Prediction of the interaction of HIV-1 integrase and its dicaffeoylquinic acid inhibitor through molecular modeling approach.

An essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is integration of the double-stranded retroviral DNA into the genome of the host cell. HIV-1 integrase, the enzyme that inserts the vital DNA into the host chromosome, is an attractive and rational target for anti-AIDS drug design because it is essential for HIV replication and there are no known counterparts in the host cell. Inhibitors of this enzyme have a great potential to complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. Natural products have provided a source of new drug candidates for anti-AIDS therapy. Dicaffeoylquinic acids, isolated from traditional medicinal plants, are a novel class of integrase inhibitors. These compounds are potent inhibitors of HIV-1 replication in cultured cell lines and catalytic activities of integrase in vitro. They are therefore promising compounds for developing new anti-AIDS drugs. To understand how the inhibitors work and therefore design more potent and specific inhibitors, we have used molecular modeling techniques to investigate the binding modes of 3,4-dicaffeoylquinic acid. Our computational modeling study demonstrated that the inhibitor of this compound on HIV integrase is likely to proceed by two different but equivalent mechanisms with one bound to the active site region of the enzyme and another docked into the binding pocket located on the other side of the catalytic site. Our study will be of help to design new pharmaceuticals for the treatment of AIDS.

Sequential combination chemotherapy in human breast cancer: a basis for increased antineoplastic activity and bone marrow protection.

These studies were designed to develop procedures that would capitalize on the growth inhibitory effects of tamoxifen (Tam) and methotrexate (MTX) in breast cancer, while protecting bone marrow with a priming dose of 5-fluorouracil (5-FU). High-dose MTX (10 microM) cytotoxicity is maintained in MCF-7 breast cancer cells but reduced in human bone marrow by a priming and nontoxic dose of 5-FU (10 microM). MTX cytotoxicity is decreased in MCF-7 breast cancer cells when the selective estrogen receptor modulator (SERM) Tam (10 microM) is administered 24 hours prior to 5-FU (10 microM) followed two hours later by MTX (early Tam) resulting in a growth rate of 57.42 +/- 4.38% of the control rate. However, when breast cancer cells are exposed to Tam 24 hours after 5-FU + MTX (late Tam), the interaction between MTX and Tam is not antagonistic, the percentage of the control is 29.47 +/- 4.54%. Bone marrow exposure to these drug combinations exhibits a protective effect to the MTX cytotoxicity, with the early Tam combination yielding 59.45 +/- 16.38% of the control for MTX alone. These studies suggest that a) Tam in combination with a priming dose of 5-FU protects bone marrow from MTX cytotoxicity, b) the interactions between Tam and MTX are sequence-dependent, c) Tam decreases the effect of MTX when Tam administration precedes MTX.

The interaction of the steroidal antagonist faslodex and methotrexate.

Faslodex (FAS, ICI 182, 780), a novel steroidal estrogen antagonist decreased high-dose methotrexate (MTX) cytotoxicity in MCF-7 breast cancer cells. When FAS is given at least 24 hr prior to MTX, the resultant interaction is antagonistic. However, when breast cancer cells are exposed to FAS 24 hr after MTX, the interaction between FAS and MTX is not antagonistic. The proliferation of cells exposed to 0.1 microM FAS and 10 microM MTX alone or in combination with FAS 24 hr prior to MTX was in the following order: FAS>FAS 24 hr prior to MTX>MTX. MTX administration 24 hr prior to FAS had the following inhibitory effects on the growth of cells: MTX 24 hr prior to FAS >MTX>FAS 24 hr prior to MTX>FAS>control (no drug exposure). To determine if the antagonistic interaction between FAS and MTX was a function of sequence and time, cells were exposed to FAS 24 hr and 36 hr prior to MTX exposure. The percentages of control rates were 42.70 +/- 4.60% and 57.89 +/- 0.55%, respectively, from a 24 hr and 36 hr exposure of FAS prior to MTX. The growth rates after 24 and 36 hr exposures to MTX alone were 30.30 +/- 0.61% and 33.11 +/- 2.57% of control rates, respectively. These studies suggest that: a) the interactions between FAS and MTX are sequence-dependent; b) FAS antagonizes the effect of MTX when FAS administration precedes MTX, and c) FAS antagonism to MTX is a function of time.

Implications for improved high-dose methotrexate therapeutic effects in cultured human breast cancer and bone marrow cells.

The cytotoxicity of high-dose methotrexate (MTX), 10 and 100 microM, and 5-fluorouracil (5-FU) combinations is independent of sequence in human MDA-MB-436 breast carcinoma cells. The growth inhibitory effects of 10 and 100 microM MTX are 22.54+/-1.56% and 16.20+/-0.74%, respectively, of the control rate. When the MTX and 5-FU concentrations are 10 microM, antiproliferative effects of MTX 2 hr before 5-FU (MTX/5-FU) and 5-FU 2 h before MTX (5-FU/MTX) are 25.17+/-1.23% and 25.60+/-1.28% of the control rate, respectively. The percentage of control rates for 5-FU alone is 94.89+/-1.35%. The growth rates of MDA-MB-436 cells in 100 microM MTX and 10 microM 5-FU are 15.19+/-0.62% (MTX/5-FU) and 16.53+/-0.85% (5-FU/MTX) of the control rate. The growth of cancer cells in the presence of 5-FU alone is 93.82+/-1.69% of the control rate. A comparison of the cell-killing effects of MTX and the nonpolyglutamable antifolate trimetrexate (TMQ) alone and in combination with 5-FU was performed to indirectly explore the role of polyglutamylation in breast cancer and bone marrow cells. The comparisons were made in equitoxic concentrations (10 microM) of MTX and TMQ and the time of exposure was the same. The inhibitory effects of TMQ, TMQ/5-FU, and 5-FU/TMQ in breast cancer cells were identical, but significantly less than MTX, MTX/5-FU, and 5-FU/MTX. The interaction between TMQ and MTX, TMQ/5-FU and MTX/5-FU, and 5-FU/TMQ and 5-FU/MTX was quantitatively similar in bone marrow. (Significant protection occurred in bone marrow cells exposed to 5-FU/TMQ and 5-FU/MTX.) Because the effects of 5-FU/MTX and 5-FU/TMQ on bone marrow were the same, it is unlikely that polyglutamylation plays a significant role in the protective effects of 5-FU. However, the greater inhibitory effect of MTX or MTX and 5-FU combinations, when compared with TMQ or TMQ and 5-FU, suggests that polyglutamylation of MTX may contribute to the cytotoxicity of this antifolate to breast cancer cells. Hence, these studies suggest that a priming and nontoxic dose of 5-FU before high-dose MTX sustains MTX cytotoxicity in breast cancer and protects against MTX toxicity to bone marrow progenitor cells.

Interaction energy analyses of folate analog binding to human dihydrofolate reductase: contribution of the antifolate substructural regions to complex stability.

The three-dimensional structure of the human dihydrofolate reductase (DHFR), methotrexate tetrazole, and NADPH ternary complex was used to model the corresponding ternary complexes with methotrexate tetrazole replaced by methotrexate, methotrexate-polyglutamate with three glutamyl residues, and 5,10-deazaaminopterin, respectively. Each complex was solvated in a 60-angstrom cube of explicit water and subjected to structural minimization followed by interaction energy analyses. Interaction energy calculations were performed for the antifolate interaction with water, NADPH, the DHFR binding site residues, the entire DHFR protein, and the solvated NADPH:DHFR complex. These studies revealed that methotrexate-polyglutamate exhibited the most stable interactions and that approximately one half of antifolate:DHFR stability could be accounted for by the interaction of the antifolate with the binding site residues. The antifolate structures were also subdivided into heterocyclic, phenyl, and glutamyl substructural regions. Interaction energies were subsequently calculated for the interactions of the subregions with water, NADPH, the DHFR binding site residues, the DHFR protein, and the solvated NADPH:DHFR complex. The glutamyl substructural region showed the greatest contribution to overall antifolate binding stability due to its interaction with the DHFR protein. The heterocyclic and phenyl substructural regions generally showed much less stable interactions. These results suggest that the primary stabilizing factor of the antifolate interaction is the interaction of glutamyl with the DHFR protein. Additionally, interaction energy analyses were performed for specific groups of atoms within the substructural regions. These studies indicated that the stability of the glutamyl interaction is due to the interaction of glutamyl oxygen atoms with the DHFR protein. In the case of the methotrexate tetrazole complex, the tetrazole nitrogens also contribute significantly to the stability of the glutamyl interaction. The carbon atoms of the heterocyclic and phenyl groups both showed more stable interactions with NADPH than with water, while the nitrogen atoms showed more stable interactions with water than with NADPH. Collectively, these results indicate that the glutamyl region is the most important in antifolate binding stability.

