My research probes the structure, function and evolution of nuclear hormone receptors (NR) and cytochrome P450 (CYP) genes involved in human toxicology and the progression of diseases ranging from cancer and kidney disease to hypertension and obesity. My laboratory focuses primarily on mammalian genes involved in vitamin A and vitamin D metabolism, including CYP1B1, CYP3A5, CYP24A1 and CYP26A1, each of which can become overexpressed in human tumors, making them important therapeutic targets for a range of human disorders. My graduate and post-doctoral work was focused primarily on characterizing the structure/function of mitochondrial CYP genes, including CYP11A1 and CYP24A1, which provided me with expertise in heterologous protein expression systems and the purification of recombinant proteins for biochemical, biophysical and x-ray crystallographic studies that underpin the structure-based drug development (SBDD) process. As a faculty member in the Dept. of Environmental and Molecular Toxicology at Oregon State University, I have shifted my focus to exploring the role of alternative gene splicing in human health, and I hope to develop new approaches for modulating both NR and CYP gene function using antisense approaches to gene therapy. Validation of this methodology may inspire the next generation of RNA-based therapeutics that circumvent toxicity issues associated with conventional, hormone-based, small molecule drugs. In recent years, I have also gained substantial experience designing and implementing 2D cell-culture assays to probe gene expression, mechanisms of toxicity and PK/PD parameters for novel therapeutic leads. I also have an extensive background in computational bioinformatics, molecular modeling and docking, untargeted metabolomics and LC/MS analysis of both vitamin D and corticosterone metabolites.
Positions and Employment
1999-2006 - Research Assistant, Dept. of Molecular Biology and Biochemistry, University of New Mexico.
2001-2006 - Research Assistant, Dept. of Toxicology, University of New Mexico – School of Pharmacy.
2002-2003 - Graduate Level Summer Research Intern, Dept. of Structural Biology, Abbott Laboratories.
2006-2011 - Postdoctoral Research Associate, Dept. of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA.
2011-2013 - Senior Research Associate, Dept. of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA.
2013-present - Assistant Professor (Sr. Research), Dept. of Environmental & Mol. Toxicology, Oregon State University.
Honors and Professional Memberships
1996 - University of New Mexico Regents’ Scholar – Full, 4-year Academic Scholarship
2001 - Bachelor of Sciences (B.S.) In Biochemistry - Magna Cum Laude.
2001 - University of New Mexico - Honors Program Graduate – Summa Cum Laude.
2001 - Inducted into UNM chapter of Phi Beta Kappa Honor Society.
2002 - Awarded Graduate Student Teaching Assistantship- UNM College Of Pharmacy.
2003 - Approved for Student Membership into the National and Mountain West Society of Toxicology.
2003 - Awarded Best Poster Presentation – 21st Mountain West Society of Toxicology Meeting.
2004 - Awarded Best Platform Presentation – 22nd Mountain West Society of Toxicology Meeting.
2004 - Awarded Red Suspender Award - Best Poster – 4th Southwest P450 Meeting.
2005 - Awarded Best Poster – MB Specialty Section – 2005 National Society of Toxicology Meeting.
2005 - Awarded Best Poster In Session - 14th International Conference on Cytochromes P450.
2005 - Awarded Doctor Of Philosophy (Ph.D.) in Biomedical Sciences – With Highest Distinction.
2009 - Named the John L. Omdahl Memorial Lecturer for 2009 – University of New Mexico-SOM.
2012 - Invited Platform Speaker at the 15th International Vitamin D Workshop, Houston, Texas.
2015 - Approved for Full Membership into the National Society of Toxicology.
2015 - Approved for Membership into the Pacific Northwest Association of Toxicologists (PANWAT).
2016 - Promoted to Graduate Teaching Faculty in OSU’s Dept. of Environmental & Mol. Toxicology.
2017 - Invited Platform Speaker at Oregon State’s 8th Annual EMT Research Day, Corvallis, Oregon.
2018 - Developed a Workshop selected for the 58th Annual National Society of Toxicology Meeting.
Contributions to Science
Biochemical Characterization of Vitamin D Metabolism and Site-Directed Mutational Analysis of the CYP24A1 Gene - My early work in the vitamin D laboratory of Dr. Jack Omdahl (University of New Mexico; Dept. of Biochemistry & Molecular Biology) was focused on optimizing protocols for the bacterial overexpression and purification of recombinant CYP24A1 and its redox partner proteins, adrenodoxin and adrenodoxin reductase, for use in a cell-free, CYP24A1 enzyme reconstitution system and UV/Vis spectral analysis. We used highly-purified proteins to characterize the substrate binding and enzyme kinetic properties of purified CYP24A1 for the first time. Historically, the characterization of mitochondrial cytochrome P450 forms, like CYP24A1, were limited by an inability to overexpress and purify sufficient quantities of these highly amphipathic, peripheral membrane proteins, and much of my early work was focused on overcoming this challenge using novel purification procedures, mutagenesis and experimental reagents. Using biochemical analysis, Met-246 and Phe-249 were identified as important active site residues coordinating proper substrate recognition. Ser-57 was later identified as a determinant of substrate recruitment using a vitamin D hormone-based affinity-labeling probe. I subsequently completed a site-directed mutagenesis study of CYP24A1 as a core component of my doctoral dissertation, which included the development of a CYP24A1 homology model that aided in the prediction of key active site residues. Using this combinatorial approach, Val-391 and Ile-500 were next identified as key structural determinants of vitamin D hormone binding, and the positioning of Met-246 and Phe-249 was further validated. Models for mitochondrial membrane insertion and substrate-access near the plasma membrane were also proposed for the first time. This early work helped to validate an array of computational approaches for CYP homology modeling and protein structure prediction, while improving basic knowledge of mitochondrial P450 structure/function, and the determinants of vitamin D hormone recognition in CYP24A Although this work was conducted under the supervision from advisors, I participated in all aspects of this research, including the hands-on expression, purification, and characterization work, for both wild-type and mutant forms of CYP24A
Omdahl JL, Bobrovnikova EV, Annalora A, Chen P, Serda R. (2003) Expression, structure-function, and molecular modeling of vitamin D P450s. J Cell Biochem. 88(2):356-62. PMID: 12520537.
Annalora AJ, Bobrovnikova-Marjon EV, Serda R, Lansing L, Chiu ML, Pastuszyn A, Iyer S, Marcus CB, Omdahl JL. (2004) Rat Cytochrome P450c24 (CYP24A1) and the Role of F249 in Substrate Binding and Catalytic Activity. Arch Biochem Biophys. 425(2):133-46.
Annalora A, Bobrovnikova-Marjon E, Serda R, Lansing L, Chiu ML, Pastuszyn A, Iyer S, Marcus CB, Omdahl JL. (2004) Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue. J Steroid Biochem Mol Biol. 89-90(1-5):159-62. PMID: 15111121.
Annalora A, Bobrovnikova-Marjon E, Pastuszyn A, Serda R, Graham SE, Marcus CB, Omdahl JL. (2007) Hybrid homology modeling and mutational analysis of cytochrome P450 C24A1 (CYP24A1) of the vitamin D pathway: Insights into substrate specificity and membrane bound structure-function. Arch Biochem Biophys. 460(2):262-73.
Structural Characterization of Mitochondrial Cytochrome P450 Genes: CYP24A1 and CYP11A1. My postdoctoral work was focused on structural biology and using x-ray crystallography to study the structure of mitochondrial P450s. Under the supervision of Dr. C. David Stout and Dr. Eric F. Johnson at the Scripps Research Institute I independently completed all phases of the project that resulted in the first, high-resolution crystal structures of CYP24A1 (PDB: 3k9v and 3k9y). This breakthrough improved our understanding of substrate recognition, membrane binding, and cofactor recruitment for mitochondrial CYP forms, and provided a highly-ordered, open form structural template for future CYP homology modeling studies. Structural insights gained from this work confirmed the proposed “cylindrical active site” of CYP24A1, and validated much of the early homology modeling work conducted by our laboratory and others. While working on structures of CYP24A1, I also participated in collaborations with Dr. Irina Pikuleva (Case Western University) to help characterize mitochondrial CYP11A1, the cytochrome P450 side chain cleavage (SCC) enzyme. To this end, I collected x-ray diffraction data sets for CYP11A1 crystals grown using my protocols, and coordinates from the CYP24A1 structure were required to solve the structure of CYP11A1 via the molecular replacement method. Subsequently, I used the structural coordinates of CYP24A1 to conduct computational docking studies of vitamin D hormone analogs, 25-Hydroxy-16-ene-23-yne-vitamin D3 and 3-epi-1α,25-dihydroxyvitamin D3, which show reduced CYP24A1-mediated metabolism compared to the native hormone. These results demonstrated that the apo-form of CYP24A1 selectively bind to secosteroids with a discrete side-chain length and polarity, and that the reversible 3-epimerization behavior of vitamin D hormones may represent a discrete mechanism for reducing the rate of CYP24A1-mediated hormone catabolism.
