The inophyllums, novel inhibitors of HIV-1 reverse transcriptase isolated from the Malaysian tree, Calophyllum inophyllum Linn.

As part of a search for novel inhibitors of HIV-1 reverse transcriptase, the acetone extract of the giant African snail, Achatina fulica, was shown to be active. Fractionation of the extract yielded inophyllums A, B, C, and E and calophyllolide (1a, 2a, 3a, 3b, and 6), previously isolated from Calophyllum inophyllum Linn., a known source of nutrition for A. fulica. From a methanol/methylene chloride extract of C. inophyllum, the same natural products in considerably greater yield were isolated in addition to a novel enantiomer of soulattrolide (4), inophyllum P (2b), and two other novel compounds, inophyllums G-1 (7) and G-2 (8). The absolute stereochemistry of inophyllum A (1a) was determined to be 10(R), 11(S), 12(S) from a single-crystal X-ray analysis of its 4-bromobenzoate derivative, and the relative stereochemistries of the other inophyllums isolated from C. inophyllum were established by a comparison of their 1H NMR NOE values and coupling constants to those of inophyllum A (1a). Inophyllums B and P (2a and 2b) inhibited HIV reverse transcriptase with IC50 values of 38 and 130 nM, respectively, and both were active against HIV-1 in cell culture (IC50 of 1.4 and 1.6 microM). Closely related inophyllums A, C, D, and E, including calophyllic acids, were significantly less active or totally inactive, indicating certain structural requirements in the chromanol ring. Altogether, 11 compounds of the inophyllum class were isolated from C. inophyllum and are described together with the SAR of these novel anti-HIV compounds.

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site.

Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.

On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex.

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.

Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity.

Several camptothecin derivatives containing a modified hydroxy lactone ring have been synthesized and evaluated for inhibition of topoisomerase I and cytotoxicity to mammalian cells. Each of the groups of the hydroxy lactone moiety, the carbonyl oxygen, the ring lactone oxygen, and the 20-hydroxy group, were shown to be critical for enzyme inhibition. For example the lactol, lactam, thiolactone, and 20-deoxy derivatives did not stabilize the covalent DNA-topoisomerase I complex. With a few exceptions, those compounds that did not inhibit topoisomerase I were not cytotoxic to mammalian cells. Two cytotoxic derivatives that did not inhibit topoisomerase I were shown to produce non-protein-associated DNA single-strand breaks and are likely to have a different mechanism of action. One of these compounds was tested for antitumor activity and was found to be inactive. The present findings, as well as other reports that the hydroxy lactone ring of camptothecin is critical for antitumor activity in vivo, correlate with the structure-activity relationships at the level of topoisomerase I and support the hypothesis that antitumor activity is related to inhibition of this target enzyme.

Camptothecin-resistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase I.

In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to camptothecin (CptR mutants) have been isolated from mutagen-treated cultures. The CptR mutants exhibited no cross-resistance towards drugs such as colchicine, vinblastine, taxol, or puromycin but showed slightly (2- to 3-fold) enhanced sensitivity towards various drugs that inhibit DNA topoisomerase II (namely teniposide, etoposide, doxorubicin, mitoxantrone, amsacrine, ellipticine), suggesting that the genetic lesion in these mutants was highly specific. In contrast to the wild-type cells, the CptR line was resistant to camptothecin-induced DNA strand breaks as measured by alkaline elution. Biochemical studies revealed that in CptR mutants the cellular activity as well as protein content of DNA topoisomerase I were reduced to about 40-50% of the level in wild-type cells. Normal levels of activity and content were observed for the related enzyme DNA topoisomerase II. Studies with DNA topoisomerase I purified from the wild-type and the mutant cells showed that the enzyme from the CptR cells was markedly resistant to camptothecin as assayed by the drug's effects either on relaxation of supercoiled DNA or on stabilization of the covalent enzyme-DNA intermediate. The presence of a camptothecin-resistant form of DNA topoisomerase I in the mutant cells provides further evidence that this enzyme is the cellular target of camptothecin. Cell hybridization studies between the CptR and CptS cells showed that the hybrids formed between these two cell lines were sensitive to camptothecin. The recessive behavior of the CptR mutation provides a plausible explanation for the reduced topoisomerase I content (about one-half of wild-type cells) of the mutant cells and also for their enhanced sensitivity towards inhibitors of topoisomerase II.

Degradation of structurally modified DNAs by bleomycin group antibiotics.

