The Rolipram-Perillyl Alcohol Conjugate (NEO214) Is A Mediator of Cell Death through the Death Receptor Pathway.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Treatment with temozolomide, standard of care for gliomas, usually results in drug resistance and tumor recurrence. Therefore, there is a great need for drugs that target GBM. NEO214 was generated by covalently linking rolipram to perillyl alcohol (POH) via a carbamate bond to form the rolipram-perillyl alcohol conjugate. We show here that NEO214 is effective against both temozolomide-sensitive and temozolomide-resistant glioma cells. Furthermore, NEO214 is effective for different mechanisms of temozolomide resistance: overexpression of MGMT (O6-methylguanine methyl-transferase); deficiency in specific mismatch repair proteins; and overexpression of base excision repair (BER) proteins. NEO214-induced cytotoxicity involves apoptosis triggered by endoplasmic reticulum (ER) stress, as well as activating the Death Receptor 5 (DR5)/TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) pathway. In vitro studies show that glioma cells treated with NEO214 express DR5 and exhibit cell death in the presence of recombinant TRAIL, a growth factor constitutively produced by astrocytes. Our in vitro 3D coculture data show that induction of DR5 in glioma cells with NEO214 and TRAIL cause tumor cell death very effectively and specifically for glioma cells. In vivo studies show that NEO214 has antitumor efficacy in orthotropic syngeneic rodent tumor models. Furthermore, NEO214 has therapeutic potential especially for brain tumors because this drug can cross the blood-brain barrier (BBB), and is effective in the TRAIL-rich astrocyte microenvironment. NEO214 is a strong candidate for use in the treatment of GBMs.

Induction of Pro-Apoptotic Endoplasmic Reticulum Stress in Multiple Myeloma Cells by NEO214, Perillyl Alcohol Conjugated to Rolipram.

Despite the introduction of new therapies for multiple myeloma (MM), many patients are still dying from this disease and novel treatments are urgently needed. We have designed a novel hybrid molecule, called NEO214, that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH), an inducer of endoplasmic reticulum (ER) stress, to rolipram (Rp), an inhibitor of phosphodiesterase-4 (PDE4). Its potential anticancer effects were investigated in a panel of MM cell lines. We found that NEO214 effectively killed MM cells in vitro with a potency that was over an order of magnitude stronger than that of its individual components, either alone or in combination. The cytotoxic mechanism of NEO214 involved severe ER stress and prolonged induction of CCAAT/enhancer-binding protein homologous protein (CHOP), a key pro-apoptotic component of the ER stress response. These effects were prevented by salubrinal, a pharmacologic inhibitor of ER stress, and by CHOP gene knockout. Conversely, combination of NEO214 with bortezomib, a drug in clinical use for patients with MM, resulted in synergistic enhancement of MM cell death. Combination with the adenylate cyclase stimulant forskolin did not enhance NEO214 impact, indicating that cyclic adenosine 3',5'-monophosphate (AMP) pathways might play a lesser role. Our study introduces the novel agent NEO214 as a potent inducer of ER stress with significant anti-MM activity in vitro. It should be further investigated as a potential MM therapy aimed at exploiting this tumor's distinct sensitivity to ER stress.

Engineered stabilization and structural analysis of the autoinhibited conformation of PDE4.

Phosphodiesterase 4 (PDE4) is an essential contributor to intracellular signaling and an important drug target. The four members of this enzyme family (PDE4A to -D) are functional dimers in which each subunit contains two upstream conserved regions (UCR), UCR1 and -2, which precede the C-terminal catalytic domain. Alternative promoters, transcriptional start sites, and mRNA splicing lead to the existence of over 25 variants of PDE4, broadly classified as long, short, and supershort forms. We report the X-ray crystal structure of long form PDE4B containing UCR1, UCR2, and the catalytic domain, crystallized as a dimer in which a disulfide bond cross-links cysteines engineered into UCR2 and the catalytic domain. Biochemical and mass spectrometric analyses showed that the UCR2-catalytic domain interaction occurs in trans, and established that this interaction regulates the catalytic activity of PDE4. By elucidating the key structural determinants of dimerization, we show that only long forms of PDE4 can be regulated by this mechanism. The results also provide a structural basis for the long-standing observation of high- and low-affinity binding sites for the prototypic inhibitor rolipram.

