Discovery of a Drug Lead Employing a Peptide Library: Inhibition of HIV-1 Tat and Viral Replication by the Tripeptide YPG-NH₂

Abstract

A library of the 8000 tripeptides derivable from coded amino acids was prepared in 20 sets of 400 using solid phase synthesis on a benzhydrylamine resin. The peptide mixtures, as C-terminal amides, were screened for inhibition of secreted alkaline phosphatase expression in a cellular (COS) system wherein a transfected SeAP gene construct was under control of the HIV-1 LTR promoter, activated by the product of a cotransfected HIV Tat gene construct. Thus, YPG-NH2 was discovered as an inhibitor of HIV-1 Tat function and then shown to block HIV replication in a CD4+ T-cell line (CEM) with IC50 = 35μM.

Synthesis and Antiviral Activity of Ethidium−Arginine Conjugates Directed Against the TAR RNA of HIV-1

“…With DNA, ethidium exhibits a slight preference for binding to pyrimidine(3‘-5‘)purine sites and particularly to CpG steps. − With different RNA hairpin segments, ethidium also binds preferentially to CpG sites proximal to a bulge. , More recent studies have revealed that, upon binding to TAR, ethidium intercalates near the unpaired A17 residue. , Molecular studies have shown that the amino groups at the 3 and 8 positions of ethidium are critical components in ethidium interactions with nucleic acids and that the geometry of the phenanthridine ring system is nearly ideal for maximizing favorable contacts with the nucleic acid bases that form the top and the bottom of the intercalation site − …”

“…36 Therefore, the development of agents which may interfere with the protein-RNA complex formation is a potential strategy for controlling the proliferation of HIV. [37][38][39][40][41][42][43][44][45][46][47][48][49] The capacity of ethidium to form stable complexes with RNA including TAR, on the one hand, and the fact that arginine and arginine derivatives bind to the pyrimidine bulge of TAR, on the other hand, prompted us to design bifunctional molecules capable of binding to both binding sites of TAR. For this purpose, we synthesized a series of molecules containing: (i) an arginine residue which would bind near the U23-C24-U25 bulge, (ii) an ethidium moiety which would intercalate between the base pairs C18-G44 and C19-G43, close to the A17 bulge, and (iii) a spacer arm connecting the two RNA-binding parts of the molecule (Chart 2).…”

Interactions of HIV-1 TAR RNA with Tat-Derived Peptides Discriminated by On-Line Acoustic Wave Detector

“…Given the occurrence of viral resistance against drugs targeting enzymes (e.g., reverse transcriptase and protease) and slow progress in the development of a vaccine, it is not surprising that the TAR−Tat system, in addition to other regulatory protein interactions, has attracted much drug discovery research . Indeed, recent times have seen the rational design of small molecules capable of blocking the binding of Tat to the TAR RNA. − A key issue with regard to the characterization of binding events between the RNA and Tat, peptides, and small molecules is the choice of a technique capable of signaling the appropriate chemistry. One such method is the gel mobility shift assay, which appears to constitute a widely used approach when it comes to the measurement of dissociation rates .…”