Novel P2X7 Antagonist Ameliorates the Early Phase of ALS Disease and Decreases Inflammation and Autophagy in SOD1-G93A Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a disease with a resilient neuroinflammatory component caused by activated microglia and infiltrated immune cells. How to successfully balance neuroprotective versus neurotoxic actions through the use of anti-inflammatory agents is still under debate. There has been a boost of awareness regarding the role of extracellular ATP and purinergic receptors in modulating the physiological and pathological mechanisms in the nervous system. Particularly in ALS, it is known that the purinergic ionotropic P2X7 receptor plays a dual role in disease progression by acting at different cellular and molecular levels. In this context, we previously demonstrated that the P2X7 receptor antagonist, brilliant blue G, reduces neuroinflammation and ameliorates some of the pathological features of ALS in the SOD1-G93A mouse model. Here, we test the novel, noncommercially available, and centrally permeant Axxam proprietary P2X7 antagonist, AXX71, in SOD1-G93A mice, by assessing some behavioral and molecular parameters, among which are disease progression, survival, gliosis, and motor neuron wealth. We demonstrate that AXX71 affects the early symptomatic phase of the disease by reducing microglia-related proinflammatory markers and autophagy without affecting the anti-inflammatory markers or motor neuron survival. Our results suggest that P2X7 modulation can be further investigated as a therapeutic strategy in preclinical studies, and exploited in ALS clinical trials.

Chemical interrogation of FOXO3a nuclear translocation identifies potent and selective inhibitors of phosphoinositide 3-kinases.

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3Kalpha. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.

Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing.

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.

Differentiation between [1,2,4]triazolo[1,5-a] pyrimidine and [1,2,4]triazolo[4,3-a]-pyrimidine regioisomers by 1H-15N HMBC experiments.

The condensation of malonoaldehyde derivatives with either a 3-amino-[1,2,4]-triazole or a 3,5-diamino-[1,2,4]-triazole precursor was studied. In agreement with previous reports, two different bicycles, namely, bearing the regioisomeric [1,2,4]triazolo[1,5-a]pyrimidine (1) or [1,2,4]triazolo[4,3-a]pyrimidine (2) structural surrogates, could be obtained. We found that, depending on the triazole precursor, only one regioisomer resulted, either of the 1 or 2 series. We also observed that these two structural surrogates could be unambiguously differentiated by indirectly measuring their (15)N chemical shifts by (1)H-(15)N HMBC experiments. The occasional conversion of [1,2,4]triazolo[4,3-a]pyrimidines to the [1,2,4]triazolo[1,5-a]pyrimidine counterparts could be unequivocally determined by (15)N NMR data.

CDK inhibitors in cancer therapy: what is next?

The pursuit for drugs that inhibit cyclin-dependent kinases (CDKs) has been an intense area of research for more than 15 years. The first-generation inhibitors, Flavopiridol and CY-202, are in late-stage clinical trials, but so far have demonstrated only modest activity. Several second-generation inhibitors are now in clinical trials. Future approaches to determine clinical benefit need to incorporate both the lessons learned from these early compounds and information recently obtained from the genetic analysis of CDKs in preclinical models. Here we discuss key concepts that should be considered when validating the clinical utility of CDK inhibitors in cancer therapy.

P2X7 antagonists for CNS indications: recent patent disclosures.

P2X7, a ligand-gated purinergic ion channel, has been at the center of intense efforts in the pharmaceutical industry in the last 15 years due to the growing appreciation of its role in inflammation. Since 2008-2009, increased focus on CNS available compounds has led to the publication of various patents on behalf of several pharmaceutical companies. This patent review aims at analyzing the recent patent literature (2008-2016) with a particular emphasis on those patents that are thought to deal with CNS penetrant compounds on the basis of their physicochemical features, the assays described in the patents and the uses these compounds are claimed for.

Structure-based drug design to the discovery of new 2-aminothiazole CDK2 inhibitors.

N-(5-Bromo-1,3-thiazol-2-yl)butanamide (compound 1) was found active (IC50=808 nM) in a high throughput screening (HTS) for CDK2 inhibitors. By exploiting crystal structures of several complexes between CDK2 and inhibitors and applying structure-based drug design (SBDD), we rapidly discovered a very potent and selective CDK2 inhibitor 4-[(5-isopropyl-1,3-thiazol-2-yl)amino] benzenesulfonamide (compound 4, IC50=20 nM). The syntheses, structure-based analog design, kinases inhibition data and X-ray crystallographic structures of CDK2/inhibitor complexes are reported.

An analysis of the binding modes of ATP-competitive CDK2 inhibitors as revealed by X-ray structures of protein-inhibitor complexes.

CDK2 is an attractive target for the design of new therapeutic antitumor agent. Numerous CDK2 inhibitors have been discovered and their crystallographic structures either in complex with CDK2 or CDK2/Cyclin A have been published. This review aims to summarize the publicly available structural characterization of CDK2/(Cyclin A) -- ligand complexes and to highlight the similarities among the binding modes of structurally diverse inhibitors.