Sequence-dependent antagonism between tamoxifen and methotrexate in human breast cancer cells.

High-dose methotrexate (MTX) cytotoxicity is decreased in MCF-7 breast cancer cells when the chemoendocrine agent tamoxifen (TAM) is given to cells 24 hours prior to MTX (early TAM). However, when breast cancer cells are exposed to TAM 24 hours after MTX (delayed TAM), MTX cytotoxicity is enhanced by TAM. The growth of cells exposed to 10 microM TAM and 10 microM MTX alone or in combination with early TAM plus MTX had the following order: TAM > TAM (early) + MTX > MTX. The percentages of control rates for TAM, MTX, and TAM (early) + MTX are 74.71 +/- 1.36%, 22.13 +/- 2.76%, and 38.17 +/- 2.75%, respectively. The inhibitory sequence from cells exposed to MTX + TAM (delayed TAM), MTX and TAM alone is MTX + TAM (delayed TAM) > MTX > TAM; and the percentages of control rates were 16.87 87% (MTX + TAM [delayed TAM]), 25.92 +/- 2.14% (MTX), and 54.08 +/- 14.79% (TAM). These studies suggest that: (a) the interactions between TAM and MTX are sequence-dependent; (b) TAM antagonizes the effect of MTX when TAM administration precedes MTX; and (c) TAM enhances the effect of MTX when TAM administration follows MTX.

5-Fluorouracil simultaneously maintains methotrexate antineoplastic activity in human breast cancer and protects against methotrexate cytotoxicity in human bone marrow.

High-dose methotrexate (MTX) cytotoxicity is maintained in MCF-7 breast cancer cells but reduced in Hs824.T human bone marrow by a priming and nontoxic 5-fluorouracil (5-FU) dose. When MCF-7 breast or Hs824.T bone marrow cells are incubated with 10 microM 5-FU and 10 microM MTX for 48 h, the growth rates of breast cancer cells were 97.59 +/- 0.97% and 21.81 +/- 3.33% of the control rate, respectively, and the growth rates of bone marrow cells were 90.61 +/- 3.71% and 29.58 +/- 2.99% of the control rate. The combinations of 5-FU 2 h prior to MTX or MTX 2 h prior to 5-FU followed by a 48 h incubation, respectively, gave growth rates of 20.96 +/- 2.44% and 19.86 +/- 2.56% of the control rate for MCF-7 cells. In bone marrow cells, the combinations of 5-FU 2 h prior to MTX or MTX 2 h prior to 5-FU followed by a 48 h incubation, respectively, gave growth rates of 79.66 +/- 7.41% (protection) and 31.39 +/- 1.77% of the control rate. Similar patterns to bone marrow emerges in platelets. These studies suggest that: a) MTX and 5-FU combination on the growth of human MCF-7 breast cancer cells is independent of sequence; and b) a priming-dose of 5-FU will protect bone marrow from MTX cytotoxicity but not breast cancer cells. Therefore, a priming and non-toxic dose of 5-FU and MTX may have maximum antineoplastic activity while at the same time provide protection to the hematopoietic system.

Molecular modelling of trimethoprim complexes of human wild-type and mutant dihydrofolate reductases: identification of two subsets of binding residues in the antifolate binding site.

