A posttranslational modification of beta-actin contributes to the slow dissociation of the spectrin-protein 4.1-actin complex of irreversibly sickled cells.

Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin.

Pharmacophore-guided lead optimization: the rational design of a non-zinc coordinating, sub-micromolar inhibitor of the botulinum neurotoxin serotype a metalloprotease.

Botulinum neurotoxins, responsible for the neuroparalytic syndrome botulism, are the deadliest of known biological toxins. The work described in this study was based on a three-zone pharmacophore model for botulinum neurotoxin serotype A light chain inhibition. Specifically, the pharmacophore defined a separation between the overlaps of several different, non-zinc(II)-coordinating small molecule chemotypes, enabling the design and synthesis of a new structural hybrid possessing a Ki=600 nM (+/-100 nM).

Biochemical genetic analysis of indanocine resistance in human leukemia.

Indanocine is a potent tubulin-binding drug that is cytotoxic to multidrug-resistant cancer cell lines. We demonstrated that indanocine specifically induces apoptosis in malignant B cells from patients with chronic lymphocytic leukemia. To address the exact biochemical basis for indanocine toxicity, an indanocine-resistant clone was selected from mutagenized CEM human lymphoblastoid cells. The resistant cells displayed a stable indanocine-resistant phenotype for at least 9 months in drug-free culture. The cloned cells are cross-resistant to colchicine and vinblastine, but not to paclitaxel, and do not have increased expression of the multidrug-resistant p170 glycoprotein. In both parental cells and cell extracts, indanocine treatment caused tubulin depolymerization. In contrast, the tubulin in the resistant clone did not depolymerize under identical conditions. Both extract mixing and cell fusion experiments suggested that a stable structural change in microtubules, rather than a soluble factor, was responsible for indanocine resistance. Sequence analysis of parental and resistant cells revealed a single point mutation in the M40 isotype of beta-tubulin at nucleotide 1050 (G-->T, Lys(350)-->Asn) in the indanocine-resistant clone, in a region close to the putative colchicine binding site.

Discovery and initial characterization of the paullones, a novel class of small-molecule inhibitors of cyclin-dependent kinases.

Analysis of the National Cancer Institute Human Tumor Cell Line Anti-Cancer Drug Screen data using the COMPARE algorithm to detect similarities in the pattern of compound action to flavopiridol, a known inhibitor of cyclin-dependent kinases (CDKs), has suggested several possible novel CDK inhibitors. 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one, NSC-664704 (kenpaullone), is reported here to be a potent inhibitor of CDK1/cyclin B (IC50, 0.4 microM). This compound also inhibited CDK2/cyclin A (IC50, 0.68 microM), CDK2/cyclin E (IC50, 7.5 microM), and CDK5/p25 (IC50, 0.85 microM) but had much less effect on other kinases; only c-src (IC50, 15 microM), casein kinase 2 (IC50, 20 microM), erk 1 (IC50, 20 microM), and erk 2 (IC50, 9 microM) were inhibited with IC50s less than 35 microM. Kenpaullone acts by competitive inhibition of ATP binding. Molecular modeling indicates that kenpaullone can bind in the ATP binding site of CDK2 with residue contacts similar to those observed in the crystal structures of other CDK2-bound inhibitors. Analogues of kenpaullone, in particular 10-bromopaullone (NSC-672234), also inhibited various protein kinases including CDKs. Cells exposed to kenpaullone and 10-bromopaullone display delayed cell cycle progression. Kenpaullone represents a novel chemotype for compounds that preferentially inhibit CDKs.

Cyclin-dependent kinases: initial approaches to exploit a novel therapeutic target.

Cyclin-dependent kinases (CDKs) have been recognized as key regulators of cell cycle progression. Alteration and deregulation of CDK activity are pathogenic hallmarks of neoplasia. Therefore, inhibitors or modulators would be of interest to explore as novel therapeutic agents in cancer, as well as other hyperproliferative disorders. Flavopiridol is a semisynthetic flavonoid that emerged from an empirical screening program as a potent antiproliferative agent that mechanistic studies demonstrated to directly inhibit CDKs 1, 2, and 4 as a competitive ATP site antagonist. Initial clinical trials have shown that concentrations that inhibit cell proliferation and CDK activity in vitro can be safely achieved in humans, and additional clinical trials will establish its clinical potential. To address the need for additional chemotypes that may serve as lead structures for drugs that would not have the toxicities associated with flavopiridol, compounds with a similar pattern of cell growth inhibitory activity in the National Cancer Institute's in vitro anticancer drug screen have been recognized by the computer-assisted pattern recognition algorithm COMPARE and then screened for anti-CDK activity in a biochemical screen. The benzodiazepine derivative NSC 664704 (7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one) was revealed by that approach as a moderately potent (IC50 0.4 microM) inhibitor of CDK2, which in initial experiments shows evidence of causing cell cycle redistribution in living cells. NSC 664704 is, therefore, a candidate for further structural optimization, guided in part by understanding of the ATP-binding site in CDK2. This approach represents one way of combining empirical screening information with structure-based design to derive novel candidate therapeutic agents directed against an important cellular target.