3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 2. Lead optimization.

Inhibitors of cyclin-dependent kinases (CDK) such as CDK2/cyclin A-E are currently undergoing clinical trials to verify their potential as new anticancer agents. In a previous article we described the lead discovery process of a 3-aminopyrazole class of CDK2/cyclin A-E inhibitors. The endpoint of this process was PNU-292137, a compound endowed with in vivo antitumor activity in a mouse tumor xenograft model. We optimized this lead compound to improve some physicochemical properties, notably solubility and plasma protein binding. This lead optimization process brought us to the discovery of (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (PHA-533533, 13), a compound with a balanced activity vs druglike profile. Compound 13 inhibited CDK2/cyclin A with a K(i) of 31 nM, counteracting tumor cell proliferation of different cell lines with an IC(50) in the submicromolar range. Solubility was improved more than 10 times over the starting lead, while plasma protein binding was decreased from 99% to 74%. With exploitation of this globally enhanced in vitro profile, 13 was more active than PNU-292137 in vivo in the A2780 xenograft model showing a tumor growth inhibition of 70%. Proof of mechanism of action was obtained in vivo by immunohistochemical analysis of tumor slices of 13-treated vs untreated animals.

FFA4/GPR120 agonists: a survey of the recent patent literature.

FFA4/GPR120, a member of the rhodopsin family of G-protein-coupled receptors (GPCRs), is becoming an important target for therapeutic intervention in several areas of disease, including metabolic diseases, inflammation and cancer. In the last few years several patents on original chemotypes have been generated by different companies. In this review an analysis of the patents in the FFA4 agonism field is presented, with an emphasis on the documents published between 2013 and mid-2015. A discussion of the biological methods used in the patents is included. The general interest in this area is growing fast as half of the existing patents on FFA4 agonists have been issued after 2013. There is, however, a need of further diversifying new chemical classes away form the current substrate-like, carboxylic acid-containing agonists.

NMR-Based screening with competition water-ligand observed via gradient spectroscopy experiments: detection of high-affinity ligands.

Water-ligand observed via gradient spectroscopy (WaterLOGSY) represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. The method is useful for the detection of compounds binding to a receptor with binding affinity in the micromolar range. The Achille's heel of the method, as with all the techniques that detect the ligand resonances, is its inability to identify strong ligands with slow dissociation rates. We show here that the use of a reference compound with a known K(D) in the micromolar range together with properly designed competition binding experiments (c-WaterLOGSY) permits the detection of strong binders. A derived mathematical expression is used for the selection of the appropriate setup NMR experimental conditions and for an approximate determination of the binding constant. The experiment requires low ligand concentration, therefore allowing its application in the identification of potential strong inhibitors that are only marginally soluble. The technique is particularly suitable for rapid screening of chemical mixtures and plant or fungi extracts.

3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 1. Lead finding.

Abnormal proliferation mediated by disruption of the normal cell cycle mechanisms is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDK) and cyclins, such as CDK2/cyclin A and CDK2/cyclin E, and inhibiting their kinase activity are regarded as promising antitumor agents to complement the existing therapies. From a high-throughput screening effort, we identified a new class of CDK2/cyclin A/E inhibitors. The hit-to-lead expansion of this class is described. X-ray crystallographic data of early compounds in this series, as well as in vitro testing funneled for rapidly achieving in vivo efficacy, led to a nanomolar inhibitor of CDK2/cyclin A (N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-naphthyl)acetamide (41), PNU-292137, IC50 = 37 nM) with in vivo antitumor activity (TGI > 50%) in a mouse xenograft model at a dose devoid of toxic effects.

Modulation of the kynurine pathway of tryptophan metabolism in search for neuroprotective agents. Focus on kynurenine-3-hydroxylase.

A novel potent and selective kynurenine-3-hydroxylase inhibitor is descibed along a preliminary evaluation in a in vivo gerbil model of its ability to increase the kynurenine and kynurenic acid concentration in both plasma and brain. These data support the notion that kynurenine-3-hydroxylase inhibitors may have a sustained therapeutic potential in those diseases characterized by unbalance in the QUIN/KYNA branches of the kynurenine pathway.

Ca(2+) release-activated Ca(2+) channel inhibitors.

Ca(2+) release-activated Ca(2+) (CRAC) channels are becoming important targets for therapeutic intervention in several areas of disease, including immunology, allergy and cancer. In parallel to the progression towards reliable methods for measuring CRAC currents and their inhibition, patents have been generated by several companies. In this Patent Review, an analysis of the patents in the CRAC channel inhibition filed is presented. A discussion of the biological methods used in the patents is included. The general interest in this area is growing fast with almost 80% of the patents issued after 2010.

Kinase inhibitors for CNS diseases: an analysis of the recent patent literature.

