Ricin inhibitors and methods for use thereof

Details Ricin inhibitors and methods for use thereof Ricin inhibitors and methods for use thereof

1998-07-17

2001-01-23

Ceperley, Mary E. (Department: 1641)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

C436S503000, C435S091500

Reexamination Certificate

active

06177280

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to protein chemistry. More specifically, the present invention relates to the identification of inhibitors of the heterodimeric cytotoxin ricin, using computer modeling of the ricin active site as a template in structure-based drug design. Moreover, the present invention relates to the use of the identified inhibitors as antidotes to ricin or to facilitate immunotoxin treatment by controlling non-specific cytotoxicity. In addition, Shiga toxin, a compound essentially identical to the protein originally isolated from
Shigella dysenteriae,
has an active protein chain that is a homologue of the ricin active chain, and catalyzes the same depurination reaction. Thus, the present invention is drawn additionally to identifying inhibitors as antidotes to Shiga toxin.
DESCRIPTION OF THE RELATED ART
Ricin is a potent heterodimeric cytotoxin easily isolated from the seeds of the castor plant,
Ricinus communis.
The protein consists of a lectin B chain, which can bind cell surfaces and is linked by disulfide bonds to an A chain (RTA), which enzymatically depurinates a key adenine residue in 28 S rRNA (Endo and Tsurugi,
J. Biol. Chem.,
262:8128-30 (1987)). Ricin is an extraordinarily toxic molecule that attacks ribosomes, thereby inhibiting protein synthesis. Ricin has an LD
50
of approximately 1 &mgr;g/kg body weight for mice, rats, and dogs and is ten times more potent against rabbits (Olsnes, S. and Pihl, A., “Toxic Lectins and Related Proteins,”
The Molecular Action of Toxins and Viruses
, pp 52-105 (1982)). The toxic dose for humans is likely to be in the &mgr;g/kg range which ranks it among the most toxic substances known.
The protein has been used extensively in the design of therapeutic immunotoxins. In these constructs, ricin, RTA, or a related toxin is chemically or genetically linked to an antibody to form a so-called “magic bullet”, which can target preferentially those cell lines carrying antigenic markers recognized by the antibody (Frankel, A. E., ed.,
Immunotoxins,
Kluwer academic publishers, Boston (1988)).
Ricin also has been used as a poison agent. The protein gained notoriety when it was used in the famous “umbrella tip” assassination of Georgy Markov and was also used in an unsuccessful attempt to poison the Soviet dissident Alexander Solzhenitsyn. More recently, ricin was prepared by a militant anti-tax group which planned to poison IRS personnel.
Given the above, there is interest in identifying or designing potent inhibitors of ricin. These inhibitors could, in principle, be used to facilitate immunotoxin treatment by helping to control nonspecific cytotoxicity, or could be used as antidotes to poison attacks. Recently there has been interest in structure-based drug design—that is, using the knowledge of protein structure to identify enzyme inhibitors. The most common paradigm for this overall process has been called an “iterative protein crystallographic algorithm” by Appelt et al.,
J. Med. Chem.
34:1925-34 (1991), where the design of inhibitors for thymidylate synthetase is described. The main concept of the approach is that the protein active site is used as a template to design or to identify complementary ligands. The identified putative ligands are ranked and tested kinetically. Promising inhibitor candidates are bound to the protein target and analyzed crystallographically for comparison with the proposed model. Additionally, alterations are made in the inhibitor to improve binding and a new round of tests is carried out on the altered compound.
A number of laboratories have used variations on this protocol to design efficacious inhibitors. For example, the search program DOCK (see Kuntz et al.,
J. Mol. Biol.
161:269-88 (1982)) was used to predict that the known anti-psychotic drug haloperidol would bind to the HIV protease (DesJarlais et al.,
