Fluorescent markers for hypoxic cells. A study of novel heterocyclic compounds that undergo bio-reductive binding.

The bioreductive metabolism and binding of nitroaromatic compounds has been suggested as a method for the identification of hypoxic tumour cells. Bound metabolites of suitable nitroaryl compounds (and some other reducible aromatic compounds) may fluoresce, offering an alternative to radiolabelling or NMR etc. as a diagnostic method. In this paper, the synthesis of some heteroaromatic nitro-compounds is given together with the results obtained from testing of these and other mainly nitroaromatic compounds in vitro as potential bioreductive fluorescent probes for hypoxic cells in tumours. Compounds were incubated with oxygenated or hypoxic mammalian cell suspensions for various times before evaluation of the cellular fluorescence from bioreductive metabolites by fluorescence microscopy and flow cytometry. Among those compounds yielding fluorescent metabolites in cells, considerable variation in hypoxic:oxic differential fluorescence was observed. The in vitro mammalian cell test system showed several of the compounds to be sufficiently promising to merit further investigation in vivo.     

Bioreductive fluorescent markers for hypoxic cells: a study of 2-nitroimidazoles with 1-substituents containing fluorescent, bridgehead-nitrogen, bicyclic systems.

The oxygen-sensitive bioreductive binding of 2-nitroimidazoles labeled with fluorescent side chains has been used to stain hypoxic mammalian cells selectively. Several novel compounds were synthesized with a 1-substituent containing a fluorescent, bicyclic system having a bridgehead-nitrogen atom. Additional amine and secondary alcohol substituents were also included in the link between the fluorophor and the nitroimidazole to improve water solubility. Their ability to discriminate between hypoxic and oxic cells was compared by flow cytometric analysis. A wide range of cellular fluorescence and hypoxic-oxic differentials in fluorescence was observed when compounds with indolizine fluorophors were incubated with cells, and one such compound was considered suitable for further evaluation in vivo. Two compounds with bimane fluorophors gave very little cellular fluorescence when incubated with hypoxic cells.     

Overcoming the hurdle of fluorescent compounds in kinase screening: a case study

In the field of screening in general and especially in the kinase area, taking into consideration throughput and cost, fluorescence- and luminescence-based assays have been developed as alternatives to radioactivity-based assays. However, fluorescence-based technologies are not devoid of pitfalls. One of the main problems is interference from autofluorescent compounds and the incidence of false-positives as exemplified here with a fluorescence polarization (FP)-based assay. Using the scintillation proximity assay as the in-house standard, we assessed several alternatives to radioactive methods, namely, the amplified luminescent proximity homogeneous assay screen (ALPHAScreen, Perkin-Elmer Life Sciences, Boston, MA), enzyme fragment complementation, FP, and nanofluidics-based fluorescence intensity. Data comparing the sensitivity, robustness, relative sensitivity to autofluorescent compounds, enzyme consumption, and relative costs of each assay for one common kinase are presented. Results obtained seem to favor the nanofluidics mobility shift assay as a method of choice, followed by the direct FP approach, using generic high-molecular-weight phosphate group-binders.     

Design,synthesis and CD4 binding studies of a fluorescent analogue of a peptide that enhances HIV-1 infectivity

We previously demonstrated that a 23-amino-acid peptide derived from the V3 loop of the surface glycoprotein of human immunodeficiency virus (HIV-I) strain MN was able to bind soluble CD4 and to enhance HIV-1 infection. Further studies suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. To facilitate identification of the complementary binding site for the peptide on cellular CD4, we designed an analogue carrying a single fluorescein moiety. The synthesis of this modified analogue presented several problems because of the presence of several amino acids in the sequence carrying potentially reactive groups in their side-chains, and the necessity of introducing only one marker per molecule in a position that would not affect biological activity. The side-chain of Lys¹⁹ was selected because separate studies demonstrated that its substitution with an uncharged amino acid does not reduce the peptide's biological activity. We compared the merits of various synthetic protocols used to condense the fluorescent marker with the peptide. Biological assays indicated that the presence of the fluorescein moiety did not compromise peptide binding to CD4; furthermore, binding of the labeled analogue was not abolished by trypsin treatment, suggesting that the peptide may interact with both CD4 and additional trypsin-resistant binding sites on the cell surface. Finally, we verified the preservation of HIV infection enhancing ability in the labeled peptide.     