Protein kinases (PKs), as members of an important target class in current pharmaceutical research, have been mostly exploited so far in therapeutic areas such as oncology and inflammation. However, basic research on some PKs as key components of molecular mechanisms underlying neurodegeneration and neuroprotection may translate into new medicines for CNS diseases in the next few years. This review is an account of recent patents dealing with kinase inhibitors primarily designed for CNS indications. CNS-directed patents on kinase modulators published after 2008 were surveyed using SciFinder(®) and public patent search engines. Some PK targets, such as GSK-3ß, CDK5, ROCK and p38α MAPK, continue to attract interest even though a clinical proof-of-concept is yet to be attained in a CNS setting. Less established PKs such as LRRK2, MLK, PAK and DAPK-1 hold promise as valuable targets of the future.

IP issues facing researchers by Gino D'Oca.

Patents are clearly one of the main drivers of innovation in pharmaceutical and medical R&D. It is increasingly important for researchers at the sharp end to be familiar with the ins and outs of the patenting process. In this feature a panel of experts from academia and industry discuss their experiences of analyzing the patent landscape and preparing applications. Interview conducted by Gino D'Oca, Managing Commissioning Editor.

Fragment-hopping-based discovery of a novel chemical series of proto-oncogene PIM-1 kinase inhibitors.

A new chemical series, triazolo[4,5-b]pyridines, has been identified as an inhibitor of PIM-1 by a chemotype hopping strategy based on a chemically feasible fragment database. In this case, structure-based virtual screening and in silico chemogenomics provide added value to the previously reported strategy of prioritizing among proposed novel scaffolds. Pairwise comparison between compound 3, recently discontinued from Phase I clinical trials, and molecule 8, bearing the selected novel scaffold, shows that the primary activities are similar (IC(50) in the 20 to 150 nM range). At the same time, some ADME properties (for example, an increase of more than 45% in metabolic stability in human liver microsomes) and the off-target selectivity (for example, an increase of more than 2 log units in IC(50)vs. FLT3) are improved, and the intellectual property (IP) position is enhanced. The discovery of a reliable starting point that fulfills critical criteria for a plausible medicinal chemistry project is demonstrated in this prospective study.

Pim 1 kinase inhibitor ETP-45299 suppresses cellular proliferation and synergizes with PI3K inhibition.

The serine/threonine Pim 1 kinase is an oncogene whose expression is deregulated in several human cancers. Overexpression of Pim 1 facilitates cell cycle progression and suppresses apoptosis. Hence pharmacologic inhibitors of Pim 1 are of therapeutic interest for cancer. ETP-45299 is a potent and selective inhibitor of Pim 1 that inhibits the phosphorylation of Bad and 4EBP1 in cells and suppresses the proliferation of several non-solid and solid human tumor cell lines. The combination of the PI3K inhibitor GDC-0941 with ETP-45299 was strongly synergistic in MV-4-11 AML cells, indicating that the combination of selective Pim kinase inhibitors and PI3K inhibitor could have clinical benefit.

Ion channel blockers for the treatment of neuropathic pain.

Neuropathic pain, a severe chronic pain condition characterized by a complex pathophysiology, is a largely unmet medical need. Ion channels, which underlie cell excitability, are heavily implicated in the biological mechanisms that generate and sustain neuropathic pain. This review highlights the biological evidence supporting the involvement of voltage-, proton- and ligand-gated ion channels in the neuropathic pain setting. Ion channel modulators at different research or development stages are reviewed and referenced. Ion channel modulation is one of the main avenues to achieve novel, improved neuropathic pain treatments. Voltage-gated sodium and calcium channel and glutamate receptor modulators are likely to produce new, improved agents in the future. Rationally targeting subtypes of known ion channels, tackling recently discovered ion channel targets or combining drugs with different mechanism of action will be primary sources of new drugs in the longer term.

Identification of potent pyrazolo[4,3-h]quinazoline-3-carboxamides as multi-cyclin-dependent kinase inhibitors.

Abnormal proliferation mediated by disruption of the mechanisms that keep the cell cycle under control is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDKs) and cyclins (Cy) and inhibiting their activity are regarded as promising antitumor agents to complement the existing therapies. An expansion of pyrazolo[4,3-h]quinazoline chemical class oriented to the development of three points of variability was undertaken leading to a series of compounds able to inhibit CDKs both in vitro and in vivo. Starting from the CDK selective but poorly soluble hit compound 1, we succeeded in obtaining several compounds showing enhanced inhibitory activity both on CDKs and on tumor cells and displaying improved physical properties and pharmacokinetic behavior. Our study led to the identification of compound 59 as a highly potent, orally bioavailable CDK inhibitor that exhibited significant in vivo efficacy on the A2780 ovarian carcinoma xenograft model. The demonstrated mechanisms of action of compound 59 on cancer cell lines and its ability to inhibit tumor growth in vivo render this compound very interesting as potential antineoplastic agent.