Proc. Natl. Acad. Sci USA
87:6644-48 (1990)). Crystallographic studies together with computer aided search methods also were used in the design of inhibitors of purine nucleoside phosphorylase. The program GRID (Goodford, P. J.,
J. Med. Chem.
28:849-57 (1985)) has been used to design two very successful inhibitors of influenza virus. Certain chemical substitutions to the sialic acid substrate were predicted to be energetically favorable, based on interactions with the known X-ray structure of the enzyme. Subsequent binding assays revealed Ki values as low as 0.2 nM. These compounds not only inhibited neuraminidase but retarded viral replication in cultured cell and animal models as well.
The X-ray structure of ricin has been solved (Montfort et al.,
J. Biol. Chem.
262:5398-03 (1987)), refined to 2.5 Å (Rutenber et al.,
Proteins
10:240-50 (1991)), and described in detail (Katzin et al.,
Proteins
10:251-59 (1991); and Rutenber and Robertus,
Proteins
10: 260-69 (1991)). The structure of RTA expressed in
Escherichia coli
has been resolved to 2.3 Å resolution for monoclinic crystals (Mlsna et al.,
Prot. Sci.
2:429-35 (1993)), and recently to 1.8 Å resolution for a tetragonal form (Weston et al.,
J. Mol. Biol.
244:410-422 (1994)).
The X-ray model of RTA allowed identification of a number of amino acids which were hypothesized to be important for substrate binding and for the depurination mechanism; these residues include Glu 177, Arg 180, Trp 211, Tyr 80 and Tyr 123. Site-directed mutagenesis of the cloned RTA gene has been used to examine the relative significance of these residues (see, e.g., Schlossman et al.,
Mol. Cell. Biol.
9:5012-21 (1989); Frankel et al.,
Mol. Cell. Biol.
10:6257-63 (1990); Ready et al.,
Proteins
10:270-78 (1991); and Kim and Robertus,
Protein Engineering
5:775-79 (1992)). In addition, Monzingo and Robertus,
J. Mol. Biol.
227:1136-45 (1992), carried out an X-ray analysis of substrate analogs in the RTA active site, examining FMP, adenyl guanosine (ApG) and guanyl adenosine (GpA). The structure of important substrate and analog bases, together with the numbering scheme used in energy minimizations, is shown in FIGS.
6
A-
6
D.
Several closely related mechanisms of action have been proposed which incorporate elements of both the structural and kinetic analyses. It is likely that the susceptible adenine base binds between tyrosines 80 and 123 while forming specific hydrogen bonds with the backbone carbonyl and amido nitrogen of Val 81 and with the carbonyl of Gly 121. In the hydrolysis, the leaving adenine is at least partially protonated by Arg 180, and Glu 177 may stabilize a putative oxycarbonium transition state or, more likely, act as a base to polarize the attacking water.
Shiga toxin, a compound essentially identical to the protein originally isolated from
Shigella dysenteriae,
has an A chain which is activated by proteolysis, generating an active A1 enzyme and an A2 fragment, which remains bound until a disulfide bond linking them is reduced. The active A1 chain (STA1) is a homologue of RTA, and catalyzes the same depurination reaction.
E. coli
strains can carry the gene for Shiga toxin, and this renders them pathogenic. Human infection by Shiga toxin lead to hemorrhagic colitis and hemolytic-uremic syndrome—commonly referred to as “hamburger disease”—a severe and often fatal form of food poisoning. It is difficult to control outbreaks of hamburger disease because antibiotics tend to lyse the bacteria, releasing the destructive toxin into the system, aggravating tissue damage and internal bleeding. An effective inhibitor of STA1 would be a powerful adjunct to treatment of hemorrhagic colitis and hemolytic-uremic syndrome.
The Shiga toxin gene has been cloned and engineered to express the enzyme. A comparison of the amino acid sequences of RTA and STA show clearly that they are homologues. An energy-minimized model of STA1 was constructed (Deresiewicz et al,
Biochemistry
31:3272-80 (1992)), and it was noted that key residues conserved among plant Ribosome Inactivating Protein (RIP) enzymes are conserved in STA1 (Katzin et al.,
Proteins
10:251-59 (1991)).
Until the pres

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