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Supplemental Methods Figure S1: Reports clinical information's about normal pregnant women, patients with complete hydatiform moles or choriocarcinoma. Figure S2: Experimental procedure. Figure S3: Quantification of EG-VEGF PROKR1 and PROKR2 protein expression in CTL, Complete hydatiform mole (CHM) and choriocarcinoma (CC) placental section. Figure S4: EG-VEGF effect on JEG3 migration, in the absence or presence of PROKR1 or PROKR2 antagonists. Figure S5: JEG3-luc injected mice exhibited arrested gestation and disorganized vascularization. Figure S6: Antibody microarray analysis. Figure S7: Characterization of the angiogenic status of sera and placentas collected from CTL, CHM and CC patients. Figure S8: Antibody angiogenic microarray analysis of sera collected from CTL, CHM and CC patients.

Purpose: Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland–derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy.Experimental Design: Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (n = 20) and in distinctive CHM (n = 38) and CC (n = 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice.Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions.Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130–40. ©2017 AACR.

Literature has revealed that the delta opioid receptor (DOR) exhibited diverse pharmacological effects on neuron and skin. In the present study, we have investigated whether the activation of DOR has hair-growth promotion effects. Compared with other opioid receptor, DOR was highly expressed in epidermal component of hair follicle in human and rodents. The expression of DOR was high in the anagen phase, but it was low in the catagen and telogen phases during mouse hair cycle. Topical application of UFP-512, a specific DOR agonist, significantly accelerated the induction of the anagen in C3H mice. Topical application of UFP-512 also increased the hair length in hair organ cultures and promoted the proliferation and the migration of outer root sheath (ORS) cells. Similarly, pharmacological inhibition of DOR by naltrindole significantly inhibited the anagen transition process and decreased hair length in hair organ cultures. Thus, we further examined whether Wnt/β-catenin pathway was related to the effects of DOR on hair growth. We found that Wnt/β-catenin pathway was activated by UFP-512 and siRNA for β-catenin attenuated the UFP-512 induced proliferation and migration of ORS cells. Collectively, result established that DOR was involved in hair cycle regulation, and that DOR agonists such as UFP-512 should be developed for novel hair-loss treatment.

Our previous studies have shown that the δ-opioid receptor (DOR) is an important neuroprotector via the regulation of PTEN-induced kinase 1 (PINK1), a mitochondria-related molecule, under hypoxic and MPP+ insults. Since mitochondrial dysfunctions are observed in both hypoxia and MPP+ insults, this study further investigated whether DOR is cytoprotective against these insults by targeting mitochondria. Through comparing DOR-induced responses to hypoxia versus MPP+-induced parkinsonian insult in PC12 cells, we found that both hypoxia and MPP+ caused a collapse of mitochondrial membrane potential and severe mitochondrial dysfunction. In sharp contrast to its inappreciable effect on mitochondria in hypoxic conditions, DOR activation with UFP-512, a specific agonist, significantly attenuated the MPP+-induced mitochondrial injury. Mechanistically, DOR activation effectively upregulated PINK1 expression and promoted Parkin’s mitochondrial translocation and modification, thus enhancing the PINK1-Parkin mediated mitophagy. Either PINK1 knockdown or DOR knockdown largely interfered with the DOR-mediated mitoprotection in MPP+ conditions. Moreover, there was a major difference between hypoxia versus MPP+ in terms of the regulation of mitophagy with hypoxia-induced mitophagy being independent from DOR-PINK1 signaling. Taken together, our novel data suggest that DOR activation is neuroprotective against parkinsonian injury by specifically promoting mitophagy in a PINK1-dependent pathway and thus attenuating mitochondrial damage.

