The major route of metabolism of the bis(heteroaryl)piperazine (BHAP) class of reverse transcriptase inhibitors (RTIs), atevirdine and delavirdine, is via oxidative N-dealkylation of the 3-ethyl- or ...3-isopropylamino substituent on the pyridine ring. This metabolic pathway is also the predominant mode of metabolism of (alkylamino)piperidine BHAP analogs (AAP−BHAPs), compounds wherein a 4-(alkylamino)piperidine replaces the piperazine ring of the BHAPs. The novel AAP−BHAPs possess the ability to inhibit non-nucleoside reverse transcriptase inhibitor (NNRTI) resistant recombinant HIV-1 RT and NNRTI resistant variants of HIV-1. This report describes an approach to preventing this degradation which involves the replacement of the 3-ethyl- or 3-isopropylamino substituent with either a 3-tert-butylamino substituent or a 3-alkoxy substituent. The synthesis, bioactivity and metabolic stability of these analogs is described. The majority of analogs retain inhibitory activities in enzyme and cell culture assays. In general, a 3-ethoxy or 3-isopropoxy substituent on the pyridine ring, as in compounds 10, 20, or 21, resulted in enhanced stabilities. The 3-tert-butylamino substituent was somewhat beneficial in the AAP−BHAP series of analogs, but did not exert a significant effect in the BHAP series. Lastly, the nature of the indole substitution sometimes plays a significant role in metabolic stability, particularly in the BHAP series of analogs.
The multifunctional HIV-1 RT (human immunodeficiency virus type 1-reverse transcriptase) enzyme possesses three main functions
including the RNA- and DNA-directed DNA polymerases and the RNase H. The ...bisheteroarylpiperazine U-87201E inhibits the two
polymerase functions but not the RNase H. Enzymatic kinetic studies of the HIV-1 RT-catalyzed RNA- and DNA-directed DNA polymerase
activities were carried out in order to determine if the inhibitor interferes with either the template:primer or the deoxyribonucleotide
triphosphate (dNTP)-binding sites of the enzyme. The data were analyzed using steady-state kinetics, considering that the
polymerase reaction is ordered in that the template:primer is added first, followed by the dNTP and that the enzyme functions
processively. The data were consistent with the model. The steady-state rate constants for the forward and backward reactions
were of similar magnitude for both the RNA- and DNA-catalyzed DNA polymerases and suggest that both functions share the same
substrate-binding sites. The dissociation constants for the enzyme-inhibitor and enzyme-substrate-inhibitor complexes were
somewhat higher for the DNA-directed DNA polymerase function as compared to the RNA directed one. This indicates that U-87201E
is a more potent inhibitor for the RNA-directed DNA polymerase than the DNA-directed DNA polymerase. The pattern of inhibition
exerted by U-87201E was noncompetitive with respect to both the nucleic acid and nucleotide-binding sites of the RT enzyme
for both the RNA- and DNA-directed DNA polymerases. Hence, U-87201E inhibits these functions by interacting with a site distinct
from the template:primer and dNTP-binding sites. HIV-2 RT was insensitive to U-87201E, demonstrating the unique sensitivity
of HIV-1 RT to this inhibitor.
The antibiotic rubradirin is structurally related to the ansamycin family of antibiotics. Most members of this group act as specific inhibitors of bacterial RNAP*. Rubradirin and its aglycone possess ...diverging modes of action. Rubradirin inhibits ribosomal functions related to the peptide chain initiation process. It does not inhibit RNAP. By contrast the aglycone of rubradirin retains moderate inhibitory activity towards ribosomal functions but acts essentially as an extremely potent inhibitor of RNAP.
The two peptide-like antibiotics ficellomycin and feldamycin impair semiconservative DNA replication but not DNA repair synthesis in bacteria. Specifically both antibiotics cause the accumulation of ...a 34S DNA species in toluenized Escherichia coli cells which lacks the capability of being integrated into larger DNA pieces and eventually the complete bacterial chromosome. Novobiocin, a known inhibitor of replicative DNA synthesis, was investigated for comparative purposes. The action of this latter antibiotic differs from the ones exerted by ficellomycin and feldamycin in the novobiocin appears to block an event associated with the initiation of Okazaki fragments. The fact that novobiocin impairs DNA gyrase suggests that this enzyme plays an essential role during the initiation of Okazaki pieces.
The bis(heteroaryl)piperazine U-88204E is a potent inhibitor of HIV-1 reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. ...Enzymatic kinetic studies of the RNA- and DNA-dependent DNA polymerases of RT were carried out in order to determine whether the inhibitor interacts directly with the template:primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The experimental results were analyzed using steady-state or Briggs-Haldane kinetics, by assuming that the template:primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The results of the analysis show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template:primer and the dNTP binding sites. The potency of U-88204E on the RNA-directed DNA polymerase activity depends on the base composition of the template:primer. The Ki values for the poly(rC):(dG)10-directed reactions were at least 7 times lower than the ones for reactions directed by poly(rA):(dT)10. The inhibitor did not inhibit the RNase H function of HIV-1 RT nor did it impair the RNA-directed DNA polymerase activity of HIV-2 RT. These data thus demonstrate the unique specificity of U-88204E for HIV-1 RT.