Fred K. Friedman

The human peroxisome proliferator-activated receptor alpha gene: identification and functional characterization of two natural allelic variants.

Peroxisome proliferator-activated receptor (PPAR)alpha-null mice have a defect in fatty acid metabolism but reproduce normally. The lack of a detrimental effect of the null phenotype in development and reproduction opens up the possibility for null or variant PPARalpha gene (PPARA) alleles in humans. To search the coding region and splice junctions for mutant and variant PPARalpha alleles, the human PPARalpha gene was cloned and characterized, and sequencing by polymerase chain reaction was carried out. Two point mutations in the human gene were found in the DNA binding domain at codons for amino acids 131 and 162. The allele containing the mutation in codon 162 (CTT to GTT, L162V) designated PPARA*3, was found at a high frequency in a Northern Indian population. Transfection assays of this mutant showed that the non-ligand dependent transactivation activity was less than one-half as active as the wild-type receptor. PPARA*3 was also unresponsive to low concentrations of ligand as compared to the wild-type PPARA*1 receptor. However, the difference is ligand concentration-dependent; at concentrations of the peroxisome proliferator Wy-14 643 > 25 microM, induction activity was restored in this variants transactivation activity to a level five-fold greater as compared with wild-type PPARA*1 with no ligand. The mutation in codon 131 (CGA to CAA, R131Q), designated PPARA*2 is less frequent than PPARA*3, and the constitutive ligand independent activity was slightly higher than PPARA*1. Increasing concentrations of Wy-14 643 activated PPARA*2 similar to that observed with PPARA*1. The biological significance of these novel PPARalpha alleles remains to be established. It will be of great interest to determine whether these alleles are associated with differential response to fibrate therapy.

DIFFERENTIAL MECHANISMS OF CYTOCHROME P450 INHIBITION AND ACTIVATION BY ALPHA -NAPHTHOFLAVONE

The anticarcinogenicity of some flavonoids has been attributed to modulation of the cytochrome P450 enzymes, which metabolize procarcinogens to their activated forms. However, the mechanism by which flavonoids inhibit some P450-mediated activities while activating others is a longstanding, intriguing question. We employed flash photolysis to measure carbon monoxide binding to P450 as a rapid kinetic technique to probe the interaction of the prototype flavonoid α-naphthoflavone with human cytochrome P450s 1A1 and 3A4, whose benzo[a]pyrene hydroxylation activities are respectively inhibited and stimulated by this compound. This flavonoid inhibited P450 1A1 binding to benzo[a]pyrene via a classical competitive mechanism. In contrast, α-naphthoflavone stimulated P450 3A4 by selectively binding and activating an otherwise inactive subpopulation of this P450 and promoting benzo[a]pyrene binding to the latter. These data indicate that flavonoids enhance activity by increasing the pool of active P450 molecules within this P450 macrosystem. Activators in other biological systems may similarly exert their effect by expanding the population of active receptor molecules.

Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells

We have synthesized three peptides from the mdm-2 binding domain of human p53, residues 12–26 (PPLSQETFSDLWKLL), residues 12–20, and 17–26. To enable transport of the peptides across the cell membrane and at the same time to maximize the active mdm-2 binding α-helical conformation for these peptides, each was attached at its carboxyl terminus to the penetratin sequence, KKWKMRRNQFWVKVQRG, that contains many positively charged residues that stabilize an α-helix when present on its carboxyl terminal end. All three peptides were cytotoxic to human cancer cells in culture, whereas a control, unrelated peptide attached to the same penetratin sequence had no effect on these cell lines. The same three cytotoxic peptides had no effect on the growth of normal cells, including human cord blood-derived stem cells. These peptides were as effective in causing cell death in p53-null cancer cells as in those having mutant or normal p53. Peptide-induced cell death is not accompanied by expression of apoptosis-associated proteins such as Bax and wafp21. Based on these findings, we conclude that the antiproliferative effects of these p53-derived peptides are not completely dependent on p53 activity and may prove useful as general anticancer agents.

Preferential induction of necrosis in human breast cancer cells by a p53 peptide derived from the MDM2 binding site

p53 is the most frequently altered gene in human cancer and therefore represents an ideal target for cancer therapy. Several amino terminal p53-derived synthetic peptides were tested for their antiproliferative effects on breast cancer cell lines MDA-MB-468 (mutant p53), MCF-7 (overexpressed wild-type p53), and MDA-MB-157 (null p53). p53(15)Ant peptide representing the majority of the mouse double minute clone 2 binding site on p53 (amino acids 12–26) fused to the Drosophila carrier protein Antennapedia was the most effective. p53(15)Ant peptide induced rapid, nonapoptotic cell death resembling necrosis in all breast cancer cells; however, minimal cytotoxicity was observed in the nonmalignant breast epithelial cells MCF-10–2A and MCF-10F. Bioinformatic/biophysical analysis utilizing hydrophobic moment and secondary structure predictions as well as circular dichroism spectroscopy revealed an α-helical hydrophobic peptide structure with membrane disruptive potential. Based on these findings, p53(15)Ant peptide may be a novel peptide cancer therapeutic because it induces necrotic cell death and not apoptosis, which is uncommon in traditional cancer therapy.

Drug-drug interactions: Effect of quinidine on nifedipine binding to human cytochrome P450 3A4

Quinidine is a known inhibitor of cytochrome P450-mediated nifedipine metabolism. The interactions of nifedipine and quinidine with human cytochrome P450 3A4, which metabolizes these drugs, were examined using the kinetics of CO binding to this P450 as a rapid kinetic probe of protein conformation and dynamics. This approach showed that nifedipine and quinidine bind to different P450 3A4 species, respectively termed species I and II, with distinct conformations. When both drugs were present simultaneously, nifedipine interacted with the quinidine-bound P450 species II, but not species I. These findings indicate that quinidine acts as an allosteric inhibitor by switching nifedipine binding from nifedipine-metabolizing species I to the nonmetabolizing species II.

Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics.

The kinetics of CO binding to cytochrome P450, as measured by the flash photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dynamics and are modulated by P450 interactions with substrates and other components of the microsomal membrane. Application of a difference method to kinetic data analysis distinguishes the kinetic behavior of individual P450 forms in the microsomal membrane. This approach shows that substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the binding rate; and/or 2) steric effects that reduce the rate. Both mechanisms are observed, the relative contributions of each varying in a substrate- and P450-dependent manner. In addition to microsomes, substrate interactions with individual P450s can be similarly probed using expressed P450s. Experiments with baculovirus-expressed human P450 3A4 show that this P450 consists of multiple conformers with distinct substrate specificities, an observation which provides a basis for its recognition of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fundamental P450-substrate interactions in a biological membrane environment.

ras-p21-induced cell transformation: unique signal transduction pathways and implications for the design of new chemotherapeutic agents.

One of the most striking discoveries over the past two decades has been the finding that a single base change at codon 12 in the human rus gene, which encodes a protein of 21 kDa, called the p21 protein (with 189 amino acids in its sequence), results in the malignant transformation of cells (1). The encoded p21 protein contains a valine in place of the normally occurring glycine at position 12 in its amino acid sequence. Transfection of the oncogene, but not its normal counterpart proto-oncogene, into NIH 3T3 cells in culture results in their malignant transformation. Microinjection of cloned, purified, oncogene-encoded (Val 12-containing) p21 protein, but not its normal counterpart protein, into NIH 3T3 cells also causes these cells to undergo cell transformation (2). Thus, the transforming agent is the rus gene-encoded p21 protein itself. Interestingly, oncogenic but not normal p21 induces maturation of frog (Xenopus luevis) oocytes (3). In the latter case, oncogenic p21 induces the progression of the oocytes through the second meiotic division. The biochemical changes induced by oncogenic p21 in all these cells appear to be the same, indicating that rus-p21 induces stereotypical events in each cell line. It is now known that there are other critical positions, such as at Gly 13, Ala 59, and Gln 61, in the polypeptide

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

Several rat liver cytochromes P-450 have been substantially purified in a one-step immunoadsorption procedure using Sepharose-bound monoclonal antibodies (MAbs) to the major forms of rat liver cytochrome P-450 induced by 3-methylcholanthrene and phenobarbital (MC-P-450 and PB-P-450, respectively). When mixed with solubilized rat liver microsomes the immunoadsorbent based on the MAb to MC-P-450 binds two polypeptides of MW 56,000 and 57,000 while the immunoadsorbent made with the MAb to PB-P-450 absorbs a species of MW 54,000. These polypeptides are readily desorbed by 0.1 M glycine (pH 3.0). Isolation of MAb-specific cytochrome P-450 isozymes by this method has applications in numerous phases of cytochrome P-450 research.

Identification of a glutathione-S-transferase effector domain for inhibition of jun kinase, by molecular dynamics.

We have recently found that the glutathione-S-transferase π-isozyme (GST-π), a cellular detoxification enzyme, potently and selectively inhibits activation of jun protein by its upstream kinase, jun kinase (JNK). This newly identified regulatory activity of GST-π is strongly inhibited by a group of agents that inhibit its enzymatic activity. Since loss of enzymatic activity in general does not correlate with loss of regulatory activity, it is likely that inhibitor binding induces changes in the structure of one or more domains of GST that block its interaction with JNK. To identify regions of GST that change conformation on the binding of inhibitors, we have performed molecular dynamics calculations on GST-π to compute its average structure in the presence and absence of the inhibitor, glutathione sulfonate. Superposition of the two average structures reveals that several regions change local structure depending upon whether the inhibitor is bound or not bound. Two of these regions, residues 36–50 and 194–201, are highly exposed. We have synthesized peptides corresponding to these two segments and find that the 194–201 sequence strongly inhibits the ability of GST-π to block the in vitro phosphorylation of jun by JNK. These results suggest that this region of GST-π is critical to its functioning as a newly discovered regulator of signal transduction.

Inhibition of human cytochrome P450 1A2 by flavones: a molecular modeling study.

Cytochrome P450 1A2 metabolizes a number of important drugs, procarcinogens, and endogenous compounds. Several flavones, a class of phytochemicals consumed in the human diet, have been shown to differentially inhibit human P450 1A2-mediated methoxyresorufin demethylase. A molecular model of this P450 was constructed in order to elucidate the molecular basis of the P450-flavone interaction. Flavone and its 3,5,7-trihydroxy and 3,5,7-trimethoxy derivatives were docked into the active site to assess their mode of binding. The site is hydrophobic and includes several residues that hydrogen bond with substituents on the flavone nucleus. The binding interactions of these flavones in the modeled active side are consistent with their relative inhibitory potentials, namely 3,5,7-trihydroxylflavone > flavone >3,5,7-trimethoxylflavone, toward P450 1A2-mediated methoxyresorufin demethylation.