Pregnancy establishment in mammals, including pigs, requires proper communication between embryos and the maternal reproductive tract. Prokineticin 1 (PROK1) has been described as a secretory protein with pleiotropic functions and as a novel tissue-specific angiogenic factor. However, despite the studies performed mainly on human cell lines and in mice, the function of PROK1 in the endometrium during early pregnancy is still not fully elucidated. We hypothesized that PROK1 contributes to pregnancy establishment in pigs. The present study is the first to report that the expression of PROK1 and its receptor (PROKR1) is elevated in the porcine endometrium during the implantation and early placentation period. PROK1 protein was detected mainly in luminal epithelial cells, glandular epithelial cells, and blood vessels in the endometrium. Using the porcine in vivo model of unilateral pregnancy, we revealed that conceptuses induced the endometrial expression of PROK1 and PROKR1. Moreover, the embryonic signal, estradiol-17β, as well as progesterone, stimulated the endometrial expression of PROK1 and PROKR1. We also evidenced that PROK1–PROKR1 signaling supports endometrial angiogenesis in pigs. The PROK1-stimulated proliferation of primary porcine endometrial endothelial (PEE) cells involved PI3K/AKT/mTOR, MAPK, cAMP, and NFKB signaling pathways. Furthermore, PROK1 via PROKR1 promoted the formation of capillary-like structures by PEE cells. PROK1 also stimulated VEGFA and PGF2α secretion, which in turn may indirectly support angiogenic changes within endometrial tissue. In summary, our study suggests that PROK1 acts as an embryonic signal mediator that regulates endometrial angiogenesis and secretory function during the implantation and early placentation period in pigs.

Vincristine (VCR) treatment is often associated to painful neuropathy. Its development is independent from antitumoral mechanism and involves neuroinflammation. We investigated the role of the chemokine prokineticin (PK)2 in a mouse model of VCR induced neuropathy using a PK-receptors (PK-R) antagonist to counteract its development. We also evaluated emotional like deficits in VCR mice. VCR (0,1 mg/kg) was i.p. injected in C57BL/6J male mice once a day for 14 consecutive days. Pain, anxiety and depressive like behaviors were assessed in animals. PK2, PK-Rs, cytokines, neuroinflammatory markers (CD68, CD11b, GFAP, TLR4) and ATF3 were evaluated in DRG, spinal cord, prefrontal cortex and hippocampus. The PK-Rs antagonist PC1, was s.c. injected (150 μg/kg) twice a day from day 7 (hypersensitivity state) until day 14. Its effect on pain and neuroinflammation was evaluated. VCR mice developed neuropathic pain but not mood alterations. After 7 days of VCR treatment we observed a neuroinflammatory condition in DRG with high levels of PK-Rs, TLR4, CD68, ATF3 and IL-1β without relevant alterations in spinal cord. At day 14, an upregulation of PK system and a marked neuroinflammation was evident also in spinal cord. Moreover, at the same time, we observed initial alterations in supraspinal brain areas. PC1 treatment significantly counteracted neuropathic pain and blunted neuroinflammation.

The development of neuropathy and of mood alterations is frequent after chemotherapy. These complications, independent from the antitumoral mechanism, are interconnected due to an overlapping in their processing pathways and a common neuroinflammatory condition. This study aims to verify whether in mice the treatment with the proteasome inhibitor bortezomib (BTZ), at a protocol capable of inducing painful neuropathy, is associated with anxiety, depression and supraspinal neuroinflammation. We also verify if the therapeutic treatment with the antagonist of the prokineticin (PK) system PC1, which is known to contrast pain and neuroinflammation, can prevent mood alterations. Mice were treated with BTZ (0.4 mg/kg three times/week for 4 weeks), mechanical allodynia and locomotor activity were evaluated over time while anxiety (dark light and marble burying test), depression (sucrose preference and swimming test) and supraspinal neuroinflammation were checked at the end of the protocol. BTZ treated neuropathic mice develop anxiety and depression. The presence of mood alterations is related to the presence of neuroinflammation and PK system activation in prefrontal cortex, hippocampus and hypothalamus with high levels of PK2 and PKR2 receptor, IL-6 and TNF-α, TLR4 and an upregulation of glial markers. PC1 treatment, counteracting pain, prevented the development of supraspinal inflammation and depression-like behavior in BTZ mice.

