Peter Kretschmer

Species selectivity of a small molecule antagonist for the CCR1 chemokine receptor.

The species specificity of a small molecule antagonist for the human CCR1 chemokine receptor, 2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (CCR1 antagonist 1), has been examined using cloned CCR1 receptors from various species. The compound was able to bind to rabbit, marmoset, and human CCR1, and was able to block the functional activation of these receptors. However, it failed to significantly displace radiolabeled macrophage inflammatory protein-1alpha (MIP-1alpha) binding to mouse CCR1 at concentrations up to 10 microM. These data suggested that the antagonist binding site is well-conserved in rabbit, marmoset and human CCR1, but not in mouse CCR1. The functional selectivity and mechanism of action for CCR1 antagonist 1 were further characterized. CCR1 antagonist 1 blocked the increase in intracellular Ca(2+) stimulated by CCR1 agonists, but had no effect on N-formyl-Met-Leu-Phe (FMLP), monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor 1alpha (SDF1alpha)-induced Ca(2+) mobilization, demonstrating functional selectivity for CCR1. Since CCR1 antagonist 1 is a functional antagonist of marmoset and rabbit CCR1 receptors, it should be possible to test its efficacy in animal models of disease.

Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models

An important negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation. FVIII molecules with stabilized activity have been generated by elimination of charged residues at the A1-A2 and A2-A3 interfaces. These molecules exhibited reduced decay rates as part of the enzymatic factor Xa generation complex and retained their activities under thermal and chemical denaturing conditions. We describe here the potency and efficacy of 1 such stability variant, D519V/E665V, derived from B domain-deleted FVIII (BDD-FVIII). The major effect of A2 stabilization was on cofactor activity. D519V/E665V potency was increased twofold by the 2-stage chromogenic assay relative to BDD-FVIII. D519V/E665V demonstrated enhanced thrombin generation responses (fivefold by peak thrombin) relative to BDD-FVIII. In vivo consequences of enhanced cofactor activity of D519V/E665V included >fourfold increased maximal platelet-fibrin deposition after laser injury and twofold increased protection from bleeding in acute and prolonged vascular injury model in hemophilia A mice. These results demonstrate that noncovalent stabilization of the FVIII A2 subunit can prolong its cofactor activity, leading to differential enhancement in clot formation over protection from blood loss in hemophilia. The FVIII molecule described here is the first molecule with clear efficacy enhancement resulting from noncovalent stabilization of the A2 domain.

Abstract A74: Estrogen receptor α‐regulated growth and transcription is dependent on X‐box binding protein‐1 in breast cancer cells

This study was designed to investigate the impact of X‐Box Binding protein 1 (Xbp1) mRNA knockdown on the estrogen‐responsive growth and gene expression in an estrogen receptor α (ERα) positive breast cancer cell line model. Xbp1 is a basic leucine zipper‐containing transcription factor that plays a key role in the unfolded protein response (UPR), a cell program that is activated by unfolded or misfolded proteins in the endoplasmic reticulum. MCF7 cells, a model of ERα positive breast cancer, express high levels of Xbp1 and are dependent on estrogen for growth and ERα regulated transcription. Previous studies have revealed that further overexpression of Xbp1 in ERα positive breast cancer cells leads to estrogen independent ERα regulated transcription and anti‐estrogen resistance, suggesting that Xbp1 expression influences ERα regulated transcription. Unknown is whether ERα requires Xbp1 as a cofactor for transcription. Here we show that Xbp1 mRNA knockdown by specific siRNAs prevents estrogen responsive growth of MCF7 cells as well as ERα regulated gene expression, while non‐specific control siRNAs had little effect. These results, together with the high expression of Xbp1 seen in ERα positive breast cancers, but not in normal mammary ductal cells, suggests that high levels of Xbp1 enable ERα tumor cells to use estrogen as a growth factor. Thus, high levels of Xbp1 expression could represent an early transformation step leading to ERα positive breast cancer, and could explain why normal ERα positive breast ductal cells have not been established as cell lines. Citation Information: Cancer Res 2009;69(23 Suppl):A74.

21. Application of Splice Acceptor Transposons to the Development of Oncolytic Viruses

Oncolytic viruses tested to date in the clinic have shown marked safety but minimal potency as monotherapies. This has prompted the development of armed therapeutic viruses where therapeutic genes that complement the lytic functions of the virus are used to enhance the potency of the oncolytic virus. A technology that can scan entire viral genomes to identify transgene insertion sites that are compatible with viral replication, however, is needed. To address this need, we have previously developed a novel transposon-based technology that enables random scanning of the Ad genome for replication-compatible therapeutic transgene insertion sites using a transposon containing a GFP gene expressed by an exogenous promoter to validate this technology.