Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.

An analysis of the activity of compounds tested in pre-clinical in vivo and in vitro assays by the National Cancer Institute's Developmental Therapeutics Program was performed. For 39 agents with both xenograft data and Phase II clinical trials results available, in vivo activity in a particular histology in a tumour model did not closely correlate with activity in the same human cancer histology, casting doubt on the correspondence of the pre-clinical models to clinical results. However, for compounds with in vivo activity in at least one-third of tested xenograft models, there was correlation with ultimate activity in at least some Phase II trials. Thus, an efficient means of predicting activity in vivo models remains desirable for compounds with anti-proliferative activity in vitro. For 564 compounds tested in the hollow fibre assay which were also tested against in vivo tumour models, the likelihood of finding xenograft activity in at least one-third of the in vivo models tested rose with increasing intraperitoneal hollow fibre activity, from 8% for all compounds tested to 20% in agents with evidence of response in more than 6 intraperitoneal fibres (P< 0.0001). Intraperitoneal hollow fibre activity was also found to be a better predictor of xenograft activity than either subcutaneous hollow fibre activity or intraperitoneal plus subcutaneous activity combined. Since hollow fibre activity was a useful indicator of potential in vivo response, correlates with hollow fibre activity were examined for 2304 compounds tested in both the NCI 60 cell line in vitro cancer drug screen and hollow fibre assay. A positive correlation was found for histologic selectivity between in vitro and hollow fibre responses. The most striking correlation was between potency in the 60 cell line screen and hollow fibre activity; 56% of compounds with mean 50% growth inhibition below 10(-7.5) M were active in more than 6 intraperitoneal fibres whereas only 4% of compounds with a potency of 10(-4) M achieved the same level of hollow fibre activity (P< 0.0001). Structural parameters of the drugs analysed included compound molecular weight and hydrogen-bonding factors, both of which were found to be predictive of hollow fibre activity.

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.

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.

Inhibition of CDKs as a therapeutic modality.

Altered cell cycle control has emerged as a recurring theme in neoplasia. Strategies that would return toward normal the altered cell cycle control present in tumor cells have appeal as novel approaches to cancer treatment. Cyclin-dependent kinases (CDKs) control the progression through the cell cycle, operating at the transition from the G2 to M and G1 to S phases, and progression through S. CDKs are regulated by a complex set of mechanisms, including the presence of activating cyclins, regulatory phosphorylations, and endogenous CDK inhibitors at "checkpoints." This overview focuses on progress in defining compounds that can antagonize directly the action of CDKs. These have emerged as various types of ATP site-directed inhibitors, including flavopiridol, N-substituted adenine derivatives, the natural product butyrolactone, staurosporine derivatives, and, more recently, the synthetic paullones. Paullones appear to be of interest in that one of the most active members of the class, 9-nitropaullone (alsterpaullone), requires relatively brief periods of exposure to living cells in order to effect lasting effects on cellular and proliferative potential. Two of these compounds, flavopiridol and UCN-01 (7-hydroxy-staurosporine), have entered early clinical trials and achieved concentrations that might potentially modulate CDK function. In the case of UCN-01, unexpected human plasma protein binding might prevent direct inhibition of CDKs but allow drug concentrations to be achieved that indirectly affect CDKs by checkpoint abrogation. Further studies with CDK inhibitors should define the expected end point of CDK inhibition more clearly in preclinical models and clinical systems, including cytostasis, apoptosis, or differentiation.

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.

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.

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.

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.

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).

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.

Antitumor pyrido[3,2-d][1]benzazepines: synthesis and in vitro activity of thiophene analogs.

The bioisosteric replacement of benzene elements in the antitumor lead structures 2 and 3 led to the thienyl substituted 5H-pyrido[3,2-d][1]benzazepin-6(7H)-ones 8a and 8b, the analogous thiolactams 4a and 4b, and the 4H-pyrido[2,3-d]-thieno[3,2-b]azepines 11 and 12 which represent a novel heterocyclic scaffold. The in vitro evaluation of 4a in a cell line based screening revealed a noteworthy antitumor activity and a remarkable selectivity for renal tumor cell lines. The cell line selectivity pattern of 4a differs distinctly from the pattern displayed by standard antitumor agents.

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.

An information-intensive approach to the molecular pharmacology of cancer.

Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.

Evaluation of selected chemotypes in coupled cellular and molecular target-based screens identifies novel HIV-1 zinc finger inhibitors.

Conservation of the Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys retroviral zinc finger sequences and their absolute requirement in both the early and late phases of retroviral replication make these chemically reactive structures prime antiviral targets. We recently reported that select 2,2'-dithiobisbenzamides (DIBAs) chemically modify the zinc finger Cys residues, resulting in release of zinc from the fingers and inhibition of HIV replication. In the current study we surveyed 21 categories of disulfide-based compounds from the chemical repository of the National Cancer Institute for their capacity to act as retroviral zinc finger inhibitors. Aromatic disulfides that exerted anti-HIV activity tended to cluster in the substituted aminobenzene, benzoate, and benzenesulfonamide disulfide subclasses. Only one thiuram derivative exerted moderate anti-HIV activity, while a number of nonaromatic thiosulfones and miscellaneous disulfide congeners were moderately antiviral. Two compounds (NSC 20625 and NSC 4493) demonstrated anti-cultures. The two compounds chemically modified the p7NC zinc fingers in two separate in vitro assays, and interatomic surface molecular modeling docked the compounds efficiently but differentially into the zinc finger domains. The combined efforts of rational drug selection, cell-based screening, and molecular target-based screening led to the identification of zinc finger inhibitors that can now be optimized by medicinal chemistry for the development of biopharmaceutically useful anti-HIV agents.

