Christine C. Dykstra

Selective inhibition of topoisomerases from Pneumocystis carinii compared with that of topoisomerases from mammalian cells.

Type I and II topoisomerase activities were partially purified from Pneumocystis carinii. The catalytic (strand-passing) activities of both enzymes were selectively inhibited by members of a series of dicationic-substituted bis-benzimidazoles compared with those of topoisomerases of mammalian (calf thymus) origin. The most active inhibitors of the parasite enzymes were also highly effective in an in vivo animal model of P. carinii pneumonia. Selected dicationic-substituted bis-benzimidazoles also strongly inhibited the induction of the topoisomerase I- and II-mediated cleavable complex, suggesting that the biologically active DNA minor groove-binding molecules inhibit the enzyme-DNA binding step of the topoisomerase reaction sequence. The apparent selectivities for the parasite enzymes and the low levels of toxicity to mammalian cells for the biologically active bis-benzimidazoles suggest that these compounds hold promise as effective therapeutic agents in the treatment of a life-threatening AIDS-related disease, P. carinii pneumonia. Images

Activity of cationically substituted bis-benzimidazoles against experimental Pneumocystis carinii pneumonia

On the basis of a previously observed correlation between the antimicrobial activity and DNA binding strength of dicationic molecules, a series of 10 dicationically substituted bis-benzimidazoles were tested for activity in the rat model of Pneumocystis carinii pneumonia. One of the compounds, 1,4-bis[5-(2-imidazolinyl)-2-benzimidazolyl]butane, was found to be more potent and less toxic than pentamidine.

Synthesis of dicationic diaryltriazines nucleic acid binding agents

- The synthesis of 2,4-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1,3,5-triazine 6a and 2,4-bis[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl]-1,3,5-triazine 6b in 3 steps from either 4-bromobenzamidine or 4-(carbamoyl)benzamidine is reported. The synthesis of 4,6-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine 9a and 4,6-bis[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine 9b in 2 steps from 1,4-dicyanobenzene is also described. The compounds 6b and 9b bind strongly to DNA model sequences and inhibit topoisomerase II from 2 microbial sources. Compounds 6a and 9a bind to both DNA and RNA model sequences whereas 6b and 9b essentially do not bind to the RNA model.

Anti-Pneumocystis carinii pneumonia activity of dicationic 2,4-diarylpyrimidines.

A synthesis of 2,4-bis-(4-amidinophenyl)pyrimidine 6, 2,4-bis-[(4-imidazolin-2-yl)phenyl)]pyrimidine 7, 2,4-bis [(4-tetrahydropyrimidinyl-2-yl)phenyl]pyrimidine 8, 2,4-bis[(4-N-n-propylamidino)phenyl]pyrimidine 9, 2,4-bis[(4-N-isopropylamidino)-phenyl]pyrimidine 10 and 2,4-bis[(4-N-isobutylamidino)phenyl]pyrimidine 11 starting from 4-bromobenzamidine and 4-bromoaceto-phenone is reported. A synthesis of 2-(4-amidinopnenyl)-4-(2-methoxy-4-amidinophenyl)pyrimidine 20, 2-[4-(imidazolin2-yl)-phenyl]-4-[2-me thoxy-4-(imidazolin-2-yl)phenyl]pyrimidine 21, and 2-[4-(N-iso-propylamidino)phenyl]-4-[2-methoxy-4-(N-isopro-pylamidino)phenyl]pyrimidine 22 beginning with 4-bromobenzamidine and 2-methoxy-4-bromoacetophenone is described. Compounds 6-11 and 20-22 all bind strongly to DNA. Compounds 6, 9-11, and 20 given at 5 mg/kg are more active and less toxic than pentamidine at its effective dose when evaluated against Pneumocystis carinii pneumonia (PCP) in the immunosuppressed rat model. Several compounds in this series are being evaluated further as potential new anti-PCP agents.

Isolation and identification of six Pneumocystis carinii genes utilizing codon bias

Pneumocystis carinii pneumonia (PCP) is a leading cause of death among AIDS patients in the United States. Our analysis of P. carinii protein-coding genes has revealed a significant A + T codon bias. Polymerase chain reaction (PCR) was utilized to isolate and identify the genes encoding calmodulin, beta-tubulin, DNA polymerase II, and RNA polymerases I, II and III from P. carinii. Primer pairs were designed to incorporate P. carinii codon preference to known conserved protein regions from other organisms. This strategy should be useful for a large variety of P. carinii genes and assist in the comprehensive analysis of the genomic structure of this important pathogen.

Cloning and characterization of the calmodulin-encoding gene from Pneumocystis carinii ☆

Complete cDNA and genomic clones for the CaM gene encoding calmodulin (CaM) from Pneumocystis carinii have been isolated from rat and characterized. The nucleotide (nt) sequence contains an open reading frame interrupted by three introns, that encodes a protein of 152 amino acids. The predicted CaM protein of P. carinii shares a high degree of homology with other known CaM proteins.

Cloning and identification of Arp1, an actin-related protein from Pneumocystis carinii

ABSTRACT. The complete Pneumocystis carinii Arp1 gene has been sequenced from two cDNA clones. The gene encodes a protein 385 bp in length with an estimated size of 45,000 kD. The A + T% for the Arp1 gene and a 900‐bp sequence upstream of the gene were 63.7% and 70.3%, respectively. These values are consistant with A + T codon preference displayed by P. carinii and are similar to values reported for other P. carinii genes. The predicted amino acid sequence of the P. carinii Arp1 protein had a similarity of 87.6% with Neurospora crassa Arp1, 82.1% similarity with vertebrate centractin, and 71.2% similarity with the Saccharomyces cerevisiae Act5p. Expression of Arp1 mRNA in P. carinii was detectable via synthesis of cDNA and subsequent PCR amplification. Affinity purified antibodies against S. cerevisiae Act5p, and canine centractin recognized both the recombinantly expressed protein and a 45,000 kD protein in P. carinii nuclear extracts. The Arp1 gene is the second member of the actin multigene family that has been identified in P. carinii.