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1.
Biomolecules ; 11(8)2021 08 12.
Article in English | MEDLINE | ID: mdl-34439861

ABSTRACT

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Biofilms/growth & development , Burkholderia cepacia/physiology , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/physiology , Crystallography, X-Ray/methods , Humans , Protein Structure, Secondary , Protein Structure, Tertiary , Virulence/physiology
2.
Bioorg Med Chem Lett ; 22(20): 6460-8, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22963766

ABSTRACT

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.


Subject(s)
Carrier Proteins/agonists , Enzyme Activation/drug effects , Indoles/chemistry , Indoles/pharmacology , Membrane Proteins/agonists , Neoplasm Proteins/agonists , Neoplasms/enzymology , Thyroid Hormones/agonists , Caco-2 Cells , Carrier Proteins/metabolism , Humans , Membrane Proteins/metabolism , Molecular Docking Simulation , Neoplasm Proteins/metabolism , Neoplasms/drug therapy , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Multimerization/drug effects , Pyruvate Kinase/metabolism , Sulfinic Acids/chemistry , Sulfinic Acids/pharmacology , Thyroid Hormones/metabolism , Thyroid Hormone-Binding Proteins
3.
ACS Med Chem Lett ; 2(2): 97-101, 2011 Feb 10.
Article in English | MEDLINE | ID: mdl-24900286

ABSTRACT

We have discovered novel benzofuran-based S1P1 agonists with excellent in vitro potency and selectivity. 1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl) azetidine-3-carboxylic acid (18) is a potent S1P1 agonist with >1000× selectivity over S1P3. It demonstrated a good in vitro ADME profile and excellent oral bioavailability across species. Dosed orally at 0.3 mg/kg, 18 significantly reduced blood lymphocyte counts 24 h postdose and demonstrated efficacy in a mouse EAE model of relapsing MS.

4.
ACS Med Chem Lett ; 2(2): 102-6, 2011 Feb 10.
Article in English | MEDLINE | ID: mdl-24900287

ABSTRACT

Optimization of a benzofuranyl S1P1 agonist lead compound (3) led to the discovery of 1-(3-fluoro-4-(5-(2-fluorobenzyl)benzo[d]thiazol-2-yl)benzyl)azetidine-3-carboxylic acid (14), a potent S1P1 agonist with minimal activity at S1P3. Dosed orally at 0.3 mg/kg, 14 significantly reduced blood lymphocyte counts 24 h postdose and attenuated a delayed type hypersensitivity (DTH) response to antigen challenge.

5.
J Comput Aided Mol Des ; 24(12): 971-91, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20976528

ABSTRACT

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Ion Transport/drug effects , Protein Folding/drug effects , Animals , Binding Sites/genetics , Cell Line , Cells, Cultured , Computer Simulation , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis/physiopathology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , HeLa Cells , High-Throughput Screening Assays , Humans , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Rats , Rats, Inbred F344 , Respiratory Mucosa/drug effects , Sequence Deletion , Small Molecule Libraries/chemistry , Structure-Activity Relationship
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