Isoform-selective inhibition of facilitative glucose transporters: elucidation of the molecular mechanism of HIV protease inhibitor binding.
Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.
From the Department of Pediatrics.
Richard C
RC
Hresko
From the Department of Pediatrics.
Thomas E
TE
Kraft
From the Department of Pediatrics.
Anatoly
A
Tzekov
Biochemistry, and.
Scott A
SA
Wildman
From the Department of Pediatrics, Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 hruz_p@kids.wustl.edu.
Paul W
PW
Hruz
D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D051246:Glucose Transport Proteins, Facilitative / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D019469:Indinavir / Q000378:metabolism / Q000494:pharmacology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D051246:Glucose Transport Proteins, Facilitative; D017320:HIV Protease Inhibitors; D020033:Protein Isoforms; D019469:Indinavir
Glucose Transport; HIV; HIV Protease Inhibitor; Homology Modeling; Insulin Resistance; Monosaccharide Transport Protein; Photoaffinity Labeling; Protein Chimeras