Anthrax Toxin Lethal Factor Inhibition Study. Principal Investigator: Elizabeth Amin. Co-Investigators: BFinzel, DHook, RJohnson, MWalters. Sponsor: NIAID (RFA PA-07-070). Sponsor Award: 1 R01 AI083234-01.
Synopsis and progress: We are collaborating with the Amin & Johnson groups in Medicinal Chemistry, and the Institute for Therapeutic Discovery & Development to discover specific inhibitors of the Anthrax Lethal Factor zinc-metalloproteinase. Identified agents may have value as post-exposure anti-infective agents. After validating our ability to structurally characterize known ligands, we are continuing to pursue co-crystallization studies with compounds identified through virtual screening of high-throughput in vitro screening, and other synthetic inhibitors being created in the Amin lab. Compound binding can induce severl distinct onformational sttes of the enzyme that we have been able to characterize structurally and classify based on hinge angle and distancce of separation of Domain 3 from the active site (Figure 1). This work in progress was initiated by Todd Geders, continud by Teresa De la Mora-Rey, and currently being pursued by Kimberly Maize.
Figure 1. Overview of Lethal Factor structure. Domain 1 (red), Domain 2 (tan), Domain 3 (green), and Domain 4 (blue) are shown as cartoons, based on structure 1PWQ. The hinge angle and inner distance measurements are depicted with maximum (1PWQ, black) and minimum (4PKQ, red) values.
K.M. Maize, E.K. Kurbanov, T. De la Mora-Rey, T.W. Geders, D.-J. Hwang, M.A. Walters, R.L. Johnson, E.A. Amin, B.C. Finzel. (2014) “Anthrax Toxin Lethal Factor Domain 3 is Highly Mobile and Responsive to Ligand Binding”. Acta Crystallogr. Sect D. Biol. Crystallogr. D70:2813-2822.
Chiu, T.-L., Solberg, J., Patil, S., Geders, T., Zhang, X., Rangarajan, S., Francis, R., Finzel, B., Walters, M., Hook, D., Amin, E. Identification of novel non-hydroxamate anthrax toxin lethal factor inhibitors by topomeric searching, docking and scoring, and in vitro screening. J. Chem. Inform. Modelling, 2009, 49:2724-36.
Biotin synthesis and biotin ligation in Mycobacterium tuberculosis. Principal Investigator: Dirk Schnappinger (Cornell University). Co-Investigators: BFinzel, CAldrich. Sponsor: NIAID (PA-10-067). Sponsor Award: 1 R01 AI091790-01
Synopsis and progress: Working in collaboaration with investigators at Cornell University and the Center for Drug Design at the University of Minnesota, the objective of this research is to characterize the consequences of inhibiting biotin synthesis or ligation in mycobacterium tuberculosis. A specific aim of this proposal requires structural characterization of potent inhibitors of the biotin protein ligase (BirA) of mTB. Our preliminary work in this area has resulted in structural characterization at high resolution of a bi-substrate mimetic bound to BirA. We have shown that the enzyme can adopt a significantly different conformation in the presence of bound inhibitors. We plan to further aid in the design of potent inhibitors by characterizing the structure of other inhibitor analogs. These novel molecules will then be characterized in cell-based assays to help validate biotin ligation as a feasible strategy for combatting multi-drug resistant mTB infections. This work in progress was initiated by Todd Geders, and is being carried on by Teresa De la Mora-Rey.
Figure 2. Biotin Protein Ligase (BirA) complex with a bi-substrate analog inhibitor. A) 1.7 Angstom difference electron density for the inhibitor. B) hydrogen bondong to target protein binding site.
Another component of this research program involves BioA, which catalyzes the second step in the biochemical synthesis of biotin in mycobacterium. The PLP-dependeant transaminase can be inhibited by formation of a covalent adduct with a inhibitor designed and synthesized in the Aldrich lab. We have succeeded in characterizing the structure of BioA in its resting state (a covalent Schiff base adduct involving the PLP and Lys-283) (Fig 3A), and in the adduct-bound state (Fig 3B). Research on this enzyme in continuing following a fragment screen, with which we have been able to identify a variety of different small molecules that bind adjacent to the PLP. This work is being continued by Ran Dai.
Figure 3. Electron Density for the Ligand in BioA complexes. A) the PLP-Lys-283 complex; B) Aromatized irreversible adduct formed with inhibitor.
R. Dai, D. Wilson, T.W. Geders, C.A. Aldrich, B.C. Finzel. (2014) "Inhibition of Mycobacterium tuberculosis Transaminase BioA by Aryl Hydrazines and Hydrazides ". ChemBioChem. 15(4):575-586.
Shi, C., Geders, T., Park, S.W., Wilson, D., Boshoff, H., Orisadipe, A., Barry, C., Schnappinger, D., Finzel, B., Aldrich, C. “Mechanism-Based Inactivation by Aromatization of the Transaminase BioA Involved in Biotin Biosynthesis in Mycobacterium tuberculosis.” J.Am.Chem.Soc., 2011, 133:18194-201.
Duckworth, B., Geders, T., Tiwari,D., Boshoff, H., Sibbald, P., Barry, C., Schnappinger,D., Finzel, B., Aldrich, C. “Bisubstrate Adenylation Inhibitors of Biotin Protein Ligase from Mycobacterium tuberculosis”. Chem. & Biol., 2011, 18:1433-41.
Using Distance Geometry to Manipulate Protein Substructures and Ligand Binding Sites. Principal Investigator: Barry Finzel.
Synopsis and Progress: Software and computational methods development program aimed at effectively utilizing distance geometry in protein and nucleic acid structure to simplify the identification and superposition of structurally similar macromolecular substructures. While the original driving force in development of these methods was for substructure searching, we have found that conserved core substructures also represent an excellent basis for the comprehensive superposition of large libraries of protein-ligand binding sites to facilitate structure-aided drug design. The DrugSite database and web-server has been created as an interface to these methods and as a resource for collaboration.
Most widely applied methods for overlaying protein structures seek to include as much homologous protein structure in the overlay a possible, to emphasize similarity. Ligands - because of their chemical dissimilarity - are necessarily omitted from any superposition, but are reoriented using the same transformation that overlays the proteins. The DrugSite approach allows user definition of very specific conserved protein-based structural motifs to drive superposition so as to maintain as much similarity in ligand overlay as possible.
DrugSite Overlay Methods
Kinase-Any Overlay Method
MMP Overlay Method
J. R. Van Voorst, B.C. Finzel. (2013) “Searching for Likeness in a Database of Macromolecular Complexes”. J. Chem Inf. Model., 53(10):2634-2647.
Finzel, B.C., R. Akavaram, A. Ragipindi, J.R. Van Voorst, M. Cahn, M. Davis, M. Pokross, S. Sheriff, E.T. Baldwin. "Conserved Core Substructures in the Overlay of Protein-Ligand Complexes". 2011, J. Chem Inf. & Modelling 51:1931-1941