The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax.

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients' clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia.

Endothelin-1 promotes vascular smooth muscle cell migration across the artery wall: a mechanism contributing to vascular remodelling and intimal hyperplasia in giant-cell arteritis.

BACKGROUND: Giant-cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries, frequently involving the temporal arteries (TA). Inflammation-induced vascular remodelling leads to vaso-occlusive events. Circulating endothelin-1 (ET-1) is increased in patients with GCA with ischaemic complications suggesting a role for ET-1 in vascular occlusion beyond its vasoactive function. OBJECTIVE: To investigate whether ET-1 induces a migratory myofibroblastic phenotype in human TA-derived vascular smooth muscle cells (VSMC) leading to intimal hyperplasia and vascular occlusion in GCA. METHODS AND RESULTS: Immunofluorescence/confocal microscopy showed increased ET-1 expression in GCA lesions compared with control arteries. In inflamed arteries, ET-1 was predominantly expressed by infiltrating mononuclear cells whereas ET receptors, particularly ET-1 receptor B (ETBR), were expressed by both mononuclear cells and VSMC. ET-1 increased TA-derived VSMC migration in vitro and α-smooth muscle actin (αSMA) expression and migration from the media to the intima in cultured TA explants. ET-1 promoted VSMC motility by increasing activation of focal adhesion kinase (FAK), a crucial molecule in the turnover of focal adhesions during cell migration. FAK activation resulted in Y397 autophosphorylation creating binding sites for Src kinases and the p85 subunit of PI3kinases which, upon ET-1 exposure, colocalised with FAK at the focal adhesions of migrating VSMC. Accordingly, FAK or PI3K inhibition abrogated ET-1-induced migration in vitro. Consistently, ET-1 receptor A and ETBR antagonists reduced αSMA expression and delayed VSMC outgrowth from cultured GCA-involved artery explants. CONCLUSIONS: ET-1 is upregulated in GCA lesions and, by promoting VSMC migration towards the intimal layer, may contribute to intimal hyperplasia and vascular occlusion in GCA.

Targeting prohibitins induces apoptosis in acute myeloid leukemia cells.

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins (PHBs). In this study, the pro-apoptotic effect of fluorizoline was assessed in two cell lines and 21 primary samples from patients with debut of acute myeloid leukemia (AML). Fluorizoline induced apoptosis in AML cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespectively of patients' clinical or genetic features. In addition, fluorizoline inhibited the clonogenic capacity and induced differentiation of AML cells. Fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. These results suggest that targeting PHBs could be a new therapeutic strategy for AML.

Carboxylation of Phenols with CO2 at Atmospheric Pressure.

A convenient and efficient method for the ortho-carboxylation of phenols under atmospheric CO2 pressure has been developed. This method provides an alternative to the previously reported Kolbe-Schmitt method, which requires very high pressures of CO2 . The addition of a trisubstituted phenol has proved essential for the successful carboxylation of phenols with CO2 at standard atmospheric pressure, allowing the efficient preparation of a broad variety of salicylic acids.

Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue.

Fluorescent antimicrobial peptides are promising structures for in situ, real-time imaging of fungal infection. Here we report a fluorogenic probe to image Aspergillus fumigatus directly in human pulmonary tissue. We have developed a fluorogenic Trp-BODIPY amino acid with a spacer-free C-C linkage between Trp and a BODIPY fluorogen, which shows remarkable fluorescence enhancement in hydrophobic microenvironments. The incorporation of our fluorogenic amino acid in short antimicrobial peptides does not impair their selectivity for fungal cells, and enables rapid and direct fungal imaging without any washing steps. We have optimized the stability of our probes in human samples to perform multi-photon imaging of A. fumigatus in ex vivo human tissue. The incorporation of our unique BODIPY fluorogen in biologically relevant peptides will accelerate the development of novel imaging probes with high sensitivity and specificity.

A Domino Oxidation/Arylation/Protodecarboxylation Reaction of Salicylaldehydes: Expanded Access to meta-Arylphenols.

A method that allows salicylaldehydes to be efficiently transformed into meta-arylated phenol derivatives through a cascade oxidation/arylation/protodecarboxylation sequence is presented. We demonstrate that the aldehyde functional group can be used as a convenient removable directing group to control site selectivity in C-H activation. Aldehydes are easily introduced into the starting materials and the group is readily cleaved after the C-H functionalization event.

A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation.

We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway.

New peptide architectures through C-H activation stapling between tryptophan-phenylalanine/tyrosine residues.

Natural peptides show high degrees of specificity in their biological action. However, their therapeutical profile is severely limited by their conformational freedom and metabolic instability. Stapled peptides constitute a solution to these problems and access to these structures lies on a limited number of reactions involving the use of non-natural amino acids. Here, we describe a synthetic strategy for the preparation of unique constrained peptides featuring a covalent bond between tryptophan and phenylalanine or tyrosine residues. The preparation of such peptides is achieved in solution and on solid phase directly from the corresponding sequences having an iodo-aryl amino acid through an intramolecular palladium-catalysed C-H activation process. Moreover, complex topologies arise from the internal stapling of cyclopeptides and double intramolecular arylations within a linear peptide. Finally, as a proof of principle, we report the application to this new stapling method to relevant biologically active compounds.