Tumour growth inhibition and anti-angiogenic effects using curcumin correspond to combined PDE2 and PDE4 inhibition.

Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis by stimulating endothelial cells. Increase in cyclic AMP (cAMP) level inhibits VEGF-induced endothelial cell proliferation and migration. Cyclic nucleotide phosphodiesterases (PDEs), which specifically hydrolyse cyclic nucleotides, are critical in the regulation of this signal transduction. We have previously reported that PDE2 and PDE4 up-regulations in human umbilical vein endothelial cells (HUVECs) are implicated in VEGF-induced angiogenesis and that inhibition of PDE2 and PDE4 activities prevents the development of the in vitro angiogenesis by increasing cAMP level, as well as the in vivo chicken embryo angiogenesis. We have also shown that polyphenols are able to inhibit PDEs. The curcumin having anti-cancer properties, the present study investigated whether PDE2 and PDE4 inhibitors and curcumin could have similar in vivo anti-tumour properties and whether the anti-angiogenic effects of curcumin are mediated by PDEs. Both PDE2/PDE4 inhibitor association and curcumin significantly inhibited in vivo tumour growth in C57BL/6N mice. In vitro, curcumin inhibited basal and VEGF-stimulated HUVEC proliferation and migration and delayed cell cycle progression at G0/G1, similarly to the combination of selective PDE2 and PDE4 inhibitors. cAMP levels in HUVECs were significantly increased by curcumin, similarly to rolipram (PDE4 inhibitor) and BAY-60-550 (PDE2 inhibitor) association, indicating cAMP-PDE inhibitions. Moreover, curcumin was able to inhibit VEGF-induced cAMP-PDE activity without acting on cGMP-PDE activity and to modulate PDE2 and PDE4 expressions in HUVECs. The present results suggest that curcumin exerts its in vitro anti-angiogenic and in vivo anti-tumour properties through combined PDE2 and PDE4 inhibition.

Microplate-based method to screen inhibitors of isozymes of cyclic nucleotide phosphodiesterase fused to SUMO.

The feasibility for microplate-based screening of inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE) was tested via the coupled action of a phosphatase on adenosine-5'-monophosphate and an improved malachite green assay of phosphate. Human full-length PDE4B2 and truncated mutant (152-528aa) were expressed in Escherichia coli via fusion to SUMO, which after purification through Ni-NTA column exhibited specific activities >0.017 U mg(-1). In the presence of proteins <30 mg L(-1), absorbance for 10 µΜ phosphate was measurable; a PDE isozyme of specific activity over 0.008 U mg(-1) after reaction for 20 min thus suited for microplate-based screening of inhibitors. By using Biotek ELX 800 microplate reader, affinities of two forms of PEDE4B2 for cAMP, rolipram and papaverine varied over three magnitudes and were consistent with those by routine assay, respectively. Hence, the proposed method was promising for high-throughput-screening of inhibitors of phosphate-releasing enzymes bearing specific activities over 0.008 U mg(-1).

Protein kinase A (PKA) pathway is functionally linked to androgen receptor (AR) in the progression of prostate cancer.

OBJECTIVES: In the present study, we investigated whether the cyclic adenosine monophosphate (cAMP)-activated protein kinase A (PKA) pathway may regulate the expression of AR and prostate-specific antigen (PSA) and whether there is a correlation between the expression of cAMP/PKA-associated genes and androgen receptor (AR) in patients with prostate cancer (CaP). MATERIALS AND METHODS: The functional studies were performed in LNCaP and PC3 cell lines. Data on the mRNA expression of sets of genes in human clinical samples, including prostate tissues from organ donors, prostate primary cancer, and metastatic cancer, were extracted from the National Center for Biotechnology Informations Gene Expression Omnibus (GEO) database. Statistical tests were applied. RESULTS: We showed that elevated levels of cAMP/PKA pathways induced an increased expression of AR and PSA proteins in LNCaP cells in the absence of androgen. A cAMP-associated phosphodiesterase-4 (PDE4) inhibitor, rolipram induced an up-regulation in AR expression, whereas a cAMP enhancer, forskolin increased PSA level without affecting AR expression. Forskolin treatment increased the level of PKA R1α in LNCaP cells, but remarkably inhibited R1α expression in aggressive PC3 cells. In patients with CaP, we found that the expression of genes encoding R1α and phosphodiesterase-4B was statistically significantly lower in the metastatic specimens than that in the primary CaP specimens or in the normal prostate tissues (P<0.01) and was reversely correlated with AR expression. Conversely, AR and PRKAR2B mRNA expressions were significantly higher in metastatic lesions than those in the primary CaP specimens or in the normal prostate tissues (P<0.01). CONCLUSION: Our study revealed a novel mechanism to precisely define the functional and clinical interrelationship between the cAMP/PKA pathway and AR signaling in the development of androgen-independent growth of CaPs and metastasis progression.