Computer-assisted molecular modelling was used to generate structures for the trimethoprim (TMP):NADPH:dihydrofolate reductase (DHFR) ternary complexes for human wild-type DHFR and for five DHFR mutants (L22R, L22F, F31S, F31W and Q35P). The mutants correspond to DHFR proteins that have been isolated from tissues exposed to chronic or high dose methotrexate (MTX) and show decreased sensitivity to antifolate inhibition. Analysis of the TMP:DHFR interactions suggest the presence of two subsets of TMP binding residues in the DHFR antifolate binding site. One subset of these residues (GLU30, PHE34, ILE60 and VAL115) are common to each DHFR complex studied and are referred to as core residues. The other TMP binding residues vary among the DHFR complexes studied and are referred to as noncore residues. The core residues exhibit a greater number of TMP contacts/residue and form more stable TMP interactions than noncore residues. Additionally, the core and noncore residues make contact with different regions of the TMP structure. Information presented here provides additional insight into the design of new agents for the improved inhibition of wild-type DHFR and the simultaneous inhibition of both wild-type and mutant DHFR molecules.

Selectivity in human breast cancer and human bone marrow using trimetrexate in combination with 5-fluorouracil.

The growth inhibitory effect of trimetrexate (TMQ) is maintained in MCF-7 breast cancer but is decreased in Hs 824.T human bone marrow cells by a priming- and non-toxic 5-fluorouracil (5-FU) dose. Incubation of MCF-7 breast cells with 10 microM TMQ alone or in combination with 10 M 5-FU (TMQ 2 h prior to 5-FU [TMQ/5-FU] or 5-FU 2 h prior to TMQ[5-FU/TMQ]) resulted in similar inhibitory effects but dissimilar effects occurred in Hs 824.T bone marrow. In breast cancer, the percentage differences among TMQ and TMQ/5-FU, TMQ and 5-FU/TMQ, and TMQ/5-FU and 5-FU/TMQ on growth rates, respectively, were 3.56%, 2.35%, and 1.68%. The percentage differences on growth rates of TMQ and TMQ/5-FU, TMQ and 5-FU/TMQ, and TMQ/5-FU and 5-FU/TMQ in bone marrow, respectively, were 5.76%, 30.03% (significant protection by 5-FU, i.e. the inhibitory effect of 5-FU/TMQ < or = TMQ), and 35.78% (sequence dependent). The growth rates of breast cancer and bone marrow cells in the presence of 5-FU were 96.03 +/- 1.17% and 94.59 +/- 1.15%, respectively, of control rates. These studies suggest that (a) TMQ and 5-FU combinations on the growth of MCF-7 breast cancer cells are independent of sequence of administration and best related to TMQ and (b) a priming- and non-toxic 5-FU dose protects against TMQ toxicity in human bone marrow while not affecting the maximum inhibitory effect of TMQ in breast cancer.

Interaction of swainsonine with lymphoid and highly perfused tissues: a pharmacokinetics explanation for sustained immunomodulation.

The kinetics of inhibition of metastasis by the immunomodulator swainsonine (SW) is effective 1 to 3 days after administration. It is likely that SW's prolonged antimetastatic effect is due to its mitogenic property (spleenocytes isolated from animals treated with SW for 42-72 hours stimulated DNA synthesis that remained elevated for up to 3 days after removal of the drug from the drinking water). An analysis of SW in lymphoid (spleen and thymus) and highly perfused tissues was undertaken to determine if SW's sustained antimetastatic effect could be correlated to its retention. C57BL/6 mice received [3H]SW in drinking water for 24-72 hours and thereafter, received SW-free drinking for 24, 48, and 72 hours. Lymphoid and highly perfused tissues were analyzed for [3H]SW. At 24, 48, and 72 hours, spleen SW levels are, respectively, at least 2.33, 2.25, and 2.00 times greater than the perfused tissue; and thymus are, respectively, 1.44, 1.50, and 1.77 as great as the perfused tissue (kidney) with the highest SW level. These studies suggest that SW is predominantly retained for at least 72 hours, in lymphoid tissue. The targeting and retention of SW for lymphoid tissue days after removal of SW from animal drinking water is consistent with a) the immunomodulatory/mitogenic property and b) the sustained antimetastatic effect attributed to SW.

A molecular model of the folate binding site of Pneumocystis carinii dihydrofolate reductase.

The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.

The reaction of bisulfite with liposomal membranes.

Bisulfite has been shown to induce leakage of encapsulated substances from liposomal vesicles. The bisulfite induced leakage of either DNP-tyrosine, potassium ferricyanide, or [3H]glycine was observed to be greater with lipsomes composed of phospholipids containing unsaturated fatty acids. The leakage of encapsulated substances from liposomes was found to be concentration dependent when incubated for a constant time interval and time dependent when incubated at a constant bisulfite concentration. In addition, bisulfite caused the leakage of approximately 5 times more [3H]glycine from unilamellar liposomes than from multilamellar liposomes. These findings are consistent with the interaction of bisulfite with liposomal membranes via reaction with sites of unsaturation.

Sulfite oxidase activity in Thiobacillus novellus.

Thiobacillus novellus shows a maximum induction of sulfite oxidase activity and a maximum growth rate as a result of supplementing the autotrophic growth medium with 4.0 microM ammonium molybdate. Cells grown in the presence of molybdate showed approximately 10-fold increases in the amount of enzyme-associated molybdenum and in the sulfite-to-cytochrome c and sulfite-to-ferricyanide reductase activities. The effect of exogenous molybdate was not discernible with cells grown in the absence of thiosulfate. Tungsten inhibited the growth of T. novellus and the expression of sulfite oxidase activity.

Purification of Thiobacillus novellus sulfite oxidase. Evidence for the presence of heme and molybdenum.

Sulfite oxidase from Thiobacillus novellus has been purified 206-fold. The enzyme reduced both ferricyanide and cytochrome c. The ferricyanide activity was 3-5% of the cytochrome c activity. During purification, the absorbance ratio of A413 nm/A280 nm showed a continual increase, suggesting the presence of heme in the T. novellus sulfite oxidase molecule. The absorption spectrum of the enzyme is very similar to that of rat liver sulfite oxidase which contains cytochrome b5 type heme. Gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate revealed a protein staining band of approximately 41,000 Da. Gel filtration chromatography of the enzyme in aqueous media indicated a molecular weight of 38,000. These results suggest that T. novellus sulfite oxidase is a monomer of approximately 40,000 Da. Moreover, analysis of the visible absorption spectrum of the purified enzyme and the co-elution of 413 and 280 nm absorbing material during high pressure liquid chromatography gel chromatography provided clear evidence for the presence of heme in T. novellus sulfite oxidase. EPR spectroscopy of the enzyme revealed a characteristic molybdenum spectrum, which was observed only in the presence of sulfite. Analysis of the T. novellus sulfite oxidase molybdenum cofactor showed a fluorescence spectrum indistinguishable from that displayed by the molybdenum cofactor of chicken liver sulfite oxidase. Therefore, it is concluded that T. novellus sulfite oxidase is a monomeric (Mr approximately 40,000) molybdohemoprotein.

Interaction of bisulfite with unsaturated fatty acids.

Sodium bisulfite reacted with unsaturated fatty acids, significantly increasing their polarity as determined by behavior on silica gel thin-layer chromatography. The ultraviolet absorption spectra of unsaturated fatty acids (due to the presence of double bonds) were abolished as a result of the reaction. Fatty acids containing more than one double bond (arachidonate, linolinate, and linoleate) reacted more rapidly with bisulfite than did oleate. When arachidonate double bonds were titrated with bisulfite there was a much larger spectral decrease with the first equivalent of bisulfite added than with each subsequent addition. Vitamin E, vitamin E nicotinate, and butylated hydroxytoluene significantly inhibited the reaction of bisulfite with unsaturated fatty acids. It is suggested that the reaction of bisulfite with unsaturated fatty acids may be a mechanism of SO2 toxicity.