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XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.

We have used an antisynthetic peptide antiserum to a murine recombinant virus gp70 to probe normal mouse tissues for immunologically related proteins. In addition to cognate gp70s, this antiserum reacts with the heterogeneous nuclear ribonucleoparticle protein A1 by virtue of a 5-amino acid epitope, PRNQG. Further structural similarity is evident both 5' and 3' of this epitope. Since the function of the heterogeneous nuclear ribonucleoprotein particles in the cell is to aid in the stabilization and processing of newly synthesized RNA, we have investigated whether this retroviral sequence exhibits any nucleic acid-binding properties by the same criteria established for the identification of heterogeneous nuclear ribonucleoprotein particles. Analysis of the peptide in a poly(eA) binding assay shows this retroviral sequence to bind with high affinity to single-stranded nucleic acid. This binding occurs in a salt-sensitive manner characteristic of single-stranded nucleic acid-binding proteins. Flanking peptides not containing this sequence generated from either the A1 or gp70 show no ability to bind single-stranded nucleic acids by this assay.

A full-length infectious molecular clone, designated HIV-K, was obtained from a recombinant bacteriophage library constructed from HI-2PIE-infected MOLT-4/8 genomic DNA. Nucleotide sequence analysis of this clone demonstrated that the rev coding region extends for nearly 69 amino acid residues past the end of most other HIV-2/SIV rev genes. In addition, the long terminal repeat (LTR) contains a deletion of 9-10 bp, depending upon the nucleotide sequence alignment parameters, upstream from the SpI binding sites. This deletion is not present in other HIV-2/SIV isolates and is not similar to previously disclosed NFkB duplications in this region. The HIV-2KR LTR displays higher levels of basal transcriptional acitivty as compared to prototypical HIV-2 isolates. HIV-2KR is capable of infecting macaque peripheral blood lymphocytes (PBMCs) in vitro and produces a productive and persistant, albeit attenuated, infection in Macacca nemestrina. These proviral constructs are useful, inter alia, for the generation of in vitro diagnostic reagents, cell transcuction vectors, and the generation of HIV-2 based packaging cell lines.