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Charles E. McKenna

Professor of Chemistry
Organic and Bioorganic Chemistry

Ph.D., 1971, University of California at San Diego
B.A., 1966, Oakland University
Office: LJS 268
Phone: (213) 740-7007
Fax: (213) 740-0930
Email: mckenna@usc.edu
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Research Focus

 

Synthesis of Bisphosphonates: Drugs and Imaging Probes

The complex formed from crystallization of human farnesyl pyrophosphate synthase (hFPPS) from a solution of racemic [6,7-dihydro-5H-cyclopental[c]pyridin-7-yl(hydroxy)methylene]bis( phosphonic acid) (NE-10501, 8), a chiral analog of the anti-osteoporotic drug risedronate, contained the R enantionmer in the enzyme active site. This enantiospecificity was assessed by computer modeling of inhibitor-active site interactions using Autodock 3. The compound 8 complex exhibited only one Mg2+ (vs. 2 typically found with similar inhibitors), which could contribute to its 100-fold higher IC50. An improved synthesis of 8 is described, which decreases the number of steps from 12 to 8 and increases the overall yield by 17-fold.

We report the synthesis of the first fluorescently labeled conjugates of risedronate (1), using an epoxide linker strategy enabling conjugation of 1 via its pyridyl nitrogen with the label (carboxyfluorescein). Unlike prior approaches to create fluorescent bisphosphonate probes, the new linking chemistry did not abolish the ability to inhibit protein prenylation in vitro, while significantly retaining hydroxyapatite affinity. The utility of a fluorescent 1 conjugate in visualizing osteoclast resorption in vitro was demonstrated.


Nucleotide Analogues: A "Toolkit" to Study DNA Polymerase

The mechanism of DNA polymerase -catalyzed nucleotidyl transfer consists of chemical steps involving primer 3'OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the - bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.


Synthesis of Peptidomemitic Prodrugs of Cidofovir

Cidofovir (HPMPC, 1), a broad-spectrum antiviral agent, is currently used to treat AIDS-related human cytomegalovirus (HCMV) retinitis and has recognized therapeutic potential for orthopox virus infections, but is limited by its low oral bioavailability. Cyclic cidofovir (2) displays decreased nephrotoxicity compared to 1, while also exhibiting potent antiviral activity. Here we describe in detail the synthesis and evaluation as prodrugs of four cHPMPC dipeptide conjugates in which the free POH of 2 is esterified by the Ser side chain alcohol group of an X-L-Ser(OMe) dipeptide: 3 (X = L-Ala), 4 (X = L-Val), 5 (X = L-Leu), and 6 (X = L-Phe). Perfusion studies in the rat establish that the mesenteric permeability to 4 is more than 20-fold greater than to 1, and the bioavailability of 4 is increased 6-fold relative to 1 in an in vivo murine model. In gastrointestinal and liver homogenates, the cHPMPC prodrugs are rapidly hydrolyzed to 2. Prodrugs 3, 4, and 5 are nontoxic at 100 µM in HFF and KB cells and in cell-based plaque reduction assays had IC50 values of 0.1-0.5 µM for HCMV and 10 µM for two orthopox viruses (vaccinia and cowpox). The enhanced transport properties of 3-6, conferred by incorporation of a biologically benign dipeptide moiety, and the facile cleavage of the Ser-O-P linkage suggest that these prodrugs represent a promising new approach to enhancing the bioavailability of 2.

When the X-L-Ser(OMe) dipeptide is conjugated with 2, a new chiral center is formed at the phosphorus atom resulting in two diastereomers. They can be detected individually by 31P NMR and separated by RP-HPLC. Geometry optimization using a 3-21G* basis set on Spartan '02 predicts a substantial difference in polarity between the two diastereomers (5.72 D vs 13.17 D). As the diastereomers are presumably separated on the C-18 HPLC column due to their differing hydrophobicity, the less hydrophobic compound is likely the R isomer, which is calculated to have a larger dipole moment than the S isomer (the slower eluting compound). It will be of future interest to determine whether this significant difference in ground state polarities is reflected in the relative rates of enzyme-mediated activation processes.


Selected publications

 

 

Chemistry Dept., USC College of Letters, Arts & Sciences