RÉSUMÉ
In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.
Sujet(s)
Adénosine triphosphate/antagonistes et inhibiteurs , Choline kinase/antagonistes et inhibiteurs , Cyclohexanes/pharmacologie , Inhibiteurs de protéines kinases/pharmacologie , Adénosine triphosphate/métabolisme , Choline kinase/métabolisme , Cyclohexanes/synthèse chimique , Cyclohexanes/composition chimique , Relation dose-effet des médicaments , Humains , Structure moléculaire , Inhibiteurs de protéines kinases/synthèse chimique , Inhibiteurs de protéines kinases/composition chimique , Relation structure-activitéRÉSUMÉ
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.
Sujet(s)
Antinéoplasiques/pharmacologie , Benzamides/pharmacologie , Découverte de médicament , Indazoles/pharmacologie , Tumeurs du poumon/traitement médicamenteux , Inhibiteurs de protéines kinases/pharmacologie , Protein-tyrosine kinases/antagonistes et inhibiteurs , Récepteurs à activité tyrosine kinase/antagonistes et inhibiteurs , Administration par voie orale , Kinase du lymphome anaplasique , Animaux , Antinéoplasiques/administration et posologie , Antinéoplasiques/composition chimique , Benzamides/administration et posologie , Benzamides/composition chimique , Barrière hémato-encéphalique/effets des médicaments et des substances chimiques , Technique de Western , Perméabilité des membranes cellulaires/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Cristallisation , Cristallographie aux rayons X , Chiens , Humains , Indazoles/administration et posologie , Indazoles/composition chimique , Tumeurs du poumon/métabolisme , Tumeurs du poumon/anatomopathologie , Souris , Souris nude , Souris SCID , Microsomes du foie/effets des médicaments et des substances chimiques , Inhibiteurs de protéines kinases/administration et posologie , Inhibiteurs de protéines kinases/composition chimique , Protéines proto-oncogènes/antagonistes et inhibiteurs , Rats , Rat Wistar , Récepteur trkA/antagonistes et inhibiteurs , Récepteur trkB/antagonistes et inhibiteurs , Récepteur trkC/antagonistes et inhibiteurs , Cellules cancéreuses en culture , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
The nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.
Sujet(s)
Antinéoplasiques/composition chimique , Isoindoles/composition chimique , Pipéridines/composition chimique , Inhibiteurs de poly(ADP-ribose) polymérases/composition chimique , Administration par voie orale , Animaux , Antinéoplasiques/administration et posologie , Antinéoplasiques/pharmacologie , Protocoles de polychimiothérapie antinéoplasique/usage thérapeutique , Biodisponibilité , Prolifération cellulaire/effets des médicaments et des substances chimiques , Dacarbazine/administration et posologie , Dacarbazine/analogues et dérivés , Tests de criblage d'agents antitumoraux , Femelle , Hétérogreffes , Tests de criblage à haut débit , Humains , Isoindoles/administration et posologie , Isoindoles/pharmacologie , Souris de lignée BALB C , Souris nude , Microsomes du foie/métabolisme , Modèles moléculaires , Transplantation tumorale , Tumeurs du pancréas/traitement médicamenteux , Tumeurs du pancréas/anatomopathologie , Pipéridines/administration et posologie , Pipéridines/pharmacologie , Inhibiteurs de poly(ADP-ribose) polymérases/administration et posologie , Inhibiteurs de poly(ADP-ribose) polymérases/pharmacologie , Rat Sprague-Dawley , Relation structure-activité , Témozolomide , Tumeurs du sein triple-négativesRÉSUMÉ
Aberrant activation of the mitogen-activated protein kinase (MAPK)-mediated pathway components, RAF-MEK-ERK, is frequently observed in human cancers and clearly contributes to oncogenesis. As part of a project aimed at finding inhibitors of B-Raf, a key player in the MAPK cascade, we originally identified a thiazole derivative endowed with high potency and selectivity, optimal in vitro ADME properties, and good pharmacokinetic profiles in rodents, but that suffers from elevated hERG inhibitory activity. An optimization program was thus undertaken, focused mainly on the elaboration of the R(1) and R(2) groups of the scaffold. This effort ultimately led to N-(4-{2-(1-cyclopropylpiperidin-4-yl)-4-[3-(2,5-difluorobenzenesulfonylamino)-2-fluorophenyl]thiazol-5-yl}-pyridin-2-yl)acetamide (20), which maintains favorable in vitro and in vivo properties, but lacks hERG liability. Besides exhibiting potent antiproliferative activity against only cell lines bearing B-Raf V600E or V600D mutations, compound 20 also intriguingly shows a weaker "paradoxical" activation of MEK in non-mutant B-Raf cells than other known B-Raf inhibitors. It also demonstrates very good efficacy in vivo against the A375 xenograft melanoma model (tumor volume inhibition >90% at 10 mg kg(-1) ); it is therefore a suitable candidate for preclinical development.