Identification of a novel inhibitor (NSC 665564) of dihydroorotate dehydrogenase with a potency equivalent to brequinar.

A novel inhibitor of dihydroorotate dehydrogenase (DHO-DH) has been discovered using data from the National Cancer Institute's in vitro drug screen. Upon analysis of cytotoxicity results from the sixty tumor cell lines used in this screen, the COMPARE program predicted that NSC 665564 was likely to have the same mechanism of inhibition as brequinar, a known potent inhibitor of DHO-DH. We validated this prediction experimentally using MOLT-4 lymphoblast and found the IC50 of brequinar (0.5 microM) and NSC 665564 (0.3 microM) were comparable and that this induced cytotoxicity was reversed by either uridine or cytidine. The enzyme target of NSC 665564 was shown to be identical to that of brequinar when incubation with each drug followed by a 1 h pulse with [14C] sodium bicarbonate resulted in cellular accumulation of [14C]N-carbamyl-L-aspartic acid and [14C]L-dihydroorotic acid, with concurrent marked depletion of CTP and UTP. The Ki's for NSC 665564 and brequinar were 0.14 and 0.24 microM, respectively, when partially purified MOLT-4 mitochondria (the site of DHO-DH) were used. These results show that mechanistic predictions obtained using correlations from the COMPARE algorithm are independent of structure since the structure of NSC 665564 is dissimilar to that of other established DHO-DH inhibitors.

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.

Site of action of two novel pyrimidine biosynthesis inhibitors accurately predicted by the compare program.

The computer algorithm COMPARE provides information regarding the biological mechanism of action of a compound. In this study, excellent correlations were obtained for 2,2'-[3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)diimino]bis- benzoic acid (redoxal) and 1-(p-bromophenyl)-2-methyl-1H- naphth[2,3-d]imidazole-4,9-dione (BNID) and two well-studied dihydroorotate dehydrogenase (DHOD) inhibitors, dichloroallyl lawsone and brequinar, in terms of antiproliferative activity against tumor cell lines in vitro. When redoxal and BNID were incubated with MOLT-4 cells for 72 hr, 50% growth inhibition was achieved at 0.7 and 3.5 microM, respectively. After 24 hr of incubation, pyrimidine triphosphate pools were shown to be decreased by 50% by redoxal (1 microM) and BNID (0.25 microM). Addition of either uridine (50 microM) or cytidine (100 microM) antagonized the cellular cytotoxicity caused by either drug; uridine corrected the UTP and CTP deficit, whereas cytidine corrected only the CTP deficit. Exposure of MOLT-4 cells to a 1 microM concentration of either drug for 18 hr followed by a 1-hr exposure to [14C]bicarbonate showed a 97% decrease of incorporation of [14C] into pyrimidine triphosphates accompanied by a 91- and 82-fold increase in radioactive incorporation into L-dihydroorotate and N-carbamyl-L-aspartate, respectively. By direct exposure of DHOD prepared from MOLT-4 cell mitochondria to a range of concentrations of the two drugs, apparent Ki values of 0.33 microM (redoxal) and 0.53 microM (BNID) were determined. These data provide direct evidence for inhibition of DHOD by redoxal and BNID in MOLT-4 lymphoblasts.

Cyclopentenyl uracil: an effective inhibitor of uridine salvage in vivo.

Cyclopentenyl uracil, a non-cytotoxic inhibitor of uridine kinase, was found to effectively block the salvage of circulating uridine by host and tumor tissues in the intact mouse. Dose-response characteristics of the inhibition were determined. Large doses (1 g/kg) of cyclopentenyl uracil were required, and the effect of a single dose fell rapidly over a 24-hr period. A sustained inhibition of uridine salvage of > 64-79% could be maintained by multiple doses of 1 g/kg given on an every 8-hr schedule. Mice given cyclopentenyl uracil (1 g/kg) every 8 hr for 5 days continued to gain weight and showed no signs of toxicity; however, the combination of cyclopentenyl uracil with a non-toxic dose of N-(phosphonacetyl)-L-aspartic acid (PALA; 200 mg/kg daily for 5 days) was lethal to mice, indicating that circulating uridine modifies the toxicity of agents that act on enzymes of the de novo pyrimidine pathway. Although the duration of action and potency of cyclopentenyl uracil are not ideal, this is the first demonstration of an effective inhibition of uridine salvage in the intact mouse with a non-cytotoxic agent. This makes possible the evaluation of concurrent inhibition of de novo and salvage routes to pyrimidine nucleotides as an approach to chemotherapy.

The discovery of novel, structurally diverse protein kinase C agonists through computer 3D-database pharmacophore search. Molecular modeling studies.

A computer protein kinase C (PK-C) pharmacophore search on 206,876 nonproprietary structures in the NCI 3D-database led to the discovery of five compounds which were found to possess PK-C binding affinities in the low micromolar range and six others having detectable, but marginal, binding affinities. Molecular modeling studies showed that in addition to the presence of the defined pharmacophore, hydrophobicity and conformational energy are the two other important factors determining the PK-C binding affinity of a compound. The modeling results were confirmed by synthetic modification of two inactive compounds, producing two active derivatives. These newly discovered, structurally diverse lead compounds are being used as the basis for further synthetic modifications aimed at more potent PK-C ligands that will compete with the phorbol esters.