Discovery of Compounds That Selectively Repress the Amyloidogenic Processing of the Amyloid Precursor Protein: Design, Synthesis and Pharmacological Evaluation of Diphenylpyrazoles.

The rationale to define the biological and molecular parameters derived from structure-activity relationships (SAR) is mandatory for the lead selection of small drug compounds. Several series of small molecules have been synthesized based on a computer-assisted pharmacophore design derived from two series of compounds whose scaffold originates from chloroquine or amodiaquine. All compounds share similar biological activities. In vivo, Alzheimer's disease-related pathological lesions are reduced, consisting of amyloid deposition and neurofibrillary degeneration, which restore and reduce cognitive-associated impairments and neuroinflammation, respectively. Screening election was performed using a cell-based assay to measure the repression of Aß1-x peptide production, the increased stability of APP metabolites, and modulation of the ratio of autophagy markers. These screening parameters enabled us to select compounds as potent non-competitive ß-secretase modulators, associated with various levels of lysosomotropic or autophagy modulatory activities. Structure-activity relationship analyses enabled us to define that (1) selectively reducing the production of Aß1-x, and (2) little Aßx-40/42 modification together with (3) a decreased ratio of p62/(LC3-I/LC3-II) enabled the selection of non-competitive ß-secretase modulators. Increased stability of CTFα and AICD precluded the selection of compounds with lysosomotropic activity whereas cell toxicity was associated with the sole p62 enhanced expression shown to be driven by the loss of nitrogen moieties. These SAR parameters are herein proposed with thresholds that enable the selection of potent anti-Alzheimer drugs for which further investigation is necessary to determine the basic mechanism underlying their mode of action.

High ligand efficiency quinazoline compounds as novel A2A adenosine receptor antagonists.

The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes.

Discovery of a cryptic site at the interface 2 of TEAD - Towards a new family of YAP/TAZ-TEAD inhibitors.

The Hippo pathway is involved in organ size control and tissue homeostasis by regulating cell growth, proliferation and apoptosis. It controls the phosphorylation of the transcription co-activator YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) in order to control their nuclear import and their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ and TEADs are dysregulated in several cancers making YAP/TAZ-TEAD interaction a new emerging anti-cancer target. We report the synthesis of a set of trisubstituted pyrazoles which bind to hTEAD2 at the interface 2 revealing for the first time a cryptic pocket created by the movement of the phenol ring of Y382. Compound 6 disrupts YAP/TAZ-TEAD interaction in HEK293T cells and inhibits TEAD target genes and cell proliferation in MDA-MB-231 cells. Compound 6 is therefore the first inhibitor of YAP/TAZ-TEAD targeting interface 2. This molecule could serve with other pan-TEAD inhibitors such as interface 3 ligands, for the delineation of the relative importance of VGLL vs YAP/TAZ in a given cellular model.

Design, Synthesis and Evaluation of a Series of 1,5-Diaryl-1,2,3-triazole-4-carbohydrazones as Inhibitors of the YAP-TAZ/TEAD Complex.

Starting from our previously reported hit, a series of 1,5-diaryl-1,2,3-triazole-4-carbohydrazones were synthesized and evaluated as inhibitors of the YAP/TAZ-TEAD complex. Their binding to hTEAD2 was confirmed by nanodifferential scanning fluorimetry, and some of the compounds were also found to moderately disrupt the YAP-TEAD interaction, as assessed by a fluorescence polarization assay. A TEAD luciferase gene reporter assay performed in HEK293T cells and RTqPCR measurements in MDA-MB231 cells showed that these compounds inhibit YAP/TAZ-TEAD activity to cells in the micromolar range. In spite of the cytotoxic effects displayed by some of the compounds of this series, they are still good starting points and can be suitably modified into an effective and viable YAP-TEAD disruptor in the future.

New piperazine multi-effect drugs prevent neurofibrillary degeneration and amyloid deposition, and preserve memory in animal models of Alzheimer's disease.

