Development of LM-41 and AF-2112, two flufenamic acid-derived TEAD inhibitors obtained through the replacement of the trifluoromethyl group by aryl rings.

The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP-TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF, Cyr61, Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF, Cyr61, Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells.

Antiproliferative and apoptotic effects of the oxidative dimerization product of methyl caffeate on human breast cancer cells.

Caffeic acid derivatives are increasingly regarded as potential oncoprotective that could inhibit both the initiation and progression of cancer. Here we have synthesized seven 1-arylnaphthalene lignans and related compounds and tested their impact on breast cancer cell growth in tissue culture. The product of the oxidative dimerization of methyl caffeate, 1-phenylnaphthalene lignan, was found to induce a strong decrease in breast cancer cell number (IC(50) ~1 µM) and was selected for further investigation. Flow cytometry analysis revealed a decrease in cell proliferation and an increase in apoptosis in both MCF-7 and MDA-MB-231 breast cancer cell lines that are representative of the two main categories of breast tumors. The 3,4-dihydroxyphenyl group probably induced the biological activity, as the control compounds lacking it had no effect on breast cancer cells. Together, our data indicate that the oxidative dimerization product of methyl caffeate can inhibit breast cancer cell growth at a concentration adequate for pharmacological use.

Development of a series of 3-hydroxyquinolin-2(1H)-ones as selective inhibitors of HIV-1 reverse transcriptase associated RNase H activity.

We report herein the synthesis of a series of 3-hydroxyquinolin-2(1H)-one derivatives. Esters and amide groups were introduced at position 4 of the basis scaffold and some modulations of the benzenic moiety were performed. Most compounds presented selective inhibitory properties in the 10-20 µM range against HIV-1 reverse transcriptase associated ribonuclease H activity, without affecting the integrase and reverse transcriptase DNA polymerase activities. Unfortunately all tested compounds exhibited high cellular cytotoxicity in cell culture which limited their applications as antiviral agents.

Progress with YAP/TAZ-TEAD inhibitors: a patent review (2018-present).

INTRODUCTION: The Hippo pathway represents a new opportunity for the treatment of cancer. Overexpression of Yes-associated protein (YAP) or transcriptional coactivator with PDZ-binding motif (TAZ) or TEAD has been demonstrated in cancers and YAP mediates resistance to cancer drugs. Since 2018, the potential of this pathway has been illustrated by numerous articles and patents and the first drugs entering in clinical trial phase 1. AREAS COVERED: This review is limited to published patent applications that have disclosed direct small-molecule inhibitors of the YAP/TAZ-TEAD interaction. EXPERT OPINION: The YAP/TAZ-TEAD transcriptional complex is a promising target for the treatment of cancer. Approximately 30 international patents (used database: Sci-finder, query: TEAD; documents: patents; period: from 2017-January 2022) that disclose TEAD transcriptional inhibitors have been filled since 2018. The mechanism of action is not always described in the patents, we can divide the drugs into three different categories: (i) external TEAD ligands; (ii) non-covalent TEAD ligands of the palmitate pocket; (iii) covalent TEAD ligands, which bind into the palmitate pocket. The first molecules in clinical trial phase 1 are non-covalent TEAD ligands. The selective TEAD ligand have also been patented, published and selectivity could be of great interest for personalized medicine.

Crosstalk between the Hippo Pathway and the Wnt Pathway in Huntington's Disease and Other Neurodegenerative Disorders.

The Hippo pathway consists of a cascade of kinases that controls the phosphorylation of the co-activators YAP/TAZ. When unphosphorylated, YAP and TAZ translocate into the nucleus, where they mainly bind to the TEAD transcription factor family and activate genes related to cell proliferation and survival. In this way, the inhibition of the Hippo pathway promotes cell survival, proliferation, and stemness fate. Another pathway can modulate these processes, namely the Wnt/ß-catenin pathway that is indeed involved in cellular functions such as proliferation and cell survival, as well as apoptosis, growth, and cell renewal. Wnt signaling can act in a canonical or noncanonical way, depending on whether ß-catenin is involved in the process. In this review, we will focus only on the canonical Wnt pathway. It has emerged that YAP/TAZ are components of the ß-catenin destruction complex and that there is a close relationship between the Hippo pathway and the canonical Wnt pathway. Furthermore, recent data have shown that both of these pathways may play a role in neurodegenerative diseases, such as Huntington's disease, Alzheimer's disease, or Amyotrophic Lateral Sclerosis. Thus, this review analyzes the Hippo pathway and the Wnt pathway, their crosstalk, and their involvement in Huntington's disease, as well as in other neurodegenerative disorders. Altogether, these data suggest possible therapeutic approaches targeting key players of these pathways.

Toward the Design of Ligands Selective for the C-Terminal Domain of TEADs.

The Hippo signaling pathway plays a fundamental role in the control of organ growth, cell proliferation, and stem cell characters. TEADs are the main transcriptional output regulators of the Hippo signaling pathway and bind to YAP and TAZ co-activators. TEAD1-4 are expressed differently, depending on the tissue and developmental level, and can be overexpressed in certain pathologies. TEAD ligands mainly target the internal pocket of the C-terminal domain of TEAD, and the first ligands selective for TEAD1 and TEAD3 have been recently reported. In this paper, we focus on the topographic homology of the TEAD C-terminal domain both externally and in the internal pocket to highlight the possibility of rationally designing ligands selective for one of the TEAD family members. We identified a novel TEAD2-specific pocket and reported its first ligand. Finally, AlphaFold2 models of full-length TEADs suggest TEAD autoregulation and emphasize the importance of the interface 2.

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay.

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design. Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target.

The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.

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.

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.

New 2-arylnaphthalenediols and triol inhibitors of HIV-1 integrase--discovery of a new polyhydroxylated antiviral agent.

A series of 13 hydroxylated 2-arylnaphthalenes have been synthesized and evaluated as HIV-1 integrase inhibitors. 7-(3,4,5-trihydroxyphenyl)naphthalene-1,2,3-triol 1c revealed chemical instability upon storage, leading to the isolation of a dimer 5c which was also tested. In the 2-arylnaphthalene series, all compounds were active against HIV-1 IN with IC50's within the 1-10 microM range, except for 1c and 5c which displayed submicromolar activity. Antiviral activity against HIV-1 replication was measured on 1b-c and 5c. Amongst the tested molecules, only 5c was found to present antiviral properties with a low cytotoxicity on two different cell lines.

Metal chelators for the inhibition of the lymphocytic choriomeningitis virus endonuclease domain.

Arenaviridae is a viral family whose members are associated with rodent-transmitted infections to humans responsible of severe diseases. The current lack of a vaccine and limited therapeutic options make the development of efficacious drugs of high priority. The cap-snatching mechanism of transcription of Arenavirus performed by the endonuclease domain of the L-protein is unique and essential, so we developed a drug design program targeting the endonuclease activity of the prototypic Lymphocytic ChorioMeningitis Virus. Since the endonuclease activity is metal ion dependent, we designed a library of compounds bearing chelating motifs (diketo acids, polyphenols, and N-hydroxyisoquinoline-1,3-diones) able to block the catalytic center through the chelation of the critical metal ions, resulting in a functional impairment. We pre-screened 59 compounds by Differential Scanning Fluorimetry. Then, we characterized the binding affinity by Microscale Thermophoresis and evaluated selected compounds in in vitro and in cellula assays. We found several potent binders and inhibitors of the endonuclease activity. This study validates the proof of concept that the endonuclease domain of Arenavirus can be used as a target for anti-arena-viral drug discovery and that both diketo acids and N-hydroxyisoquinoline-1,3-diones can be considered further as potential metal-chelating pharmacophores.

Reaction of rosmarinic acid with nitrite ions in acidic conditions: discovery of nitro- and dinitrorosmarinic acids as new anti-HIV-1 agents.

