Mutants of thymidylate synthase and uses thereof

Details Mutants of thymidylate synthase and uses thereof Mutants of thymidylate synthase and uses thereof

1999-08-04

2002-07-09

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S252300, C435S320100, C435S325000, C536S023200

Reexamination Certificate

active

06416987

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of gene therapy and biochemical pharmacology. More specifically, the present invention relates to mutants of human enzyme thymidylate synthase and uses thereof.
2. Description of the Related Art
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the rate limiting step in the sole de novo biosynthesis pathway to thymidylate, which is necessary for DNA synthesis and repair (Carreras et al., 1995). The mechanism of TS activity involves the reductive methylation of the substrate, 2′-deoxyuridine 5′-monophosphate (dUMP) by transfer of a methylene group from the cofactor, 5,10-methylenetetrahydrofolate (CH
2
H
4
folate), to generate 2′-deoxythymidine 5′-monophosphate (dTMP) and 7,8-dihydrofolate (H
2
folate). The inhibition of the TS pathway results in a thymineless state, which is toxic to rapidly dividing cells which have a high dTTP demand for DNA synthesis. This cytotoxicity is caused by DNA fragmentation and misincorporation of dUTP due to dTTP depletion. If there is enough supplied exogenous thymidine, cells survive through the salvage pathway depending on the use of thymidine kinase (TK). However, in normal tissues and in some tumor cells, the concentrations of circulating thymidine may not be sufficient to keep cells normally growing (Touroutoglou et al., 1996).
As a consequence, TS is an attractive target for anti-cancer drug design due to its crucial role in maintaining pools of thymidylate for DNA synthesis. Since the 1950s, many analogues of both the pyrimidine substrate (dUMP) and folate cofactor (CH
2
H
4
folate) have been synthesized and tested as potential anti-cancer therapeutics. However, although a number of inhibitors that tightly bind to TS were discovered, before 1995, 5-fluorouracil (5-FU) was the sole TS-targeted drug approved for clinical application. In vivo, 5-FU is metabolized to 5-fluoro-2-deoxyuridylate (FdUMP) that subsequently occupies the dUMP binding site forming a ternary complex with the enzyme and the folate cofactor, resulting in inhibition of TS. As the three-dimensional structures of TS have been revealed, the folate binding site in TS has been explored for the design of highly specific inhibitors (Jackman et al., 1995b), and have led to the emergence of novel folate analogues, such as tomudex (ZD1694), BW1843U89, AG331 and AG337 etc. These agents as the new generation TS-directed inhibitors have entered clinical trail in recent years. The approval of tomudex for treatment of advanced colorectal cancer in the United Kingdom occurred last year.
The major blood folate is 5-methyl-tetrahydrofolate (5-CH
3
—H
4
folate), which enters cells via membrane transports [or called reduced folate carriers (RFC)]. Once inside the cell, 5-CH
3
—H
4
folate is metabolized by methionine synthase to tetrahydrofolate. This coenzyme is converted by serine hydroxymethyltransferase to 5,10-methylene-tetrahydrofolate and also polyglutamated by folylpolyglutamate synthase (FPGS) to become 5,10-methylene-tetrahydrofolate polyglutamates [CH
2
H
4
folate(Glu)
n
]. CH
2
H
4
folate(Glu)
n
, as a cofactor, donates its one-carbon unit and two electrons to the reductive methylation reaction converting dUMP to dTMP. Dihydrofolate (H
2
folate) is a product of this process, which requires the sequential action of dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase in order to resynthesize CH
2
H
4
folate(Glu)
n
. Inhibition of DHFR by methotrexate (MTX) may lead to an accumulation of folates in the inactive H
2
folate form, resulting in depletion of CH
2
H
4
folate and dTMP.
There is much interest in correlating enzyme structure and function using mutagenesis. To date, several hundred mutations have been made in
L. casei, E. coli
and human TS (Climie et al., 1990b; Michael et al., 1990). Most of mutations in
L. casei