Alzheimer's Disease is a devastating dementing disease involving amyloid deposits, neurofibrillary tangles, progressive and irreversible cognitive impairment. Today, only symptomatic drugs are available and therapeutic treatments, possibly acting at a multiscale level, are thus urgently needed. To that purpose, we designed multi-effects compounds by synthesizing drug candidates derived by substituting a novel N,N'-disubstituted piperazine anti-amyloid scaffold and adding acetylcholinesterase inhibition property. Two compounds were synthesized and evaluated. The most promising hybrid molecule reduces both the amyloid pathology and the Tau pathology as well as the memory impairments in a preclinical model of Alzheimer's disease. In vitro also, the compound reduces the phosphorylation of Tau and inhibits the release of Aß peptides while preserving the processing of other metabolites of the amyloid precursor protein. We synthetized and tested the first drug capable of ameliorating both the amyloid and Tau pathology in animal models of AD as well as preventing the major brain lesions and associated memory impairments. This work paves the way for future compound medicines against both Alzheimer's-related brain lesions development and the associated cognitive impairments.

Contribution of the Endosomal-Lysosomal and Proteasomal Systems in Amyloid-ß Precursor Protein Derived Fragments Processing.

Aß peptides, the major components of Alzheimer's disease (AD) amyloid deposits, are released following sequential cleavages by secretases of its precursor named the amyloid precursor protein (APP). In addition to secretases, degradation pathways, in particular the endosomal/lysosomal and proteasomal systems have been reported to contribute to APP processing. However, the respective role of each of these pathways toward APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length wild type APP or the ß-secretase-derived C99 fragment (ß-CTF) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and Aß peptides were studied. In this report, we show that APP-CTFs produced from endogenous or overexpressed full-length APP are mainly processed by γ-secretase and the endosomal/lysosomal pathway, while in sharp contrast, overexpressed C99 is mainly degraded by the proteasome and to a lesser extent by γ-secretase.

A phenotypic approach to the discovery of compounds that promote non-amyloidogenic processing of the amyloid precursor protein: Toward a new profile of indirect ß-secretase inhibitors.

Dysregulation of the Amyloid Precursor Protein (APP) processing leading to toxic species of amyloid ß peptides (Aß) is central to Alzheimer's disease (AD) etiology. Aß peptides are produced by sequential cleavage of APP by ß-secretase (BACE-1) and γ-secretase. Lysosomotropic agent, chloroquine (CQ), has been reported to inhibit Aß peptide production. However, this effect is accompanied by an inhibition of lysosome-mediated degradation pathways. Following on from the promising activity of two series of APP metabolism modulators derived from CQ, we sought to develop new series of compounds that would retain the inhibitory effects on Aß production without altering lysosome functions. Herein, we applied a ligand-based pharmacophore modeling approach coupled with de novo design that led to the discovery of a series of biaryl compounds. Structure-activity relationship studies revealed that minor modifications like replacing a piperidine moiety of compound 30 by a cyclohexyl (compound 31) allowed for the identification of compounds with the desired profile. Further studies have demonstrated that compounds 30 and 31 act through an indirect mechanism to inhibit ß-secretase activity. This work shows that it is possible to dissociate the inhibitory effect on Aß peptide secretion of CQ-derived compounds from the lysosome-mediated degradation effect, providing a new profile of indirect ß-secretase inhibitors.

Toward the Discovery of a Novel Class of YAP⁻TEAD Interaction Inhibitors by Virtual Screening Approach Targeting YAP⁻TEAD Protein⁻Protein Interface.

