Dual inhibition of REV-ERBß and autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells.

REV-ERBα and REV-ERBß nuclear receptors regulate several physiological processes, including circadian rhythm and metabolism. A previous study reported the REV-ERBα gene to be co-overexpressed with ERBB2 in breast cancer cell lines. Surprisingly, we found that several tumor types, including a number of breast cancer cell lines, predominantly express the REV-ERBß variant. This pattern was independent of ERBB2 and ER status, and opposite to that of non-cancer mammary epithelial HMEC cells, in which REV-ERBα was the major variant. Consistent with this molecular profile, REV-ERB target genes in both circadian and metabolic pathways were derepressed upon silencing of REV-ERBß, but not REV-ERBα. Strikingly, we found that REV-ERBß is a determinant of sensitivity to chloroquine, a clinically relevant lysosomotropic agent that suppresses autophagy. The cytoprotective function of REV-ERBß appears to operate downstream of autophagy blockade. Through compound screening, we identified ARN5187, a novel lysosomotropic REV-ERBß ligand with a dual inhibitory activity toward REV-ERB-mediated transcriptional regulation and autophagy. Remarkably, although ARN5187 and chloroquine share similar lysosomotropic potency and have a similar effect on autophagy inhibition, ARN5187 is significantly more cytotoxic. Collectively, our results reveal that dual inhibition of REV-ERBß and autophagy is an effective strategy for eliciting cytotoxicity in cancer cells. Furthermore, our discovery of a novel inhibitor compound of both REV-ERB and autophagy may provide a scaffold for the discovery of new multifunctional anticancer agents.

Anandamide transport inhibition by ARN272 attenuates nausea-induced behaviour in rats, and vomiting in shrews (Suncus murinus).

BACKGROUND AND PURPOSE: To understand how anandamide transport inhibition impacts the regulation of nausea and vomiting and the receptor level mechanism of action involved. In light of recent characterization of an anandamide transporter, fatty acid amide hydrolase-1-like anandamide transporter, to provide behavioural support for anandamide cellular reuptake as a facilitated transport process. EXPERIMENTAL APPROACH: The systemic administration of the anandamide transport inhibitor ARN272 ([(4-(5-(4-hydroxy-phenyl)-3,4-diaza-bicyclo[4.4.0]deca-1(6),2,4,7,9-pentaen-2-ylamino)-phenyl)-phenylamino-methanone]) was used to evaluate the prevention of LiCl-induced nausea-induced behaviour (conditioned gaping) in rats, and LiCl-induced emesis in shrews (Suncus murinus). The mechanism of how prolonging anandamide availability acts to regulate nausea in rats was explored by the antagonism of cannabinoid 1 (CB1) receptors with the systemic co-administration of SR141716. KEY RESULTS: The systemic administration of ARN272 produced a dose-dependent suppression of nausea-induced conditioned gaping in rats, and produced a dose-dependent reduction of vomiting in shrews. The systemic co-administration of SR141716 with ARN272 (at 3.0 mg·kg(-1)) in rats produced a complete reversal of ARN272-suppressed gaping at 1.0 mg·kg(-1). SR141716 alone did not differ from the vehicle solution. CONCLUSIONS AND IMPLICATIONS: These results suggest that anandamide transport inhibition by the compound ARN272 tonically activates CB1 receptors and as such produces a type of indirect agonism to regulate toxin-induced nausea and vomiting. The results also provide behavioural evidence in support of a facilitated transport mechanism used in the cellular reuptake of anandamide.

Computational methods in the discovery and design of BACE-1 inhibitors.

BACE-1 is a membrane associated aspartyl protease and is one of the enzymes responsible for the hydrolysis of the amyloid precursor protein. Due to its central role in the generation of the amyloid-ß peptide, it is considered as a primary drug target for Alzheimer's disease. BACE-1 has been the focus of many drug discovery programs aimed at identifying inhibitors that effectively block this enzyme and trigger the sought therapeutic effects. Thanks to the availability of a large number of crystal structures of the catalytic domain of this enzyme, computational methods, ranging from molecular dynamics simulations, quantum mechanical calculations and ligand docking, have played a fundamental role in almost every hit discovery and hit optimization campaign performed on this target. The present article reviews the latest computational modeling and drug discovery efforts that have been carried out on this target.

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase 1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation,sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Polo-like kinases inhibitors.

Polo-like kinases (PLKs) are a family of serine/threonine kinases that play crucial roles in multiple stages of mitosis. PLK1 is the most studied member of the family. It is overexpressed in a wide spectrum of cancer types and is a promising target in oncology. Most of PLK1 inhibitors are ATP-competitive. Despite the structural similarities among various kinases, several inhibitors are selective. Some areas of the PLK1 active site are important for selectivity against other kinases. These include a small pocket formed by Leu 132 in the hinge region, a bulky phenylalanine and a small cysteine at the bottom and in the roof of the ATP pocket, respectively, and an unusual concentration of positively charged residues in the solvent-exposed region. Many ATP-competitive inhibitors are heterocyclic systems able to interact with the unique features of the PLK1 binding site. Other inhibitors target regions outside the ATP pocket, such as the substrate binding domain or a hydrophobic pocket, formed when the kinase is in the inactive conformation. An alternative approach to obtain specificity and to overcome drug resistance often associated with kinase inhibitors is the inhibition of the polo-box domain (PBD) of PLK1. The PBD is unique for the family of PLKs and is essential for PLK functions; so it is a useful target for the development of selective and potent inhibitors for clinical uses. In this review some PLK inhibitors are reported, focusing on chemical structures, structure-activity-relationships (SAR) and biological activities. The great potential of these compounds could open promising perspectives. Moreover, a combination of polo-like kinases inhibitors with other anticancer drugs might offer new opportunities for cancer therapy.

Irreversible protein kinase inhibitors.

Targeting cancer with small molecule irreversible inhibitors of kinases represents an emerging challenge in drug discovery. Irreversible inhibitors bind to kinase active site in a covalent and irreversible form, most frequently by reacting with a nucleophilic cysteine residue, located near the ATP binding pocket. The most common mechanism is the Michael reaction, that refers to the addition of a nucleophile, such as cysteine, to an α,ß unsaturated carbonyl. The nucleophile reacts at the electrophilic ß-position to form an adduct; as a result the inhibitor irreversibly blocks binding of ATP to the kinase, rendering the kinase inactive. Different cysteine-reactive groups have been evaluated, an acrylamide or a substituted acrylamide moiety are the Michael acceptors of choice. There are some advantages for the irreversible kinase inhibition. These compounds are highly selective because they target a specific cysteine and only a limited number of kinases has a cysteine at the corresponding position. Another advantage is that covalent bond formation can overcome competition with the high endogenous concentration of ATP. A further motivation for designing irreversible inhibitors is their longer duration of action respect to conventional inhibitors. In fact, once bound to enzyme, these compounds do not readily dissociate and the inhibition continues even after the inhibitor leaves the circulation. Moreover, these inhibitors have the potential to overcome and prevent the emergence of acquired resistance conferred by mutations. In this review examples of irreversible inhibitors are reported, focusing on chemical structures, SAR and biological activities. The great potential of these compounds could open new and promising perspectives for a broader application of this approach.

Non-ATP competitive protein kinase inhibitors.

Protein kinases represent an attractive target in oncology drug discovery. Most of kinase inhibitors are ATP-competitive and are called type I inhibitors. The ATP-binding pocket is highly conserved among members of the kinase family and it is difficult to find selective agents. Moreover, the ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy between IC50s measured by biochemical versus cellular assays. The non-ATP competitive inhibitors, called type II and type III inhibitors, offer the possibility to overcome these problems. These inhibitors act by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. In the DFG-out form, the phenylalanine side chain moves to a new position. This movement creates a hydrophobic pocket available for occupation by the inhibitor. Some common features are present in these inhibitors. They contain a heterocyclic system that forms one or two hydrogen bonds with the kinase hinge residue. They also contain a hydrophobic moiety that occupies the pocket formed by the shift of phenylalanine from the DFG motif. Moreover, all the inhibitors bear a hydrogen bond donor-acceptor pair, usually urea or amide, that links the hinge-binding portion to the hydrophobic moiety and interacts with the allosteric site. Examples of non ATP-competitive inhibitors are available for various kinases. In this review small molecules capable of inducing the DFG-out conformation are reported, especially focusing on structural feature, SAR and biological properties.