were produced by cassette mutagenesis (Wells et al., 1985; Climie et al., 1990a). The synthetic
L. casei
TS gene was engineered by creating over 30 unique restriction sites about equally spaced throughout the entire gene, providing “replacements sets” in which several target amino acids were replaced by a large number of substitutions. Another approach involving the introduction of an amber stop codon were adopted to generate multiple mutants of
E. coli
TS (Michaels et al., 1990; Kim et al., 1992). Using these approaches the various mutants in either
L. casei
or
E. coli
system were first screened for catalytic activity of TS by genetic complementation in a TS-deficient
E. coli
host, and then mutants of interest characterized by kinetic studies. A few mutants of human TS and their expressed enzymes in mammalian cells have also been studied. The mutant human TSs were also tested to complementation of the growth of TS-negative
E. coli
stains in the absence of thymine to determine if the activity of an altered enzyme is sufficient to support growth. However, the correlation of a mutant human TS and drug resistance can not be interpreted by this complementation study in a bacterial system and mammalian cells lacking TS are required. The three-dimensional structure of human TS has provided the impetus to generate mutants of human TS having novel enzyme properties such as drug resistance.
Prediction of properties of enzymes obtained by site-directed mutations is poor. When the enzyme accommodates a single amino acid substitution, readjustment of neighboring residues may occur, resulting in structural plasticity. TS is one of best examples for observing this phenomenon. In general, TS can tolerate amino acid substitutions even in a highly conserved residue that is important for enzyme structure or function. In a few cases, a single amino acid replacement causing dramatic change in properties of TS was also found. By reviewing the mutations already made, it was found that highly conserved residues are hot spots for amino acid substitutions (Carreras et al., 1995), and there are a few residues such as Arg50, Glu87, Trp109, Cys195, Arg215, Asp218, and Tyr258 especially sensitive to substitution (Stroud et al., 1993). All of these residues are in the substrate or folate binding site.
Cys195 (ec146, lc198) involved in the binding of 2′-deoxyuridylate as well as initiating the catalytic process could only be modified to Ser for
E. coli
TS and still retain activity, albeit severely diminished activity. None of the comparable
L. casei
mutants showed detectable activity (Dev et al., 1988; Climie et al., 1990b). Conserved Arg residues at positions 50, 215, 175, and 176 form a positively charged binding surface for the phosphate anion of dUMP. In
L. casei
, For Arg175, another completely conserved residue could be replaced by a neutral (Ala, Thr), positive (Lys). or negative (Glu) amino acid without drastic changes in substrate binding or catalytic activity (Santi et al, 1990). Most substitutions for Arg176 of either
E. coli
or
L. casei
TS result in little impairment of function. In contrast, Arg218 could not resist any amino acid shifts.
The Arg50 loop, having less than 1.0 Å movement and reorientation upon Arg50 (ec21, lc23) binding to the phosphate of dUMP, is a highly conserved region. For Arg50, only four amino acids (Gly, Pro, Ser, and His) in
E. coli
TS and three residues (Val, Ile, and Gln) in
L. casei
TS are substitutable with retention of 10-50% of the wild-type activity (Zhang et al., 1990; Michaels et al., 1990). Asp49 (ec20, lc22) is quite sensitive to mutagenesis, except for replacements by the two polar (Cys, Ser) and one acidic (Glu) residues, all
E. coli
Asp49 mutants do not complement growth of TS-negative cells. In
E. coli
TS, Thr51 (ec22, lc24) tolerates substitutions of Pro, Ser, Tyr, Gln and Lys. Surprisingly, contrary to those neighbor residues, Gly52 (ec23, lc: His52) accepts any mutations. This residue has apparent reorientation upon the formation of ternary TS complex (Kim et al., 1992).
Trp109 (ec80, lc82) and Asn112 (ec: Trp83, lc: Trp85) are highly

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