Quinazolines as cyclin dependent kinase inhibitors.

Quinazolines have been identified as inhibitors of CDK4/D1 and CDK2/E. Aspects of the SAR were investigated using solution-phase, parallel synthesis. An X-ray crystal structure was obtained of quinazoline 51 bound in CDK2 and key interactions within the ATP binding pocket are defined.

A cyclin D1/cyclin-dependent kinase 4 binding site within the C domain of the retinoblastoma protein.

Phosphorylation of the retinoblastoma protein (Rb) by the cyclin D1/cyclin-dependent kinase (cdk) 4 complex (cdk4/D1) is a key regulatory step for maintaining the orderly progression of the cell cycle. The B domain of Rb contains a site that recognizes and binds the LXCXE motif found in D-type cyclins. This interaction is important for phosphorylation of Rb by cdk4/D1, although in vitro the Rb C domain alone is efficiently phosphorylated by cdk4/D1. A mutation in the C domain of Rb, L901Q, has been identified that completely abolishes cdk4/D1 phosphorylation of the isolated C domain. By contrast, the L901Q mutation has no effect on phosphorylation by either cyclin E/cdk2 or cyclin B/cdk1, suggesting that the interaction between L901Q and cdk4/D1 is specific. Introduction of the L901Q mutation into Rb containing the A, B, and C domains results in phosphorylation becoming predominantly dependent on the LXCXE binding region. However, when the LXCXE binding region of Rb is mutated, phosphorylation becomes dependent on the L901 site within the C domain. The L901 binding site can supplant the LXCXE binding site for the cdk4/D1-dependent phosphorylation of S780 and S795 but not S807/S811. Despite the limited homology between C domains of Rb, p107, and p130, the L901 site is conserved and introduction of the L925Q mutation into the isolated C domain of p107 also inhibits phosphorylation by cdk4/D1. These data support a model for cdk4/D1 recognizing two independent binding sites in Rb and suggests a conservation of this C domain binding motif for cyclin D1/cdk4 kinase among the Rb family of proteins.

Defining the substrate specificity of cdk4 kinase-cyclin D1 complex.

cdk4 kinase-cyclin D1 complex (cdk4/D1) does not phosphorylate all of the sites within retinoblastoma protein (Rb) equally. Comparison of five phosphorylation sites within the 15 kDa C domain of Rb indicates that Ser795 is the preferred site of phosphorylation by cdk4/D1. A series of experiments has been performed to determine the properties of this site that direct preferential phosphorylation. For cdk4/D1, the preferred amino acid at the third position C-terminal to the phosphorylated serine/threonine is arginine. Substitution of other amino acids, including a conservative change to lysine, has dramatic effects on the rates of phosphorylation. This information has been used to mutate less favorable sites in Rb, converting them to sites that are now preferentially phosphorylated by cdk4/D1. A conserved site at Ser842 in the related pocket protein p107 is also preferentially phosphorylated by cdk4/D1. Although Rb and p107 differ significantly in sequence, the Rb Ser795 site can replace the p107 Ser842 site without affecting the rate of phosphorylation. These results suggest that although a determinant of specificity resides in the sequences surrounding the phosphorylated site, the structural context of the site is also a critical parameter of specificity.

Genetically engineered bacteria to identify and produce anti-viral agents.

We have prepared a strain of Escherichia coli that expresses both the HIV protease and a Tet protein which has been modified to contain the HIV protease recognition sequence. When the protease is expressed, the bacteria will not grow in the presence of tetracycline. However, when the protease is inhibited the bacteria can grow in tetracycline containing media (Block and Grafstrom 1990). We have selected spontaneously arising Tet resistant mutants and have screened them for those that could be producing an inhibitor of HIV protease. The problems in the construction of this strain and the characterization of the various Tetr mutants are discussed.

The DNA binding properties of the MutL protein isolated from Escherichia coli.

The mutL gene of Escherichia coli, which is involved in the repair of mispaired and unpaired nucleotides in DNA, has been independently cloned and the gene product purified. In addition to restoring methyl-directed DNA repair in extracts prepared from mutL strains, the purified MutL protein binds to both double and single stranded DNA. The affinity constant of MutL for unmethylated single stranded DNA was twice that of its affinity constant for methylated single stranded DNA and methylated or unmethylated double stranded DNA. The binding of MutL to double stranded DNA was not affected by the pattern of DNA methylation or the presence of a MutHLS-repairable lesion.

Novel bacteriological assay for detection of potential antiviral agents.

A prototype assay for the initial screening of potential antiviral agents that uses bacterial growth on selective media is described. The human immunodeficiency virus (HIV) protease recognition sequence was inserted into the tetracycline resistance (Tet) protein encoded by plasmid pBR322 of Escherichia coli. Expression of both the HIV protease and the modified Tet protein prevented growth in the presence of tetracycline. However, inhibition of the HIV protease restored tetracycline resistance. Thus, potential HIV protease inhibitors can be identified by their ability to confer tetracycline resistance to this bacterial strain. The assay is simple, rapid, and inexpensive, and this concept can be applied to the search for inhibitors of other viral proteases.

