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Deborah J Lee

Publications and source records attributed to Deborah J Lee.

9 recordsLinked to original sources
PubMed2006

Design, synthesis, and biological evaluation of chicoric acid analogs as inhibitors of HIV-1 integrase.

A series of analogs of the potent HIV-1 integrase (HIV IN) inhibitor chicoric acid (CA) was designed with the intention of ameliorating some of the parent natural product's undesirable properties, in particular its toxicity, instability, and poor membrane permeability. More than 70 analogs were synthesized and assayed for three types of activity: (1) the ability to inhibit 3'-end processing and strand transfer reactions using recombinant HIV IN in vitro, (2) toxicity against the CD4+ lymphoblastoid cell line, MT2, and (3) anti-HIV activity against HIV(LAI). CA analogs lacking one of the carboxyl groups of CA and with 3,4,5-trihydroxycinnamoyl sidechains in place of the caffeoyl group of CA exhibited the most potent inhibition of HIV replication and end-processing activity. Galloyl-substituted derivatives also displayed very potent in vitro and in vivo activities, in most cases exceeding the inhibitory effects of CA itself. Conversely, analogous monocarboxy caffeoyl analogs exhibited only modest inhibition, while the corresponding 3,4-dihydroxybenzoyl-substituted compounds were devoid of activity.

Anti-HIV Agents↗
PubMed2006

Preliminary mapping of a putative inhibitor-binding pocket for human immunodeficiency virus type 1 integrase inhibitors.

Molecular modeling studies have identified a putative human immunodeficiency virus (HIV) integrase (IN) inhibitor-binding pocket for l-chicoric acid (l-CA) and other inhibitors of IN (C. A. Sotriffer, H. Ni, and A. McCammon, J. Med. Chem. 43:4109-4117, 2000). By using site-directed mutagenesis of several amino acid residues identified by modeling studies, a common inhibitor-binding pocket on IN was confirmed for l-CA and the diketo acid L-731,988. Specifically, the single mutations E92K, Q148A, K156A, K156R, G140S, and G149S, as well as the double mutations C65S-K156N and H67D-G140A were evaluated for their effects on enzymatic activity and inhibitor susceptibility. Each recombinant IN was attenuated for 3'-end processing and strand transfer activities. Most proteins were also attenuated for disintegration; the IN that contained K156R and C65S-K156N, however, displayed disintegration activity similar to that of IN from HIV(NL4-3). All mutant IN proteins demonstrated decreased susceptibility to l-CA, while all mutant proteins except E92K and K156R demonstrated resistance to L-731,988. These data validate the computer modeling data and demonstrate that l-CA and L-731,988 share an overlapping inhibitor-binding pocket that involves amino acids Q148, C65, and H67. The resistance studies confirm that L-731,988 fills one-half of the inhibitor-binding pocket and binds to Q148 but excludes E92, while l-CA fills the entire binding groove and thus interacts with E92. These results provide "wet laboratory" evidence that molecular models of the HIV IN inhibitor-binding pocket can be used for drug discovery.

Binding Sites↗
PubMed2004

L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro.

The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.

Acetoacetates↗
PubMed2004

Human immunodeficiency virus type 1 (HIV-1) integrase: resistance to diketo acid integrase inhibitors impairs HIV-1 replication and integration and confers cross-resistance to L-chicoric acid.

