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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.

Affinity selection screening of macrocycle libraries derived from DNA-programmed chemistry identified XIAP BIR2 and BIR3 domain inhibitors that displace bound pro-apoptotic caspases. X-ray cocrystal structures of key compounds with XIAP BIR2 suggested potency-enhancing structural modifications. Optimization of dimeric macrocycles with similar affinity for both domains were potent pro-apoptotic agents in cancer cell lines and efficacious in shrinking tumors in a mouse xenograft model.

The prominent role of IAPs in controlling cell death and their overexpression in a variety of cancers has prompted the development of IAP antagonists as potential antitumor therapies. We describe the identification of a series of heterodimeric antagonists with highly potent antiproliferative activities in cIAP- and XIAP-dependent cell lines. Compounds 15 and 17 further demonstrate curative efficacy in human melanoma and lung cancer xenograft models and are promising candidates for advanced studies.

Cellular levels of inhibitor of apoptosis (IAP) proteins are elevated in multiple human cancers and their activities often play a part in promoting cancer cell survival by blocking apoptotic pathways, controlling signal transduction pathways and contributing to resistance. These proteins function through interactions of their BIR (baculoviral IAP repeat) protein domains with pathway components and these interactions are endogenously antagonized by Smac/Diablo (second mitochondrial activator of caspases/direct IAP binding protein with low isoelectric point). This report describes development of synthetic smac mimetics (SM) and compares their binding, antiproliferative and anti-tumor activities. All dimeric antagonists inhibit in vitro smac tetrapeptide binding to recombinant IAP proteins, rescue IAP-bound caspase-3 activity and show anti-proliferative activity against human A875 melanoma cells. One heterodimeric SM, SM3, binds tightly to IAP proteins in vitro and slowly dissociates (greater than two hours) from these protein complexes compared to the other antagonists. In addition, in vitro SM anti-proliferation potency is influenced by ABCB1 transporter (ATP-binding cassette, sub-family B; MDR1, P-gp) activities and one antagonist, SM5, does not appear to be an ABCB1 efflux pump substrate. All dimeric smac mimetics inhibit the growth of human melanoma A875 tumors implanted in athymic mice at well-tolerated doses. One antagonist, SM4, shows broad spectrum in vivo anti-tumor activity and modulates known pharmacodynamic markers of IAP antagonism. These data taken together demonstrate the range of diverse dimeric IAP antagonist activities and supports their potential as anticancer agents.

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.

A lambda phage clone containing a full-length HIV-2 provirus, designated HIV-2KR, was obtained from the genomic DNA of Molt4 clone 8 (Molt4/8) lymphoblastic cells infected with the HIV-2PEI2 strain. HIV-2KR is genetically distinct from known HIV-2 isolates, possessing both a unique deletion in the LTR promoter region, and a long rev reading frame. It is replication competent in vitro after transfection into Molt4/8 cells, replicates in a variety of established human T lymphoblastic (Molt-3, Molt4/8, SupT1, H9, C8166) and myelomonocytic (U937) cell lines, and displays prominent cytopathic effects on infection of Molt4/8 cells, reflecting usage of both CCR5 and CXCR4 coreceptors. In addition, HIV-2KR was found to be infectious for human and Macaca nemestrina peripheral blood lymphocytes, and primary human monocyte-macrophage cultures. Intravenous inoculation of cell-free virus into M. nemestrina resulted in infection characterized by transient, low-level viremia and modest temporary decline in CD4 lymphocyte numbers, making HIV-2KR the first HIV-2 molecular clone reported to be infectious for this primate species.

Heterotrimeric G proteins transduce multiple growth-factor-receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress-activated protein kinases. Herein we report on the identification of a novel p53 target gene (A28-RGS14) that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overexpression of A28-RGS14p protein inhibits both Gi- and Gq-coupled growth-factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mammalian cells. Thus, through the induction of A28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways.

TRANSCRIPTIONAL activation of target genes represents an important component of the tumour-suppressor function of p53 and provides a functional link between p53 and various growth-regulatory processes, including cell cycle progression (p21/WAF1)1a¤-3, DNA repair (GADD45)4 and apoptosis (bax)5. Here we use a differential cloning approach to identify the gene encoding insulin-like growth factor binding protein 3 (IGF-BP3) as a novel p53-regulated target gene. Induction of IGF-BP3 gene expression by wild-type but not mutant p53 is associated with enhanced secretion of an active form of IGF-BP3 capable of inhibiting mitogenic signalling by the insulin-like growth factor IGF-1. Our results indicate that IGF-BP3 may link p53 to potential novel autocrine/paracrine signalling pathways and to processes regulated by or dependent on IGF(s), such as cellular growth, transformation and survival.

Human immunodeficiency virus 2 (HIV-2) ISY and the newly derived HIV-2KR are infectious molecular clones that yield viruses differing markedly in their abilities to infect and/or induce syncytia in various T- and monocytoid-cell lines. Chimeric viruses were constructed from these two viral genomes to localize the genetic determinants of some of these properties. Envelope sequences, particularly those spanning the CD4 binding site, appear to be critical for the ability of HIV-2KR to infect MOLT-4 clone 8 and SupT1 cells and to efficiently infect the H9 cell line. On the other hand, multiple determinants may contribute to cytopathicity (gp41 and nef) in H9 cells and replication efficiency in monocytic (THP-1) cells.

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.

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.

An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells. The integrated provirus has a total length of 9472 base pairs. Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident. In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses. Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV. Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus.