The long term objective of this project is to develop potent, safe an specific pharmacological inhibitors for interferon-gamma that are useful in the treatment of septic hock and autoimmune diseases such as multiple sclerois and Type 1 diabetes. The focus of the research is an oligonucleotide based inhibitor that specifically blocks a multitude of interferon-gamma effects, including the induction of the major histocompatibility complex Class II DR and Class I and ICAM-1 proteins in several cell types. This lead oligonucleotide also blocks the significant synergy between interferon-gamma and tumor necrosis factor-aalpha in mixture. It inhibits the binding of interferon-gamma to its cell surface receptor complex and thereby blocks downstream signaling by receptor associated kinases. Because our preliminary results support the feasibility of constructing oligonucleotide-based inhibitors for interferon-gamma, we propose to identify the molecular mechanisms of this inhibitory activity and use the resulting information to engineer an even more potent inhibitor for interferon-gamma. Accordingly, the specific aims of this project are to: i) identify the amino acid residues and nucleic acid bases that interact at the site of action, ii) use the mechanistic information to synthesize more potent inhibitors for interferon-gamma, iii) characterize the potency, activity, specificity and selectivity more potent inhibitors for interferon-gamma, iii) characterize the potency, activity, specificity and selectivity profiles of the newly synthesized inhibitors, iv) determine the effects of these inhibitors on human peripheral blood derived immune cells and to test the hypothesis that these inhibitors will drive the immune system to favor a humoral or TH2-like response and, v) test the inhibitors in a generalized Shwartzman model for septic shock. We will use epitope disruption and lysine protection assays to identify the amino acids of interferon-gamma that constitute the binding site. Hydroxyl radical footprinting and dimethylsulfate protection assays will be used to identify the nucleic acid bases involved in binding. The mechanistic information will then be used to optimize the length, sequence and backbone composition of these interferon-gamma inhibitory oligonucleotides. These studies will provide critical mechanistic information on the pharmacological and molecular basis for interferon-gamma inhibitory oligonucleotide activity. More importantly, the results from this model system should be generalizable to the design of oligonucleotide inhibitors for other protein, particularly cytokines.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
Application #
Study Section
Pharmacology A Study Section (PHRA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
State University of New York at Buffalo
Other Health Professions
Schools of Pharmacy
United States
Zip Code
Wong, Diane; Modi, Reshma; Ramanathan, Murali (2003) Assessment of Markov-dependent stochastic models for drug administration compliance. Clin Pharmacokinet 42:193-204
Ramanathan, M; Weinstock-Guttman, B; Nguyen, L T et al. (2001) In vivo gene expression revealed by cDNA arrays: the pattern in relapsing-remitting multiple sclerosis patients compared with normal subjects. J Neuroimmunol 116:213-9
Ramanathan, M (2000) Pharmacokinetic variability and therapeutic drug monitoring actions at steady state. Pharm Res 17:589-92
Balasubramanian, V; Ramanathan, M (2000) Glycosaminoglycans alter the conformation of interferon-gamma. Cytokine 12:466-71
Nguyen, L T; Ramanathan, M; Weinstock-Guttman, B et al. (2000) Detection of cytochrome P450 and other drug-metabolizing enzyme mRNAs in peripheral blood mononuclear cells using DNA arrays. Drug Metab Dispos 28:987-93
Parikh, H H; Li, W C; Ramanathan, M (1999) Evaluation of an alternative to the Kolmogorov-Smirnov test for flow cytometric histogram comparisons. J Immunol Methods 229:97-105
Ramanathan, M (1999) A method for estimating pharmacokinetic risks of concentration-dependent drug interactions from preclinical data. Drug Metab Dispos 27:1479-87
Ramanathan, M (1999) An application of Ito's lemma in population pharmacokinetics and pharmacodynamics. Pharm Res 16:584-6
Balasubramanian, V; Nguyen, L T; Balasubramanian, S V et al. (1998) Interferon-gamma-inhibitory oligodeoxynucleotides alter the conformation of interferon-gamma. Mol Pharmacol 53:926-32
Ramanathan, M (1998) Analysis of intracellular Th1 cytokine secretion data using parametric methodology. Cytometry 32:147-56

Showing the most recent 10 out of 12 publications