Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 μM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a β phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h β half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.

A18 CVX-045, the product of a chemical fusion of a recombinant humanized monoclonal antibody and two anti-angiogenic peptides, is the first in a unique class of fusion proteins. In single dose pharmacokinetic studies, the elimination half-life of CVX-045 increased as test species increased in size. CVX-045 had the longest half-life (T½) in monkeys (80 hr) followed by the rat (65 hr) and the mouse (50 hr). Anti-tumor activity of CVX-045 was evaluated in A549, A431, and HT-29 human xenograft models. Cells were implanted subcutaneously in female nu/nu mice, and tumors were staged to 300-400 mm3 prior to initiation of weekly treatments. CVX-045 (10 mg/kg) significantly reduced A549 and A43I tumor growth 73% (day 49) and 51 % (day 22), respectively, but was not effective in the HT-29 xenograft (10 or 30 mg/kg). CVX-045 demonstrated significant anti-angiogenic activity, reducing tumor microvessel density 51% in A549, 49% in A431, and 36% in HT-29 xenografts. Co-treatment with CVX-045 (30 mg/kg) plus oral sunitinib (15 mg/kg) decreased HT-29 tumor growth rate significantly (p

Potent library. Anthrax lethal factor (LF) is a zinc-dependent metalloprotease involved in the rapid development of the deadly infection caused by Bacillus anthracis. Blocking its action is a plausible method to mitigate the deleterious effects of late stage infection. We report the inhibition of LF by tetrahydro-isoquinoline polyphenolic compounds, such as 5 a, which were identified by screening a combinatorial library that was generated by Pictet–Spengler reaction. We also report the identification of commercially available polyphenolic inhibitors against LF. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2005/z500009_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Functional profiling technologies using arrayed collections of genome-scale siRNA and cDNA arrayed libraries enable the comprehensive global analysis of gene function. However, the current repertoire of high-throughput detection methodologies has limited the scope of cellular phenotypes that can be studied. In this report, we describe the systematic identification of mammalian growth-regulatory factors achieved through the integration of automated microscopy, pattern recognition analysis, and cell-based functional genomics. The effects of 7364 human and mouse proteins, encoded by individually arrayed cDNAs, upon proliferation and viability in U2OS osteosarcoma cells were evaluated in a live-cell, kinetic assay using quantitative image analysis. Overexpression of more than 86 cDNAs (1.15%) conferred dramatic increases in the proliferation, as determined cell enumeration. These included several known growth regulators, as well as previously uncharacterized ones (LRRK1, Ankrd25). In addition, novel functional roles for two genes (5033414D02Rik, 2810429O05Rik), now termed Gatp1 and Gatp2, respectively, were identified. Further analysis demonstrated that these encoded proteins promoted cellular proliferation and transformation in primary cells. Conversely, cells depleted for Gatp1 underwent apoptosis upon serum reduction, suggesting that Gatp1 is essential for cell survival under growth-factor-restricted conditions. Taken together, our findings offer new insight into the regulation of cellular growth and proliferation, and demonstrate the value and feasibility of assessing cellular phenotypes through genome-level computational image analysis.

The anthrax lethal factor (LF), a Zn-dependent endopeptidase, is considered the dominant virulence factor of anthrax. Because pharmacological inhibition of the catalytic activity of LF is considered a plausible mechanism for preventing the lethality of anthrax, a high-throughput screening experiment based on LF-catalyzed cleavage of a fluorescent substrate was performed to identify novel inhibitors of LF. The RNA-targeting antibiotics, neomycin B and some synthetic dimeric aminoglycosides, were found to be nanomolar active-site inhibitors of LF.