Methyl-directed repair of frameshift heteroduplexes in cell extracts from Escherichia coli.

The methyl-directed DNA repair efficiency of a series of M13mp9 frameshift heteroduplexes 1, 2, or 3 unpaired bases was determined by using an in vitro DNA mismatch repair assay. Repair of hemimethylated frameshift heteroduplexes in vitro was directed to the unmethylated strand; was dependent on MutH, MutL, and MutS; and was equally efficient on base insertions and deletions. However, fully methylated frameshift heteroduplexes were resistant to repair, while totally unmethylated substrates were repaired with no strand bias. Hemimethylated 1-, 2-, or 3-base insertion and deletion heteroduplexes were repaired by the methyl-directed mismatch repair pathway as efficiently as the G.T mismatch. These results are consistent with earlier in vivo studies and demonstrate the involvement of methyl-directed DNA repair in the efficient prevention of frameshift mutations.

In vivo studies of repair of 2-aminopurine in Escherichia coli.

The repair of the base analog 2-aminopurine has been studied in vivo by using a temperature-sensitive mutant of the cloned mutH gene of Escherichia coli. Our results suggest that the lethal event in killing of dam mutants by 2-aminopurine does not result simply from incorporation of 2-aminopurine into the DNA and its subsequent repair. Furthermore, a 10-fold increase in the level of 2-aminopurine incorporated into the DNA of a dam mutH double mutant has little effect on the mutation frequency of this strain. An alternative mechanism for the mutagenicity of 2-aminopurine in E. coli is proposed.

Nucleotide sequence of the Escherichia coli mutH gene.

The complete nucleotide sequence of mutH gene from E. coli has been determined. Based on the deduced amino acid sequence, the MutH protein has a molecular weight of 25.4 kdaltons in agreement with the previous estimates based on SDS-polyacrylamide gel electrophoresis of the purified protein. Deletion analysis of the DNA sequences upstream of mutH has identified the promoter region for this gene. Two independently isolated temperature sensitive alleles of the mutH gene have also been sequenced. One mutation results in an amino acid change at position 27 (thr to leu) while the other occurs at position 156 (asp to asn).

The repair of pyrimidine dimers via a DNA-glycosylase mechanism.

The "UV endonuclease" isolated either from M. luteus or bacteriophage T4 infected E. coli (the denV gene product) consists of two enzymatic activities on a single polypeptide chain: a pyrimidine dimer-DNA glycosylase and an AP endonuclease. The repair of pyrimidine dimers by this enzyme is initiated by the cleavage of the N-glycosylic bond of the 5' pyrimidine of the dimer that leaves the cyclobutane dimer still attached to the DNA through the N-glycosylic bond of the 3' pyrimidine of the dimer. This reaction results in the formation of an apyrimidinic site in the DNA. The second step in this repair pathway is the endonucleolytic cleavage of the DNA 3' to the AP site by the associated AP endonuclease. As a result, the nicked DNA contains DNA damage on both sides of the incision site: an apyrimidinic moiety on the 3' end and a thymine-thymidylate dimer on the 5' end. The enzymes prefer double stranded DNA over single stranded DNA, and thymine over cytosine at the 5' position of the dimer. The AP endonuclease activity prefers the AP site created by the pyrimidine dimer-DNA glycosylase on UV irradiated DNA over either apurinic or apyrimidinic DNA. This repair mechanism appears to be operative in vivo since DNA intermediates containing thymine-thymidylate dimer sites have been detected in UV irradiated T4 infected E. coli and in UV irradiated M. luteus. The cloned denV gene partially complements the UV repair deficient uvr A, B, C strains of E. coli.

The characteristics of DNA methylation in an in vitro DNA synthesizing system from mouse fibroblasts.

An in vitro DNA synthesizing system from mouse fibroblasts has been used to study DNA methylation. DNA methylation occurs in two phases, one at the replication fork and the other farther behind it. Although 4% of the dCMP residues in mouse cell DNA are mdCMP, only 1.7% of the total [alpha 32P]dCMP in newly replicated DNA is methylated in vitro. No methylation of Okazaki fragments was detected. Nearest neighbor analysis of the newly replicated DNA revealed that, although 40% of the CpG dinucleotides were methylated, significant amounts of cytosine methylation were also found in CpC, CpT, and CpA dinucleotides.

Cloning of mutH and identification of the gene product.

A specialized lambda transducing phage carrying the mutH gene and several deletion derivatives of this phage were characterized by restriction enzyme analysis. This analysis localized the mutH gene to a small region of bacterial DNA on the transducing phage and facilitated the subsequent cloning of this gene into the multicopy plasmid pBR322. The mutH gene is contained entirely on a 1.5-kb HindIII fragment as judged by the ability of plasmids carrying this fragment to complement mutH- alleles on the bacterial chromosome. Using recombinant plasmids containing the 1.5-kb HindIII fragment, we identified an Mr 25000 protein as the product of the mutH gene in an in vitro transcription-translation system as well as in maxicells. Various deletion derivatives of the mutH-containing plasmids that exhibit a Mut- phenotype also have lost the Mr 25000 protein.

