AIDSWEEKLY Plus, Monday, 5 May 1997
Daniel J. DeNoon, Senior Editor
A new compound can block HIV replication by interfering with the RNA-binding activity of a viral regulatory protein known as Tat.
Because this is the first report of an antiviral drug that specifically targets a protein/RNA interaction, the discovery represents a novel pharmaceutical approach to antimicrobial and antitumor agents.
The compound, dubbed CGP64222, was created by a researchers at Novartis Ltd. Pharmaceuticals, Basel, Switzerland, and the Medical Research Council, Cambridge, England.
"CGP64222 is to our knowledge the first example of an antiviral compound that selectively inhibits a protein/RNA interaction," wrote Novartis researcher Francois Hamy and colleagues.
Hamy et al. reported their findings in the Proceedings of the National Academy of Sciences ("An Inhibitor of the Tat/TAR RNA Interaction That Effectively Suppresses HIV-1 Replication," PNAS, 1997;94:3548-53).
Tat is crucial to the HIV life cycle: after the virus has integrated itself into the host-cell genome, it needs the protein to achieve high-level replication. Without Tat, HIV remains virtually quiescent.
Tat acts by binding to a 59-nucleotide RNA loop called the trans-activation response element or TAR. This structure is well conserved among HIV-1 subtypes.
Hamy et al. began their search for a compound able to block the Tat/TAR interaction by creating what they called a combinatorial peptoid library.
"Peptoids are isomers of peptides in which all the side chains are carried by the backbone nitrogens (N-substituted glycines)," they explained. "Peptoids are more flexible than peptides because intramolecular CO-HN hydrogen bonds are removed and the steric interactions that induce secondary structure are different. For pharmacological applications, peptoids have the advantage of being stabilized after enzymatic degradation."
They began by deciding to limit their search to compounds with four constant C-terminal d-amino acid residues, the sequence d-Lys-d-Lys-d-Arg-d-Pro-amide. They then randomized five residue positions to contain one of 20 building blocks comprising a wide range of functional chemical groups.
For each of the five variable positions (labelled A through E), Hamy et al. determined which of the building blocks made the most effective side chains. When all of the optimal side chains had been discovered - a process that involved the division of 3.2 million compounds into 20 sublibraries of 160,000 compounds each - CGP64222 was the result.
"An aminohexyl residue was optimal at position A; Narg [N- 3-guanidopropylglycine] residues were optimal at positions B, C, and E, and an N-benzylglycine (Nphe) residue was the optimal residue at position D," Hamy et al. wrote.
Molecular modeling showed that CGP64222 induced a conformational change in TAR. The five essential residues of the compound bind the major groove of TAR RNA. This groove is the Tat binding site.
A series of in vitro experiments showed that:
* Nanomolar concentrations of CGP64222 could block the formation Tat/TAR RNA complexes.
* In a Tat-dependent transactivation assay, CGP64222 specifically inhibited Tat activity at 10-30 (micro)M concentrations.
* 30 (micro)M concentrations of CGP64222 completely suppressed the replication of HIV-1 (strain LAV) in primary human lymphocytes when added to cultures six hours after infection. The drug had no effect on lymphocyte viability or proliferation.
"We have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, both in vitro and in vivo," Hamy et al. concluded.
The authors suggested that their findings pave the way for an entirely new type of antimicrobial and antitumor agent.
"In addition to the Tat/TAR interaction, there are numerous viral and cellular processes that depend on critical protein/RNA interactions," they wrote. "As the chemistry for synthesizing small molecules capable of recognizing specific RNA sequences becomes more refined, RNA targets will find increasing importance in drug discovery."
The corresponding author for this study is Francois Hamy, Novartis Ltd. Pharmaceuticals, Pharma Research, CH-4002 Basel, Switzerland.
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