Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3–20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30–240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.

The mechanism of action of a novel antiproliferative compound LY290181 [2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-carbonitrile] was characterized. LY290181 is a potent inhibitor of cell proliferation, producing 50% inhibition of vascular smooth muscle, endothelial, Chinese hamster ovary, HeLa, and human erythroleukemia cells at concentrations of 8-40 nM. Cell cycle analysis showed that LY290181 caused accumulation of smooth muscle cells at the G2/M phase and induced mitotic arrest in Chinese hamster ovary cells and HeLa cells. At low concentrations (3-30 nM), LY290181 blocked transition of cells from metaphase to anaphase and disrupted mitotic spindle organization. At high concentrations (>/=100 nM), LY290181 produced a concentration-dependent loss of cytoplasmic and spindle microtubules. LY290181 inhibited the polymerization of purified bovine brain microtubule protein into microtubules, and it depolymerized preformed microtubules. Using tubulin-1-anilino-8-naphthalene sulfonate complex fluorescence, we have shown that LY290181 directly interacted with tubulin in a unique manner. These studies show that LY290181 induces cell growth arrest in prometaphase/metaphase, and tubulin appears to be its molecular target.

No abstract

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds that disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89000 small molecules under nonreplicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that disrupt intrabacterial pH homeostasis and also showed some activity against nonreplicating bacteria in a 4-stress model, but with no (or greatly reduced) activity against replicating bacteria. The compounds selected were two benzamide sulfonamides, a benzothiadiazole, a bissulfone, and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against nonreplicating bacteria in buffer. Four of the five compounds demonstrated increased activity under low pH conditions. None of the five compounds acted as ionophores or as general disrupters of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.

Dipyridamole inhibited smooth muscle cell proliferation in vitro. This activity was attenuated by serum proteins. Locally delivered dipyridamole inhibited cell replication in arteries and intimal thickening after balloon injury. These results suggest that although systemic treatment with dipyridamole may not be efficacious because of inadequate serum levels, its antiproliferative action on smooth muscle cells may reduce restenosis when the drug is delivered locally after coronary angioplasty.

No abstract

FMPD [6-fluoro-10-[3-(2-methoxyethyl)-4-methyl-piperazin-1-yl]-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene] is a potential novel antipsychotic with high affinity for dopamine D 2 (K i ϭ 6.3 nM), 5-HT 2A (K i ϭ 7.3 nM), and 5-HT 6 (K i ϭ 8.0 nM) human recombinant receptors and lower affinity for histamine H 1 (K i ϭ 30 nM) and 5-HT 2C (K i ϭ 102 nM) human recombinant receptors than olanzapine. Oral administration of FMPD increased rat nucleus accumbens 3,4-dihyroxyphenylacetic acid concentrations (ED 200 ϭ 6 mg/kg), blocked 5-HT 2A agonist-induced increases in rat serum corticosterone levels (ED 50 ϭ 1.8 mg/kg), and inhibited the ex vivo binding of [ 125 I]SB-258585 [4-iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide] to striatal 5-HT 6 receptors (ED 50 ϭ 10 mg/kg) but failed to inhibit ex vivo binding of [ 3 H]pyrilamine to hypothalamic histamine H 1 receptors at doses of up to 30 mg/kg. In electrophysiology studies, acute administration of FMPD selectively elevated the number of spontaneously active A10 (versus A9) dopamine neurons and chronic administration selectively decreased the number of spontaneously active A10 (versus A9) dopamine neurons. FMPD did not produce catalepsy at doses lower than 25 mg/kg p.o. In Fos-induction studies, FMPD had an atypical antipsychotic profile in the striatum and nucleus accumbens and increased Fos expression in orexin-containing neurons of the hypothalamus. FMPD produced only a transient elevation of prolactin levels. These data indicate that FMPD is an orally available potent antagonist of dopamine D 2 , 5-HT 2A , and 5-HT 6 receptors and a weak antagonist of H 1 and 5-HT 2C receptors. FMPD has the potential to have efficacy in treating schizophrenia and bipolar mania with a low risk of treatmentemergent extrapyramidal symptoms, prolactin elevation, and weight gain. Clinical trials are needed to test these hypotheses.Olanzapine is effective in treating schizophrenia and bipolar mania (Beasley et al., 1996;Tohen et al., 2000). It is classified as an atypical antipsychotic in that it has therapeutic effects on the positive, negative, and cognitive symptoms of schizophrenia while having a low propensity to produce extrapyramidal symptoms (EPS) and prolactin elevation. However, many patients have observed weight gain associated with the treatment of olanzapine and other atypical antipsychotics (Wirshing, 2001).It has been hypothesized that blockade of the histamine H 1 receptor plays a critical role in antipsychotic treatmentemergent weight gain. Histamine has long been associated with the control of food intake. Histamine activates postsynaptic H 1 receptors in the ventromedial and para-ventricular nucleus of the hypothalamus to suppress feeding (Sakata et al., 1988;Ookuma et al., 1989), and H 1 knock-out mice develop diet-induced and age-related obesity (Masaki et al., 2001). In addition, a significant correlation between affinity for the human H 1 receptor and weight gain has been reported for antipsychotics (Kroeze et al., 2003). Thus, an a...

The role of serinekhreonine kinase casein kinase-11 (CK-II) in vascular smooth muscle cell proliferation and inhibition by heparin was investigated. cDNAs for a and p subunits of CK-II from rabbit vascular smooth muscle cells were cloned and sequenced. A strong evolutionary conservation was found at amino acid and nucleotide levels in CK-II from rabbit and human. Treatment of smooth muscle cells with a specific antisense oligonucleotide to CK-II blocked stimulation of DNA synthesis in response to PDGF. Addition of a known inhibitor of CK-I1 to cultures of smooth muscle cells also blocked DNA synthesis. Mitogenic stimulation of growth arrested quiescent cultures of smooth muscle cells with PDGF produced a2-3-fold increase in CK-II activity.Heparin was a potent inhibitor of smooth muscle cell derived CK-II in vitro and attenuated the stimulation of CK-I1 activity in response to PDGF in intact cells. lntracellular localization studies showed that heparin and CK-I1 were localized in the nucleus. These results suggest a potential role of CK-II in signal transduction mediating smooth muscle cell growth. Modulation of CK-II activity by heparin and its co-localization in the nucleus suggest that heparin may effect CK-II activity in intact cells. 0 1993 Wiley-Liss, Inc.