5 research outputs found
Identification and validation of G protein-coupled receptors modulating flow-dependent signaling pathways in vascular endothelial cells
Vascular endothelial cells are exposed to mechanical forces due to their presence at the interface between the vessel wall and flowing blood. The patterns of these mechanical forces (laminar vs. turbulent) regulate endothelial cell function and play an important role in determining endothelial phenotype and ultimately cardiovascular health. One of the key transcriptional mediators of the positive effects of laminar flow patterns on endothelial cell phenotype is the zinc-finger transcription factor, krüppel-like factor 2 (KLF2). Given its importance in maintaining a healthy endothelium, we sought to identify endothelial regulators of the KLF2 transcriptional program as potential new therapeutic approaches to treating cardiovascular disease. Using an approach that utilized both bioinformatics and targeted gene knockdown, we identified endothelial GPCRs capable of modulating KLF2 expression. Genetic screening using siRNAs directed to these GPCRs identified 12 potential GPCR targets that could modulate the KLF2 program, including a subset capable of regulating flow-induced KLF2 expression in primary endothelial cells. Among these targets, we describe the ability of several GPCRs (GPR116, SSTR3, GPR101, LGR4) to affect KLF2 transcriptional activation. We also identify these targets as potential validated targets for the development of novel treatments targeting the endothelium. Finally, we highlight the initiation of drug discovery efforts for LGR4 and report the identification of the first known synthetic ligands to this receptor as a proof-of-concept for pathway-directed phenotypic screening to identify novel drug targets
PK/PD Disconnect Observed with a Reversible Endothelial Lipase Inhibitor
Screening of a small set of nonselective
lipase inhibitors against
endothelial lipase (EL) identified a potent and reversible inhibitor, <i>N</i>-(3-(3,4-dichlorophenyl)propyl)-3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide
(<b>5</b>; EL IC<sub>50</sub> = 61 nM, EL<sub>HDL</sub> IC<sub>50</sub> = 454 nM). Deck mining identified a related hit, <i>N</i>-(3-(3,4-dichlorophenyl)propyl)-4-hydroxy-1-methyl-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>6a</b>; EL IC<sub>50</sub> = 41 nM, EL<sub>HDL</sub> IC<sub>50</sub> = 1760 nM). Both compounds
were selective against lipoprotein lipase (LPL) but nonselective versus
hepatic lipase (HL). Optimization of compound <b>6a</b> for
EL inhibition using HDL as substrate led to <i>N</i>-(4-(3,4<b>-</b>dichlorophenyl)butan-2-yl)-1-ethyl-4-hydroxy-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>7c</b>; EL IC<sub>50</sub> = 148 nM, EL<sub>HDL</sub> IC<sub>50</sub> = 218 nM) having improved
PK over compound <b>6a</b>, providing a tool molecule to test
for the ability to increase HDL-cholesterol (HDL-C) levels in vivo
using a reversible EL inhibitor. Compound <b>7c</b> did not
increase HDL-C in vivo despite achieving plasma exposures targeted
on the basis of enzyme activity and protein binding demonstrating
the need to develop more physiologically relevant in vitro assays
to guide compound progression for in vivo evaluation
Phenylimidazoles as Potent and Selective Inhibitors of Coagulation Factor XIa with in Vivo Antithrombotic Activity
Novel inhibitors of FXIa containing
an (<i>S</i>)-2-phenyl-1-(4-phenyl-1<i>H</i>-imidazol-2-yl)ethanamine
core have been optimized to provide
compound <b>16b</b>, a potent, reversible inhibitor of FXIa
(<i>K</i><sub>i</sub> = 0.3 nM) having in vivo antithrombotic
efficacy in the rabbit AV-shunt thrombosis model (ID<sub>50</sub> =
0.6 mg/kg + 1 mg kg<sup>–1</sup> h<sup>–1</sup>). Initial
analog selection was informed by molecular modeling using
compounds <b>11a</b> and <b>11h</b> overlaid onto the
X-ray crystal structure of tetrahydroquinoline <b>3</b> complexed
to FXIa. Further optimization was achieved by specific modifications
derived from careful analysis of the X-ray crystal structure of the
FXIa/<b>11h</b> complex. Compound <b>16b</b> was well
tolerated and enabled extensive pharmacologic evaluation of the FXIa
mechanism up to the ID<sub>90</sub> for thrombus inhibition