Comparative affinity of steroidal and non-steroidal antioestrogens, cholesterol derivatives and compounds with a dialkylamino side chain for the rat liver antioestrogen binding site.

Steroidal and non-steroidal antioestrogens, steroidal compounds with (disubstituted) dialkyl amino side chain, cholesterol derivatives, histaminic and (anti)-progestational compounds were tested for their ability to compete with [3H]tamoxifen for the specific antioestrogen binding site (AEBS) in the post-mitochondrial fraction of rat liver homogenates. Relative binding affinity was highest for compounds with diethylamino or pyrrolidino ethoxy side chains. Affinity decreased with shortening of this side chain. No connection could be established between the carbon backbone of the compound and affinity, except for the presence of (sometimes aromatic) ring structures. Steroidal ring structures do not seem to be necessary for binding. The cholesterol derivatives, tested in our laboratory, showed very little affinity for the rat liver AEBS. Histamine, melatonin and the (anti)-progestational compounds showed no affinity for the AEBS; and we therefore conclude that the AEBS is not identical to receptors for these compounds. Results of these experiments could be useful in other investigations on the development of resistance of breast cancer to antioestrogens.     

Structural evaluation of the agonistic action of a vitamin D analog with two side chains binding to the nuclear vitamin D receptor

The vitamin D receptor (VDR) is one of the endocrine members of the nuclear receptor superfamily and has a characteristic high affinity for its natural ligand 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. From a mechanistic point of view, the most interesting analog of 1alpha,25(OH)2D3 is the one that carries two side chains, referred to as Gemini. In this study, molecular dynamics (MD) simulations of the Gemini-VDR complex were performed that demonstrated that the binding of a ligand with a 25% increased volume does not disturb the overall structure of the ligand-binding domain (LBD). It was found that one of the two side chains takes exactly the same position as the single side chain of the natural ligand, which suggests that the molecular mechanism of the agonism of Gemini is identical to that of 1alpha,25(OH)2D3. VDR single and double point mutants at L227, A303, I313, and L397 and in vitro and ex vivo assessment of their agonistic action confirmed the predictions of the MD simulations. Moreover, it was found that the second side chain of Gemini can choose between two binding positions within the ligand-binding pocket of the VDR. These two newly identified "corners" were characterized most specifically by the amino acids pairs L227/A303 and I313/L397. Therefore, Gemini is an important model compound that allows further insight into the molecular actions of the VDR but is, in parallel, also a promising precursor for the design of even more potent 1alpha,25(OH)2D3 analogs.     

UV-spectrophotometry in drug control. 39. Spectrophotometric behavior of substances with chromophores and auxochromes in aliphatic chains, in saturated heterocyclics and the heterocyclic compounds: furan, imidazole, thiazole, thiadiazole and oxadiazole

The spectra of 22 drug substances with chromophores and auxochromes in the aliphatic chain, in the saturated heterocyclus and in heterocyclic compounds (furan, imidazole, thiazole, thiadiazole and oxadiazole) underwent a systematic investigation and the results obtained were evaluated. Influences of substituents and solvents on shifts of the E, K, B and R bands were discussed.     

Nicotine and behavioral markers of risk for schizophrenia: a double-blind, placebo-controlled, cross-over study.

We investigated the effect of nicotine on three behavioral markers of risk for schizophrenia: sustained attention (using the Continuous Performance Task (CPT)), antisaccade performance, and smooth pursuit. Smooth pursuit was investigated in two conditions, one in which attention was enhanced (monitoring target changes) and one in which attention was not enhanced (no monitoring). Patients with schizophrenia (n = 15) and controls (n = 14) were given a 14-mg nicotine patch in a double-blind, placebo-controlled, crossover design and plasma nicotine concentrations were monitored. Nicotine concentrations were similar in both groups. A Group x Drug interaction (p <.02) on CPT hits indicated that nicotine improved sustained attention in patients but not in controls. Nicotine significantly decreased antisaccade errors (p <.01) in both groups. A Drug x Monitoring condition interaction (p <.01) on pursuit gain indicated that nicotine significantly increased pursuit gain in the no-monitoring condition in patients and controls equally, but did not improve pursuit in the monitoring condition. Thus, improvement in pursuit may have been mediated via an effect on attention rather than by an effect on oculomotor function per se. In patients, the magnitude of improvement in attention on nicotine was correlated with the improvement on eye movement tasks. Thus, nicotine improves performance on both attention and oculomotor markers of risk for schizophrenia, possibly via common mechanisms.     

Cytotoxicity of organic compounds against ovarian cancer cells: a quantitative structure-activity relationship study

The interest in the application of structure-activity relationships has steadily increased in recent decades. In the present paper, we have discussed the cytotoxicity of various sets of organic compounds against ovarian cancer cells by the formulation of a total number of 11 QSARs. Hydrophobicity is found to be one of the most important determinants of activity followed by steric parameters. Parabolic correlation with hydrophobicity is an encouraging example, where the optimal hydrophobicity is well-defined. We believe that this may be the predictive model to narrow the synthetic challenges in order to yield very specific OVCAR-3 inhibitors. On the basis of this model, we can predict three compounds that may be the next synthetic target. Cross-validation and Y-randomization tests were used to validate all the QSAR models.     

Generation of suppressor cells in normal rats by treatment with spirogermanium, a novel heterocyclic anticancer drug

Daily oral administration of spirogermanium to Lewis rats resulted in the generation of radiation-resistant (2000 Rad) suppressor cells which inhibited the proliferative response of normal spleen cells to an optimum concentration of concanavalin A. These suppressor cells became evident after three to six days of spirogermanium administration. After one day's treatment, although no suppressor cells could be detected, the response of these cells to concanavalin A was less than 50% of controls. Experiments designed to characterize the cell type(s) responsible for this suppression resulted in the finding that T cell-'depleted' populations of spleen cells were more suppressive than T cell-'enriched' populations. The induction of suppressor cells by spirogermanium and the previously described activity in the adjuvant arthritic rat model suggest therapeutic potential for autoimmune diseases.     

Investigation of nicotine binding to THP-1 cells: evidence for a non-cholinergic binding site

Nicotine is known to modulate immune function, but reports have produced conflicting evidence as to whether nicotinic acetylcholine (nACh) receptors are responsible for these effects. This study was designed to examine the identity of nicotine-binding sites on immune cells using a human leukaemic monocytic cell line, THP-1, that is known to have functions that are modulated by nicotine. Binding studies were performed on THP-1 whole cells using [3H]nicotine as a probe to analyse any possible nicotine-binding sites on these cells. Saturation analysis of THP-1 cells revealed the presence of 2 distinct binding sites; one with a K(d1) of 3.5 +/- 2.1 x 10(-9) M and a B(max1) of 4100 +/- 560 sites/cell (designated the high-affinity site) and the other with a K(d2) of 27 +/- 9.2 x 10(-9) M and a B(max2) of 11,600 +/- 630 sites/cell (low-affinity site). Competition analysis revealed that one site had an affinity to a range of cholinergic ligands including epibatidine and cytisine. When saturation analysis of [3H](-)-nicotine to THP-1 cells was performed in the presence of 1 x 10(-6) M epibatidine, only one binding site was detected. Comparisons of K(d) and B(max) values showed that the high-affinity site was not occluded by epibatidine. No drugs tested displayed any affinity for the high-affinity site except the two enantiomers of nicotine. The high-affinity site was shown to be stereoselective for the (+)-enantiomer of nicotine as shown by K(i) values produced by competition analysis in the presence of 1 x 10(-6) M epibatidine. These values were 5.7 +/- 0.32 x 10(-11) M and 1.9 +/- 4.9 x 10(-9) M for (+)-nicotine and (-)-nicotine, respectively. This study presents evidence for a possible non-cholinergic binding site that may play a role in the mechanism of immunomodulation by nicotine.     

General pseudoreceptor model for sweet compounds: a semiquantitative prediction of binding affinity for sweet-tasting molecules

The chemical structures of sweet compounds are very different, ranging from sugars to amino acids and peptides or other compounds such as saccharin. The biological mechanism underlying the generation of sweet taste is still unknown, although in the past few years much research has provided evidence for the existence of a true chemoreception process, mediated by receptor proteins on the taste buds. In particular, the initial step of the process involves the reversible binding of the sweet compounds to their receptor(s). In this work, we have investigated this binding via a pseudoreceptor model, which has been developed using a training set of 24 compounds belonging to different families including sugars, peptides, and other intensive sweeteners. This model provided a correlation coefficient (r(2)) of 0.985 between the calculated and the experimental free energies of binding, which are related to the molar relative sweetness, for the training set and is able to predict semiquantitatively free energies of ligand binding for an independent set of five test ligand molecules within 0.3-2.1 kcal mol(-1) of the experimental values.     