Chemical synthesis and biological properties of pyridine epothilones.

BACKGROUND: Numerous analogs of the antitumor agents epothilones A and B have been synthesized in search of better pharmacological profiles. Insights into the structure-activity relationships within the epothilone family are still needed and more potent and selective analogs of these compounds are in demand, both as biological tools and as chemotherapeutic agents, especially against drug-resistant tumors. RESULTS: A series of pyridine epothilone B analogs were designed, synthesized and screened. The synthesized compounds exhibited varying degrees of tubulin polymerization and cytotoxicity properties against a number of human cancer cell lines depending on the location of the nitrogen atom and the methyl substituent within the pyridine nucleus. CONCLUSIONS: The biological screening results in this study established the importance of the nitrogen atom at the ortho position as well as the beneficial effect of a methyl substituent at the 4- or 5-position of the pyridine ring. Two pyridine epothilone B analogs (i.e. compounds 3 and 4) possessing higher potencies against drug-resistant tumor cells than epothilone B, the most powerful of the naturally occurring epothilones, were identified.

Cross-resistance analysis and molecular modeling of nonnucleoside reverse transcriptase inhibitors targeting drug-resistance mutations in the reverse transcriptase of human immunodeficiency virus.

Oxathlin carboxanilide analogs (UC) and alpha APA, compounds recognized as nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI), were evaluated for activity against the human immunodeficiency virus (HIV-1) and drug-resistant variants. These NNRTIs are structurally diverse but potent inhibitors of HIV-1 with efficacy in the nanomolar to low micromolar concentrations. They interact at a specific site in the pain domain of the p66 subunit of RT. Treatment of HIV-1 infected cell cultures with UC compounds resulted in the selection of drug-resistant viruses bearing specific amino acid changes at 100, 101, 103, 106, and/or 181. Since Y181C and L1001 are the most commonly observed resistance-engendering mutations, RT enzymatic analysis was correlated with molecular modeling to glean information on the structural interactions between these NNRTIs and RT. Information derived from these studies will facilitate rational drug design and the selection of complementary anti-HIV drugs for combination therapy.

Cortical EEG changes during the self-administration of phencyclinoids.

Female rats, implanted with cerebrocortical EEG recording electrodes, were trained to self-administer cocaine and then ketamine under a fixed ratio 10 schedule of reinforcement, during limited access sessions. Periodically, a single unit dose of either phencyclidine (0.5 mg/kg), ketamine (4 mg/kg) or 1-(1-phenylcyclohexyl)morpholine (PCM 4 mg/kg) was substituted for ketamine, while the cortical EEG was recorded. Spectrum quantities of samples of EEG, taken immediately before and after each injection, were subjected to a discriminant analysis. For each drug, the preinjection state of the EEG could be classified separately from the postinjection state, using specific EEG spectrum quantities from the global frequency range (0.1-20 Hz). Furthermore, the relevant EEG parameters, which described the change from pre- to postinjection states, were unique for each drug (phencyclidine: total power and complexity; ketamine: peak frequency, relative power and mobility; PCM: all parameters except peak frequency), indicating potential differences in the EEG, occurring with a level of intake of drug which was controlled by the subject. Overall, these data serve to model the changes in the EEG that occur during the self-administration of three phencyclinoids. Furthermore, the combination of EEG spectrum analysis with discriminant analysis is useful in detecting subtle differences in the effects of these three drugs on the EEG.

Haloperidol pretreatment unmasks the kappa opioid effects of U-50, 488H on cortical EEG and EEG power spectra in rats.

Adult female Sprague-Dawley rats were implanted with chronic cortical EEG electrodes and intravenous cannulae. U-50, 488H injection (5.0 mg/kg) produced initial EEG desynchrony and EEG spectral power that was mainly distributed over the zero to 10 Hz range, including a relatively small spectral peak in the 4-6 Hz band. In contrast, following haloperidol pretreatment (0.1 mg/kg), U-50, 488H injection produced high-voltage EEG bursts and a predominant EEG spectral peak in the 4-6 Hz band. These effects of U-50, 488H after haloperidol pretreatment were identical to those previously demonstrated with the benzomorphan kappa agonist ethylketocyclazocine. Thus, haloperidol pretreatment unmasked the kappa opioid effects of U-50, 488H.

All-atom models for the non-nucleoside binding site of HIV-1 reverse transcriptase complexed with inhibitors: a 3D QSAR approach.

