Dr. Ernesto Freire
Joint-Appointed Faculty
Professor, Biology

Johns Hopkins University
Department of Biology
3400 North Charles Street
114 Biology East
Baltimore, MD 21218
410-516-7743 Office
410-516-6469 Fax
ef@jhu.edu

Structure-Based Thermodynamics of Molecular Recognition

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:

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.

Selected Publications:

• Ernesto Freire A thermodynamic approach to the optimization of drug candidates (2009) Chem Biol Drug Design  In Press
• Regnier Thomas, Diganta Sarma,Koushi Hidaka, Usman Bacha, Ernesto Freire, Yoshio Hayashi and Yoshiaki Kiso, New Developments for the Design, Synthesis and Biological Evaluation of Potent SARS-CoV 3CLpro Inhibitors (2009) Bioorganic & Medicinal Chemistry Letters 19, 2722-2727
• Chunlong Ma, Alexei Polishchuk, Yuki Ohigashi, Amanda Stouffer, Arne Schön, Emma Magavern, James D. Lear, Ernesto Freire, Robert A. Lamb, William F. DeGrado, and Lawrence H. Pinto Identification of the Functional Core of the Influenza A Virus A/M2 Proton-Selective Ion Channel (2009) Proceedings of the National Academy of Sciences 106:12283-12288
• Brower, E. T., Schon, A., Klein, J. C., and Freire, E. Binding Thermodynamics of the N-Terminal Peptide of the CCR5 Coreceptor to HIV-1 Envelope Glycoprotein gp120 (2009) Biochemistry 48, 779-785
• Freire, E. ITC: Affinity is not everything (2009) European Pharmaceutical Review 14, 44-47
• Freire, E.  A Thermodynamic Platform for Drug Discovery and Optimization (2008)  in Towards Drugs of the Future, pages 13-22 Kruse, C.G. and Timmerman, H. Eds.
• Hidaka, K., Kimura, T., Ruben, A. J., Uemura, T., Kamiya, M., Kiso, A., Okamoto, T., Tsuchiya, Y., Hayashi, Y., Freire, E. and Kiso, Y. (2008) Antimalarial activity enhancement in hydroxymethylcarbonyl (HMC) isostere-based dipeptidomimetics targeting malarial aspartic protease plasmepsin.  Bioorganic and Medicinal Chemistry,  16 10049-10060
• Navid Madani, Arne Schon, Amy M. Princiotto, Judith M. LaLonde, Joel R. Courter, Takahiro Soeta, Danny Ng, Liping Wang, Evan T. Brower, Shi-Hua Xiang, Young Do Kwon, Chih-chin Huang, Richard Wyatt, Peter D. Kwong, Ernesto Freire, Amos B. Smith III and Joseph Sodroski (2008) Small-Molecule CD4 Mimics Interact with a Highly Conserved Pocket on HIV-1 gp120.  Structure 11, 1689-1701
• Freire, E., Schön, A., Velazquez-Campoy, A. (2009) Isothermal Titration Calorimetry: General Formalism Using Binding Polynomials.  Methods in Enzymology.  455, 127- 155
• Freire, E. (2008) Do Enthalpy and Entropy Distinguish First in Class From Best in Class?  Drug Discovery Today 13, 869-874
• Freire, E. A new era for microcalorimetry in drug development (2008) American   Pharmaceutical Review 11, 118-123
• Bacha, U., Barrila, J., Gabelli, S.B., Kiso, Y. Amzel, L.M. Freire, E. (2008) Development of Broad-Spectrum Halomethyl Ketone Inhibitors Against Coronavirus Main Protease 3CLpro Chem. Biol. Drug. Design 72, 34-49
• Hosahudya Gopi, M.Umashankara, Vanessa Pirrone, Judith LaLonde, Navid Madani, Ferit Tuzer, Sabine Baxter, Isaac Zentne, Simon Cocklin, Navneet Jawanda, Shendra R. Miller, Arne Schön, Jeffrey C. Klein, Ernesto Freire, Fred C. Krebs, Amos B. Smith III, Joseph Sodroski and Irwin Chaiken (2008) Structural Determinants for Affinity Enhancement of a Dual Antagonist Peptide Entry Inhibitor of Human Immunodeficiency Virus Type-1 J. Medicinal Chem.  51 2638-2647
• Cremades, N., Velazquez-Campoy, A., Freire, E. and Sancho, J. The Flavodoxin from Helicobacter pylori: Structural Determinants of Thermostability and FMN Cofactor Binding (2008) Biochemistry 47 627-639
• Bower, E., Bacha, U.M., Kawasaki, Y. and Freire, E. Inhibition of HIV-2 Protease by HIV-1 Protease Inhibitors in Clinical Use (2008) Chem. Biol. Drug. Design 71 298-305
• Schon, A. and Freire, E. Strategies for targeting HIV-1 envelope glycoprotein gp120 in the development of new antivirals (2007) Future Medicine 1, 223-229
• Freire, E. A new era for microcalorimetry in drug development (2007) European   Pharmaceutical Review 5, 73-78
• Lafont, V., Armstrong, A.A., Ohtaka, H., Kiso, Y., Amzel, M. and Freire, E. (2007) Compensating Enthalpic and Entropic Changes Hinder Binding Affinity Optimization. Chem. Biol. Drug. Design 69, 413-422
• Koushi Hidaka, Tooru Kimura, Yumi Tsuchiya, Mami Kamiya, Adam J. Ruben,  Ernesto Freire, Yoshio Hayashi and Yoshiaki Kiso (2007) Additional interaction of allophenylnorstatine-containing tripeptidomimetics with malarial aspartic protease plasmepsin II. Bioorg. Medicinal Chem. Letters 17, 3048-3052