The organization of biological structures, including the folding of a protein into its native conformation, the association of supra-molecular complexes, or the binding of a ligand to a receptor are controlled by the energy changes involved in their formation. In my laboratory, we are interested in the elucidation of the relationships between structure and energetics, and their applications to molecular design. In particular, we are interested in developing new methods for structure-based drug design and for the design of proteins with altered stability and function. Toward those ends, we have developed a very accurate structural parameterization of the folding and binding energetics aimed at predicting the stability of proteins and the binding affinity of ligands from structure. This parameterization is being used in the development and implementation of new algorithms for the design of medically important molecules. The research program in this laboratory is multidisciplinary and covers all aspects of the design process, including molecular biology, structural studies, energetic determinations, software development and computational analysis. Currently, our research projects include: HIV-1 Protease Inhibition. One of the most important side effects in the treatment of HIV-1 infection is the appearance of viral strains encoding for mutant protease molecules that are resistant to the inhibitors currently in clinical use. One of the goals of this project is to elucidate the molecular origins of resistance and to use that knowledge in the development of new types of inhibitors. Cooperative Interactions in Proteins. Proteins are not static objects but highly dynamic entities undergoing constant conformational fluctuations. As such, any rigorous description of their structural and functional behavior is of necessity statistical in nature. This laboratory has developed a statistical thermodynamic formalism that correctly accounts for the properties of the native state ensemble of proteins as reported by NMR-detected hydrogen exchange experiments. This approach is being used to investigate how perturbations initiated at specific sites (e.g. by ligand binding) are transmitted to other sites in the protein molecule. For this project the systems of choice have been SH3 and SH2 domains since these ubiquitous protein modules are involved in many cellular signaling processes. The knowledge gained in this project will provide the basis for the design of "smart" ligands. Structural Parameterization of the Folding and Binding Energetics. A permanent endeavor in this laboratory is the improvement of the structural parameterization of the energetics. This project involves the refinement of the parametric equations that relate the quantities that define the Gibbs energy (enthalpy, entropy, heat capacity changes and their different subcomponents) to structural parameters. Since one of the main components of the parameterization is a joint database of systems for which high resolution structures and high resolution thermodynamic data are available, this project includes a careful determination of the folding/unfolding thermodynamics and binding interactions of selected protein systems. Antibiotic Resistance. The emergence of bacterial strains that are resistant to traditional antibiotic therapies is a major health problem. In this project, we are working in the development of b -lactamase inhibitors. b -lactamase is a bacterial enzyme responsible for degrading penicillin-type antibiotics and rendering them inactive. Thus, b -lactamase inhibition provides a way to restore antibiotic potency. New Antimalarial Targets. More than 300 million people worldwide are infected with malaria each year. The problem has been compounded by the appearance of Plasmodium strains that are resistant to conventional therapies. A new potential target is the parasite enzyme plasmepsin, an aspartic protease involved in the degradation of the hemoglobin of the infected victims. In this project we are pursuing novel strategies to develop plasmepsin inhibitors.
Freire, E. Drug Design Against Heterogeneous Targets: A Major Challenge in Post-Genomic Medicine (2002) Nature Biotech. 20 15-16 Nezami, A., Luque, I. Kimura, T., Kiso, Y. and Freire, E. Identification and Characterization of Allophenylnorstatine-Based Inhibitors of Plasmepsin II, an Anti-Malarial Target (2002) Biochemistry 41 2273-2280 Ohtaka, H., Velazquez-Campoy, A. and Freire, E. Overcoming Drug Resistance in HIV-1 Chemotherapy: The Binding Thermodynamics of Amprenavir and TMC-126 to Wild Type and Drug-Resistant Mutants of the HIV-1 Protease (2002) Protein Science 11 1908-1916 Velazquez-Campoy, A., Vega, S. and Freire, E. Amplification of the Effects of Drug-Resistance Mutations by Background Polymorphisms in HIV-1 Protease from African Subtypes (2002) Biochemistry, 41 8613-8619 Nezami, A. and Freire, E. The Integration of Genomic and Structural Information in the Development of High Affinity Plasmepsin Inhibitors (2002) Int. Journal Parasitology, 32 1669-1676 Velazquez-Campoy, A. and Freire, E. Isothermal Titration Calorimetry: Measuring Intermolecular Interactions (2002) Cold Spring Harbor Laboratory Series, In Press Dowd, C. S., Leavitt, S., Babcock, G., Godillot, A.P., Van Ryk, D., Canziani, G. A., Sodroski, J., Freire, E., Chaiken, I. M. -Turn Phe in HIV-1 Env binding site of CD4 and CD4 mimetic miniprotein enhances Env binding affinity but is not required for activation of co-receptor/17b site (2002) Biochemistry, 41 7038-7046 Luque, I. and Freire, E. Structural Parameterization of the Binding Enthalpy of Small Ligands (2002) Proteins, 49 181-190 Schon, A., Ingaramo, M. and Freire, E. The Binding of HIV-1 Protease Inhibitors to Human Serum Proteins (2003) Biophysical Chemistry In Press Muzammil, S. Ross, P. and Freire, E. A Major Role for a Set of Non-Active Site Mutations in the Development of HIV-1 Protease Drug Resistance (2003) Biochemistry 42 631-638
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