Several molecular modeling techniques were used to generate an all-atom molecular model of a receptor binding site starting only from Ca atom coordinates. The model consists of 48 noncontiguous residues of the non-nucleoside binding site of HIV-1 reverse transcriptase and was generated using a congeneric series of nevirapine analogs as structural probes. On the basis of the receptor-ligand atom contacts, the program HINT was used to develop a 3D quantitative structure activity relationship that predicted the rank order of binding affinities for the series of inhibitors. Electronic profiles of the ligands in their docked conformations were characterized using electrostatic potential maps and frontier orbital calculations. These results led to the development of a 3D stereoelectronic pharmacophore which was used to construct 3D queries for database searches. A search of the National Cancer Institute's open database identified a lead compound that exhibited moderate antiviral activity.

Paullones, a series of cyclin-dependent kinase inhibitors: synthesis, evaluation of CDK1/cyclin B inhibition, and in vitro antitumor activity.

The paullones represent a novel class of small molecule cyclin-dependent kinase (CDK) inhibitors. To investigate structure-activity relationships and to develop paullones with antitumor activity, derivatives of the lead structure kenpaullone (9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one, 4a) were synthesized. Paullones with different substituents in the 2-, 3-, 4-, 9-, and 11-positions were prepared by a Fischer indole reaction starting from 1H-[1]benzazepine-2,5(3H,4H)-diones 5. Selective substitutions at either the lactam or the indole nitrogen atom were accomplished by treating kenpaullone with alkyl halides in the presence of sodium hydride/THF or potassium hydroxide/acetone, respectively. S-Methylation of the kenpaullone-derived thiolactam 18 yielded the methylthioimidate 19, which gave the hydroxyamidine 20 upon reaction with hydroxylamine. The new paullones were tested both in a CDK1/cyclin B inhibition assay and in the in vitro antitumor cell line-screening program of the National Cancer Institute (NCI). With respect to the CDK1/cyclin B inhibition, electron-withdrawing substituents in the 9-position as well as a 2,3-dimethoxy substitution on the paullone basic scaffold turned out to be favorable. A 9-trifluoromethyl substituent was found to be equivalent to the 9-bromo substituent of kenpaullone. Replacement of the 9-bromo substituent of kenpaullone by a 9-cyano or 9-nitro group produced a substantial increase in enzyme-inhibiting potency. Substitutions in other positions or the replacement of the lactam moiety led to decreased CDK1 inhibition. Noteworthy in vitro antitumor activities (GI(50) values between 1 and 10 microM) were found with the 9-bromo-2,3-dimethoxy-7,12-dihydroindolo[3, 2-d][1]benzazepin-6(5H)-one (4t), its 9-trifluoromethyl analogue 4u, the 12-Boc-substituted paullone15, and the methylthioimidate 19, respectively. The 9-nitro-7,12-dihydroindolo[3, 2-d][1]benzazepin-6(5H)-one (4j, named alsterpaullone) showed a high CDK1/cyclin B inhibitory activity (IC(50) = 0.035 microM) and exceeded the in vitro antitumor potency of the other paullones by 1 order of magnitude (log GI(50) mean graph midpoint = -6.4 M).

Comparative electroencephalographic and behavioral effects of phencyclidine, (+)-SKF-10,047 and MK-801 in rats.

Female Sprague-Dawley rats prepared with chronic i.v. cannulas and/or cerebrocortical electrodes were administered sequentially increasing doses of phencyclidine (PCP, 0.1-6.4 mg/kg/injection), (+)-SKF-10,047 [(+)-N-allynormetazocine] (0.4-25.6 mg/kg/injection) or MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate] (0.01-0.64 mg/kg/injection). Effects on overt behavior, cortical EEG power spectra, locomotor activity and rotarod performance were assessed. Quantitative EEG spectral parameters (peak, mean and edge frequency; total and relative power; time domain descriptors mobility and complexity) were analyzed from the global frequency range of 1 to 50 Hz. Increasing doses of each drug produced increases in EEG spectra power from 1 to 50 Hz which was associated with a slowing of the peak frequency. PCP and MK-801 produced decreases in the mean frequency, mobility and edge frequency whereas (+)-SKF-10,047 produced increases in these spectral parameters. Moreover, (+)-SKF-10,047 increased complexity whereas MK-801 decreased complexity and PCP did not change this parameter. Total spectral power from 20 to 50 Hz was increased by (+)-SKF-10,047 and PCP, but was not changed by MK-801. Each drug increased spontaneous locomotor activity. At the highest doses, PCP and MK-801 decreased activity whereas (+)-SKF-10,047 was lethal. Each drug disrupted rotarod performance. The rank order of potency for each effect was: MK-801 greater than PCP greater than (+)-SKF-10,047. The data indicate that subtle differences in the effects of these drugs can be detected using EEG power spectral analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

A pseudoreceptor docking study of 4,5-alpha-epoxymorphinans with a range of dielectric constants.

