Structural requirements of sphingosine molecules for inhibition of DNA primase: biochemical and computational analyses.
Using 28 chemically well-defined compounds containing D-erythro-sphingosine and its analogues, we analyzed structure-activity relationships for DNA primase inhibition. Biochemical studies demonstrated a positively charged amino group at C2 and a long aliphatic chain to be absolutely required for inhibition. Whereas C2-amino group is intact, sphingosine 1-phosphate was totally inactive. This result could be due to cancellation of positive charge of the amino group by the interaction with negatively charged C1-phosphate, since simulations with the software INSIGHT II showed these two groups to be close enough to interact. The hydroxyl group at C3 and trans-double bond at C4-C5 were also found to be important for the inhibition. Dehydroxylation of C3, as well as saturation or cis-conversion of the trans-double bond led to decrease of inhibitory activity. Despite saturation of the double bond, introduction of a hydroxyl group into C4 of dihydrosphingosine resulted in restoration of inhibition. Conversion of the double bond into a triple bond did not abolish but rather enhanced the inhibitory activity. Among sphingosine stereoisomers, the naturally occurring D-erythro-sphingosine proved to be the strongest inhibitor. To ascertain the contribution of the total conformation to the inhibition, especially of the long aliphatic chain, we constructed a 3D-quantitative structure-activity relationship model using the computer program Catalyst/HipHop on the basis of information described above. Analysis of the hypothesis model for active compounds revealed that the orientation of aliphatic chain, represented by the dihedral angle of C2-3-4-5, correlated well with the inhibition. Modifications such as deletion of the hydroxyl group at C3 or saturation of the C4-C5 double bond caused shifts in the dihedral angle of C2-3-4-5, with concomitant decrease in inhibitory activity.
Ito
Y
Y
Equipment Center for Research and Education, Laboratory of Cancer Cell Biology, School of Health Science, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan. yitoh@tsuru.med.nagoya-u.ac.jp
Tamiya-Koizumi
K
K
Koide
Y
Y
Nakagawa
M
M
Kawade
T
T
Nishida
A
A
Murate
T
T
Takemura
M
M
Suzuki
M
M
Yoshida
S
S
D000818:Animals; D002417:Cattle; D004256:DNA Polymerase I / Q000378:metabolism; D019915:DNA Primase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015394:Molecular Structure; D021281:Quantitative Structure-Activity Relationship; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013950:Thymus Gland / Q000201:enzymology
D019915:DNA Primase; D004256:DNA Polymerase I; D013110:Sphingosine