Salicylic acids as readily available starting materials for the synthesis of meta-substituted biaryls.

Salicylic acids are shown to be readily available and versatile starting materials that easily undergo a tandem arylation-protodecarboxylation process under Pd-catalysis. The corresponding meta-arylphenols can subsequently be easily transformed into a variety of meta-functionalized biaryls, highlighting the versatility of this approach to access this structural motif.

A trifluorinated thiazoline scaffold leading to pro-apoptotic agents targeting prohibitins.

A new class of small molecules, with an unprecedented trifluorothiazoline scaffold, were synthesized and their pro-apoptotic activity was evaluated. With an EC50 in the low micromolar range, these compounds proved to be potent inducers of apoptosis in a broad spectrum of tumor cell lines, regardless of the functional status of p53. Fast structure-activity relationship studies allowed the preparation of the strongest apoptosis-inducing candidate. Using a high performance affinity purification approach, we identified prohibitins 1 and 2, key proteins involved in the maintenance of cell viability, as the targets for these compounds.

Overriding ortho-para selectivity via a traceless directing group relay strategy: the meta-selective arylation of phenols.

The direct functionalization of phenols at the ortho and para position is generally facilitated by the electron-donating nature of the hydroxyl group. Accessing meta-functionalized phenols from the parent phenols, on the other hand, generally requires lengthy synthetic sequences. Here, we report the first methodology for the one-pot direct meta-selective arylation of phenols. This methodology is based on a traceless directing group relay strategy. In this process carbon dioxide is used as a transient directing group which facilitates a palladium catalyzed arylation meta to the phenol hydroxyl group with iodoarenes. This transformation proceeds with complete meta-selectivity and is compatible with a variety of functional groups both in the phenol and in the iodoarene coupling partner.

Evolution of a multicomponent system: computational and mechanistic studies on the chemo- and stereoselectivity of a divergent process.

The evolution of a ternary molecular system (imine, diene and nitrile) is analyzed to disclose the pathways leading to a divergent synthetic outcome. The Lewis acid catalyzed reaction between cyclohexadiene, 2-phenyl-indol-3-one and acetonitrile yields the imino-Diels-Alder adduct as the major product together with minor amounts of the Mannich-Ritter-amidine product. The experimental and computational data show that the relative orientation of the initial reactants dictates the synthetic outcome. The exo approach between imine and diene leads to the Diels-Alder adduct in a concerted process, whereas the endo mode leads to a polarized intermediate, which is trapped by acetonitrile to yield the multicomponent adduct.

Multicomponent reactions for de novo synthesis of BODIPY probes: in vivo imaging of phagocytic macrophages.

Multicomponent reactions are excellent tools to generate complex structures with broad chemical diversity and fluorescent properties. Herein we describe the adaptation of the fluorescent BODIPY scaffold to multicomponent reaction chemistry with the synthesis of BODIPY adducts with high fluorescence quantum yields and good cell permeability. From this library we identified one BODIPY derivative (PhagoGreen) as a low-pH sensing fluorescent probe that enabled imaging of phagosomal acidification in activated macrophages. The fluorescence emission of PhagoGreen was proportional to the degree of activation of macrophages and could be specifically blocked by bafilomycin A, an inhibitor of phagosomal acidification. PhagoGreen does not impair the normal functions of macrophages and can be used to image phagocytic macrophages in vivo.

Synthesis of C-2 arylated tryptophan amino acids and related compounds through palladium-catalyzed C-H activation.

Tryptophan (Trp) and tryptophan derivatives are C2-arylated. A C-H activation process allows the preparation of both protected and unprotected arylated-Trp amino acids, directly from the amino acid precursor and aryl iodides. The obtained compounds are suitable for standard solid-phase peptide synthesis.

Exploration of forbidden Povarov processes as a source of unexpected reactivity: a multicomponent Mannich-Ritter transformation.

When a door closes, a window opens! The use of geometrically or electronically restricted imines for Povarov-type processes does not afford the anti-Bredt tetrahydroquinolines, but leads instead to highly functionalized structures through novel reaction pathways (see picture; LA=Lewis acid). The exploration of "forbidden" routes constitutes a valuable approach in the search for new multicomponent reactions.

Novel triazolopyridylbenzamides as potent and selective p38α inhibitors.

A new class of p38α inhibitors based on a biaryl-triazolopyridine scaffold was investigated. X-ray crystallographic data of the initial lead compound cocrystallised with p38α was crucial in order to uncover a unique binding mode of the inhibitor to the hinge region via a pair of water molecules. Synthesis and SAR was directed towards the improvement of binding affinity, as well as ADME properties for this new class of p38α inhibitors and ultimately afforded compounds showing good in vivo efficacy.

Multicomponent reaction access to complex quinolines via oxidation of the Povarov adducts.

The tetrahydroquinolines obtained through the Povarov multicomponent reaction have been oxidized to the corresponding quinoline, giving access to a single product through a two-step sequence. Several oxidizing agents were studied and manganese dioxide proved to be the reagent of choice, affording higher yields, cleaner reactions and practical protocols.