Thirteen 4,5-epoxymorphinan mu agonists with established analgesic action were docked into an Asp-Lys-His-Phe pseudoreceptor complex under a range of distance-dependent dielectric conditions. The number of compounds with potential energies of the docked complexes that agreed in rank order with corresponding analgesic potencies was determined for each condition. Two dielectric conditions, n-decane (1.991) and ethanol (24.3), enabled the greatest number of compounds to relate to their pseudoreceptors with each having 9 and 8 successes respectively. Both of these conditions demonstrated unique influences on the types of structures that were successfully docked. For example, the morphine stereoisomer alpha-isomorphine, the geometric isomer B/C trans-morphine, and the 8-position-substituted gamma-isomorphine were successes in the n-decane condition, whereas the ethanol condition produced the substituted codeine derivatives dihydrocodeinone and dihydroxycodeinone. These findings emphasize the importance of dielectric influence when developing force-field modeled quantitative structure-activity relationships for a closely related homologous series.

Use of 3D QSAR methodology for data mining the National Cancer Institute Repository of Small Molecules: application to HIV-1 reverse transcriptase inhibition.

A three-dimensional (3D) stereoelectronic pharmacophore developed from a 3D quantitative structure-activity relationship (QSAR) investigation formed the basis of the development of a two-phase data-mining methodology to uncover novel leads to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase at the nonnucleoside binding site. The database searching phase employed a field search for ligand requirements (such as log P, molecular volume) that were accessible from the database keys. Next, a 3D database search was performed that used an automated fitting procedure and the calculation of several binding parameters. These binding parameters were used to test the hits by a discriminant function that was previously trained to recognize active from inactive analogs. During the structural evaluation phase of the methodology, conformational properties and complementary receptor features of the hits were examined by 2D and 3D evaluations, which were followed by molecular modeling investigations. When this method was applied to a test database, an improvement from 6.4% to 100% active analogs was achieved.

A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.

The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on beta-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired beta-tubulin mutations (beta274(Thr-->Ile) and beta282(Arg-->Gln)) located in the atomic model of alphabeta-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure-activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug-tubulin interactions that should assist in the rational design of agents targeting tubulin.

2-Substituted paullones: CDK1/cyclin B-inhibiting property and in vitro antiproliferative activity.

9-Trifluoromethyl-paullones with a carbon chain in the 2-position were synthesized by palladium-catalyzed coupling reactions of a 2-iodoprecursor with terminal alkenes or alkynes, respectively. The introduction of a 2-cyanoethyl substituent led to a significant enhancement of CDK1/cyclin B inhibiting property and in vitro antiproliferative activity.

Structure-based design modifications of the paullone molecular scaffold for cyclin-dependent kinase inhibition.

A congeneric series of paullones were characterized using a 3-D QSAR with cyclin-dependent kinase 1 (CDK1) inhibition data. A homology model of CDK1-cyclin B was developed from the crystal structure of CDK2-cyclin A, which subsequently served as the basis for the structure-based design. Paullones were docked into the ATP binding site of the CDK1-cylin B models and were optimized with molecular mechanics. Hydropathic analyses of the paullone-CDK1 complexes were performed after the atom types were assigned based on each ligand's electronic properties calculated from quantum mechanics. Hydropathic descriptors formed a significant multiple regression equation that predicts paullone IC50 data. The results indicate that the combination of hydropathic descriptors with molecular mechanics geometries are sufficient to design overt steric and chemical complementarity of the ligands. However, the electronic properties derived from quantum mechanics helped direct synthetic chemistry efforts to produce ligands that promote better charge transfer and strengthen hydrogen bonding as facilitated by resonance stabilization. Compounds with low affinity for CDK1 were poor charge acceptors and made less than ideal hydrogen bonding arrangements with the receptor. These considerations led to the prediction that structures such as 9-cyanopaullone would be considerably more potent than the parent compound, a finding supported by enzyme inhibition data. Also, 9-nitropaullone emerged as a paullone which also had similar potency in enzyme inhibition as well as a favorable anti-proliferative activity profile in living cells.

Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP-competitive, inhibitors of the cell cycle regulating cyclin-dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase-3beta (GSK-3beta) (IC50: 4-80 nM) and the neuronal CDK5/p25 (IC50: 20-200 nM). These two enzymes are responsible for most of the hyperphosphorylation of the microtubule-binding protein tau, a feature observed in the brains of patients with Alzheimer's disease and other neurodegenerative 'taupathies'. Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK-3beta. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK-3beta in Alzheimer's disease. Alsterpaullone also inhibits the CDK5/p25-dependent phosphorylation of DARPP-32 in mouse striatum slices in vitro. This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders.