Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

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 ß phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h ß half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

Stabilizers of the Max homodimer identified in virtual ligand screening inhibit Myc function.

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.

Identification of novel mammalian growth regulatory factors by genome-scale quantitative image analysis.

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.

Transcriptional repression of MMP-1 by p21SNFT and reduced in vitro invasiveness of hepatocarcinoma cells.

p21SNFT (21 kDa small nuclear factor isolated from T cells) is a human basic leucine zipper transcription factor that can repress AP-1-mediated transcription. We show here that overexpression of p21SNFT in HepG2 cells leads to repression of matrix metalloproteinase-1 by 70-80%. p21SNFT interacted with Jun at the matrix metalloproteinase-1 promoter -88 Ets/AP-1 enhancer element, where Jun is known to activate transcription via interaction with Fos and Ets proteins. When p21SNFT/Jun dimers bound the element in the presence of Ets, DNA was protected differently than when Fos was paired with Jun. The data suggest a difference in overall conformation between p21SNFT-containing and Fos-containing complexes that may be involved in the repression of matrix metalloproteinase-1 by p21SNFT. Overexpression of p21SNFT led to a reduction in invasiveness of HepG2 cells through type I collagen and reconstituted basement membrane, an effect similar to that obtained via direct immunodepletion of matrix metalloproteinase-1. The results indicate that the mechanism of repression of matrix metalloproteinase-1 by p21SNFT may be exploited in inhibiting pathological matrix remodeling during cancer progression in vivo.

Inhibition of the proteolytic activity of anthrax lethal factor by aminoglycosides.

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.

Correlation of transcriptional repression by p21(SNFT) with changes in DNA.NF-AT complex interactions.

p21(SNFT) (21-kDa small nuclear factor isolated from T cells) is a novel human protein of the basic leucine zipper family. The overexpression of p21(SNFT) leads to the significant and specific repression of transcription from the interleukin-2 promoter as well as from several essential activator protein 1 (AP-1)-driven composite promoter elements. One example is the distal nuclear factor of activated T cells (NF-AT)/AP-1 element where the AP-1 (Fos/Jun) basic leucine zipper heterodimer interacts with members of the NF-AT family. p21(SNFT) has been shown to replace Fos in dimerization with Jun on a consensus AP-1 binding site (12-O-tetradecanolyphorbol-13-acetate response element (TRE)) and to interact with Jun and NF-AT at the distal NF-AT/AP-1 enhancer element. A detailed biochemical analysis presented here compares interactions involving p21(SNFT) with those involving Fos. The results demonstrate that a p21(SNFT)/Jun dimer binds a TRE similarly to AP-1 and like AP-1 binds cooperatively with NF-AT at the NF-AT/AP-1 composite element. However, Fos interacts significantly more efficiently than p21(SNFT) with Jun and NF-AT, and the replacement of Fos by p21(SNFT) in the trimolecular complex drastically alters protein-DNA contacts. The data suggest that p21(SNFT) may repress transcriptional activity by inducing a unique conformation in the transcription factor complex.