK family kinase inhibitor, dehydrocrenatidine, that inhibits JAK-STAT3–dependent DU145 and MDA-MB-468 cell survival and induces cell apoptosis. Dehydrocrenatidine represses constitutively activated JAK2 and STAT3, as well as interleukin-6–, interferon- α– , and interferon- –stimulated JAK activity, and STAT phosphorylation, and suppresses STAT3 and STAT1 downstream gene expression. Dehydrocrenatidine inhibits JAKs-JH1 domain overexpression–induced STAT3 and STAT1 phosphorylation. In addition, dehydrocrenatidine inhibits JAK2-JH1 kinase activity in vitro. Importantly, dehydrocrenatidine does not show significant effect on Src overexpression and epidermal growth factor–induced STAT3 activation. Our results indicate that dehydrocrenatidine is a JAK-specific inhibitor.

Description: Hypoxia and oxidative stress are critical factors in carcinogenesis and exist throughout cancer development; however, the underlying mechanisms are far from clear. Here, for the first time to our knowledge, we reported that neuroglobin (Ngb), an intracellular hexa-coordinated globin serving as an oxygen/reactive oxygen species (ROS) sensor, functions as a tumor suppressor in hepatocelluar carcinoma (HCC). Ngb protein and mRNA expression were significantly down-regulated in tumor tissues, compared with its adjacent non-tumor tissues of human HCC samples and normal liver tissues. Knock-down of Ngb by RNA interference promoted human HCC cell line (HepG2) growth and proliferation, G0/G1-S transition in vitro, and tumor growth in vivo. On the contrary, overexpression of Ngb suppressed HepG2 cell growth and proliferation, G0/G1-S transition, colony formation in vitro, and tumorigenicity in vivo. These results established a tumor suppressor function of Ngb in HCC. The underlying mechanisms were further investigated. Overexpression of Ngb suppressed Raf/MEK/extracellular signal-regulated kinase (Erk), whereas knockdown of Ngb enhanced Raf/MEK/Erk activation in HepG2 cells in vitro and in vivo. Glutathione S-transferase pull-down showed that Ngb interacted with c-Raf-1 in HepG2 cells. Overexpression of Ngb suppressed serum- and H 2 O 2 -stimulated Erk activation in HepG2 cells. Pharmacological inhibition of Erk activation abolished the proliferative effect of Ngb knockdown in HepG2 cells. Mutation of Ngb at its oxygen-binding site (H64L) abolished the inhibitory effects of Ngb on Erk activation and HepG2 cell proliferation. Therefore, we propose that Ngb controls HCC development by linking oxygen/ROS signals to oncogenic Raf/mitogen-activated protein kinase (MAPK)/Erk signaling. Our data suggest that neuroglobin could be a new target for cancer therapy.

Description: Retigabine (RTG, [ethyl N -[2-amino-4-[(4-fluorophenyl)methyl]amino] phenyl] carbamate]) is a first-in-class antiepileptic drug that acts by potentiating neuronal KCNQ potassium channels; however, it has less than optimal brain distribution. In this study, we report that P-RTG (ethyl N -[2-amino-4-((4-fluorobenzyl)(prop-2-ynyl)amino)phenyl]carbamate), an RTG derivative that incorporates a propargyl group at the N position of the RTG linker, exhibits an inverted brain distribution compared with RTG. The brain-to-plasma concentration ratio of P-RTG increased to 2.30 compared with 0.16 for RTG. However, the structural modification did not change the drug’s potentiation potency, subtype selectivity, or RTG molecular determinants on KCNQ channels. In addition, in cultured hippocampal neurons, P-RTG exhibited a similar capability as RTG for suppressing both induced and spontaneous action potential firing. Notably, P-RTG antiepileptic activity in the maximal electroshock (MES)-induced mouse seizure model was significantly enhanced to a value 2.5 times greater than that of RTG. Additionally, the neurotoxicity of P-RTG in the rotarod test was comparable with that of RTG. Collectively, our results indicate that the incorporation of a propargyl group significantly improves the RTG brain distribution, supporting P-RTG as a promising antiepileptic drug candidate. The strategy for improving brain-to-plasma distribution of RTG might be applicable for the drug development of other central nervous system diseases.

Description: Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [ 125 I]iodoarylazidoprazosin in a concentration-dependent manner (IC 50 = 0.24 μM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function.

Description: Bicyclol [4,4'-dimethoxy-5,6,5',6'-bis(methylenedioxy)-2-hydroxy-methyl-2'-methoxycarbonyl biphenyl] is a synthetic hepatoprotectant widely used in clinical practice, but resistance to this treatment is often observed. We found that the hepatoprotective effect of bicyclol was greatly compromised in female and castrated male mice. This study was to dissect the molecular basis behind the sex difference, which might underlie the clinical uncertainty. We compared bicyclol-induced hepatoprotection between male and female mice using acute liver damage models. Inducible knockout by the Cre/loxp system was used to decipher the role of heat shock transcription factor 1 (HSF1). Functional experiments, western blot, and histopathological analysis were used to determine the key causative factors which might antagonize bicyclol in female livers. HSF1 activation and heat shock protein 70 (Hsp70) expression, which were responsible for bicyclol-induced hepatoprotection, were compromised in female and castrated male livers. Compromised HSF1 activation was a result of HSF1 phosphorylation at serine 303, which was catalyzed by glycogen synthase kinase 3 β (GSK3 β ). Testosterone was necessary for bicyclol to inhibit hepatic GSK3 β activity. Administration of testosterone or GSK3 β inhibitors restored bicyclol-induced protection in females. Bicyclol induces sex-specific hepatoprotection based on a sex-specific HSF1/Hsp70 response, in which testosterone and GSK3 β play key roles. Because a lot of patients suffering from liver diseases have very low testosterone levels, our results give a possible explanation for the clinical variation in bicyclol-induced hepatoprotection, as well as practicable solutions to improve the effect of bicyclol.

Description: Antibody-drug conjugates (ADCs) have achieved great success in cancer therapy in recent years. Some peptidyl microtubule inhibitors consisting of natural and unnatural amino acids, such as monomethyl auristatin E (MMAE) and F (MMAF), are extremely cytotoxic and have been used as a payload in ADCs. However, their precise molecular interaction with tubulin and microtubules remains unclear. We determined the crystal structures of tubulin in complex with three ultra-potent peptidyl microtubule inhibitors [MMAE, taltobulin (HTI- 286), and tubulysin M] at 2.5 Å. Our data showed that the three peptides bound to the vinca domain and shared a common and key pharmacophore containing two consecutive hydrophobic groups (Val, Ile-like side chain). These groups protruded in opposite directions into hydrophobic pockets on the tubulin β and α subunits. Nitrogen and oxygen atoms from the same backbone formed hydrogen bonds with Asn329 from the α subunit and Asp179 from the β subunit in a direction normal to the surface formed by the aforementioned hydrophobic groups. In addition, our crystal structure data indicated that tubulysin M bound to the β subunit alone, providing a structural explanation for its higher affinity. We also compared the conformations of two representative structurally different vinca domain compounds, ustiloxin D and vinblastine, with those of the aforementioned peptidyl ligands, and found that they shared a similar pharmacophore. Our findings lay a foundation for the rational design of novel vinca domain ligands and may facilitate the development of microtubule inhibitors with high specificity, affinity, and efficiency as payloads for ADCs in cancer therapy.

Description: Hepatocellular carcinoma (HCC) is the fifth most common and the third most deadly malignant tumor worldwide. Hypoxia and related oxidative stress are heavily involved in the process of HCC development and its therapies. However, direct and accurate measurement of oxygen concentration and evaluation of hypoxic effects in HCC prove difficult. Moreover, the hypoxia-mediated mechanisms in HCC remain elusive. Here, we summarize recent major evidence of hypoxia in HCC lesions shown by measuring partial pressure of oxygen (pO 2 ), the clinical importance of hypoxic markers in HCC, and recent advances in hypoxia-related mechanisms and therapies in HCC. For the mechanisms, we focus mainly on the roles of oxygen-sensing proteins (i.e., hypoxia-inducible factor and neuroglobin) and hypoxia-induced signaling proteins (e.g., matrix metalloproteinases, high mobility group box 1, Beclin 1, glucose metabolism enzymes, and vascular endothelial growth factor). With respect to therapies, we discuss mainly YQ23, sorafenib, 2-methoxyestradiol, and celastrol. This review focuses primarily on the results of clinical and animal studies.

Description: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were reported to express in the well-known vasomotor region, rostral ventrolateral medulla (RVLM), and can be inhibited by propofol. However, whether HCN channels in RVLM contribute to propofol-induced cardiovascular depression remains unclear. We recorded the hemodynamic changes when either continuous intravenous infusions or microinjections of propofol and ZD-7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride; HCN channel blocker) in RVLM. Expressions of HCN channels in RVLM neurons of mice of different ages were examined by quantitative real-time polymerase chain reaction and Western blotting. The effects of propofol and ZD-7288 on HCN channels and the excitability of RVLM neurons were examined by electrophysiological recording. Propofol (1.25, 2.5, 5, and 7.5 mg/kg per minute, i.v., 10 minutes) decreased mean arterial pressure (MAP) and heart rate (HR) in a concentration-dependent manner in wild-type mice that were markedly attenuated in HCN1 knockout mice. Bilateral microinjection of propofol (1%, 0.1 μ l) in RVLM caused a sharp and pronounced drop in MAP and HR values, which were abated by pretreatment with ZD-7288. In electrophysiological recording, propofol (5, 10, and 20 μ M) concentration-dependently inhibited HCN current, increased input resistance, decreased firing rate, and caused membrane hyperpolarization in RVLM neurons. These actions of propofol were attenuated by ZD-7288 pretreatment. The mRNA and protein level of HCN channels increased in an age-dependent manner, which may contribute to the age-dependent increase in the sensitivity to propofol. Our results indicated that the inhibition of HCN channels in RVLM neurons may contribute to propofol-induced cardiovascular inhibition.

Description: Conventional drug discovery efforts at the β 2 -adrenoceptor ( β 2 AR) have led to the development of ligands that bind almost exclusively to the receptor’s hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein–coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first β 2 AR small-molecule positive allosteric modulators (PAMs)—compound (Cmpd)-6 [( R )- N -(4-amino-1-(4-( tert -butyl)phenyl)-4-oxobutan-2-yl)-5-( N -isopropyl- N -methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human β 2 ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied β 2 AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and β -arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the β 2 AR and potentiates downstream cAMP production and receptor recruitment of β -arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the β 2 AR compared with the closely related β 1 AR. Structure–activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the β 2 AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.