Intrinsically disordered protein YAP (yes-associated protein) interacts with TEADs transcriptional factors family (transcriptional enhancer associated domain) creating three interfaces. Interface 3, between the Ω-loop of YAP and a shallow pocket of TEAD was identified as the most important TEAD zone for YAP-TEAD interaction. Using the first X-ray structure of the hYAP50⁻71-hTEAD1209⁻426 complex (PDB 3KYS) published in 2010, a protein-protein interaction inhibitors-enriched library (175,000 chemical compounds) was screened against this hydrophobic pocket of TEAD. Four different chemical families have been identified and evaluated using biophysical techniques (thermal shift assay, microscale thermophoresis) and in cellulo assays (luciferase activity in transfected HEK293 cells, RTqPCR in MDA-MB231 cells). A first promising hit with micromolar inhibition in the luciferase gene reporter assay was discovered. This hit also decreased mRNA levels of TEAD target genes.

Design and synthesis of 2,6-disubstituted-8-amino imidazo[1,2a]pyridines, a promising privileged structure.

Imidazo[1,2a]pyridines have gained much interest in the field of medicinal chemistry research. In the aim of accessing new privileged structure, we decided to design and synthesize 8-aminated-imidazo[1,2a]pyridines substituted on positions 2 and 6. This scaffold, rarely found in the literature, was obtained via palladium-catalyzed coupling reactions (Suzuki reaction or N-hydroxysuccinimidyl activated ester method) and tested on adenosine receptor A2A. We demonstrated how incorporation of an exocyclic amine enhanced affinity towards this receptor while maintaining low cytotoxicity.

New phenylaniline derivatives as modulators of amyloid protein precursor metabolism.

The chloroquinoline scaffold is characteristic of anti-malarial drugs such as chloroquine (CQ) or amodiaquine (AQ). These drugs are also described for their potential effectiveness against prion disease, HCV, EBV, Ebola virus, cancer, Parkinson or Alzheimer diseases. Amyloid precursor protein (APP) metabolism is deregulated in Alzheimer's disease. Indeed, CQ modifies amyloid precursor protein (APP) metabolism by precluding the release of amyloid-beta peptides (Aß), which accumulate in the brain of Alzheimer patients to form the so-called amyloid plaques. We showed that AQ and analogs have similar effects although having a higher cytotoxicity. Herein, two new series of compounds were synthesized by replacing 7-chloroquinolin-4-amine moiety of AQ by 2-aminomethylaniline and 2-aminomethylphenyle moieties. Their structure activity relationship was based on their ability to modulate APP metabolism, Aß release, and their cytotoxicity similarly to CQ. Two compounds 15a, 16a showed interesting and potent effect on the redirection of APP metabolism toward a decrease of Aß peptide release (in the same range compared to AQ), and a 3-10-fold increased stability of APP carboxy terminal fragments (CTFα and AICD) without obvious cellular toxicity at 100 µM.

Molecular Features of the YAP Inhibitor Verteporfin: Synthesis of Hexasubstituted Dipyrrins as Potential Inhibitors of YAP/TAZ, the Downstream Effectors of the Hippo Pathway.

Porphyrin derivatives, in particular verteporfin (VP), a photosensitizer initially designed for cancer therapy, have been identified as inhibitors of the YAP-TEAD interaction and transcriptional activity. Herein we report the efficient convergent synthesis of the dipyrrin half of protoporphyrin IX dimethyl ester (PPIX-DME), in which the sensitive vinyl group was created at the final stage by a dehydroiodination reaction. Two other dipyrrin derivatives were synthesized, including dipyrrin 19 [(Z)-2-((3,5-dimethyl-4-vinyl-2H-pyrrol-2-ylidene)methyl)-3,5-dimethyl-4-vinyl-1H-pyrrole], containing two vinyl groups. We found that VP and dipyrrin 19 showed significant inhibitory effects on TEAD transcriptional activity in MDA-MB-231 human breast cancer cells, whereas other compounds did not show significant changes. In addition, we observed a marked decrease in both YAP and TAZ levels following VP treatment, whereas dipyrrin 19 treatment primarily decreased the levels of YAP and receptor kinase AXL, a downstream target of YAP. Together, our data suggest that, due to their chemical structures, porphyrin- and dipyrrin-related derivatives can directly target YAP and/or TAZ proteins and inhibit TEAD transcriptional activity.