RÉSUMÉ
While several therapeutic options exist, the need for more effective, safe, and convenient treatment for a variety of autoimmune diseases persists. Targeting the Janus tyrosine kinases (JAKs), which play essential roles in cell signaling responses and can contribute to aberrant immune function associated with disease, has emerged as a novel and attractive approach for the development of new autoimmune disease therapies. We screened our compound library against JAK3, a key signaling kinase in immune cells, and identified multiple scaffolds showing good inhibitory activity for this kinase. A particular scaffold of interest, the 1H-pyrrolo[2,3-b]pyridine series (7-azaindoles), was selected for further optimization in part on the basis of binding affinity (Ki) as well as on the basis of cellular potency. Optimization of this chemical series led to the identification of VX-509 (decernotinib), a novel, potent, and selective JAK3 inhibitor, which demonstrates good efficacy in vivo in the rat host versus graft model (HvG). On the basis of these findings, it appears that VX-509 offers potential for the treatment of a variety of autoimmune diseases.
Sujet(s)
Maladies auto-immunes/traitement médicamenteux , Composés hétérobicycliques/composition chimique , Janus kinase 3/antagonistes et inhibiteurs , Valine/analogues et dérivés , Animaux , Lignée cellulaire , Bases de données chimiques , Chiens , Femelle , Maladie du greffon contre l'hôte/traitement médicamenteux , Maladie du greffon contre l'hôte/immunologie , Haplorhini , Composés hétérobicycliques/pharmacocinétique , Composés hétérobicycliques/pharmacologie , Humains , Kinase Janus-2/composition chimique , Janus kinase 3/composition chimique , Mâle , Souris , Souris de lignée BALB C , Souris de lignée CBA , Microsomes du foie/métabolisme , Modèles moléculaires , Rats , Rats de lignée LEW , Rat Sprague-Dawley , Stéréoisomérie , Relation structure-activité , Valine/composition chimique , Valine/pharmacocinétique , Valine/pharmacologieRÉSUMÉ
VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.
Sujet(s)
Antiviraux/pharmacologie , DNA-directed RNA polymerases/antagonistes et inhibiteurs , Virus de la grippe A/effets des médicaments et des substances chimiques , Protéines virales/antagonistes et inhibiteurs , Administration par voie orale , Animaux , Biodisponibilité , Lignée cellulaire , Chiens , Cellules HEK293 , Humains , Grippe humaine/traitement médicamenteux , Grippe humaine/virologie , Cellules rénales canines Madin-Darby , Mâle , Souris , Souris de lignée BALB C , Infections à Orthomyxoviridae/traitement médicamenteux , Infections à Orthomyxoviridae/virologieRÉSUMÉ
UNLABELLED: The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication. IMPORTANCE: The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.
Sujet(s)
Hepacivirus/enzymologie , Hepacivirus/physiologie , Interactions hôte-pathogène , Protéine A de réplication/métabolisme , Protéines virales non structurales/métabolisme , Réplication virale , Humains , Protéines mutantes/génétique , Protéines mutantes/métabolisme , Liaison aux protéines , ARN viral/métabolisme , Délétion de séquence , Protéines virales non structurales/génétiqueRÉSUMÉ
Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167-179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002.Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases.
Sujet(s)
4H-1-Benzopyran-4-ones/métabolisme , Antienzymes/métabolisme , Morpholines/métabolisme , Conformation des protéines , Protein-Serine-Threonine Kinases/composition chimique , Protein-Serine-Threonine Kinases/métabolisme , Protéines proto-oncogènes/composition chimique , Protéines proto-oncogènes/métabolisme , Adénosine/composition chimique , Adénosine/métabolisme , Séquence d'acides aminés , Sites de fixation , 4H-1-Benzopyran-4-ones/composition chimique , Cyclic AMP-Dependent Protein Kinases/composition chimique , Cyclic AMP-Dependent Protein Kinases/génétique , Antienzymes/composition chimique , Humains , Liaison hydrogène , Ligands , Modèles moléculaires , Données de séquences moléculaires , Structure moléculaire , Morpholines/composition chimique , Phosphatidylinositol 3-kinases/composition chimique , Phosphatidylinositol 3-kinases/génétique , Liaison aux protéines , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Protein-Serine-Threonine Kinases/génétique , Protéines proto-oncogènes/antagonistes et inhibiteurs , Protéines proto-oncogènes/génétique , Protéines proto-oncogènes c-pim-1 , Alignement de séquences , Staurosporine/composition chimique , Staurosporine/métabolismeRÉSUMÉ
FLT3 is a type III receptor tyrosine kinase that is thought to play a key role in hematopoiesis. Certain classes of FLT3 mutations cause constitutively activated forms of the receptor that are found in significant numbers of patients with acute myelogenous leukemia (AML). The mutations occur either in the activation loop, for example, as point mutations of Asp835 or as internal tandem duplication (ITD) sequences in the juxtamembrane (JM) domain. To further understand the nature of FLT3 autoinhibition and regulation, we have determined the crystal structure of the autoinhibited form of FLT3. This structure shows the autoinhibitory conformation of a complete JM domain in this class of receptor tyrosine kinases. The detailed inhibitory mechanism of the JM domain is revealed, which is likely utilized by other members of type III receptor tyrosine kinases.