Annalora AJ, Goodin DB, Hong WX, Zhang Q, Johnson EF, Stout CD. (2010) Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism. J Mol Biol. 396: 441-451.
Mast N, Annalora AJ, Lodowski DT, Palczewski K, Stout CD, Pikuleva IA. (2011) Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1. J Biol Chem. 286(7):5607-13.
Rhieu SY, Annalora AJ, Wang G, Flarakos CC, Gathungu RM, Vouros P, Sigüeiro R, Mouriño A, Schuster I, Palmore GT, Reddy GS. (2013) Metabolic stability of 3-epi-1α,25-dihydroxyvitamin D3 over 1α,25- dihydroxyvitamin D3: metabolism and molecular docking studies using rat CYP24A1. J Cell Biochem. 114(10):2293-305. PMID: 23606409.
Rhieu SY, Annalora AJ, Laporta E, Welsh J, Itoh T, Yamamoto K, Sakaki T, Chen TC, Uskokovic MR, Reddy GS. (2014) Potent Antiproliferative Effects of 25-Hydroxy-16-ene-23-yne-vitamin D(3) That Resists the Catalytic Activity of Both CYP27B1 and CYP24A1. J Cell Biochem. 115(8):1392-402. PMID: 24535953.
Alternative Gene Splicing and Antisense Drug Development. While much of my early work was focused on addressing discrete questions related to cytochrome P450 (CYP) structure/function for drug discovery purposes, my current work is focused more on elaborating the role that alternative gene splicing plays in regulating the cell-specific expression and function of both Nuclear Receptors and CYP genes linked to cancer, obesity and hypertension. We recently completed a comprehensive review of alternative gene splicing in the Cytochrome P450 superfamily that describes how limitations in GWAS and personalized medicine arise from insufficient information regarding the tissue-specific expression of key metabolic genes sensitive to alternative splicing. In this regard, we have recently developed a US patent (PCT/US2016/050239) for antisense oligomers targeting variant forms of CYP3A5, and we are preparing a suite of publications describing the development of this antisense technology. We hope to develop an improved roadmap for precision medicine by improving the characterization of the dynamic CYP transcriptome. Our recent publication in Oncotarget represents the translational potential of this research approach, as the tumor-specific transcriptome of the Ewing’s family of tumors (EFT) was studied using advanced computational methods, and we identified several new cancer-specific molecular targets for EFT drug development, including CYP4F22.
Annalora AJ, Marcus CB, Iversen PL. (2017) Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity to Augment Gene Function and Redirect Human Drug Metabolism. Drug Metab Dispos. 2017 Feb 10. pii: dmd.116.073254. doi: 10.1124/dmd.116.073254. (Review)
Annalora AJ, O'Neil S, Bushman JD, Summerton JE, Marcus CB, Iversen PL. (2018) A k-mer based transcriptomics approach for antisense drug discovery targeting the Ewing's family of tumors. Oncotarget. 2018 Jul 17;9(55):30568-30586. doi: 10.18632/oncotarget.25736.
Iversen PL, Marcus CB, Annalora AJ (2018) Clinical and preclinical pharmacokinetics and pharmacodynamics of AVI-4557, a novel antisense oligonucleotide inhibitor of human CYP3A4. (In Review).
Annalora AJ, Jozic M, Marcus CB, Iversen PL. (2019) Alternative splicing of the vitamin D receptor modulates target gene expression and promotes ligand-independent functions. Toxicol Appl Pharmacol. 364:55-67. doi: 10.1016/j.taap.2018.12.009.