Persistent cAMP signaling by TSH receptors revealed by phosphodiesterase inhibition.

BACKGROUND: It is controversial whether persistent signaling by the thyrotropin (TSH) receptor (TSHR) is cell-type specific. We reported persistent TSHR signaling in human embryonic kidney 293 (HEK293) cells expressing human TSHRs (HEK-TSHRs), whereas another group reported persistent signaling in mouse thyroid follicles but not in HEK293 cells. Herein, we test this hypothesis directly. METHODS: We used two methods to measure persistent signaling in HEK-TSHRs and confirm our previous observations. In Method 1, we used a chemiluminescent immunoassay to measure intracellular cAMP accumulation over 30-60 min by adding a phosphodiesterase inhibitor to the incubation medium. In Method 2, we used an intracellular biosensor to record cAMP levels continuously. RESULTS: Using Method 1, we show that TSHR signals persistently in human thyrocytes and human osteosarcoma U2OS-TSHR cells. Using Method 1 in HEK-TSHRs, we show that after 5 min, the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) increases cAMP to 2.5 pmol/well, TSH increases cAMP to 1.6 pmol/well, but IBMX added 30 min after TSH withdrawal increases cAMP to 105 pmol/well. Using Method 2 in HEK-TSHRs, we confirm that without IBMX, TSH causes a transient increase in cAMP and 30 min after TSH withdrawal, IBMX increases cAMP in cells pretreated with TSH more rapidly and to a higher level than IBMX added to cells not pre-exposed to TSH. Lastly, using Method 2, we show that in HEK-TSHRs phosphodiesterases types 3 and 4 are involved in degrading cAMP as the specific inhibitors Rolipram and Milrinone expose persistent TSHR signaling. CONCLUSIONS: We conclude that persistent TSHR activation occurs in human thyrocytes, U2OS-TSHR cells and HEK-TSHRs; it is not cell-type specific but is revealed by inhibiting phosphodiesterases.

Lipidic nanocapsule drug delivery: neuronal protection for cochlear implant optimization.

OBJECTIVE: Sensorineural hearing loss leads to the progressive degeneration of spiral ganglion cells (SGC). Next to postoperative fibrous tissue growth, which should be suppressed to assure a close nerve-electrode interaction, the density of healthy SGC is one factor that influences the efficiency of cochlear implants (CI), the choice of treatment for affected patients. Rolipram, a phosphodiesterase-4 inhibitor, has proven neuroprotective and anti-inflammatory effects and might also reduce SGC degeneration and fibrosis, but it has to pass the cellular membrane to be biologically active. METHODS: Lipidic nanocapsules (LNC) can be used as biodegradable drug carriers to increase the efficacy of conventional application methods. We examined the biological effects of rolipram and LNC's core encapsulated rolipram on SGC and dendritic cell (DC) tumor necrosis factor-α (TNF-α) production in vitro and on SGC survival in systemically-deafened guinea pigs in vivo. RESULTS: Our results prove that rolipram does not have a beneficial effect on cultured SGC. Incorporation of rolipram in LNC increased the survival of SGC significantly. In the DC study, rolipram significantly inhibited TNF-α in a dose-dependent manner. The rolipram-loaded LNC provided a significant cytokine inhibition as well. In vivo data do not confirm the in vitro results. CONCLUSION: By transporting rolipram into the SGC cytoplasm, LNC enabled the neuroprotective effect of rolipram in vitro, but not in vivo. This might be due to dilution of test substances by perilymph or an inadequate release of rolipram based on differing in vivo and in vitro conditions. Nevertheless, based on in vitro results, proving a significantly increased neuronal survival when using LNC-rolipram compared to pure rolipram and pure LNC application, we believe that the combination of rolipram and LNC can potentially reduce neuronal degeneration and fibrosis after CI implantation. We conclude that rolipram is a promising drug that can be used in inner ear therapy and that LNC have potential as an inner ear drug-delivery system. Further experiments with modified conditions might reveal in vivo biological effects.

