Inhibition of RPTPß/ζ reduces chronic ethanol intake in adolescent mice and modulates ethanol effects on hippocampal neurogenesis and glial responses in a sex-dependent manner.

Pleiotrophin (PTN) is a cytokine that modulates ethanol drinking and reward and regulates glial responses in different contexts. PTN is an inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ. Inhibition of RPTPß/ζ reduces binge-like drinking in adult male mice. Whether inhibition of RPTPß/ζ is effective in reducing ethanol consumption during adolescence and in both sexes remained to be studied. In this work, male and female adolescent mice underwent an intermittent access to ethanol (IAE) 2-bottle choice protocol. Treatment with MY10 (60 mg/kg, i.g.), a small-molecule RPTPß/ζ inhibitor, reduced chronic 3-week ethanol consumption only in male mice. We detected an ethanol-induced overall decrease in hippocampal GFAPir and Iba1ir, independently of the treatment received, suggesting that RPTPß/ζ is not key in the regulation of IAE-induced glial responses. However, we found a significant negative correlation between the size of microglial cells and the number of hippocampal neuronal progenitors only in male mice after IAE. This correlation was disrupted by treatment with MY10 before each drinking session, which may be related to the ability of MY10 to regulate the intensity of the perineuronal nets (PNNs) in the hippocampus in a sex-dependent manner. The data show for the first time that inhibition of RPTPß/ζ reduces chronic voluntary ethanol consumption in adolescent mice in a sex-dependent manner. In addition, we show evidence for sex-specific differences in the effects of IAE on glial responses and hippocampal neurogenesis, which may be related to different actions of the RPTPß/ζ signalling pathway in the brains of male and female mice.

Receptor protein tyrosine phosphatase ß/ζ regulates loss of neurogenesis in the mouse hippocampus following adolescent acute ethanol exposure.

Adolescence is a critical period for brain maturation in which this organ is more vulnerable to the damaging effects of ethanol. Administration of ethanol in mice induces a rapid cerebral upregulation of pleiotrophin (PTN), a cytokine that regulates the neuroinflammatory processes induced by different insults and the behavioral effects of ethanol. PTN binds Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ and inhibits its phosphatase activity, suggesting that RPTPß/ζ may be involved in the regulation of ethanol effects. To test this hypothesis, we have treated adolescent mice with the RPTPß/ζ inhibitor MY10 (60 mg/kg) before an acute ethanol (6 g/kg) administration. Treatment with MY10 completely prevented the ethanol-induced neurogenic loss in the hippocampus of both male and female mice. In flow cytometry studies, ethanol tended to increase the number of NeuN+/activated Caspase-3+ cells particularly in female mice, but no significant effects were found. Ethanol increased Iba1+ cell area and the total marked area in the hippocampus of female mice, suggesting sex differences in ethanol-induced microgliosis. In addition, ethanol reduced the circulating levels of IL-6 and IL-10 in both sexes, although this reduction was only found significant in males and not affected by MY10 treatment. Interestingly, MY10 alone increased the total marked area and the number of Iba1+ cells only in the female hippocampus, but tended to reduce the circulating levels of TNF-α only in male mice. In summary, the data identify a novel modulatory role of RPTPß/ζ on ethanol-induced loss of hippocampal neurogenesis, which seems unrelated to glial and inflammatory responses. The data also suggest sex differences in RPTPß/ζ function that may be relevant to immune responses and ethanol-induced microglial responses.

Design and Synthesis of Water-Soluble and Potent MMP-13 Inhibitors with Activity in Human Osteosarcoma Cells.

Osteoarthritis is a degenerative disease, often resulting in chronic joint pain and commonly affecting elderly people. Current treatments with anti-inflammatory drugs are palliative, making the discovery of new treatments necessary. The inhibition of matrix metalloproteinase MMP-13 is a validated strategy to prevent the progression of this common joint disorder. We recently described polybrominated benzotriazole derivatives with nanomolar inhibitory activity and a promising selectivity profile against this collagenase. In this work, we have extended the study in order to explore the influence of bromine atoms and the nature of the S1' heterocyclic interacting moiety on the solubility/selectivity balance of this type of compound. Drug target interactions have been assessed through a combination of molecular modeling studies and NMR experiments. Compound 9a has been identified as a water-soluble and highly potent inhibitor with activity in MG-63 human osteosarcoma cells.

Role of Receptor Protein Tyrosine Phosphatase ß/ζ in Neuron-Microglia Communication in a Cellular Model of Parkinson's Disease.

