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1.
J Biol Chem ; 295(15): 5136-5151, 2020 04 10.
Article in English | MEDLINE | ID: mdl-32132173

ABSTRACT

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 µm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).


Subject(s)
Apolipoproteins A/antagonists & inhibitors , Apolipoproteins A/metabolism , Fibrin/metabolism , Kringles/drug effects , Small Molecule Libraries/pharmacology , Amino Acid Sequence , High-Throughput Screening Assays , Humans , Ligands , Models, Molecular , Protein Binding , Protein Domains , Sequence Homology
2.
ACS Sens ; 4(12): 3166-3174, 2019 12 27.
Article in English | MEDLINE | ID: mdl-31724395

ABSTRACT

Biomarkers serve as indicators of disease progression or therapeutic response of an medical intervention, and means for enabling a reliable and sensitive biomarker detection are therefore vital in clinical settings. Most biosensor assays require high-affinity interactions in combination with an enzyme or fluorescent tag to enable detection and frequently employ extensive washing procedures prior to signal readout. Attempts to overcome this limitation by using natural biological partners tend to be demanding, because their very low affinity is frequently not compatible with the need of reaching low limits of detection (LODs), especially for circulating biomarkers that possess short half-lives. To address these challenges, we developed a label-free surface plasmon resonance (SPR) platform for the detection of neuregulin 1 (NRG1) using ErbB4-modified liposomes offering both signal amplification and affinity enhancement via functional multivalent interactions. Through the functional avidity interaction between NRG1 and ErbB4, an LOD of 3.5 picomolar was reached, which is about 60-fold higher than traditional SPR and miniaturized immunoassays. The biosensor displays also an 8-fold higher sensitivity when compared with a single-molecule immunoassay employing the natural binding partner rather than a high-affinity antibody as one of the interaction partners. In fact, the liposome-induced avidity between NRG1 and ErbB4 offered an LOD that was comparable to that obtained using a high-affinity antibody and enabled detection of NRG1 in plasma with a LOD of 36 pM. Employing the liposome-enhanced platform in conjunction with a low-affinity biomarker receptor thus enables the assessment of the functional state of the biomarker at competitive LODs and eliminates the need for high-affinity antibodies.


Subject(s)
Biosensing Techniques/methods , Liposomes/chemistry , Neuregulin-1/analysis , Surface Plasmon Resonance/methods , Antibodies, Immobilized/immunology , Antibodies, Monoclonal/immunology , Biomarkers/analysis , Biomarkers/metabolism , HEK293 Cells , Humans , Immunoassay/methods , Limit of Detection , Nanoparticles/chemistry , Neuregulin-1/immunology , Neuregulin-1/metabolism , Receptor, ErbB-4/analysis , Receptor, ErbB-4/metabolism
3.
Nat Chem Biol ; 15(4): 348-357, 2019 04.
Article in English | MEDLINE | ID: mdl-30718815

ABSTRACT

We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kß. On the basis of X-ray crystallography, hydrogen-deuterium exchange-mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.


Subject(s)
Class Ib Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Adenosine Triphosphatases , Binding Sites , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class I Phosphatidylinositol 3-Kinases/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Mutagenesis, Site-Directed , Phthalimides , Protein Binding , Protein Conformation , Protein Isoforms/physiology , Protein Kinase Inhibitors , Substrate Specificity
4.
Bioorg Med Chem ; 19(10): 3039-53, 2011 May 15.
Article in English | MEDLINE | ID: mdl-21515056

ABSTRACT

Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats.


Subject(s)
Acetyl-CoA Carboxylase/antagonists & inhibitors , Diabetes Mellitus, Type 2/drug therapy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Obesity/drug therapy , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Acetyl-CoA Carboxylase/metabolism , Animals , Crystallography, X-Ray , Diabetes Mellitus, Type 2/enzymology , Drug Design , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Fatty Acids/metabolism , Humans , Liver/drug effects , Liver/enzymology , Male , Malonyl Coenzyme A/metabolism , Mice , Mice, Inbred C57BL , Models, Molecular , Obesity/enzymology , Rats , Rats, Zucker , Small Molecule Libraries/pharmacokinetics , Small Molecule Libraries/therapeutic use , Structure-Activity Relationship
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