Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.

Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation

The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed IBa recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Involvement of the amygdala in memory storage: Interaction with other brain systems

There is extensive evidence that the amygdala is involved in affectively influenced memory. The central hypothesis guiding the research reviewed in this paper is that emotional arousal activates the amygdala and that such activation results in the modulation of memory storage occurring in other brain regions. Several lines of evidence support this view. First, the effects of stress-related hormones (epinephrine and glucocorticoids) are mediated by influences involving the amygdala. In rats, lesions of the amygdala and the stria terminalis block the effects of posttraining administration of epinephrine and glucocorticoids on memory. Furthermore, memory is enhanced by posttraining intra-amygdala infusions of drugs that activate β-adrenergic and glucocorticoid receptors. Additionally, infusion of β-adrenergic blockers into the amygdala blocks the memory-modulating effects of epinephrine and glucocorticoids, as well as those of drugs affecting opiate and GABAergic systems. Second, an intact amygdala is not required for expression of retention. Inactivation of the amygdala prior to retention testing (by posttraining lesions or drug infusions) does not block retention performance. Third, findings of studies using human subjects are consistent with those of animal experiments. β-Blockers and amygdala lesions attenuate the effects of emotional arousal on memory. Additionally, 3-week recall of emotional material is highly correlated with positron-emission tomography activation (cerebral glucose metabolism) of the right amygdala during encoding. These findings provide strong evidence supporting the hypothesis that the amygdala is involved in modulating long-term memory storage.

Public–private interaction in pharmaceutical research

We empirically examine interaction between the public and private sectors in pharmaceutical research using qualitative data on the drug discovery process and quantitative data on the incidence of coauthorship between public and private institutions. We find evidence of significant reciprocal interaction, and reject a simple “linear” dichotomous model in which the public sector performs basic research and the private sector exploits it. Linkages to the public sector differ across firms, reflecting variation in internal incentives and policy choices, and the nature of these linkages correlates with their research performance.

Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.