Human placental apurinic/apyrimidinic endonuclease. Its isolation and characterization.

An endonuclease from human placenta has been purified to apparent homogeneity, which acts specifically on DNA containing either apurinic or apyrimidinic sites. The isolation procedure, which results in a 20,000-fold purification and an overall yield of 15%, employs chromatography on a gel of octyl succinic anhydride coupled to agarose by diaminohexane spacers, isoelectric focusing, Sephadex G-75 chromatography, and DNA agarose affinity chromatography. Under conditions in which proteolysis is minimized, this enzyme appears to be the major species of apurinic/apyrimidinic endonuclease. The endonuclease is a monomeric protein with an apparent Mr = 37,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 7.4-7.6, requires Mg2+, is partially stimulated by Mn2+, and is inhibited by EDTA. It has no detectable exonuclease or phosphomonoesterase activity.

Enzymatic repair of pyrimidine dimer-containing DNA. A 5' dimer DNA glycosylase: 3'-apyrimidinic endonuclease mechanism from Micrococcus luteus.

A pyrimidine dimer-DNA glycosylase has been purified 20,000-fold from Micrococcus luteus. The enzyme is a single polypeptide chain with Mr = 18,000 that acts specifically on pyrimidine dimers, preferring those in double-stranded DNA to those in single-stranded DNA. The glycosylase cleaves the 5' residue of a pyrimidine dimer generating an apyrimidinic site and a mixed pyrimidine/pyrimidine nucleotide dimer. Under conditions of substrate excess, dimers containing a 5'-thymine are preferred to those with a 5'-cytosine residue. The glycosylase has an associated apyrimidinic/apurinic (AP) endonuclease that prefers apyrimidinic sites at the site of glycosylase action to either apurinic or apyrimidinic residues. This endonuclease is a Class I AP endonuclease in that it cleaves 3' to the AP site generating a 3'-deoxyribose moiety and a 5'-phosphate.

Human placental apurinic/apyrimidinic endonuclease. Mechanism of action.

The mechanism of action of the homogeneous preparation of human placental apurinic/apyrimidinic (AP) endonuclease, described in the previous paper (Shaper, N. L., Grafstrom, R. H., and Grossman, L. (1982) J. Biol. Chem. 257, 13455-13458), has been investigated in detail. This enzyme cleaves apyrimidinic DNA both 5' and 3' to the site of damage in a ratio of 60:40, respectively. Even though this enzyme can cleave on both sides of an internal AP site, it does not release deoxyribose 5-phosphate from terminal AP sites. However, a compound, tentatively identified as alpha, beta unsaturated deoxyribose 5-phosphate, is nonenzymatically released only from 5'-terminal AP sites, presumably by a beta-elimination mechanism.

Cleavage of pyrimidine dimers in specific DNA sequences by a pyrimidine dimer DNA-glycosylase of M. luteus.

Pyrimidine dimer formation in response to UV radiation is governed by the thymine content of the potential dimer and the two flanking nucleotides. An enzymatic activity can be purified from Micrococcus luteus that cleaves the N-glycosyl bond between the 5' pyrimidine of a dimer and the corresponding sugar without rupture of a phosphodiester bond. We propose that strand scission at a dimer site by the M. luteus enzyme requires two activities, a pyrimidine dimer DNA-glycosylase and an apyrimidinic/apurinic endonuclease.

Covalent association of protein with replicative form DNA of parvovirus H-1.

The double-stranded replicative form (RF) DNA of the autonomous parvovirus H-1 can be isolated from infected cells in a covalent complex with protein. The protein is present on most or all of the RF DNA, including actively replicating molecules, and is associated with the 5'-terminal endonuclease Hae III fragments of both the viral and complementary strands of RF. The size of the protein is estimated to be 60,000-70,000 daltons from its effect on buoyant density of DNA. DNA with covalently bound protein has not been found in H-1 virions.

The incorporation of uracil into animal cell DNA in vitro.

In the presence of dUTP, net DNA synthesis in vitro is substantially reduced. Small DNA fragments that arise during in vitro DNA synthesis in the presence of dUTP are produced as a result of dUMP incorporation and subsequent post-replication excision repair process initiated by uracil-DNA-glycosylase. The size of the fragments is dependent upon the amount of dUMP incorporated, but unlike the normal 4S intermediates of DNA synthesis, these repair products are not precursors to high molecular weight DNA but are further degraded. The high levels of dUTPase as well as the presence of RNA primers on most nascent DNA pieces (Tseng and Goulian, 1977) suggest that repair of uracil-containing DNA does not contribute to the generation of the small, nascent DNA pieces found during DNA synthesis in this in vitro system.