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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.

A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue 11, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimately cell apoptosis, inhibits growth of human melanoma and colorectal cell lines at low nanomolar concentrations. Furthermore, compound 11 demonstrated significant antitumor activity in the A875 human melanoma xenograft model at doses as low as 2 mg/kg on a q3d schedule.

Bivalent heterodimeric IAP antagonists that incorporate (R)-tetrahydroisoquinoline in the P3′ subunit show high affinity for the BIR2 domain and demonstrated potent IAP inhibitory activities in biochemical and cellular assays. Potent in vivo efficacy was observed in a variety of human tumor xenograft models. The bivalent heterodimeric molecule 3 with a P3–P3′ benzamide linker induced pharmacodynamic markers of apoptosis and was efficacious when administered intravenously at a dose of 1 mg/kg to mice harboring A875 human melanoma tumors. Analog 5, with a polyamine group incorporated at the P2′ thiovaline side chain exhibited antiproliferative activity against the P-gp expressing HCT116/VM46 cell line.

Exposure of mammalian cells to hypoxia, radiation and certain chemotherapeutic agents promotes cell cycle arrest and/or apoptosis. Activation of p53 responsive genes is believed to play an important role in mediating such responses. In this study we identified a novel gene, PA26, which maps to chromosome 6q21 and encodes at least three transcript isoforms, of which two are differentially induced by genotoxic stress (UV, gamma-irradiation and cytotoxic drugs) in a p53-dependent manner. A functional p53-responsive element was identified in the second intron of the PA26 gene, in consistance with a mechanism of transcriptional induction of the PA26 gene by p53. No clues to its functions were revealed by sequence analysis, although pronounced negative regulation by serum factors argues for a potential role of PA26 in growth regulation. Immunological analysis suggests that PA26 protein(s) is localized to the cell nucleus. Our results suggest that the PA26 gene is a novel p53 target gene with properties common to the GADD family of growth arrest and DNA damage-inducible stress-response genes, and, thus, a potential novel regulator of cellular growth.

The ability of the p53 protein to act as a sequence-specific transcriptional activator suggests that genes induced by p53 may encode critical mediators of p53 tumor suppression. Using a tetracycline-regulated p53 expression system and cDNA library subtraction procedure, we identified several p53-induced gene transcripts in human Saos-2 osteosarcoma cells that are novel on the basis of their size, regulation, and low abundance. Wild-type p53-dependent induction of these transcripts was observed in cells that are growth arrested by p53, as well as in cells that undergo apoptosis upon expression of an inducible wild-type p53 transgene. These results show that p53 activates the expression of numerous response genes and suggest that multiple effectors may play a role in mediating cellular functions of p53.

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.

Two molecular clones of feline immunodeficiency virus were compared. The first clone, 34TF10, was from a Petaluma, Calif., isolate; the second, PPR, was isolated from a cat in the San Diego, Calif., area. The cats from which the isolates were obtained suffered from chronic debilitating illnesses. The two molecular clones differed in their in vitro host cell range. The 34TF10 clone infected the Crandall feline kidney and G355-5 cell lines, but replicated less efficiently on feline peripheral blood leukocytes. In contrast, the PPR clone productively infected the primary feline peripheral blood leukocytes but not Crandall feline kidney or G355-5 cells. The 34TF10 and PPR clones had an overall sequence identity of 91%. The env gene was the least conserved (85% at the amino acid level). Additionally, the potential open reading frame for a Tat-like protein, ORF 2, contained a stop codon in the 34TF10 isolate which was not found in the PPR clone. This truncation did not prevent in vitro or in vivo replication of 34TF10. Two splice acceptor sites were identified in the 34TF10 clone. One was 5' to the beginning of the putative tat open reading frame, and the other was 5' to the putative vif product. Both of these acceptor sites were conserved in the PPR clone. The long terminal repeats of the viruses were 7% divergent between the two clones, with a lack of conservation in putative NF-kappa B, LBP-1, and CCAAT enhancer-promoter sites.

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.