Alexander Doemling

Two Strategies for the Development of Mitochondrion-Targeted Small Molecule Radiation Damage Mitigators

PURPOSE To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondrion-targeted small molecules. METHODS AND MATERIALS We evaluated the ability of nitroxide-linked alkene peptide isostere JP4-039, the nitric oxide synthase inhibitor-linked alkene peptide esostere MCF201-89, and the p53/mdm2/mdm4 protein complex inhibitor BEB55 to mitigate radiation effects by clonogenic survival curves with the murine hematopoietic progenitor cell line 32D cl 3 and the human bone marrow stromal (KM101) and pulmonary epithelial (IB3) cell lines. The p53-dependent mechanism of action was tested with p53(+/+) and p53(-/-) murine bone marrow stromal cell lines. C57BL/6 NHsd female mice were injected i.p. with JP4-039, MCF201-89, or BEB55 individually or in combination, after receiving 9.5 Gy total body irradiation (TBI). RESULTS Each drug, JP4-039, MCF201-89, or BEB55, individually or as a mixture of all three compounds increased the survival of 32D cl 3 (p = 0.0021, p = 0.0011, p = 0.0038, and p = 0.0073, respectively) and IB3 cells (p = 0.0193, p = 0.0452, p = 0.0017, and p = 0.0019, respectively) significantly relative to that of control irradiated cells. KM101 cells were protected by individual drugs (p = 0.0007, p = 0.0235, p = 0.0044, respectively). JP4-039 and MCF201-89 increased irradiation survival of both p53(+/+) (p = 0.0396 and p = 0.0071, respectively) and p53(-/-) cells (p = 0.0007 and p = 0.0188, respectively), while BEB55 was ineffective with p53(-/-) cells. Drugs administered individually or as a mixtures of all three after TBI significantly increased mouse survival (p = 0.0234, 0.0009, 0.0052, and 0.0167, respectively). CONCLUSION Mitochondrial targeting of small molecule radiation mitigators decreases irradiation-induced cell death in vitro and prolongs survival of lethally irradiated mice.

NMR‐Based Screening of Membrane Protein Ligands

Membrane proteins pose problems for the application of NMR‐based ligand‐screening methods because of the need to maintain the proteins in a membrane mimetic environment such as detergent micelles: they add to the molecular weight of the protein, increase the viscosity of the solution, interact with ligands non‐specifically, overlap with protein signals, modulate protein dynamics and conformational exchange and compromise sensitivity by adding highly intense background signals. In this article, we discuss the special considerations arising from these problems when conducting NMR‐based ligand‐binding studies with membrane proteins. While the use of 13C and 15N isotopes is becoming increasingly feasible, 19F and 1H NMR‐based approaches are currently the most widely explored. By using suitable NMR parameter selection schemes independent of or exploiting the presence of detergent, 1H‐based approaches require least effort in sample preparation because of the high sensitivity and natural abundance of 1H in both, ligand and protein. On the other hand, the 19F nucleus provides an ideal NMR probe because of its similarly high sensitivity to that of 1H and the lack of natural 19F background in biologic systems. Despite its potential, the use of NMR spectroscopy is highly underdeveloped in the area of drug discovery for membrane proteins.

Identification of cancer-specific COPI inhibitors and their associated apoptotic cell death pathways

The COPI complex is a heptameric complex involved in multiple aspects of intravesicular transport. Recently, Shtutman et al. identified the ζ1 subunit of the COPI complex as a potential target for cancer therapy1. The ζ subunit of COPI is coded for by two genes, COPZ1 and COPZ2. In normal tissue, both COPZ genes are expressed and the protein products are functional isoforms within the COPI complex. In numerous different cancers, COPZ2 is silenced and the cells must depend on the ζ1 isoform for COPI function. When ζ1 is inhibited by siRNAs, cancer cells undergo apoptotic cell death while normal cells, which retain the ζ2 isoform, survive. Thus, the ζ1 subunit of the COPI complex is a potential target for cancer therapy. Here we present our work to develop and test small molecule inhibitors of ζ1-containing COPI complexes, as well as the identification of the apoptotic cell death pathways activated upon ζ1 depletion with siRNAs. We have identified a small molecule which disrupts the formation of COPI vesicles in biochemical and immunofluorescence studies, and also kills cells in a ζ1-specific manner. In addition we have shown that the unfolded protein response (UPR) is activated by ζ1 depletion but that it is not the primary activator of apoptotic cell death in cancer cells. The identification of small molecules which can inhibit ζ1-containing COPI will be a valuable tool for cancer therapy as well as the study of COPI biology. In addition, elucidation of the mechanism of activation and execution of apoptosis upon COPI depletion will provide insight into methods for combination therapy. Citation Format: David J. Oliver, Anusha Chaparala, Emeka Okafor, Carlos J. Camacho, Alexander S. Doemling, Felix T. Wieland, Igor B. Roninson, Michael Shtutman. Identification of cancer-specific COPI inhibitors and their associated apoptotic cell death pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5104. doi:10.1158/1538-7445.AM2014-5104