Sujet(s)
Antinéoplasiques/composition chimique , Inhibiteurs de protéines kinases/composition chimique , Protéines proto-oncogènes B-raf/antagonistes et inhibiteurs , Sulfonamides/composition chimique , Thiazoles/composition chimique , Administration par voie orale , Animaux , Antinéoplasiques/pharmacologie , Antinéoplasiques/usage thérapeutique , Sites de fixation , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Canal potassique ERG1 , Canaux potassiques éther-à-go-go/antagonistes et inhibiteurs , Canaux potassiques éther-à-go-go/métabolisme , Humains , MAP Kinase Kinase 1/génétique , MAP Kinase Kinase 1/métabolisme , Souris , Souris de lignée BALB C , Souris nude , Phosphorylation/effets des médicaments et des substances chimiques , Liaison aux protéines , Inhibiteurs de protéines kinases/pharmacologie , Inhibiteurs de protéines kinases/usage thérapeutique , Structure tertiaire des protéines , Protéines proto-oncogènes B-raf/génétique , Protéines proto-oncogènes B-raf/métabolisme , Sulfonamides/usage thérapeutique , Sulfonamides/toxicité , Thiazoles/pharmacologie , Thiazoles/usage thérapeutique , Thiazoles/toxicité , Transplantation hétérologueRÉSUMÉ
We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.
Sujet(s)
Antinéoplasiques/pharmacologie , Kinase Janus-2/antagonistes et inhibiteurs , Tumeurs expérimentales/traitement médicamenteux , Inhibiteurs de protéines kinases/pharmacologie , Pyrimidines/pharmacologie , Pyrroles/pharmacologie , Animaux , Antinéoplasiques/synthèse chimique , Antinéoplasiques/composition chimique , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Cristallographie aux rayons X , Relation dose-effet des médicaments , Tests de criblage d'agents antitumoraux , Humains , Kinase Janus-2/métabolisme , Souris , Souris SCID , Modèles moléculaires , Structure moléculaire , Tumeurs expérimentales/anatomopathologie , Inhibiteurs de protéines kinases/synthèse chimique , Inhibiteurs de protéines kinases/composition chimique , Pyrimidines/synthèse chimique , Pyrimidines/composition chimique , Pyrroles/synthèse chimique , Pyrroles/composition chimique , Relation structure-activité , Spécificité du substratRÉSUMÉ
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). HCN isoforms respond differently to cAMP; binding of cAMP shifts activation of HCN2 and HCN4 by 17 mV but shifts that of HCN1 by only 2-4 mV. To explain the peculiarity of HCN1, we solved the crystal structures and performed a biochemical-biophysical characterization of the C-terminal domain (C-linker plus CNBD) of the three isoforms. Our main finding is that tetramerization of the C-terminal domain of HCN1 occurs at basal cAMP concentrations, whereas those of HCN2 and HCN4 require cAMP saturating levels. Therefore, HCN1 responds less markedly than HCN2 and HCN4 to cAMP increase because its CNBD is already partly tetrameric. This is confirmed by voltage clamp experiments showing that the right-shifted position of V(½) in HCN1 is correlated with its propensity to tetramerize in vitro. These data underscore that ligand-induced CNBD tetramerization removes tonic inhibition from the pore of HCN channels.
Sujet(s)
AMP cyclique/métabolisme , Ouverture et fermeture des portes des canaux ioniques/physiologie , Canaux ioniques/composition chimique , Canaux ioniques/métabolisme , Multimérisation de protéines/physiologie , Animaux , AMP cyclique/composition chimique , AMP cyclique/génétique , Humains , Canaux contrôlés par les nucléotides cycliques et activés par l'hyperpolarisation , Canaux ioniques/génétique , Ovocytes , Canaux potassiques , Isoformes de protéines/composition chimique , Isoformes de protéines/génétique , Isoformes de protéines/métabolisme , Structure tertiaire des protéines , Xenopus laevisRÉSUMÉ
As part of our drug discovery effort, we identified and developed 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as PLK1 inhibitors. We now report the optimization of this class that led to the identification of NMS-P937, a potent, selective and orally available PLK1 inhibitor. Also, in order to understand the source of PLK1 selectivity, we determined the crystal structure of PLK1 with NMS-P937. The compound was active in vivo in HCT116 xenograft model after oral administration and is presently in Phase I clinical trials evaluation.