Drug-eluting microfibrous patches for the local delivery of rolipram in spinal cord repair.

Spinal cord injury (SCI) remains a major challenge for regenerative medicine. Following SCI, axon growth inhibitors and other inflammatory responses prevent functional recovery. Previous studies have demonstrated that rolipram, an anti-inflammatory and cyclic adenosine monophosphate preserving small molecule, improves spinal cord regeneration when delivered systemically. However, more recent studies showed that rolipram has some adverse effects in spinal cord repair. Here, we developed a drug-delivery platform for the local delivery of rolipram into the spinal cord. The potential of drug-eluting microfibrous patches for continuous delivery of high and low-dose rolipram concentrations was characterized in vitro. Following C5 hemisections, athymic rats were treated with patches loaded with low and high doses of rolipram. In general, animals treated with low-dose rolipram experienced greater functional and anatomical recovery relative to all other groups. Outcomes from the high-dose rolipram treatment were similar to those with no treatment. In addition, high-dose treated animals experienced reduced survival rates suggesting that systemic toxicity was reached. With the ability to control the release of drug dosage locally within the spinal cord, drug-eluting microfibrous patches demonstrate the importance of appropriate local release-kinetics of rolipram, proving their usefulness as a therapeutic platform for the study and repair of SCI.

Design, synthesis, and biological evaluation of new phosphodiesterase type 4 inhibitors.

The design, synthesis, and biological evaluation of new phosphodiesterase type 4 inhibitors, which possess new templates instead of a cyclohexane ring, are described. The mode of interaction with the enzyme is discussed based on the structure-activity relationship (SAR) data obtained for the synthesized inhibitors. Furthermore, the roles of three pharmacophores, a catechol moiety, a nitrile moiety, and acidic moieties, are discussed using in silico docking studies. More detailed biological evaluations of selected compounds are also presented.

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Crystal structures of the catalytic domain of phosphodiesterase 4B complexed with AMP, 8-Br-AMP, and rolipram.

Phosphodiesterase catalyzes the hydrolysis of the intracellular second messenger 3',5'-cyclic AMP (cAMP) into the corresponding 5'-nucleotide. Phosphodiesterase 4 (PDE4), the major cAMP-specific PDE in inflammatory and immune cells, is an attractive target for the treatment of asthma and COPD. We have determined crystal structures of the catalytic domain of PDE4B complexed with AMP (2.0 A), 8-Br-AMP (2.13 A) and the potent inhibitor rolipram (2.0 A). All the ligands bind in the same hydrophobic pocket and can interact directly with the active site metal ions. The identity of these metal ions was examined using X-ray anomalous difference data. The structure of the AMP complex confirms the location of the catalytic site and allowed us to speculate about the detailed mechanism of catalysis. The high-resolution structures provided the experimental insight into the nucleotide selectivity of phosphodiesterase. 8-Br-AMP binds in the syn conformation to the enzyme and demonstrates an alternative nucleotide-binding mode. Rolipram occupies much of the AMP-binding site and forms two hydrogen bonds with Gln443 similar to the nucleotides.

Dissecting the cofactor-dependent and independent bindings of PDE4 inhibitors.

