Ligand screening and design by X-ray crystallography

Details Ligand screening and design by X-ray crystallography Ligand screening and design by X-ray crystallography

1999-03-05

2001-10-02

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C117S927000

Reexamination Certificate

active

06297021

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
X-ray crystallography is useful for identifying ligands that bind target receptor molecules and for designing ligands with improved biological activity for the target receptor.
BACKGROUND OF THE INVENTION
X-ray crystallography (crystallography) is an established, well-studied technique that provides what can be best described as a three-dimensional picture of what a molecule looks like in a crystal. Scientists have used crystallography to solve the crystal structures for many biologically important molecules. Many classes of biomolecules can be studied by crystallography, including, but not limited to, proteins, DNA, RNA and viruses. Scientists have even reported the crystal structures of biomolecules that carry ligands within its receptors (a “ligand-receptor complex”).
Given a “picture” of a target biomolecule or a ligand-receptor complex, scientists can look for pockets or receptors where biological activity can take place. Then scientists can experimentally or computationally design high-affinity ligands (or drugs) for the receptors. Computational methods have alternatively been used to screen for the binding of small molecules. However, these previous attempts have met with limited success. Several problems plague ligand design by computational methods. Computational methods are based on estimates rather than exact determinations of the binding energies, and rely on simple calculations when compared with the complex interactions that exist within a biomolecule. Moreover, computational models require experimental confirmation which often expose the models as false positives that do not work on the real target.
Moreover, experimental high-affinity ligand design based on a “picture” of the ligand-receptor complex has been limited to biomolecules that already have known ligands. Finally, scientists only recently reported the crystallographic study of interactions between organic solvents and target biomolecules. Allen et al.,
J. Phys. Chem
., v. 100, pp. 2605-11 (1996). However, these studies are limited to mapping solvent sites rather than ligand sites. It would be desirable to directly identify potential ligands, and to obtain detailed information on how the ligand binds and changes in the target biomolecule. In addition, methods for identifying and/or designing ligands which possess biological and/or pharmaceutical activity with respect to a given target molecule would be desireable.
BRIEF SUMMARY OF THE INVENTION
Crystallography can be used to screen and identify compounds that are not known ligands of a target biomolecule for their ability to bind the target. The method (hereinafter “CrystaLEAD™”) comprises obtaining a crystal of a target biomolecule; exposing the target to one or more test samples that are potential ligands of the target; and determining whether a ligand/biomolecule complex is formed. The target is exposed to potential ligands by various methods, including but not limited to, soaking a crystal in a solution of one or more potential ligands or co-crystallizing a biomolecule in the presence of one or more potential ligands.
In a further embodiment, structural information from the ligand/receptor complexes found are used to design new ligands that bind tighter, bind more specifically, have better biological activity or have better safety profile than known ligands.
In a preferred embodiment, libraries of “shape-diverse” compounds are used to allow direct identification of the ligand-receptor complex even when the ligand is exposed as part of a mixture. This avoids the need for time-consuming de-convolution of a hit from the mixture. Here, three important steps are achieved simultaneously. The calculated electron density function directly reveals the binding event, identifies the bound compound and provides a detailed 3-D structure of the ligand-receptor complex. In one embodiment, once a hit is found, one could screen a number of analogs or derivatives of the hit for tighter binding or better biological activity by traditional screening methods. Another embodiment uses the hit and information about structure of the target to develop analogs or derivatives with tighter binding or better biological activity. In yet another embodiment, the ligand-receptor complex is exposed to additional iterations of potential ligands so that two or more hits can be linked together to make a more potent ligand.


REFERENCES:
patent: 5112755 (1992-05-01), Heyneker et al.
Schevitz et al., “Structure-Based Design of the First Potent and Selective Inhibitor of Human Non-Pancreatic Secretory Phospholipase A2”, Nature Struct. Biol., 2(6), 458-465, Jun. 1995.*
Chen et al., “3-D Structure of a Mutant (Asp 101-Ser) of E. coli Alkaline Phosphatase with Higher Catalytic Activity”, Prot. Eng., 5(7), 605-610, Oct. 1992.*
Thunnissen et al., “X-Ray Structure of Phospholipase A2 Complexed with a Substrate-Derived Inhibitor”, Nature, 347(6294), 689-691, Oct. 1990.*
J. Zheng, et al., Biochemistry, vol. 32, No. 9, pp. 2154-2161, 1993.
A. Hausrath, et al., The Journal of Biological Chemistry, vol. 269, No. 29, Issue of Jul. 22, pp. 18839-18842, 1994.
Verlinde, et al., “Antitrypanosomiasis Drug Development Based on Structures of Glycolytic Enzymes”, Structured-Based Drug Design, 1997, Chapter 15, pp. 365-394.
J. Hajdu, et al., Nature, vol. 329, pp. 178-181, 1987.
P.M. Colman, Current Opinion in Structural Biology, vol. 4, pp. 868-874, 1994.
J. Greer, et al., Journal of Medicinal Chemistry, vol. 37, No. *, pp. 1035-1054, 1994.
C. Verlinde, et al., Structure, vol. 2, pp. 577-587, 1984.
S. B. Shuker, et al., Science, vol. 274, pp. 1531-1534, 1996.
C. Verlinde, et al., Journal of Computer-Aided Molecular Design, vol. 6, pp. 131-147, 1992.
P. Verde, et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 4727-4731, 1984.
M. Nagai, et al., Gene, vol. 36 (1-2), pp. 183-188, 1985.
G. Spraggon, et al., Structure, vol. 3 No. 7, pp. 681-691, 1995.
T. J. Pilot-Matias, et al., Gene, vol. 128, pp. 219-225, 1993.
A. Granelli-Piperno, et al., J. Exp. Med., vol. 148, pp. 223-234, 1978.
J. Vialard, et al., Journal of Virology, vol. 64(1), pp. 37-50, 1990.
G. Claeson, et al., Haemostasis, vol., 7, pp. 76-78, 1978.
E. Menegatti, et al., J. Enzyme Inhibition, vol. 2, pp. 249-259, 1989.
J. Navaza, Acta Crystallographica, A50, pp. 157-163, 1994.
K. Allen, et al., J. Phys. Chem. 1996 100, pp. 2605-2611.
Ford et al., “Crystal Structure of a Lysozyme-Tetrasaccharide Lactone Complex,” J. Mol. Biol. 1974, vol. 88, pp. 349-371.
Alber et al., “Crystal Structure of Elastase-Substrate Complex at -55° C,” Nature, Sep. 23, 1976, vol. 263, No. 5575, pp. 297-300.
Fink et al., “X-Ray Cryoenzymology,” Adv. Enz A. Meister Ed Wiley & Sons. New York, 1981, vol. 52, pp. 177-246.
Howell et al., “Functional Role of Aspartic Acid-27 in Dihydrofolate Reductase Revealed by mutagenesis,” Science, Mar. 7, 1986, vol. 231, pp. 1123-1128.
Verlinde et al., “In Search of New Lead Compounds for Trypanosomiasis Drug Design: Protein Structure-Based Linked-Fragment Approach,” J. Computer-Aided Mol. Des., Apr. 1992, vol. 6, No. 2, pp. 131-147.
Thaisrivongs et al., “Structure-Based Design of HIV Protease Inhibitors: 5,6-Dihydro-4-hydroxy-2-pyrones as Effective, Nonpeptidic Inhibitors,” J. Med. Chem., Nov. 8, 1996. vol. 39, No. 23, pp. 4630-4642.

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