ABSTRACT
To develop agents for the treatment of infections caused by Mycobacterium tuberculosis, a novel phenotypic screen was undertaken that identified a series of 2-N-aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis. Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro. The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo.
Subject(s)
Anti-Bacterial Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Thiazoles/pharmacology , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Dose-Response Relationship, Drug , Humans , Macrophages/drug effects , Macrophages/microbiology , Mice , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/chemistryABSTRACT
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.
Subject(s)
Autoimmune Diseases/drug therapy , Heterocyclic Compounds, 2-Ring/chemistry , Janus Kinase 3/antagonists & inhibitors , Valine/analogs & derivatives , Animals , Cell Line , Databases, Chemical , Dogs , Female , Graft vs Host Disease/drug therapy , Graft vs Host Disease/immunology , Haplorhini , Heterocyclic Compounds, 2-Ring/pharmacokinetics , Heterocyclic Compounds, 2-Ring/pharmacology , Humans , Janus Kinase 2/chemistry , Janus Kinase 3/chemistry , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Microsomes, Liver/metabolism , Models, Molecular , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity Relationship , Valine/chemistry , Valine/pharmacokinetics , Valine/pharmacologyABSTRACT
Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD (MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity (Ki â¼ 25 µM) and antimycobacterial activity (MIC80) â¼ 40-80 µM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. These findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.
Subject(s)
Antitubercular Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/enzymology , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Amino Acid Sequence , Antitubercular Agents/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mycobacterium tuberculosis/drug effects , Nicotinamide-Nucleotide Adenylyltransferase/antagonists & inhibitors , Nicotinamide-Nucleotide Adenylyltransferase/chemistry , Protein Conformation , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
Pictet-Spengler condensation of aldehydes or alpha-keto-esters with 4-(2-anilinophenyl)-7-azaindole (11) or deazapurine (12) gave high yields of the 3,4-fused cyclic compounds. SAR studies, by varying the substituted benzaldehyde components, lead to the discovery of a series of potent JAK2 kinase inhibitors.
Subject(s)
Indoles/chemistry , Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Purines/chemistry , Benzaldehydes/chemistry , Binding Sites , Cell Line , Crystallography, X-Ray , Drug Discovery , Humans , Janus Kinase 2/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
The synthesis and characterization of a novel polycyclic azaindole based derivative is disclosed, and its binding to JAK2 is described. The compound is further evaluated for its ability to block the EPO/JAK2 signaling cascade in vitro and in vivo.
Subject(s)
Aza Compounds/chemistry , Aza Compounds/pharmacology , Indoles/chemistry , Indoles/pharmacology , Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Aza Compounds/chemical synthesis , Aza Compounds/pharmacokinetics , Cell Line, Tumor , Crystallography, X-Ray , Erythropoietin/metabolism , Indoles/chemical synthesis , Indoles/pharmacokinetics , Janus Kinase 2/metabolism , Lactams, Macrocyclic/chemical synthesis , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/pharmacokinetics , Lactams, Macrocyclic/pharmacology , Male , Mice , Mice, Inbred BALB C , Mice, SCID , Models, Molecular , Molecular Conformation , Protein Conformation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effectsABSTRACT
Constitutive activation of the EPO/JAK2 signaling cascade has recently been implicated in a variety of myeloproliferative disorders including polycythemia vera, essential thrombocythemia and myelofibrosis. In an effort to uncover therapeutic potential of blocking the EPO/JAK2 signaling cascade, we sought to discover selective inhibitors that block the kinase activity of JAK2. Herein, we describe the discovery and structure based optimization of a novel series of 2-amino-pyrazolo[1,5-a]pyrimidines that exhibit potent inhibition of JAK2.
Subject(s)
Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Crystallography, X-Ray , Drug Discovery , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Signal Transduction/drug effects , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.