Sujet(s)
Antinéoplasiques/pharmacologie , Protéines du cycle cellulaire/antagonistes et inhibiteurs , Inhibiteurs de protéines kinases/pharmacologie , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Protéines proto-oncogènes/antagonistes et inhibiteurs , Pyrazoles/pharmacologie , Quinazolines/pharmacologie , Administration par voie orale , Animaux , Antinéoplasiques/synthèse chimique , Antinéoplasiques/composition chimique , Prolifération cellulaire/effets des médicaments et des substances chimiques , Essais cliniques de phase I comme sujet , Concentration inhibitrice 50 , Souris , Souris nude , Structure moléculaire , Tumeurs/traitement médicamenteux , Inhibiteurs de protéines kinases/synthèse chimique , Inhibiteurs de protéines kinases/composition chimique , Pyrazoles/synthèse chimique , Pyrazoles/composition chimique , Quinazolines/synthèse chimique , Quinazolines/composition chimique , Relation structure-activité , Tests d'activité antitumorale sur modèle de xénogreffe ,RÉSUMÉ
CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.
Sujet(s)
Indoles/antagonistes et inhibiteurs , Indoles/composition chimique , Protein-arginine N-methyltransferases/antagonistes et inhibiteurs , Protein-arginine N-methyltransferases/composition chimique , Pyrazoles/antagonistes et inhibiteurs , Pyrazoles/composition chimique , Séquence d'acides aminés , Domaine catalytique/effets des médicaments et des substances chimiques , Cristallographie aux rayons X , Antienzymes/composition chimique , Antienzymes/métabolisme , Antienzymes/pharmacologie , Humains , Indoles/métabolisme , Données de séquences moléculaires , Liaison aux protéines/effets des médicaments et des substances chimiques , Protein-arginine N-methyltransferases/métabolisme , Pyrazoles/métabolismeRÉSUMÉ
Cdc7 kinase is a key regulator of the S-phase of the cell cycle, known to promote the activation of DNA replication origins in eukaryotic organisms. Cdc7 inhibition can cause tumor-cell death in a p53-independent manner, supporting the rationale for developing Cdc7 inhibitors for the treatment of cancer. In this paper, we conclude the structure-activity relationships study of the 2-heteroaryl-pyrrolopyridinone class of compounds that display potent inhibitory activity against Cdc7 kinase. Furthermore, we also describe the discovery of 89S, [(S)-2-(2-aminopyrimidin-4-yl)-7-(2-fluoro-ethyl)-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one], as a potent ATP mimetic inhibitor of Cdc7. Compound 89S has a Ki value of 0.5 nM, inhibits cell proliferation of different tumor cell lines with an IC50 in the submicromolar range, and exhibits in vivo tumor growth inhibition of 68% in the A2780 xenograft model.
Sujet(s)
Antinéoplasiques/pharmacologie , Protéines du cycle cellulaire/antagonistes et inhibiteurs , Inhibiteurs de protéines kinases/pharmacologie , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Pyridones/pharmacologie , Administration par voie orale , Animaux , Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacocinétique , Lignée cellulaire tumorale , Chromatographie en phase liquide à haute performance , Chiens , Découverte de médicament , Humains , Spectroscopie par résonance magnétique , Inhibiteurs de protéines kinases/composition chimique , Inhibiteurs de protéines kinases/pharmacocinétique , Pyridones/composition chimique , Pyridones/pharmacocinétique , Rats , Rat Wistar , Spectrométrie de masse ESI , Spectrophotométrie UV , Relation structure-activitéRÉSUMÉ
The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.
Sujet(s)
Protéines/antagonistes et inhibiteurs , Protéines/composition chimique , bêta-Caténine/antagonistes et inhibiteurs , Sites de fixation , Cristallographie aux rayons X , Humains , Modèles moléculaires , Mutation , Tumeurs/génétique , Conformation des protéines , Logiciel , Interface utilisateur , bêta-Caténine/génétiqueRÉSUMÉ
The interaction of beta-catenin with T-cell factor (Tcf) 4 plays a central role in the Wnt signaling pathway and has been discussed as a possible site of intervention for the development of anti-cancer drugs. In this study, we performed Ala-scanning mutagenesis of all Tcf4 residues in the Tcf-beta-catenin interface and studied the binding energetics of these mutants using isothermal titration calorimetry. Binding of Tcf4 was found to be highly cooperative. Single site mutations of most Tcf4 residues resulted in a significant reduction in binding enthalpies but in similar binding constants as compared with wild type Tcf4. Interestingly, this was also true for residues that are disordered in the reported crystal structures. The mutation D16A caused the largest reduction in binding constant (50-fold) accompanied by a large unfavorable enthalpy change (DeltaDeltaHobs) of +8 kcal/mol at 25 degrees C. In contrast, the mutation of the Tcf residues Glu24 and Glu28, which have been proposed as an interaction hot spot due to their location in a field of strong positive electrostatic potential on the beta-catenin surface (charge button), resulted only in a significant reduction of binding enthalpies, which were largely compensated for by unfavorable entropic contributions to the binding. Other mutations that significantly reduced Tcf binding constants were D11A and alanine mutations of the hydrophobic residues Leu41, Val44, and Leu48. The measured thermodynamic data are discussed with the available structural information of Tcf-beta-catenin crystal structures and allow us to propose possible sites for development of Tcf antagonists.