Pleiotrophin (PTN) is a neurotrophic factor that regulates glial responses in animal models of different types of central nervous system (CNS) injuries. PTN is upregulated in the brain in different pathologies characterized by exacerbated neuroinflammation, including Parkinson's disease. PTN is an endogenous inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ, which is abundantly expressed in the CNS. Using a specific inhibitor of RPTPß/ζ (MY10), we aimed to assess whether the PTN/RPTPß/ζ axis is involved in neuronal and glial injury induced by the toxin MPP+. Treatment with the RPTPß/ζ inhibitor MY10 alone decreased the viability of both SH-SY5Y neuroblastoma cells and BV2 microglial cultures, suggesting that normal RPTPß/ζ function is involved in neuronal and microglial viability. We observed that PTN partially decreased the cytotoxicity induced by MPP+ in SH-SY5Y cells underpinning the neuroprotective function of PTN. However, MY10 did not seem to modulate the SH-SY5Y cell loss induced by MPP+. Interestingly, we observed that media from SH-SY5Y cells treated with MPP+ and MY10 decreases microglial viability but may elicit a neuroprotective response of microglia by upregulating Ptn expression. The data suggest a neurotrophic role of microglia in response to neuronal injury through upregulation of Ptn levels.

New Dual CK2/HDAC1 Inhibitors with Nanomolar Inhibitory Activity against Both Enzymes.

Four potent CK2 inhibitors derived from CX-4945 are described. They also provided nanomolar activity against HDAC1, therefore having promising utility as dual-target agents for cancer. The linker length between the hydroxamic acid and the CX-4945 scaffold plays an important role in dictating balanced activity against the targeted enzymes. The seven-carbon linker (compound 15c) was optimal for inhibition of both CK2 and HDAC1. Remarkably, 15c showed 3.0 and 3.5 times higher inhibitory activity than the reference compounds CX-4945 (against CK2) and SAHA (against HDAC1), respectively. Compound 15c exhibited micromolar activity in cell-based cytotoxic assays against multiple cell lines.

Multitarget Anticancer Agents Based on Histone Deacetylase and Protein Kinase CK2 inhibitors.

The design of multitarget drugs (MTDs) has become an innovative approach for the search of effective treatments in complex diseases such as cancer. In this work, we communicate our efforts in the design of multi-targeting histone deacetylase (HDAC) and protein kinase CK2 inhibitors as a novel therapeutic strategy against cancer. Using tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT) as scaffolds for CK2 inhibition, and a hydroxamate to coordinate the zinc atom present in the active site of HDAC (zinc binding group, ZBG), new multitarget inhibitors have been designed and synthesized. According to the in vitro assays, N-Hydroxy-6-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)hexanamide (11b) is the most interesting compound, with IC50 values of 0.66; 1.46 and 3.67 µM. for HDAC6; HDAC1 and CK2; respectively. Cellular assays on different cancer cell lines rendered promising results for N-Hydroxy-8-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)octanamide (11d). This inhibitor presented the highest cytotoxic activity, proapoptotic capability, and the best mitochondria-targeting and multidrug-circumventing properties, thus being the most promising drug candidate for further in vivo studies.

Molecular Imaging Probes Based on Matrix Metalloproteinase Inhibitors (MMPIs).

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases which are secreted or anchored in the cell membrane and are capable of degrading the multiple components of the extracellular matrix (ECM). MMPs are frequently overexpressed or highly activated in numerous human diseases. Owing to the important role of MMPs in human diseases, many MMP inhibitors (MMPIs) have been developed as novel therapeutics, and some of them have entered clinical trials. However, so far, only one MMPI (doxycycline) has been approved by the FDA. Therefore, the evaluation of the activity of a specific subset of MMPs in human diseases using clinically relevant imaging techniques would be a powerful tool for the early diagnosis and assessment of the efficacy of therapy. In recent years, numerous MMPIs labeled imaging agents have emerged. This article begins by providing an overview of the MMP subfamily and its structure and function. The latest advances in the design of subtype selective MMPIs and their biological evaluation are then summarized. Subsequently, the potential use of MMPI-labeled diagnostic agents in clinical imaging techniques are discussed, including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging (OI). Finally, this article concludes with future perspectives and clinical utility.

Inhibition of RPTPß/ζ blocks ethanol-induced conditioned place preference in pleiotrophin knockout mice.

Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that are upregulated in the prefrontal cortex after alcohol administration and have been shown to reduce ethanol drinking and reward. PTN and MK are endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ. Interestingly, pharmacological inhibition of RPTPß/ζ reduces ethanol consumption and blocks ethanol-induced conditioned place preference (CPP) in wild type mice. Since PTN-knockout (Ptn-/-) mice are more sensitive to the conditioning effects of alcohol, we aimed to test the effects of MY10, a small-molecule inhibitor of RPTPß/ζ, on ethanol-induced CPP in Ptn-/- mice. The data presented here demonstrate for the first time that a regular dose of MY10, known to block ethanol consumption and reward in wild type mice, also blocks the rewarding effects of ethanol in the more vulnerable individuals lacking PTN, the endogenous inhibitor of RPTPß/ζ. In addition, since MY10 readily penetrates the blood brain barrier (BBB), we tested its effects in a series of behavioural tests in Ptn+/+ and Ptn-/- mice. The data indicate that MY10 does not cause gross behavioural effects in wild type mice. However, MY10 tended to induce anxiolytic effects in Ptn-/- mice in the elevated plus maze paradigm. Overall, the data indicate that MY10 rescues Ptn-/- mice from their increased susceptibility to the conditioning effects of ethanol and may induce anxiolytic effects in individuals with reduced or absent PTN functions. Further studies are needed to confirm the potential of pharmacological inhibition of RPTPß/ζ as a new therapeutic strategy in the treatment of anxiety-related disorders.

From a MMP2/CK2 multitarget approach to the identification of potent and selective MMP13 inhibitors.

In this article, we describe our efforts in the search of MMP2/CK2 dual targeting inhibitors. We have followed a rational drug design approach based on our experience in the selective inhibition of these two enzymes. We have successfully obtained highly active MMP2 (10, IC50 = 70 nM; 11, IC50 = 100 nM) and CK2 (16a, IC50 = 500 nM) inhibitors. However, structural fine tuning of these small molecules to simultaneously target both enzymes turned out to be an unattainable goal. Unexpectedly, we were lucky to identify new and selective MMP13 inhibitors (10, IC50 = 3.7 nM and 11, IC50 = 5.6 nM) with a novel TBB-derived scaffold. These compounds constitute an interesting starting point for further optimization.

Pharmacological inhibition of Receptor Protein Tyrosine Phosphatase ß/ζ (PTPRZ1) modulates behavioral responses to ethanol.

Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that are upregulated in the prefrontal cortex after alcohol administration and have been shown to reduce ethanol drinking and reward. PTN and MK are the endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ (a.k.a. PTPRZ1, RPTPß, PTPζ), suggesting a potential role for this phosphatase in the regulation of alcohol effects. To determine if RPTPß/ζ regulates ethanol consumption, we treated mice with recently developed small-molecule inhibitors of RPTPß/ζ (MY10, MY33-3) before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with RPTPß/ζ inhibitors, particularly with MY10, drank less ethanol than controls. MY10 treatment blocked ethanol conditioned place preference, showed limited effects on ethanol-induced ataxia, and potentiated the sedative effects of ethanol. We also tested whether RPTPß/ζ is involved in ethanol signaling pathways. We found that ethanol treatment of neuroblastoma cells increased phosphorylation of anaplastic lymphoma kinase (ALK) and TrkA, known substrates of RPTPß/ζ. Treatment of neuroblastoma cells with MY10 or MY33-3 also increased levels of phosphorylated ALK and TrkA. However, concomitant treatment of neuroblastoma cells with ethanol and MY10 or MY33-3 prevented the increase in pTrkA and pALK. These results demonstrate for the first time that ethanol engages TrkA signaling and that RPTPß/ζ modulates signaling pathways activated by alcohol and behavioral responses to this drug. The data support the hypothesis that RPTPß/ζ might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.

Development of inhibitors of receptor protein tyrosine phosphatase ß/ζ (PTPRZ1) as candidates for CNS disorders.

A new series of blood-brain barrier permeable molecules designed to mimic the activity of Pleiotrophin in the CNS has been designed and synthesized. These compounds exert their action by interacting with the intracellular domain PD1 of the Protein Tyrosine-Phosphatase Receptor Z1 (PTPRZ1), and inhibiting its tyrosine phosphatase activity. The most potent compounds 10a and 12b (IC50 = 0,1 µM) significantly increase the phosphorylation of key tyrosine residues of PTPRZ1 substrates involved in neuronal survival and differentiation, and display protective effects against amphetamine-induced toxicity. Docking and molecular dynamics experiments have been used to analyze the binding mode and to explain the observed selectivity against PTP1B. An In vivo experiment has demonstrated that 10a can cross the BBB, thus promoting the possibility of moving forward these candidates for the development of drugs for the treatment of CNS disorders, such as drug addiction and neurodegenerative diseases.

In silico identification and in vivo characterization of small molecule therapeutic hypothermia mimetics.

