RÉSUMÉ
Resistance to ß-lactam antibiotics in Gram-negatives producing metallo-ß-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with Ki values in the µM to sub-µM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the potentiation activity of the compounds was limited by poor outer membrane penetration or efflux. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E. coli clinical strain toward several ß-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs.
Sujet(s)
Bases de Schiff/pharmacologie , Thiones/pharmacologie , Triazoles/pharmacologie , Inhibiteurs des bêta-lactamases/pharmacologie , bêta-Lactamases/métabolisme , Cristallographie aux rayons X , Escherichia coli/effets des médicaments et des substances chimiques , Protéines Escherichia coli/métabolisme , Cellules endothéliales de la veine ombilicale humaine , Humains , Tests de sensibilité microbienne , Liaison aux protéines , Pseudomonas aeruginosa/composition chimique , Bases de Schiff/synthèse chimique , Bases de Schiff/métabolisme , Thiones/synthèse chimique , Thiones/métabolisme , Triazoles/synthèse chimique , Triazoles/métabolisme , Inhibiteurs des bêta-lactamases/synthèse chimique , Inhibiteurs des bêta-lactamases/métabolismeRÉSUMÉ
CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2ß subunits is essential for substrate selectivity. The CK2α/CK2ß interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2ß interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.
Sujet(s)
Casein Kinase II/antagonistes et inhibiteurs , Holoenzymes/métabolisme , Inhibiteurs de protéines kinases/composition chimique , Adénosine triphosphate/métabolisme , Sites de fixation , Casein Kinase II/métabolisme , Domaine catalytique , Lignée cellulaire tumorale , Mouvement cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Cristallographie aux rayons X , Holoenzymes/composition chimique , Humains , Cinétique , Simulation de docking moléculaire , Peptides/composition chimique , Peptides/métabolisme , Phosphorylation , Inhibiteurs de protéines kinases/métabolisme , Inhibiteurs de protéines kinases/pharmacologie , Sous-unités de protéines/antagonistes et inhibiteurs , Sous-unités de protéines/métabolisme , Spécificité du substrat , Résonance plasmonique de surfaceRÉSUMÉ
Protein CK2 has gained much interest as an anticancer drug target in the past decade. We had previously described the identification of a new allosteric site on the catalytic α-subunit, along with first small molecule ligands based on the 4-(4-phenylthiazol-2-ylamino)benzoic acid scaffold. In the present work, structure optimizations guided by a binding model led to the identification of the lead compound 2-hydroxy-4-((4-(naphthalen-2-yl)thiazol-2-yl)amino)benzoic acid (27), showing a submicromolar potency against purified CK2α (IC50 = 0.6 µM). Furthermore, 27 induced apoptosis and cell death in 786-O renal cell carcinoma cells (EC50 = 5 µM) and inhibited STAT3 activation even more potently than the ATP-competitive drug candidate CX-4945 (EC50 of 1.6 µM vs 5.3 µM). Notably, the potencies of our allosteric ligands to inhibit CK2 varied depending on the individual substrate. Altogether, the novel allosteric pocket was proved a druggable site, offering an excellent perspective to develop efficient and selective allosteric CK2 inhibitors.
Sujet(s)
Benzoates/pharmacologie , Inhibiteurs de protéines kinases/pharmacologie , Thiazoles/pharmacologie , Régulation allostérique , Site allostérique , Antinéoplasiques/synthèse chimique , Antinéoplasiques/métabolisme , Antinéoplasiques/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Benzoates/synthèse chimique , Benzoates/métabolisme , Casein Kinase II/antagonistes et inhibiteurs , Casein Kinase II/métabolisme , Lignée cellulaire tumorale , Humains , Ligands , Simulation de docking moléculaire , Structure moléculaire , Naphtyridines/pharmacologie , Phénazines , Promédicaments/synthèse chimique , Promédicaments/métabolisme , Promédicaments/pharmacologie , Liaison aux protéines , Inhibiteurs de protéines kinases/synthèse chimique , Inhibiteurs de protéines kinases/métabolisme , Relation structure-activité , Thiazoles/synthèse chimique , Thiazoles/métabolismeRÉSUMÉ
CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non-ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD NMR, circular dichroism spectroscopy, and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC50 of 3.4 µM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anticancer drugs.
Sujet(s)
Casein Kinase II/antagonistes et inhibiteurs , Inhibiteurs de protéines kinases/composition chimique , Thiazoles/composition chimique , Régulation allostérique , Site allostérique/génétique , Casein Kinase II/génétique , Casein Kinase II/métabolisme , Humains , Cinétique , Simulation de docking moléculaire , Structure moléculaire , Mutation , Naphtyridines/composition chimique , Naphtyridines/métabolisme , Phénazines , Liaison aux protéines/génétique , Inhibiteurs de protéines kinases/métabolisme , Stabilité protéique , Relation structure-activité , Température , Thiazoles/métabolismeRÉSUMÉ
Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α') subunits and two regulatory (ß) subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2ß subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2ß. In search of compounds inhibiting this critical protein-protein interaction, we previously designed an active cyclic peptide (Pc) derived from the CK2ß carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way.
RÉSUMÉ
Efforts were made to improve a series of potent dual ABL/SRC inhibitors based on a 7-azaindole core with the aim of developing compounds that demonstrate a wider activity on selected oncogenic kinases. Multi-targeted kinase inhibitors (MTKIs) were then derived, focusing on kinases involved in both angiogenesis and tumorigenesis processes. Antiproliferative activity studies using different cellular models led to the discovery of a lead candidate (6z) that combined both antiangiogenic and antitumoral effects. The activity of 6z was assessed against a panel of kinases and cell lines including solid cancers and leukemia cell models to explore its potential therapeutic applications. With its potency and selectivity for oncogenic kinases, 6z was revealed to be a focused MTKI that should have a bright future in fighting a wide range of cancers.