Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases

Abstract

Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases alpha and beta and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (K(i)=0.51 microM and K(i)=0.53 microM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby K(m) remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.

The (5Z)‐5‐Pentacosenoic and 5‐Pentacosynoic Acids Inhibit the HIV‐1 Reverse Transcriptase

“…Since RT inhibitors prevent the RNA-DNA conversion, a vital role of the viral life cycle, it is no wonder that more than half the currently approved HIV-1 antiretrovirals are RT inhibitors [21]. There are two classes of RT inhibitors, namely nucleoside/nucleotide retrotranscriptase inhibitors (NRTIs) and non-nucleoside retrotranscriptase inhibitors (NNRTIs) [15, 25-27]. Together they are the backbone of the highly active-antiretroviral therapy (HAART) regime [24], the standard treatment that usually combines two NRTIs with one NNRTI or with one protease inhibitor (PI) [14, 16, 24].…”

“…The HAART regime currently used is not potent enough to completely suppress virus replication to the point of eradication due to the development of drug-resistance [13, 25]. It is still necessary to develop new less cytotoxic antiretrovirals for the treatment of this disease.…”

Actinobacteria Associated with the Marine Sponges <i>Cinachyra</i> sp., <i>Petrosia</i> sp., and <i>Ulosa</i> sp. and Their Culturability

“…Petrosia sp. ( Demospongiae , Petrosiidae ), a common Mediterranean sponge, produces several bioactive polyacetylenes including acetylenic strongylodiols ( 20 , 21 , 35 ) and the steroid IPL-576,092, which is an antiasthmatic compound under clinical study ( 23 ). Ulosa sp.…”

Molecular Flexibility and Discontinuous Translocation of a Non-templated Polymerase

“…A, Defining the translocatory motif. Each series of 30mer oligonucleotides is numbered to the left(1)(2)(3)(4)(5) and separated by a horizontal crimson line. Each individual oligonucleotide is lettered (A -I).…”