Type 4 phosphodiesterases (PDE4s) are metallohydrolases that catalyze the hydrolysis of cAMP to AMP. At the bottom of its active site lie two divalent metal ions in a binuclear motif which are involved in both cAMP binding and catalysis [(2000) Science 288, 1822-1825; (2000) Biochemistry 39, 6449-6458]. Using a SPA-based equilibrium [(3)H]rolipram binding assay, we have determined that Mg(2+), Mn(2+), and Co(2+) all mediated a high-affinity (K(d) between 3 and 8 nM) and near stoichiometric (R)-rolipram binding to PDE4. In their absence, (R)-rolipram binds stoichiometrically to the metal ion-free apoenzyme with a K(d) of approximately 150 nM. The divalent cation dose responses in mediating the high-affinity rolipram/PDE4 interaction mirror their efficacy in catalysis, suggesting that both metal ions of the holoenzyme are involved in mediating the high-affinity (R)-rolipram/PDE4 interaction. The specific rolipram binding to the apo- and holoenzyme is differentially displaced by cAMP, AMP, and other inhibitors, providing a robust tool to dissect the components of metal ion-dependent and independent PDE4/ligand interactions. cAMP binds to the holoenzyme with a K(s) of 1.9 microM and nonproductively to the apoenzyme with a K(d) of 179 microM. In comparison, AMP binds to the holo- and apoenzyme with K(d) values of 7 and 11 mM, respectively. The diminished Mg(2+)-dependent component of AMP binding to PDE4 suggests that most of the Mg(2+)/phosphate interaction in the cAMP/PDE4 complex is disrupted upon the hydrolysis of the cyclic phosphoester bond, leading to the rapid release of AMP.

Diazepam and rolipram differentially inhibit cyclic AMP-specific phosphodiesterases PDE4A1 and PDE4B3 in the mouse.

Cyclic AMP is hydrolyzed by members of at least eight classes of cyclic nucleotide phosphodiesterases (PDEs). Although it has been reported that cyclic AMP PDE activity in mammalian tissues can be inhibited by benzodiazepines, it has not been conclusively demonstrated that members of the class of cyclic AMP-specific, rolipram-inhibitable PDEs (PDE4s) are targets for these drugs. Moreover, no PDE4s expressed in mice have been characterized. To address these issues, we isolated two cDNAs representing homologues of PDE4A1 and PDE4B3 from a mouse brain library. After transient transfection in human embryonic kidney (HEK) 293 cells, the mouse PDEs hydrolyzed cyclic AMP with a low K(m) and were inhibited by rolipram; both are properties typical of other mammalian PDE4 enzymes. In addition, we found that diazepam inhibited cyclic AMP hydrolysis by the mouse PDE4 subtypes. Interestingly, PDE4B was significantly more sensitive to inhibition by both rolipram and diazepam than the PDE4A subtype. This is the first demonstration that recombinantly expressed PDE4s are inhibited by diazepam, and should facilitate future studies with mouse models of depression and anxiety.

Phosphodiesterase 4 inhibitors, structurally unrelated to rolipram, as promising agents for the treatment of asthma and other pathologies.

An increase of cyclic adenosine and guanosine monophosphate (cAMP and cGMP) level can be achieved by inhibition of phosphodiesterases (PDEs), which are the enzymes responsible for the conversion of these second messengers into the corresponding 5-monophosphate inactive counterparts. The high heterogeneity in PDE families and in their tissue distribution, as well as their different functional role, make these enzymes very attractive targets for medicinal chemists. The PDE 4 family is particularly abundant in immunocompetent cells, where an increase of cAMP leads to the inhibition of the synthesis and release of pro-inflammatory mediators, cytokines and active oxygen species. Moreover PDE 4 inhibitors are able to reduce bronchial smooth muscle tone in vitro and show bronchodilatory effects in vivo. Thus, the current therapy for asthma, which is based on a combination of beta(2) agonists and corticosteroids, could be replaced by treatment with PDE 4 inhibitors. This review mainly covers PDE 4 inhibitors structurally related to xanthines and Nitraquazone, which appear to be very attractive models for the synthesis of novel PDE 4 inhibitors potentially useful for the treatment of asthma, chronic pulmonary obstructive disease and some autoimmune diseases. These compounds could be devoid of the central side-effects (nausea, vomiting, headache) of the archetypal Rolipram, which hampered its development as a drug. The review also highlights the novel structural classes of PDE 4 inhibitors recently reported in the literature.