Rosmarinic acid was reacted with nitrite ions under acidic conditions to give 6'-nitro- and 6',6''-dinitrorosmarinic acids according to the reaction time. Both compounds were active as HIV-1 integrase inhibitors at the submicromolar level. They also inhibited the viral replication in MT-4 cells with modest and similar selectivity indexes. The nitration of rosmarinic acid strongly improves the anti-integrase inhibition and the antiviral activity without increasing the cellular toxicity.

Synthesis and antiviral properties of some polyphenols related to Salvia genus.

An efficient synthesis of the acid part of salvianolic acid E 2 is described. Compound 2 was obtained from vanillin in 10 steps and 21% overall yield. During the synthesis of 2 an unexpected 5-oxo-4b,9b-dihydroindano[1,2-b]benzofuran rac-12 was isolated. Both compounds together with the acid part of salvianolic acid D were active as HIV-1 integrase inhibitors at the submicromolar level. But they did not inhibit the replication of the virus on MT-4 cells.

Synthesis and biological activities of a series of 4,5-diaryl-3-hydroxy-2(5H)-furanones.

A series of thirteen 4,5-diaryl-3-hydroxy-2(5H)-furanones were synthesized. They were evaluated for their antioxidant potencies and inhibitory properties of 5-lipoxygenase, cyclooxygenases, HIV-1 integrase and PC3 cell proliferation. New hits were discovered either in the anti-proliferation test or in the HIV anti-integrase test.

The total synthesis of fukiic acid, an HIV-1 integrase inhibitor.

A successful synthesis of fukiic acid is described in 7% overall yield (6 steps from veratraldehyde). rac-Fukiic acid was found to be a potent inhibitor of HIV-1 integrase but did not reveal any antiviral activity in the MT-4 cells assay.

Targeting Transcriptional Enhanced Associate Domains (TEADs).

Transcriptional enhanced associate domain (TEAD) proteins are the downstream effectors of the Hippo signaling pathway that regulate cell proliferation and stem cell functions. TEADs are unable to activate transcription and require the help of coactivators such as YAP, TAZ, VgLL, and p160 proteins. The expression of TEAD family is up-regulated in many cancer types including gastric, colorectal, breast, and prostate cancers, which is correlated with poor survival in patients. Pharmacological modulators of TEADs could therefore find application in cancer treatment and regenerative medicine. In this review, we present the very recent available structures of TEADs with or without coactivators or inhibitors and discuss the potential therapeutic application of their ligands.

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.

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.

Inhibition of hepatitis B virus replication by N-hydroxyisoquinolinediones and related polyoxygenated heterocycles.

We previously reported low sensitivity of the hepatitis B virus (HBV) ribonuclease H (RNaseH) enzyme to inhibition by N-hydroxyisoquinolinedione (HID) compounds. Subsequently, our biochemical RNaseH assay was found to have a high false negative rate for predicting HBV replication inhibition, leading to underestimation of the number of HIDs that inhibit HBV replication. Here, 39 HID compounds and structurally related polyoxygenated heterocycles (POH), N-hydroxypyridinediones (HPD), and flutimides were screened for inhibition of HBV replication in vitro. Inhibiting the HBV RNaseH preferentially blocks synthesis of the positive-polarity DNA strand and causes accumulation of RNA:DNA heteroduplexes. Eleven HIDs and one HPD preferentially inhibited HBV positive-polarity DNA strand accumulation. EC50s ranged from 0.69 µM to 19 µM with therapeutic indices from 2.4 to 71. Neither the HIDs nor the HPD had an effect on the ability of the polymerase to elongate DNA strands in capsids. HBV RNaseH inhibition by the HIDs was confirmed with an improved RNaseH assay and by detecting accumulation RNA:DNA heteroduplexes in HBV capsids from cells treated with a representative HID. Therefore, the HID scaffold is more promising for anti-HBV drug discovery than we originally reported, and the HPD scaffold may hold potential for antiviral development. The preliminary structure-activity relationship will guide optimization of the HID/HPDs as HBV inhibitors.