The diketo acids are potent inhibitors of human immunodeficiency virus (HIV) integrase (IN). Mutations in IN, T66I, S153Y, and M154I, as well as T66I-S153Y and T66I-M154I double mutations, confer resistance to diketo acids (D. J. Hazuda et al., Science 287:646-650, 2000). The effects of these IN mutations on viral replication, enzymatic activity, and susceptibility to other HIV inhibitors are reported herein. By immunofluorescence assay and real-time PCR, all mutant viruses demonstrated a modest delay in viral spread compared to that of reference HIV. These viruses also showed a statistically significant defect in integration without defects in reverse transcription. Recombinant IN containing S153Y, T66I, and M154I-T66I mutations had an approximately twofold decrease in both disintegration and 3'-end-processing-strand transfer activities in vitro. In contrast, IN containing M154I demonstrated a greater than twofold increase in specific activity in both reactions. All mutant HIVs were resistant to l-chicoric acid, a dicaffeoyltartaric acid IN inhibitor, both in tissue culture and in biochemical assays, yet remained susceptible to the reverse transcriptase inhibitors zidovudine and nevirapine. Thus, IN mutations conferring resistance to the diketo acids can yield integration defects, attenuated catalysis in vitro, and cross-resistance to l-chicoric acid.

Caffeic Acids↗
PubMed2003

Human immunodeficiency virus type-1 integrase containing a glycine to serine mutation at position 140 is attenuated for catalysis and resistant to integrase inhibitors.

L-chicoric acid (L-CA) is a potent inhibitor of HIV integrase (IN) in vitro. In this report, the effects of a glycine to serine mutation at position 140 (G140S) on HIV IN and its effects on IN inhibitor resistance are described. HIV containing the G140S mutation showed a delay in replication. Using real-time polymerase chain reaction, the delay was secondary to a failure in integration. The mutant protein (IN(G140S)) was attenuated approximately four-fold for catalysis under equilibrium conditions compared to wild-type IN (IN(WT)) and attenuated five-fold in steady-state kinetic analysis of disintegration. Fifty percent inhibitory concentration assays were performed with IN inhibitors against both IN proteins in disintegration and strand transfer reactions. IN(G140S) was resistant to both L-CA and L-731,988, a diketoacid. HIV containing the mutation was resistant to both inhibitors as well. The G140S mutation attenuates IN activity and confers resistance to IN inhibitors, suggesting that diketoacids and L-CA interact with a similar binding site on HIV IN.

Acetoacetates↗
PubMed2003

Replication kinetics for divergent type 1 human immunodeficiency viruses using quantitative SYBR green I real-time polymerase chain reaction.

A quantitative and sensitive measure of human immunodeficiency virus type 1 (HIV-1) replication is quantitative real-time polymerase chain reaction (PCR). Real-time PCR using SYBR green I and oligonucleotide primers that amplify early, intermediate, and late products of reverse transcription were optimized to measure HIV-1 replication of clade A, B, C, and D HIV-1 isolates in peripheral blood lymphocytes and in both transformed and viral-transformed CD4+ lymphocyte cell lines. Real-time PCR can detect HIV-1 replication as early as 1 hr postinfection and demonstrates that in established cell lines cDNA can be detected as early as 4 hr postinfection. The first round of HIV-1 replication in established cell lines is complete between 12 and 24 hr postinfection. Furthermore, real-time PCR can detect HIV-1 replication in fewer than 0.1% of cells. Patient isolates replicated at different rates in peripheral blood lymphocytes, with viral cDNA peaking between 48 and 120 hr, depending on the virus being studied. Real-time PCR differentiated the mechanisms of action of drugs targeted at HIV-1 entry, reverse transcription, and proteolytic processing and identified differences in the kinetics of reverse transcription between zidovudine-sensitive and zidovudine-resistant HIV in the presence of zidovudine. In summary, real-time PCR using SYBR green I dye is a sensitive, quantitative, and reproducible measure of replication kinetics for a variety of group M HIV-1 isolates.

Benzothiazoles↗
PubMed2002

Inhibition of human immunodeficiency virus type 1 isolates by the integrase inhibitor L-731,988, a diketo Acid.

L-731,988 inhibits human immunodeficiency virus (HIV) replication through integrase. In this study, approximately 600 nM L-731,988 inhibited the replication of 12 HIV type 1 isolates from multiple clades, including primary isolates and cloned viruses. These data suggest that diketo acids or their derivatives may prove useful on a worldwide basis in treating HIV infection.

Acetoacetates↗