Exploring interactions of endocrine-disrupting compounds with different conformations of the human estrogen receptor alpha ligand binding domain: a molecular docking study

Endocrine-disrupting compounds (EDCs) accumulating in nature are known to interact with nuclear receptors. Especially important is the human estrogen receptor alpha (hERalpha), and several EDCs are either known or suspected to influence the activity of the ligand-binding domain (LBD). We here present a comparative docking study of both well-known hERalpha ligands and small organic compounds, including selected polychlorinated biphenyls (PCBs), plasticizers, and pesticides, that are all potentially endocrine-disrupting,into different conformations of the hERalpha LBD. Three newly found quasi-stable structures of the hERalhpa LBD are examined along with three crystallographic conformations of the protein, either theapo structure or using a protein structure with a bound agonist or antagonist ligand. The possible interactions between the protein and the potentially EDCs are described. It is found that most suspected EDCs can bind in the steroid binding cavity, interacting with at least one of the two hydrophilic ends of the steroid binding site. DDE, DDT, and HPTE are predicted to bind most strongly to the hERalpha LBD. It is predicted that these compounds can interact with the three conformations of hERalpha LBD with comparable affinities.The metabolic hydroxylation of aromatic compounds is found to lead to an increase in the binding affinity of PCBs as well as DDT. Docking into the quasi-stable conformations of the hERalpha LBD leads to computed binding affinities similar to or better than those calculated for the three X-ray structures, revealing that the new structures may be of importance for assessing the function of the influence of EDCs on nuclear receptors.     

Competitive binding of two drugs for a single binding site on albumin: a circular dichroic study

Sulphaethidole has been previously shown to have two classes of binding site on bovine serum albumin (BSA). Only the primary binding site is capable of inducing optical activity in the drug. Drugs that bind to the same primary binding site as sulphaethidole, but do not become optically active themselves on binding, reduce the size of the circular dichroism signal in competitive binding studies with sulphaethidole. The binding constant for such antagonists can be calculated. It appears that a range of acidic drugs share the same primary binding site on bovine serum albumin; basic drugs do not compete for this site.     

Platelet aggregation inhibiting and anticoagulant effects of oligoamines, XVII: Oligoamines with fluorescent properties. Part A: Fluorescent bridged nitrogen functions.

Eleven dimethanamines and one disydnonimine with fluorescent properties have been synthesized. All of them show antiplatelet activities (IC50, Born-test) in concentrations between 14-75 mumol/L. Five of them inhibited fibrin formation induced by thromboplastin by more than 75% in a 200 mu molar concentration. Both effect do not run parallel. The most space consuming fluorophores show the smallest inhibition of the platelet aggregation. Best results were obtained with an azulene, acenaphthene or naphthalene moiety between the two basic nitrogen functions.     

Novel 4-anilinoquinazolines with C-7 basic side chains: design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors

We have previously shown that 4-anilinoquinazolines can be potent inhibitors of vascular endothelial growth factor (VEGF) receptor (Flt-1 and KDR) tyrosine kinase activity. A novel subseries of 4-anilinoquinazolines that possess basic side chains at the C-7 position of the quinazoline nucleus have been synthesized. This subseries contains potent, nanomolar inhibitors of KDR (median IC(50) 0.02 microM, range 0.001-0.04 microM), which are comparatively less potent vs Flt-1 tyrosine kinase (median IC(50) 0.55 microM, range 0.02-1.6 microM). The compounds also retain some inhibitory activity against the tyrosine kinase associated to the endothelial growth factor receptor (EGFR) (median IC(50) 0.2 microM, range 0.075-0.8 microM) but demonstrate selectivity vs that associated to the FGF receptor 1 (median IC(50) 2.5 microM, range 0.9-19 microM). This selectivity profile is also evident in a growth factor-stimulated human endothelial cell (HUVEC) proliferation assay (i.e., inhibition of VEGF > EGF > FGF), with inhibition of VEGF-induced proliferation being achieved at nanomolar concentrations (median IC(50) 0.06 microM). Further examination of compound 2 (ZD6474) in recombinant enzyme assays revealed excellent selectivity for the inhibition of KDR tyrosine kinase (IC(50) 0.04 microM) vs the kinase activity of erbB2, MEK, CDK-2, Tie-2, IGFR-1R, PDK, PDGFRbeta, and AKT (IC(50) range: 1.1 to >100 microM). Anilinoquinazolines possessing basic C-7 side chains exhibited markedly improved aqueous solubility over previously described anilinoquinazolines possessing neutral C-7 side chains (up to 500-fold improvement at pH 7.4). In addition, aqueous solubility of the neutral fraction present at pH 7.4 of the basic subseries of anilinoquinazoline proved to be higher than that of the neutral analogue 1 (ZD4190). Oral administration of representative compounds to mice (50 mg/kg) produced plasma levels between 0.2 and 3 microM at 24 h after dosing. Our development candidate 2 demonstrated a very attractive in vitro profile combined with excellent solubility (330 microM at pH 7.4) and good oral bioavailability in rat and dog (> 80 and > 50%, respectively). This compound demonstrated highly significant, dose-dependent, antitumor activity in athymic mice. Once daily oral administration of 100 mg/kg of compound 2 for 21 days inhibited the growth of established Calu-6 lung carcinoma xenografts by 79% (P < 0.001, Mann Whitney rank sum test), and substantial inhibition (36%, P < 0.02) was evident with 12.5 mg/kg/day.     