[The radical treatment of hepatic hydatidosis with deep and multiple locations. The role of new technologies particularly in the case of multiple locations]. / Sul trattamento radicale dell'idatidosi epatica a localizzazione profonda e multipla. Ruolo delle nuove tecnologie, specie in caso di localizzazioni multiple.

Advanced technologies (intraoperative ultrasonography, CT scan, argon coagulator ...) have changed the surgical approach of liver hydatid disease, allowing even multiple or deeply located cysts to be detected and treated successfully. Authors report a series of 4 patients with single (3) or multiple (1) unilocular hepatic cysts; and 1 patient with thoraco-pulmonary hydatid recurrent disease. Treatments of choice and surgical techniques are described. No infective compliances occurred. The mean period of hospitalization was 19 days (ranging between 10 days and 4 weeks). The longest hospitalization was observed in a patient with a post-operative biliary fistula at low out put. Total cysto-pericystectomy is emphasized as the gold standard procedure in the treatment of non complicated unilocular hydatid cysts of the liver. Modern means of investigation and technical equipment make it feasible and safe even in unfavorable localizations, allowing radical removal of the cysts preserving in the meantime all the surrounding liver parenchyma.

[Injury of the popliteal artery & the popliteal branches: analysis of 31 consecutive cases]. / I traumi dell'arteria poplitea e dei rami infrapoplitei: analisi di 31 casi consecutivi.

In complex traumas of lower limbs it is fundamental the reducing of the time of ischemia to reduce the number of failures in repairing surgery and the percentage of the demolishing of necessity surgery. The experience of the authors is based on 31 complex traumas of lower limbs with distal vascular injuries to Hunter's canal, which were associated to bone, nervous and muscular lesions. In total we are treated 37 vascular injuries by interposition of venous autografts in 37 cases, lateral pacth in one case ant suture T-T in two cases. The protection of the reconstructed vases, in case of a concomitant loss of substance, was entrused to microvascular flaps for 7 times latissimus dorsi, iliac crest and fascio-cutaneous for 5 times posterior reversed with distal baset and for three times antero-lateral, which in three cases of them needed successively a dermoepidermic grafts. The bone injuries, which were treated by external fixation, intramedullary nailing, plate and screws, just in two cases became worse in pseudoarthroses and just in one in osteomyelitis. The reparation of nervous injuries had bad results just in two cases. The percentage of saving of the limb it was about 83.9%.

[Significance of antibiotic prophylaxis in infection prevention in biliary surgery. Personal experience]. / Significato della profilassi antibiotica nella prevenzione delle infezioni in chirugia biliare. Esperienza personale.

Postoperative infections are one of the most frequent causes of morbidity in surgical patients. In biliary tract surgery the risk of septic complications is essentially increased by opening the bile ducts. Aim of this paper is to evaluate indications and effectiveness of short-term prophylaxis in preventing infective complications of biliary surgery. Authors report a personal six years review regarding 530 patients operated for biliary tract diseases. The patients were divided into two groups: the first one (n. 245) operated under short-term prophylaxis; the second one (n. 285) treated only postoperativelly with antibiotics. The results obtained show a significatively higher rate of infective complications in the second group of patients. There is a little difference between the groups in the case of elective cholecystectomy, whereas there is a great difference in case of non-elective surgery, bile ducts opening, external biliary drainages placement or sphincteropapillotomy. Authors furthermore emphasize how surgical results are conditioned by individual risk factors, especially obesity and diabetes.