Bleomycin-mediated DNA strand scission has been shown to be diminished at certain sequences in proximity to 5-methylcytidines. We have investigated the molecular basis of this observed diminution using selective bleomycin (BLM) modifications at the C-terminus. Of the four different bleomycin congeners investigated, only bleomycin A2 and bleomycin BAPP were substantially affected by cytidine methylation. We have also examined the effect of other DNA modifications on bleomycin-mediated strand scission. Methylation at the N6 position of adenosine resulted in diminution of DNA cleavage by all four bleomycin congeners. The presence of bulky 5-(glucosyloxy)methyl groups in the major groove of T4 DNA had little effect on the efficiency of DNA strand scission mediated by bleomycin A2 or B2, suggesting the absence of important steric interactions between Fe(II).BLM and DNA in the major groove. In contrast, DNA cleavage mediated by bleomycin congeners was very sensitive to a major DNA conformational change, the B----Z transition. Salt and MgCl2 titrations of the DNA copolymers poly(dG-dC).poly(dG-dC) and poly(dG-MedC).poly(dG-MedC) demonstrated that bleomycin A2 and B2 did not cleave Z-DNA efficiently. In addition, circular dichroism titrations of these copolymers revealed that both bleomycin congeners increased the cation concentration necessary to induce the B----Z transition, implying that bleomycin preferentially binds to and stabilizes B-form DNA. These results are consistent with a model in which cytidine methylation at appropriate sequences of DNA is sufficient to induce subtle conformational changes that render the helix unreceptive to cleavage by some bleomycin congeners.

DNA methylation diminishes bleomycin-mediated strand scission.

Three DNA duplexes differing substantially in sequence were derived from pBR322 plasmid DNA and supercoiled SV40 DNA by digestion with appropriate restriction endonucleases. Following treatment with the restriction methylase HhaI (recognition sequence: GCGC) or HhaI and HpaII (CCGG), the unmethylated and methylated DNAs were compared as substrates for the antitumor agent bleomycin. Bleomycin-mediated strand scission was shown to diminish substantially at a number of sites in proximity to the methylated cytidine moieties, especially where multiple sites had been methylated within a DNA segment of limited size. Detailed analysis of the DNA substrates revealed that both strands of DNA within a methylated region became more refractory to cleavage by bleomycin and that the protective effect could extend as many as 14 base pairs in proximity to the 5-methylcytidine moieties. Among the methylated DNA segments that became more resistant to bleomycin cleavage was a HpaII site of SV40 DNA, methylation of which has previously been shown to diminish the synthesis of the major late viral capsid protein following microinjection into Xenopus laevis oocytes. Study of the cleavage reaction at varying salt levels suggested that diminished bleomycin strand scission may be due, at least in part, to local conformational changes of the DNA to Z form (or other non-B-form structures). The results are generally consistent with the hypothesis that one mechanism for the expression of selective therapeutic action by certain DNA damaging agents could involve the recognition of specific methylation patterns.

New orientations of ancestral, "long interspersed repeated sequences" (LINES) in human DNA.

Three sets of long, interspersed repeated sequences (LINES) are described in human DNA. Each set contains two cleavage sites for the restriction endonuclease, XbaI. One set, called the Xba 850 LINES was detected only in gibbons, apes and man but is related in sequence to a more ancestral LINES family, the Kpn 1200 LINES, and in fact some Xba 850 LINES members retained the ancestral spacing of KpnI cleavage sites. The facts that the Xba 850 LINES appear as a subset of the Kpn 1200 LINES and vice versa and that the Xba 850 LINES are restricted to a smaller phylogenetic group than the Kpn 1200 LINES prompted the speculation that the Xba 850 LINES originated by a relatively recent amplification of one or a few Kpn 1200 LINES sequences.

Quantitation of RNA-dependent platelet DNA polymerase in patients with myeloproliferative disorders.

Platelets from 28 patients with the myeloproliferative diseases (MPD) polycythaemia vera (9), essential thrombocythaemia (6), myelofibrosis with myeloid metaplasia (5) and chronic myelogenous leukaemia (8) were examined for an RNA-dependent DNA polymerase activity using standardized conditions permitting highly reproducible quantitation. Low levels of activity were detected in platelets of normal individuals, but platelets of nearly all MPD patients (25/28) possessed higher levels. The polymerase activity correlated with diagnosis (P = 0.001) and did not correlate with platelet counts (P greater than 0.2). Quantitation of this RNA-dependent DNA polymerase activity may be a useful parameter in the diagnosis of myeloproliferative disorders.