Inhibition of the Human Immunodeficiency Virus Type 1 Integrase by Guanosine Quartet Structures

Abstract

An oligonucleotide (T30177) composed entirely of deoxyguanosine and thymidine has previously been shown to fold upon itself in the presence of potassium into a highly stable four-stranded DNA structure containing two stacked deoxyguanosine quartets (G4s). T30177 also protects host cells from the cytopathic effects of human immunodeficiency virus type 1 (HIV-1). We report that this G4 oligonucleotide is the most potent inhibitor of HIV-1 integrase identified to date, with IC50 values in the nanomolar range. Both the number of quartets formed and the sequence of the loops between the quartets are important for optimal activity. T30177 binds to HIV-1 integrase without being processed and blocks the binding of the normal viral DNA substrate to the enzyme. The normal DNA substrate was not able to compete off T30177 binding to HIV-1 integrase, indicating a tight binding of G4s to the enzyme. Experiments with truncated HIV-1 integrases indicate that the N-terminal region containing a putative zinc finger is required for inhibition by T30177 and that T30177 binds better to full-length or deletion mutant integrases containing the zinc finger region than to a deletion mutant consisting of only the central catalytic domain. The N-terminal region of integrase alone is able to bind efficiently to T30177, but not the linear viral DNA substrate, in the presence of zinc. Hence, G4s represent the first class of compounds that inhibit HIV-1 integrase by interacting with the enzyme N-terminal domain. The greater inhibitory potency of T30177 in buffer containing magnesium versus manganese suggests that divalent metal ion coordination along the phosphodiester backbone may play a role in the inhibitory activity. T30177 inhibited HIV-2 integrase with similar potency as HIV-1 but inhibited feline and simian immunodeficiency virus integrases at higher concentrations, suggesting selectivity can be achieved. We propose that novel AIDS therapies could be based upon guanosine quarters as inhibitors of HIV-1 integrase.

G-Quartet Oligonucleotides

“…Potassium with the optimal size to interact within a G-octamer greatly promotes the formation of G-quartet structures and increases their stability. G-quartet oligodeoxynucleotides (GQ-ODNs) have been suggested to play a critical role in several biological processes including modulation of telomere activity (30), inhibition of human thrombin (31), HIV infection (32), HIV-1 integrase activity (33)(34)(35), human nuclear topoisomerase 1 activity (36), and DNA replication in vitro (37). On the basis of the structure and mechanism of Stat3 activation, G-quartet-forming oligonucleotides were developed recently to block Stat3 activity within cancer cells (27).…”

An Inhibitory Monoclonal Antibody Binds at the Turn of the Helix-Turn-Helix Motif in the N-terminal Domain of HIV-1 Integrase

“…Some of the compounds bind to the catalytic core domain (28 -31); the binding sites of others have not been identified. However, because they are essential for activity, both the N-terminal and the C-terminal domains of the HIV-1 IN may be considered as possible targets for anti-AIDS drug design (32,33).…”

“…Nevertheless, these results indicate that binding of mAb17 or Fab17 increases the solubility of HIV-1 integrase. [22][23][24][25][26][27][28][29][30][31][32][33][34][35]. To map the binding region more precisely, and to investigate whether any other structural alterations could be detected as a consequence of binding, we developed a protein footprint technique using controlled proteolysis of the G t -IN(1-49)-Fab17 complex, combined with peptide mapping by MALDI-TOF mass spectrometry.…”

“…One type of inhibitor, guanosine quartets, was found to require the N-terminal domain for binding (33). The recognition site of mAb17 may represent a novel target in this domain for the development of inhibitory drugs.…”

Synthesis of 8‐Aza‐3‐deazaisoguanosine by a Novel Ring Closure of Dinitriles by Sodium Alkoxides.

“…8 G4 or isoG4 containing structures may act as negative regulators of telomerase elongation in vivo due to their ability to inhibit telomerase in vitro 9 and it was demonstrated that such ODNs are potent inhibitors of HIV-1 integrase. 10 Seela et al 11 have recently established that the isoG quartet is generally more stable than the G-quartet on the first defined tetraplex species; however, structural changes of a heterocyclic base resulted in somewhat lower stability for 7-deazaisoguanine quartet. 11b…”