Sujet(s)
Protéines du cytosquelette/métabolisme , Transactivateurs/métabolisme , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Séquence d'acides aminés , Animaux , Calorimétrie , Cristallographie , Humains , Interactions hydrophobes et hydrophiles , Données de séquences moléculaires , Liaison aux protéines , Structure secondaire des protéines , Structure tertiaire des protéines , Transduction du signal/physiologie , Facteurs de transcription TCF , Protéine-2 de type facteur-7 de transcription , Facteurs de transcription/composition chimique , bêta-CaténineRÉSUMÉ
Water-ligand observed via gradient spectroscopy (WaterLOGSY) represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. The method is useful for the detection of compounds binding to a receptor with binding affinity in the micromolar range. The Achille's heel of the method, as with all the techniques that detect the ligand resonances, is its inability to identify strong ligands with slow dissociation rates. We show here that the use of a reference compound with a known K(D) in the micromolar range together with properly designed competition binding experiments (c-WaterLOGSY) permits the detection of strong binders. A derived mathematical expression is used for the selection of the appropriate setup NMR experimental conditions and for an approximate determination of the binding constant. The experiment requires low ligand concentration, therefore allowing its application in the identification of potential strong inhibitors that are only marginally soluble. The technique is particularly suitable for rapid screening of chemical mixtures and plant or fungi extracts.
Sujet(s)
Évaluation préclinique de médicament/méthodes , Ligands , Protéines/composition chimique , Eau/composition chimique , Algorithmes , Fixation compétitive , Champignons/composition chimique , Humains , Spectroscopie par résonance magnétique , Extraits de plantes/composition chimique , Liaison aux protéines , Sérumalbumine/composition chimique , Tryptophane/analogues et dérivés , Tryptophane/composition chimiqueRÉSUMÉ
Normal peripheral nerve myelination depends on Schwann cell-basal lamina interactions. An important component of Schwann cell basal lamina is laminin-predominantly laminins 2 and 4. Mutations in the alpha 2 chain common to these two isoforms are associated with dysmyelination in mouse (dy) and man (congenital muscular dystrophy). Thus, laminin 2 and 4 receptors are also likely to be important for myelin formation. Several laminin 2/4 receptors are detected at the basal lamina surface of myelin-forming Schwann cells, namely, α6ß4 and α6ß1 integrins and dystroglycan. The evidence linking these receptors to myelination is suggestive, but not conclusive. Genetic studies have not yet confirmed a role for these molecules in myelin formation. Natural or targeted inactivation of α6, ß4, and ß1 integrins and of dystroglycan have profound effects on other tissues causing embryonic or perinatal death before myelination. Therefore, to conditionally inactivate these receptors specifically in myelin-forming Schwann cells, we have constructed and initially characterized a P0-Cre transgene that activates Cre-mediated recombination of loxP-containing genes in peripheral nerve.
RÉSUMÉ
We have previously shown that increased dosage of the mouse protein zero gene (Mpz) causes a dysmyelinating neuropathy in transgenic (Tg80) mice. To ask whether the dysmyelination is dose dependent, we inbred one of the Tg80 lines and compared the resulting phenotype in homozygous and heterozygous mice. Whereas heterozygous mice (30% overexpression) have only transient peripheral nerve hypomyelination at two weeks after birth and normal myelin at four weeks after birth, homozygous mice demonstrated more severely hypomyelinated nerves. In the latter, many Schwann cells had achieved a one-to-one relationship with large axons but formed no myelin at four weeks after birth. Expression analysis confirmed a doubling of Mpz overexpression in the sciatic nerves of the homozygous mice. Thus, a threshold exists for Mpz overexpression, above which dysmyelination results. These data have important implications for replacement therapy in Charcot-Marie-Tooth 1B neuropathies due to loss of P0 function.