The bacterial acquisition of aminoglycoside 3'-phosphotransferases [APH(3')s] is the leading mechanism for their resistance to aminoglycoside antibacterials. A multidisciplinary approach has been outlined for the study of these enzymes, which capitalizes on the earlier mechanistic findings from this laboratory.
Four specific aims are outlined.
Specific Aim 1 describes two molecules which are made up of an aminoglycoside linked covalently to a nucleotide via a variable length tether as probes of the mec hanism of APH(3')s. These molecules are expected to delineate whether the enzymic reaction proceeds by a dissociative or associative mechanism. Furthermore, these molecules are expected to be potent inhibitors for this family of enzymes.
Specific Aim 2 proposes to investigate in a series of nuclear magnetic resonance (NMR) experiments using 13C-labeled kanamycin A (an aminoglycoside), ATP and AMP, the solution structures for ATP and aminoglycoside bound in the active sites of these enzymes. These structures will be important for mechanistic reasons, and should help develop non-aminoglycoside analogs as inhibitors of these enzymes in the future. Three amino acids are identified as important sites for mutagenesis, and other sites are being identified for these experiments currently. Judiciously designed mutant variants of APH(3')s have been identified for study as the Specific Aim 3 to further our mechanistic and structural understanding of these important bacterial enzymes.
Specific Aim 4 centers on the development of a new class of aminoglycoside antibiotics which would serve as substrates for APH(3')s, but the product of phosphorylation is self-regenerating by the facile release of the phosphoryl group as inorganic phosphate. Therefore, the molec ules would hydrolyze ATP in a catalytic process driven by APH(3')s. Hence, the molecules are expected not to be inactivated by APH(3')s, and should prove to be antibacterial by either their binding to the bacterial ribosome (the normal mechanism of bacte rial death by aminoglycosides) or by in vivo depletion of the nucleotide triphosphate pool in the cytoplasm. Successful completion of these efforts, in conjunction with our earlier findings, would make the APH(3') the best studied members of the family of aminoglycoside-modifying enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI032245-06
Application #
2003755
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1992-02-01
Project End
2000-01-31
Budget Start
1997-02-01
Budget End
1998-01-31
Support Year
6
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202