Synthesis and evaluation of novel aldose reductase inhibitors: Effects on lens protein kinase Cgamma.
OBJECTIVE
To synthesize novel aldose reductase inhibitors (ARI) that will normalize losses in protein kinase Cgamma (PKCgamma) observed during diabetes and galactosemia.


METHODS
ARI were synthesized as tricyclic pyrones 1-6 (HAR-1 through HAR-6) from 3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran and (5aS,7S)-7-isopropenyl-3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran and were tested by inhibition of aldose reductase enzyme activity in vitro and by inhibition of polyol formation in lens epithelial cells in culture. Identified compounds were further tested in galactosemic rat lens in vivo for (a) normalized PKCgamma levels by Western blot, (b) reduction of phosphorylation of the gap junction protein Cx46 by analyses of co-immunoprecipitated proteins, and (c) by normalization of gap junction activity as measured by dye transfer.


RESULTS
HAR-1 (1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran-3-acetic acid) was identified as an ARI with IC50 for aldose reductase inhibition at 2 nM. Polyol accumulation in lens epithelial cells was reduced by 80% at 10 microM. Rats fed 40% galactose for 9 days had an 80% reduction in PKCgamma levels which were normalized by HAR-1 at 100 mg/kg/day, fed orally. Phosphorylation of Cx46 was increased by 50% and this was normalized in HAR-1 treated rats (6 day treatment). Gap junction activity of galactosemic rats was reduced by 55% and this was normalized by HAR-1 in six day-treated rats.


CONCLUSIONS
HAR-1 is a novel ARI which normalized losses of PKCgamma, changes in Cx46 phosphorylation, and gap junction activity.
Lewis
S
S
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Karrer
J
J
Saleh
S
S
Chan
X
X
Tan
Z
Z
Hua
D
D
McGill
J
J
Pang
Y P
YP
Fenwick
B
B
Brightman
A
A
Takemoto
D
D
D000085:Acetates / Q000138:chemical synthesis / Q000494:pharmacology*; D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors*; D000818:Animals; D001578:Benzopyrans / Q000138:chemical synthesis / Q000494:pharmacology*; D015153:Blotting, Western; D017630:Connexins / Q000378:metabolism; D003920:Diabetes Mellitus / Q000201:enzymology; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D004847:Epithelial Cells / Q000378:metabolism; D005690:Galactose / Q000008:administration & dosage; D005693:Galactosemias / Q000201:enzymology; D017629:Gap Junctions / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007904:Lens Cortex, Crystalline / Q000378:metabolism; D007908:Lens, Crystalline / Q000187:drug effects* / Q000201:enzymology; D015394:Molecular Structure; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism
C433083:1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano(4,3-b)(1)benzopyran-3-acetic acid; D000085:Acetates; D001578:Benzopyrans; D017630:Connexins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011108:Polymers; C082917:GJA3 protein, human; C024617:polyol; D012694:Serine; D000449:Aldehyde Reductase; C087857:protein kinase C gamma; D011493:Protein Kinase C; D005690:Galactose