Identification and in vitro characterization of a new series of potent and highly selective G9a inhibitors as novel anti-fibroadipogenic agents.

A new series of in vitro potent and highly selective histone methyl transferase enzyme G9a inhibitors was obtained. In particular, compound 2a, one the most potent G9a inhibitor identified, was endowed with >130-fold selectivity over GLP and excellent ligand efficiency. Therefore, it may represent a valuable tool compound to validate the role of highly selective G9a inhibitors in different pathological conditions. When 2a was characterized in vitro in cellular models of skeletal muscle differentiation, a relevant increase of myofibers' size and reduction of the fibroadipogenic infiltration were observed, further confirming the therapeutic potential of selective G9a inhibitors for the treatment of Duchenne muscle dystrophy.

Discovery of a Novel Series of Potent SHP2 Allosteric Inhibitors.

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is the first reported nonreceptor oncogenic tyrosine phosphatase connecting multiple signal transduction cascades and exerting immunoinhibitory function through the PD-1 checkpoint receptor. As part of a drug discovery program aimed at obtaining novel allosteric SHP2 inhibitors, a series of pyrazopyrazine derivatives bearing an original bicyclo[3.1.0]hexane basic moiety on the left-hand side region of the molecule were identified. We report herein the discovery process, the in vitro pharmacological profile, and the early developability features of compound 25, one of the most potent members of the series.

Discovery of a Novel Series of Imidazopyrazine Derivatives as Potent SHP2 Allosteric Inhibitors.

Protein tyrosine phosphatase SHP2 is an oncogenic protein that can regulate different cytokine receptor and receptor tyrosine kinase signaling pathways. We report here the identification of a novel series of SHP2 allosteric inhibitors having an imidazopyrazine 6,5-fused heterocyclic system as the central scaffold that displays good potency in enzymatic and cellular assays. SAR studies led to the identification of compound 8, a highly potent SHP2 allosteric inhibitor. X-ray studies showed novel stabilizing interactions with respect to known SHP2 inhibitors. Subsequent optimization allowed us to identify analogue 10, which possesses excellent potency and a promising PK profile in rodents.

[11C]CHDI-626, a PET Tracer Candidate for Imaging Mutant Huntingtin Aggregates with Reduced Binding to AD Pathological Proteins.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein.

The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Use of 'dilute-and-shoot' liquid chromatography-high resolution mass spectrometry in preclinical research: application to a DMPK study of perhexiline in mouse plasma.

This work describes a simple, sensitive and rapid liquid chromatography-high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma. Only 5 µL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution ('dilute-and-shoot'). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r(2)=0.990) ranging from 0.305 ng/mL (LLOQ) to 10000 ng/mL. Matrix effect was limited to 6.1%. The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample. The combination of the 'dilute-and-shoot' approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.

Discovery and Characterization of Novel Anti-schistosomal Properties of the Anti-anginal Drug, Perhexiline and Its Impact on Schistosoma mansoni Male and Female Reproductive Systems.

BACKGROUND: Schistosomiasis, one of the world's greatest human neglected tropical diseases, is caused by parasitic trematodes of the genus Schistosoma. A unique feature of schistosome biology is that the induction of sexual maturation as well as the maintenance of the differentiation status of female reproductive organs and egg production, necessary for both disease transmission and pathogenesis, are strictly dependent on the male. The treatment and most control initiatives of schistosomiasis rely today on the long-term application of a single drug, praziquantel (PZQ), mostly by campaigns of mass drug administration. PZQ, while very active on adult parasites, has much lower activity against juvenile worms. Monotherapy also favors the selection of drug resistance and, therefore, new drugs are urgently needed. METHODS AND FINDINGS: Following the screening of a small compound library with an ATP-based luminescent assay on Schistosoma mansoni schistosomula, we here report the identification and characterization of novel antischistosomal properties of the anti-anginal drug perhexiline maleate (PHX). By phenotypic worm survival assays and confocal microscopy studies we show that PHX, in vitro, has a marked lethal effect on all S. mansoni parasite life stages (newly transformed schistosomula, juvenile and adult worms) of the definitive host. We further demonstrate that sub-lethal doses of PHX significantly impair egg production and lipid depletion within the vitellarium of adult female worms. Moreover, we highlighted tegumental damage in adult male worms and remarkable reproductive system alterations in both female and male adult parasites. The in vivo study in S. mansoni-patent mice showed a notable variability of worm burdens in the individual experiments, with an overall minimal schistosomicidal effect upon PHX treatment. The short PHX half-life in mice, together with its very high rodent plasma proteins binding could be the cause of the modest efficacy of PHX in the schistosomiasis murine model. CONCLUSIONS/SIGNIFICANCE: Overall, our data indicate that PHX could represent a promising starting point for novel schistosomicidal drug discovery programmes.

Structure-activity relationship and properties optimization of a series of quinazoline-2,4-diones as inhibitors of the canonical Wnt pathway.

Wnt signaling pathway plays a critical role in numerous cellular processes, including tumor initiation, proliferation, invasion/infiltration, metastasis formation and resistance to chemotherapy. In a drug discovery project aimed at the identification of inhibitors of the canonical Wnt pathway, we selected a series of quinazoline 2,4-diones as starting point for the therapeutic treatment of glioblastoma multiforme. Despite of poor physico-chemical properties of hit compound 1, our medicinal chemistry effort allowed the discovery and characterization of lead compound 33 (SEN461), with improved ADME profile, good bioavailability and active in vitro and in vivo in glioblastoma, gastric and sarcoma tumors.

Brain penetration assessment in vivo: a reliable and simple method in anesthetized rats at steady state.

BACKGROUND: For CNS drugs, brain disposition is of critical importance during drug discovery. In vitro methods are used early followed by more predictive in vivo methods later on in the drug discovery process. Current in vivo methods are costly, have long turnover times or do not measure brain disposition at steady state. NEW METHOD: A new method to evaluate drug brain disposition in vivo was developed in anaesthetized rats. Seven reference compounds were administered as an initial IV bolus (loading dose) followed by IV infusion for 4.5 h in order to obtain a steady state plasma concentration before brain sampling. The loading dose was estimated from a preliminary single dose IV pharmacokinetic study and was found to successfully bring plasma concentrations to steady state for compounds exhibiting either mono- or bi-compartmental pharmacokinetics. RESULTS: Using this method, a steady state lasting at least 2h was obtained, thus making the in vivo method robust with respect to differences in the pharmacokinetics and/or blood-to-brain equilibration rate of the compounds tested. The method produced highly reproducible results, with substantial advantages in terms of cost, turnaround time and animal welfare. COMPARISON WITH EXISTING METHODS: The results agreed with those reported in other, more elaborate preclinical models and in humans, enabling brain disposition to be assessed in a simple, efficient and robust in vivo model for new chemical entities. CONCLUSIONS: Introducing the presented method in drug discovery allows brain disposition to be assessed earlier in the drug discovery pipeline and thus facilitate the selection of potent and penetrant CNS drugs.