Hypothermia has been proved to have a beneficial effect on several pathologies. CIRBP is one of the so termed cold-shock proteins involved in this process. In this work, we have detected small molecules capable of modulating the activity of CIRBP in the absence of a cold stimulus, by High Throughput Virtual Screening (HTVS) of the Diversity Set IV of the NCI and 15 compounds of our in-house data base. Fifteen compounds were selected from the HTVS to carry out a second screening through a cell-based Western blot assay. This assay, together with molecular modeling studies allowed us to select compound zr17-2 for an in vivo experiment, which showed an interesting increase of CIRBP expression in several organs of experimental animals. Therefore, we have demonstrated that the effect of hypothermia can be mimicked by small molecules, which can be developed as first-in-class new drugs for the treatment of several diseases.

Design and synthesis of potent hydroxamate inhibitors with increased selectivity within the gelatinase family.

MMP-2 is a validated target for the development of anticancer agents. Herein we describe the synthesis of a new series of potent phenylalanine derived hydroxamates, with increased MMP-2/MMP-9 selectivity compared to analogous hydroxamates described previously. Docking and molecular dynamics experiments have been used to account for this selectivity, and to clarify the role of the triazole ring in the binding process.

An integrated computational and experimental approach to gaining selectivity for MMP-2 within the gelatinase subfamily.

Looking for water-soluble inhibitors of matrix metalloproteinase-2 (MMP-2 or gelatinase A), we have previously reported compound 1, a potent MMP-2 inhibitor with a promising selectivity over the structurally homologous MMP-9 (gelatinase B). Here we report the results of Molecular Dynamics (MD) simulations for both gelatinases (MMP-2 and MMP-9), and for the corresponding MMP/1 complexes, in an attempt to shed light on the observed selectivity between the two enzymes. These studies indicated a higher plasticity of MMP-2 at the S1' pocket and suggested an induced-fit effect at the "back door" of this pocket. On the basis of these observations, we designed 11 a-d to aid further discrimination between MMP-2 and MMP-9. Those compounds displayed notably lower inhibitory activities against MMP-9; in particular, 11 b proved to be over 100 times more active against MMP-2 than against MMP-9. MD simulations of the MMP/11 b complexes and thermodynamic integration calculations provided structural insight and relative binding energies consistent with the experimentally observed activity data. These findings demonstrate that structural differences in the S1' pocket bottom permit an improvement in selectivity in the inhibition of MMP-2 over that of MMP-9; this is of great relevance for future structure-based drug design because MMP-2 is a validated target for cancer therapy, whereas MMP-9 plays both detrimental and protective roles in cancer. This study also supports the need to consider the dynamics of the S1' pocket in order to achieve selectivity in the inhibition of MMPs.

Progress towards water-soluble triazole-based selective MMP-2 inhibitors.

Water solubility is a key aspect that needs to be addressed to obtain drug-like compounds. In an effort to improve the water solubility of our recently reported nanomolar matrix metalloproteinase type 2 (MMP-2) inhibitors based on triazole-substituted hydroxamates, we synthesized a new series of α-sulfone, α-tetrahydropyran and α-piperidine, α-sulfone clicked hydroxamates and determined their inhibitory activities against both MMP-2 and MMP-9. The best results were found for 13e, a water-soluble compound that displays a low nanomolar activity against MMP-2 and is 26-fold less active against MMP-9. This finding allowed us to pursue in vitro permeability through the Caco-2 monolayer and opened the possibility of carrying out further preclinical investigations. Docking and MD simulations have been performed in order to rationalize the biological results. The inhibitory activity of this compound against a panel of ten MMPs was determined showing an interesting MMP-2/MMP-1, -8, and -14 selectivity profile. The cytotoxicity and anti-invasive activity of the compounds on highly metastatic human fibrosarcoma tumor cells (HT1080) were determined, showing, at 10 µM concentration, a decrease in cell invasiveness up to 80%.

Potent "clicked" MMP2 inhibitors: synthesis, molecular modeling and biological exploration.

A new series of MMP2 inhibitors is described, following a fragment-based drug design approach. One fragment containing an azide group and a well known hydroxamate Zinc Binding Group in a α-sulfone, α-tetrahydropyrane scaffold, has been synthesized. Water-LOGSY, STD and competition-STD experiments indicate that this fragment binds to the active site of the enzyme. A click chemistry reaction was used to connect the azide to lipophilic alkynes selected to interact selectively with the S1' subunit of MMP2, as shown by docking and molecular dynamic experiments of the designed compounds. The most potent compounds 18 and 19 displayed an IC(50) of 1.4 and 0.3 nM against MMP2 respectively, and showed negligible activity towards MMP1 and MMP7, two metalloproteinases which have a shallow S1' subsite. Compound 18 also showed a promising selectivity profile against some antitarget metalloproteinases, such as MMP8, and considerably less activity against MMP14 (IC(50) = 65 nM), and MMP9 (IC(50) = 98 nM), other MMPs characterized by having a deep S1' pocket and, therefore, more similar to MMP2.