Novel aminoglycosides for drug development
Currently, the fight against HIV essentially relies on multitherapies targeting two out of the three viral enzymes, namely the reverse transcriptase and the protease. However, the high mutation and recombination rates of HIV, together with its high replication level, allows an easy selection of drug-resistant viruses, thus reinforcing the urgent need for targeting new viral molecules required for HIV-1 replication. As for all retroviruses, HIV-1 encapsidate their genome as a dimer of two single stranded RNAs that are non-covalently linked close to their 5´-ends (scheme 1). This dimerization is initiated by a highly conserved stem-loop located in the 5´-untranslated region (UTR) of the viral genome called Dimerization Initiation Site (DIS). It has been shown that alteration of the DIS affects dimerization and, in turn, several important steps of the viral replication, such as encapsidation and reverse transcription, resulting in a dramatic loss of viral infectivity. The DIS initiates the genome dimerization by forming a loop-loop complex (also called ‘‘kissing complex’’). It was shown that, in vitro, the DIS kissing-complex is subsequently converted into a more stable extended duplex by the viral NCp7 nucleocapsid protein. However, its presence in vivo is not assessed.
Scheme 1. (a) Schematic secondary structure of the 5’-untranslated region of HIV-1 RNA and proposed dimerization mechanism. The DIS sequence is shown in blue. (b) Sequence and secondary structure of the HIV-1 subtype A 23 nucleotide fragment used in structural studies. The six nucleotide self-complementary sequence is bolded. Sequence modifications found in subtypes B and F are depicted with black and white boxes respectively.
The most striking feature of the DIS “kissing complex” structure was revealed by X-ray structures. Indeed, crystal structures showed very surprising and unexpected similarities between the DIS “kissing complex” and the bacterial 16 S RNA ribosomal A site, which is targeted by aminoglycoside antibiotics. Our data, including high-resolution crystal structures of several DIS/drug complexes, demonstrate that aminoglycosides tightly bind the DIS “kissing complex” with significantly higher affinity in comparison to their natural target, the ribosomal A site. Importantly, binding was observed in vitro, as well as ex vivo on the complete viral genome in viral particles or in HIV-infected human cells. Recent in vitro results have shown that aminoglycoside binding induces a strong stabilization of the DIS dimer, thus interfering with maturation of the viral RNA dimer and with reverse transcription.
Currently, we have designed two approaches for interfering with HIV replication cycle. The first approach was devoted to the use of dimeric aminoglycosides, designed from DIS X ray structures. The second one, that started very recently and is absolutely without precedent in the literature, relies upon on the utilization of the DIS-DIS kissing complex as template for click reaction.
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