Role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase: implications for increasing the activity against sensitive and multidrug-resistant leukaemia HL60 cells

The aim of this study was to examine the role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially with regard to multidrug resistant (MDR) tumour cells. It was found that at a high NADPH concentration (500 μmol/l), the anthracenedione agent ametantrone, with an unmodified quinone structure, was susceptible to CPR-dependent reductive activation. In contrast, it was shown that compounds with modified quinone grouping (benzoperimidine BP1, anthrapyridone CO1 and pyrazolopyrimidoacridine PPAC2) did not undergo reductive activation by CPR. This suggests that the presence of a modified quinone function is the structural factor excluding reductive activation of antitumour anthraquinone derivatives and analogues by CPR. In the second part of the work, the ability of antitumour anthraquinone derivatives and analogues to inhibit the growth of the human promyelocytic, sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. A significant increase in the activity of ametantrone with an unmodified quinone structure after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells, whereas in the case of quinone-modified compounds (BP1, CO1 and PPAC2), the presence of the activation system had no effect on their activity against the sensitive and MDR tumour cells examined.     

Application of a dynamic in vitro gastrointestinal tract model to study the availability of food mutagens, using heterocyclic aromatic amines as model compounds.

The TNO gastro-Intestinal tract Model (TIM) is a dynamic computer-controlled in vitro system that mimics the human physiological conditions in the stomach and small intestine. In the current TIM physiological parameters such as pH, temperature, peristaltic movements, secretion of digestion enzymes, bile and pancreatic juices, and absorption of digested products-by removal through dialysis-was simulated. Heterocyclic aromatic amines (HAA; viz. IQ, MeIQ, MeIQx and PhIP) were used as model compounds for food mutagens, and the passage through TIM was investigated for each of these compounds separately. Subsequently, the influence of a matrix and different rates of passage on the availability for absorption and distribution were studied in experiments with prepared meat, supplemented with MeIQx. Samples taken at various time points from the jejunal and ileal dialysates and from the lumen at the end of the small intestine (ileal delivery) were tested for the presence of mutagenic activity in the Ames test with Salmonella typhimurium strain TA98 as indicator, in the presence of mammalian metabolic activation (rat S9 mix). The results show that, comparable with the human in vivo situation, all four HAA are quickly removed (approx. 50% in 2 hr; approx. 95% in 6 hr) and mainly recovered from the lumen into the jejunal and ileal dialysates (94% of recovery). Only 5+/-1.5% is recovered in the chyme at the end of the small intestine. When MeIQx was added to meat, its availability for absorption was slower, although the influence of the gastrointestinal passage time on the availability of MeIQx was more pronounced than this matrix effect. More MeIQx was found in the jejunal dialysate (23%; P<0.01) and less in the ileal delivery (8%; P<0.01) when simulating the gastrointestinal passage of solid meals was compared to simulating that of liquid meals. The present experiments demonstrate that TIM can be applied to study in vitro the availability of heterocyclic aromatic amines in the gastrointestinal tract. More generally, these studies indicate that TIM shows promise as a useful tool for various research purposes dealing with the availability for absorption of mutagenic as well as antimutagenic components in food.