Protein misfolding and amyloid formation are associated with various human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and Type-2 Diabetes mellitus (T2DM). No disease-modifying therapeutics are available for them. Despite the lack of sequence homology between the corresponding proteins, aromatic residues are recognized as common key motifs in the formation and stabilization of amyloid structures via π-π stacking. Thus, targeting aromatic recognition interfaces could be a useful approach for inhibiting amyloid formation as well as disrupting the preformed amyloid fibrils. Combining experimental and computational approaches, we demonstrated the anti-amyloidogenic effect of naphthoquinone-tryptophan-based hybrid molecules toward PHF6 (τ-derived aggregative peptide), Amyloid β (Aβ42), and human islet amyloid polypeptide (hIAPP) implicated in AD and T2DM, respectively. These hybrid molecules significantly inhibited the aggregation and disrupted their preformed fibrillar aggregates in vitro, in a dose-dependent manner as evident from Thioflavin T/S binding assay, CD spectroscopy, and electron microscopy. Dye leakage assay from LUVs and cell-based experiments indicated that the hybrid molecules inhibit membrane disruption and cytotoxicity induced by these amyloids. Furthermore, in silico studies provided probable mechanistic insights into the interaction of these molecules with the amyloidogenic proteins in their monomeric or aggregated forms, including the role of hydrophobic interaction, hydrogen bond formation, and packing during inhibition of aggregation and fibril disassembly. Our findings may help in designing novel therapeutics toward AD, T2DM, and other proteinopathies based on the naphthoquinone derived hybrid molecules.

Prokineticin 2 (PK2) is a secreted protein involved in several pathological and physiological processes, including the regulation of inflammation, sickness behaviors, and the circadian rhythm. Recently, it was reported that PK2 is associated with the pathogenesis of collagen-induced arthritis in mice. However, whether PK2 influences the pathogenesis of rheumatoid arthritis (RA) or osteoarthritis (OA) remains unknown. In this study, we collected synovial tissue, plasma, synovial fluid, and fibroblast-like synoviocytes (FLS) from RA and OA patients to analyze the role of PK2 using immunohistochemistry, ELISAs, and tissue superfusion studies. PK2 and its receptors prokineticin receptor (PKR) 1 and 2 were expressed in RA and OA synovial tissues. PKR1 expression in RA synovial tissue was downregulated compared with OA synovial tissue. The PK2 concentration was higher in RA synovial fluid than in OA synovial fluid but similar between RA and OA plasma. PK2 suppressed the production of IL-6 from TNFα-prestimulated OA-FLS, and this effect was attenuated in TNFα-prestimulated RA-FLS. This phenomenon was accompanied by the upregulation of PKR1 in OA-FLS and the downregulation of PK2 and PKR1 in RA-FLS. This study provides a new model to explain some aspects underlying the chronicity of inflammation in RA.

Endocrine gland derived vascular endothelial growth factor (EG-VEGF) is a canonical member of the prokineticin (PROKs) family. It acts via the two G-protein coupled receptors, namely PROKR1 and PROKR2. We have recently demonstrated that EG-VEGF is highly expressed in the human placenta, contributes to placental vascularization and growth and that its aberrant expression is associated with pregnancy pathologies including preeclampsia and fetal growth restriction. These findings strongly suggested that antagonization of its receptors may constitute a potential therapy for the pregnancy pathologies. Two specific antagonists of PROKR1 (PC7) and for PROKR2 (PKRA) were reported to reverse PROKs adverse effects in other systems. In the view of using these antagonists to treat pregnancy pathologies, a proof of concept study was designed to determine the biological significances of PC7 and PKRA in normal pregnancy outcome. PC7 and PKRA were tested independently or in combination in trophoblast cells and during early gestation in the gravid mouse. Both independent and combined treatments uncovered endogenous functions of EG-VEGF. The independent use of antagonists distinctively identified PROKR1 and PROKR2-mediated EG-VEGF signaling on trophoblast differentiation and invasion, thereby enhancing feto-placental growth and pregnancy outcome. Thus, our study provides evidence for the potential safe use of PC7 or PKRA to improve pregnancy outcome.

Prokineticin 2 (PK2) is a secreted protein involved in several pathological and physiological processes, including the regulation of inflammation, sickness behaviors, and circadian rhythms. Recently, it was reported that PK2 is associated with the pathogenesis of collagen-induced arthritis in mice. However, the role of PK2 in the pathogenesis of rheumatoid arthritis (RA) or osteoarthritis (OA) remains unknown. In this study, we collected synovial tissue, plasma, synovial fluid, and synovial fibroblasts (SF) from RA and OA patients to analyze the function of PK2 using immunohistochemistry, enzyme-linked immunosorbent assays, and tissue superfusion studies. PK2 and its receptors prokineticin receptor (PKR) 1 and 2 were expressed in RA and OA synovial tissues. PKR1 expression was downregulated in RA synovial tissue compared with OA synovial tissue. The PK2 concentration was higher in RA synovial fluid than in OA synovial fluid but similar between RA and OA plasma. PK2 suppressed the production of IL-6 from TNFα-prestimulated OA-SF, and this effect was attenuated in TNFα-prestimulated RA-SF. This phenomenon was accompanied by the upregulation of PKR1 in OA-SF. This study provides a new model to explain some aspects underlying the chronicity of inflammation in RA.

Three series of arylbenzimidazole derivatives 3–40, 45 have been simply synthesized and tested for their antioxidant capacity. The 2-arylbenzimidazoles were tested against various radicals by the DPPH, FRAP and ORAC tests and showed different activity profiles. It has been observed that the number and position of the hydroxy groups on the 2-aryl portion and the presence of a diethylamino group or a 2-styryl group are related to a good antioxidant capacity. Furthermore, benzimidazoles showed satisfactory SPF values ​​in vitro compared to the commercial PBSA filter, proving to have a good photoprotective profile. In particular, 2-arylbenzimidazole-5-sulphonic acids 15 and 38, the 2-styryl-benzimidazole 45 showed broad spectrum solar protection against UVA and UVB rays. The antiproliferative effect of the benzimidazoles was tested on human skin melanoma Colo-38 cells. The styrylbenzimidazole 45 exhibited antiproliferative effect at low micromolar concentration against Colo-38 cells and very low antiproliferative activity on normal HaCat keratinocyte cells.

Herein we report the synthesis of a new series of aromatic sulfamates designed considering the sulfonamide COX-2 selective inhibitors celecoxib and valdecoxib as lead compounds. These latter were shown to possess important human carbonic anhydrase (CA, EC 4.2.1.1) inhibitory properties, with the inhibition of the tumor-associated isoform hCA IX likely being co-responsible of the celecoxib anti-tumor effects. Bioisosteric substitution of the pyrazole or isoxazole rings from these drugs with the pyrazoline one was considered owing to the multiple biological activities ascribed to this latter heterocycle and paired with the replacement of the sulfonamide of celecoxib and valdecoxib with its equally potent bioisoster sulfamate. The synthesized derivatives were screened for the inhibition of four human carbonic anhydrase isoforms, namely hCA I, II, IV, and IX. All screened sulfamates exhibited great potency enhancement in inhibiting isoform II and IV, widely involved in glaucoma (KIs in the range of 0.4–12.4 nM and 17.7 and 43.3 nM, respectively), compared to the lead compounds, whereas they affected the tumor-associated hCA IX as potently as celecoxib.

A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na+-translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found that the FMN transfer reaction robustly occurred when NqrC and ApbE were genetically targeted in the cytosol of live mammalian cells. Moreover, NqrC expression in the endoplasmic reticulum (NqrC-ER) induced the retro-translocation of NqrC to the cytosol, leading to the proteasome-mediated ER-associated degradation of NqrC, which is considered to be an innate immunological response toward the bacterial protein. This unexpected cellular process of NqrC-ER could be exploited for the construction of an in cellulo proteasome inhibitor screening system, and our proposed approach yielded substantially improved results compared to a previous method. In addition, a truncated version of RnfG (half-RnfG...

Background Neuropathy is a dose-limiting side effect of many chemotherapeutics, including bortezomib. The mechanisms underlying this condition are not fully elucidated even if a contribution of neuroinflammation was suggested. Here, we investigated the role of a chemokine family, the prokineticins (PKs), in the development of bortezomib-induced peripheral neuropathy (BIPN), and we used a PK receptor antagonist to counteract the development and progression of the pathology. Methods Neuropathy was induced in male C57BL/6J mice by using a protocol capable to induce a detectable neuropathic phenotype limiting systemic side effects. The presence of allodynia (both mechanical and thermal) and thermal hyperalgesia was monitored over time. Mice were sacrificed at two different time points: 14 and 28 days after the first bortezomib (BTZ) injection. At these times, PK system activation (PK2 and PK-Rs), macrophage and glial activation markers, and cytokine production were evaluated in the main station involved in pain transmission (sciatic nerve, DRG, and spinal cord), and the effect of a PK receptors antagonist (PC1) on the same behavioral and biochemical parameters was assessed. Structural damage of DRG during BTZ treatment and an eventual protective effect of PC1 were also evaluated. Results BTZ induces in mice a dose-related allodynia and hyperalgesia and a progressive structural damage to the DRG. We observed a precocious increase of macrophage activation markers and unbalance of pro- and anti-inflammatory cytokines in sciatic nerve and DRG together with an upregulation of GFAP in the spinal cord. At higher BTZ cumulative dose PK2 and PK receptors are upregulated in the PNS and in the spinal cord. The therapeutic treatment with the PK-R antagonist PC1 counteracts the development of allodynia and hyperalgesia, ameliorates the structural damage in the PNS, decreases the levels of activated macrophage markers, and prevents full neuroimmune activation in the spinal cord. Conclusions PK system may be a strategical pharmacological target to counteract BTZ-induced peripheral neuropathy. Blocking PK2 activity reduces progressive BTZ toxicity in the DRG, reducing neuroinflammation and structural damage to DRG, and it may prevent spinal cord sensitization.

Two series of indole derivatives 4-17, 20-22 were easily prepared and assayed for their radical-scavenging ability. Arylidene-1H-indole-2-carbohydrazones showed different extent antioxidant activity in DPPH, FRAP and ORAC assays. Good antioxidant activity is related to the number and position of hydroxyl groups on the arylidene moiety as well as to the presence of methoxy or 4-(diethylamino) group. On the contrary low antioxidant activity is showed by the isomeric 1H-indol-2-yl(methylene)-benzohydrazides. Furthermore, hydrazones 4-17 showed photoprotective capacities with satisfactory in vitro SPF as compared to the commercial PBSA sunscreen filter. The indole 16 and 17, showing the best antioxidant and photoprotective profile, were included in different formulation and their topical release was evaluated. Varying the formulation composition, it was possible to optimize skin adsorption and solubility of the active indole in the formulation. The antiproliferative effect of the hydrazones 4-17 was tested on human erythroleukemia K562 and melanoma Colo-38 cells. Hydrazones 11, 16 and 17 showed growth inhibition at sub micromolar concentrations on both cell lines. These results indicate indole hydrazones as potential multifunctional molecules especially in the treatment of neoplastic diseases being the good antioxidant properties of 16 and 17 correlated to their high antiproliferative activity.

Growing evidences demonstrate that chemokines and chemokine receptors are up-regulated in resident central nervous system cells during Alzheimer's disease contributing to neuroinflammation and neurodegeneration. Prokineticin 2 belongs to a new family of chemokines which recently emerged as a critical player in immune system and inflammatory diseases. Since pharmacological blockade in vitro of the prokineticin system is able to antagonize Amyloid β-induced neurotoxicity, the aim of the present study was to investigate in vivo effects of prokineticin receptor antagonist PC1 on memory impairment in a rodent model of Alzheimer's disease. Rats were intracerebroventricular infused with Aβ1-42 and behavioral responses as well as the expression profile in hippocampus of prokineticin 2 and its receptors were investigated. Results demonstrated that Aβ1–42-infused rats developed significant memory impairments together with a marked up-regulation of both prokineticin 2 and its receptors in hippocampal neurons and astrocytes. Treatment with PC1 significantly improved learning capability of Aβ1–42-infused rats restoring the balance of prokineticin system. This study pointed to a neuroprotective role of PC1 on Aβ1-42-induced memory deficits that could be ascribed to the ability of PC1 to modulate rat hippocampal prokineticin system and to recover the impaired Aβ1-42-induced neurogenesis. This suggests that prokineticin system antagonism could be considered as a new approach for the study of AD etiopathology.

Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 μM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10–100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathways.

Perinatal hypoxic-ischemic (HI) brain injury results in death or profound long-term neurologic disability in both children and adults. However, there is no effective pharmacological therapy due to a poor understanding of HI events, especially the initial triggers for hypoxic-ischemic injury such as disrupted ionic homeostasis and the lack of effective intervention strategy. In the present study, we showed that neonatal brains undergo a developmental increase in the disruption of K+ homeostasis during simulated ischemia, oxygen-glucose deprivation (OGD) and neonatal HI cortex has a triple phasic response (earlier attenuation, later enhancement, and then recovery) of disrupted K+ homeostasis to OGD. This response partially involves the activity of the δ-opioid receptor (DOR) since the earlier attenuation of ischemic disruption of K+ homeostasis could be blocked by DOR antagonism, while the later enhancement was reversed by DOR activation. Similar to DOR activation, acupuncture, a strategy to promote DOR activity, could partially reverse the later enhanced ischemic disruption of K+ homeostasis in the neonatal cortex. Since maintaining cellular K+ homeostasis and inhibiting excessive K+ fluxes in the early phase of hypoxic-ischemic insults may be of therapeutic benefit in the treatment of ischemic brain injury and related neurodegenerative conditions, and since many neurons and other cells can be rescued during the "window of opportunity" after HI insults, our first findings regarding the role of acupuncture and DOR in attenuating ischemic disruption of K+ homeostasis in the neonatal HI brain suggest a potential intervention therapy in the treatment of neonatal brain injury, especially hypoxic-ischemic encephalopathy.

Herein we report on a new series of hydrazidoureidobenzensulfonamides investigated as inhibitors of the cytosolic human (h) hCA I and II isoforms, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The reported derivatives contain a 4-substituted piperidine fragment in which the hydrazidoureido linker has been involved as spacer between the benzenesulfonamide fragment which binds the zinc ion from the active site, and the tail of the inhibitor. Depending on the substitution pattern at the piperidine ring, low nanomolar inhibitors were detected against hCA II, hCA IX and hCA XII, making the new class of sulfonamides of interest for various pharmacologic applications.

In the search of multifunctional compounds we designed benzimidazole derivatives endowed with phenolic hydroxy groups and a hydrazone moiety as potential radical-scavenger and the antioxidant agents. The target molecules have been prepared by a simple synthetic procedure and tested for their antioxidant activity by DPPH, FRAP, and ORAC test, for photoprotective activity against UV rays and for antiproliferative activity against Colo-38 melanoma cells. Furthermore, two different dermocosmetic formulations were prepared with the compounds endowed with the best antioxidant and photoprotective profile and their release from formulation evaluated using Franz Cells system. High antioxidant activity is related to the presence of at least two hydroxy groups on arylidene moiety of benzimidazoles. Structure activity analysis revealed that the position of hydroxy groups is crucial for antioxidant activity as well as the presence of a 2-hydroxy-4-(diethylamino)arylidene group. The same correlation pattern was found to be related to photoprotective activity resulting in an UVA Protection Factor better than the commercial solar filter PBSA and antiproliferative activity against melanoma cells without producing cytotoxicity on normal keratinocytes. The release analysis indicated that high antioxidant activities are achieved with limited release at concentration compatible with the use as UV sunscreen filter.

Two new piperazinyl-ureido single ring aryl sulfamate-based inhibitor series were designed against the emerging oncology drug target steroid sulfatase (STS), for which there are existing potent steroidal and non-steroidal agents in clinical trials. 4-(Piperazinocarbonyl)aminosulfamates (5–31) were obtained by reacting 4-hydroxyarylamines with phenylchloroformate, subsequent sulfamoylation of the resulting hydroxyarylcarbamates and coupling of the product with 1-substituted piperazines. Pyrimidinyl-piperazinourea sulfamates (35–42) were synthesized by pyrimidine ring closure of 4-Boc-piperazine-1-carboxamidine with 3-(dimethylamino)propenones, deprotection and coupling with the sulfamoylated building block. Target ureidosulfamates 5–31 and 35–42 were evaluated both as STS inhibitors in vitro using a lysate of JEG-3 human placenta choriocarcinoma cell line and in a whole cell assay. SAR conclusions were drawn from both series. In series 35–42 the best inhibitory activity is related to the presence of a benzofuryl on the pyrimidine ring. In series 5–31 the best inhibitory activity was shown by the ureas bearing 4-chlorophenyl, 3,4-dichlorophenyl groups or aliphatic chains at the piperazino 4-nitrogen displaying IC50 in the 33–94 nM concentration range. Final optimization to the low nanomolar level was achieved through substitution of the arylsulfamate ring with halogens. Four halogenated arylsulfamates of high potency were achieved and two of these 19 and 20 had IC50 values of 5.1 and 8.8 nM respectively and are attractive for potential in vivo evaluation and further development. We demonstrate the optimization of this new series to low nanomolar potency, employing fluorine substitution, providing potent membrane permeant inhibitors with further development potential indicating piperazinyl-ureido aryl sulfamate derivatives as an attractive new class of STS inhibitors.

Four new series of aromatic sulfamates were synthesized and investigated for the inhibition of four human (h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I, II, IX, and XII. The reported derivatives, obtained by a sulfamoylation reaction of the corresponding phenolic precursors, bear 3,5-diarylpyrazoline moieties as spacers between the benzenesulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor. Pyrazolines are biologically privileged scaffolds, endowed with versatile biological activity, such as an anti-proliferative action. The derivatives were tested for the inhibition of the cytosolic, hCA I and II (off target isoforms) and the trans-membrane, tumor-associated hCA IX and XII enzymes (anticancer drug targets). Generally, hCA I was not effectively inhibited, whereas many low nanomolar inhibitors were evidenced against hCA II (KIs in the range of 0.42–90.1 nM), IX (KIs in the range of 0.72–63.6 nM), and XII (KIs in the range of 0.88–85.2 nM). The best substitution fragments at the pyrazoline ring included for CA II a 4-sulfamic group on the 3-aryl and halogens on the 5-aryl or a methoxy group on the 3-aryl and a 4-sulfamate group on the 5-aryl; for CA IX and CA XII they included the sulfamic group on the 3- or 4-position of the 5-aryl and an electronwithdrawing group on the 4-postion of the 3-aryl ring.

New benzofuranhydrazones 3–12 were easily prepared and assayed for their radical-scavenging ability. Hydrazones 3–12 showed different extent antioxidant activity in DPPH, FRAP and ORAC assays. Good antioxidant activity is related to the number and position of hydroxyl groups on the arylidene moiety. High antioxidant activity is showed by the 2-hydroxy-4-(diethylamino)benzylidene derivative 11. Furthermore, hydrazones 3–12 showed photoprotective capacities with satisfactory in vitro SPF as compared to the commercial PBSA sunscreen filter. The antiproliferative effects of the hydrazones 3–12 was tested on erythroleukemia K562 and Colo-38 melanoma human cells. All the compounds showed growth inhibition in the micromolar to sub micromolar concentration range. If taken together these results points to benzofuran hydrazones as potential multifunctional molecules especially in the treatment of neoplastic diseases being the good antioxidant properties of 5, 7 and 11 correlated to their high antiproliferative activity.

There is emerging evidence suggesting that neurotoxic insults and hypoxic/ischemic injury are underlying causes of Parkinson's disease (PD). Since PTEN-induced kinase 1 (PINK1) dysfunction is involved in the molecular genesis of PD and since our recent studies have demonstrated that the δ-opioid receptor (DOR) induced neuroprotection against hypoxic and 1-methyl-4-phenyl-pyridimium (MPP

Herein we report the synthesis of a new series of aromatic sulfamates investigated for the inhibition of four human (h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I, II, IV, and IX. The reported derivatives, obtained by a sulfamoylation reaction of the corresponding phenolic precursors, bear arylthiazolin-4-one moieties as spacers between the benzenesulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor. Thiazolin-4-ones are biologically privileged scaffolds, endowed with versatile biological activity, such as an anti-proliferative action. Phenolic precursors, also evaluated for CA inhibition, did not exhibit noteworthy efficacy in inhibiting the screened hCAs, whereas low nanomolar inhibitors were evidenced within the sulfamates subset mainly against hCA II (K I s in the range of 28.7–84.3 nM) and IX (K I s in the range of 17.6–73.3 nM). The variety of substituents appended at the outer aromatic portion almost generally reduced the inhibitory efficacy against isoforms II and IV, increasing instead that against the tumor-associated isoform IX.

Purpose: Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland–derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy. Experimental Design: Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (n = 20) and in distinctive CHM (n = 38) and CC (n = 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice. Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions. Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130–40. ©2017 AACR.

Background and Purpose Hypoxia/reoxygenation induces synthesis of reactive oxygen species (ROS) which can attack macromolecules and cause brain injury. The transcription factor, nuclear factor (erythroid-derived 2)-like 2, (Nrf2), ia potent activator of genes with an antioxidant responsive element and Nrf2 can counteract oxidative injury by increasing expression of several antioxidative genes in response to ROS stress. Here, we show that activation of the δ-opioid receptor (DOR) increasedNrf2 protein expression and translocation, thereby leading to cytoprotection. Experimental Approach We used HEK293t cells exposed to 0.5% O2 for 16 h and then reoxygenated for 4 h as a model of hypoxia-reperfusion (H/R) injury. Real time PCR, Western blotting, siRNA and immunohistochemical techniques were used to follow Nrf2 expression and activity. Cell viability and damage (as LDH leakage) were also measured. Key Results H/R injury triggered Nrf2 translocation into the nucleus and up-regulated expression of several downstream genes, relevant to antioxidation, such as NAD(P)H:quinone oxidoreductase (NQO1). Incubation with the DOR agonist UFP-512 enhanced Nrf2 protein expression and translocation and up-regulated its downstream genes in normoxia and further increased Nrf2 expression and translocation after H/R, protecting the cells against loss of viability and damage. The effect of UFP-512 on Nrf2 nuclear translocation was blocked by the DOR antagonist, naltrindole. Also, DOR–mediated cytoprotection was strongly inhibited after transfection of HEK293t cells with Nrf2 siRNA. Conclusions and Implications The DOR agonist UFP-512 was cytoprotective against H/R injury and this effect was partly dependent on DOR-mediated increase in Nrf2 function.

Background and Purpose Astrocytic excitatory amino acid transporters (EAATs) regulate extracellular glutamate concentrations and play a role in preventing neuroexcitotoxicity. As the δ-opioid receptor (DOP receptor) is neuroprotective against excitotoxic injury, we determined whether DOP receptor activation up-regulates EAAT expression and function. Experimental Approach We measured mRNA and protein expression of EAAT1, EAAT2 and EAAT3 in cultured mouse astrocytes exposed to a specific DOP receptor agonist (UFP-512) with or without a DOP receptor antagonist, DOP receptor siRNA or inhibitors of PKC, PKA, PI3K, p38, MAPK, MEK and ERK, and evaluated the function of EAATs by measuring glutamate uptake. Key Results Astrocytic DOP receptor mRNA and protein were suppressed by DOP receptor siRNA knockdown. DOP receptor activation increased mRNA and protein expression of EAAT1 and EAAT2, but not EAAT3, thereby enhancing glutamate uptake of astrocytes. DOP receptor-induced EAAT1 and EAAT2 expression was largely reversed by DOP receptor antagonist naltrindole or by DOP receptor siRNA knockdown, and suppressed by inhibitors of MEK, ERK and p38. DOP receptor-accelerated glutamate uptake was inhibited by EAAT blockers, DOP receptor siRNA knockdown or inhibitors of MEK, ERK or p38. In contrast, inhibitors of PKA, PKC or PI3K had no significant effect on DOP receptor-induced EAAT expression. Conclusions and Implications DOP receptor activation up-regulates astrocytic EAATs via MEK-ERK-p38 signalling, suggesting a critical role for DOP receptors in the regulation of astrocytic EAATs and protection against neuroexcitotoxicity. As decreased EAAT expression contributes to pathophysiology in many neurological diseases, including amyotrophic lateral sclerosis, our findings present a new platform for potential treatments of these diseases.