Small molecule inhibitors of the XIAP protein-protein interaction

The X-linked inhibitor of apoptosis proteins (XIAP) is thought to play a key role in the unchecked proliferation of cancer cells by interfering with the signaling cascade leading to cell death. The structure and mechanism of XIAP has been widely investigated and characterized over the past few years, to the point where this may be the best understood apoptosis protein inhibitor. As a result, XIAP is viewed as an attractive target for the treatment of cancer. To date, several research groups have reported on the discovery of small molecule inhibitors of this protein. This review focuses on the discovery and optimization of these leads.     

Small molecule inhibitors of KDR (VEGFR-2) kinase: an overview of structure activity relationships

The Kinase insert Domain containing Receptor (KDR), alternatively referred to as VEGFR-2, is a receptor for Vascular Endothelial Growth Factors (VEGFs) and functions as a key regulator of angiogenesis, the process by which new capillaries are created from preexisting blood vessels. The induction of angiogenesis, or the "angiogenic switch," is a critical step in tumor progression, and inhibitors of KDR have been demonstrated both to induce tumor regression and reduce metastatic potential in preclinical models. In the last few years, medicinal chemists have expanded the kinase selectivity profile of known inhibitor classes to include KDR, and also identified novel classes of KDR inhibitors. This review presents structure activity relationships (SAR) of small molecule inhibitors of KDR, with an emphasis on the pharmacophore elements of the scaffolds employed. Binding hypotheses based on X-ray crystallographic analyses will also be described. Additionally, the efficacy of representative compounds in in vitro and in vivo models of tumor progression and angiogenesis are discussed.     

The Bcl-2 homology domain 3 (BH3) mimetic ABT-737 reveals the dynamic regulation of bad, a proapoptotic protein of the Bcl-2 family, by Bcl-xL

The proteins of the B-cell lymphoma 2 (Bcl-2) family are important regulators of apoptosis under normal and pathological conditions. Chemical compounds that block the antiapoptotic proteins of this family have been introduced, such as 4-[4-[(4'-Chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide (ABT-737), a BH3-mimetic that neutralizes Bcl-2 and Bcl-xL. In this study, we used ABT-737 to explore the dynamic regulation of Bcl-2 proteins in living cells of different origins. Using ABT-737 as well as RNA interference or the application of growth factors, we examined the impact of the functional availability of the antiapoptotic proteins Bcl-2 and Bcl-2-extra large (Bcl-xL) on the Bcl-2 network. We report that ABT-737 increases the expression of Bcl-2-associated death promoter (Bad), a proapoptotic partner of the proteins Bcl-2 and Bcl-xL. Our observations indicate that Bad overexpression induced by ABT-737 results from the control of its normally rapid protein turnover, leading to the stabilization of this protein. We demonstrate the relevance of Bad post-translational regulation by Bcl-xL to the physiological setting using RNA interference against Bcl-xL as well as the application of epidermal growth factor, a growth factor that promotes the dissociation of Bad from Bcl-xL. Our results highlight a new facet of the mode of action of the antiapoptotic proteins Bcl-2 and Bcl-xL consisting of the regulation of the stability of the protein Bad. Finally, our results shed light on the mode of action of ABT-737, currently the best characterized inhibitor of the antiapoptotic proteins of the Bcl-2 family, and bear important implications regarding its use as an anticancer drug.     

Arachidonic acid-induced pleurisy (ARIP): an in vivo model for testing 5-lipoxygenase inhibition

Arachidonic acid-induced pleurisy in the rat was evaluated for testing inhibitors of arachidonic acid (AA) metabolism. The model involves the administration of AA intrapleurally and the determination of LTB4 and PGE2 as indicators of 5-lipoxygenase and cyclooxygenase activities in the exudate/wash. This model is suitable for the in vivo evaluation of potential inhibitors of the 5-lipoxygenase pathway but not the cyclooxygenase pathway of AA cascade.     

2D-QSAR and HQSAR of the inhibition of calcineurin-NFAT signaling by blocking protein-protein interaction with N-(4-oxo-1 (4H)-naphthalenylidene)benzenesulfonamide analogues

The inhibition of calcineurin-NFAT signaling by blocking protein-protein interaction with N-(4-oxo-1(4H)-naphthalenylidene)benzenesulfonamide analogues was studied in order to obtain mechanistic information about the effects of structural modification and molecular design of immunomodulation agents. The study was carried out by quantitative structure-activity relationship (QSAR) analysis using 2D-QSAR and hologram QSAR (HQSAR) methods. The statistical results of the two models showed the best prediction and fitness (r2 > 0.900) for the inhibition activities. The inhibitory activities from the 2D-QSAR models were dependent upon the electronic affinity of electron acceptor and optimum dipole moment (DM opt = 4.491 Debye). In addition, the HQSAR model provided information about which structural distinctions could be significant contributors the inhibition.     

In vivo inhibition of hepatic lipogenesis in the rat by cyclandelate (3, 3′, 5-trimethylcyclohexanylmandelate)

Rates of hepatic lipogenesis were measured in vivo in rats by incorporation into lipids of [³H] from injected [³H]H₂O 17 hr after a single oral dose of cyclandelate (3, 3′,5-trimethylcyclohexanylmandelate, a vasoactive substance). Cyclandelate administration resulted in a significant inhibition (40–60%) of both sterol and fatty acid synthesis in the livers which was independent of the 3.2-fold diurnal variation in the rates of hepatic sterol and fatty acid synthesis. The inhibition of accumulation of newly synthesized fatty acid in intestine also reached statistical significance. The accumulation of newly synthesized sterol was significantly depressed in serum but did not result in any change in the concentration of serum total cholesterol. These results are interpreted in terms of the inhibitory effect of cyclandelate on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase previously reported by us (Biochem. Pharmac. 32, 649, 1983).     

Bactericidal effect of cethromycin (ABT-773) in an immunocompetent murine pneumococcal pneumonia model

The efficacy of cethromycin was assessed against isolates of Streptococcus pneumoniae in the presence of neutrophils. Comparison with data from our previous neutropenic model revealed that the presence of neutrophils enhanced the bacteriostatic and bactericidal effect of cethromycin by an average of two- to four-times, respectively.     

Small molecule antagonists of the corticotropin releasing factor (CRF) receptor: recent medicinal chemistry developments

Antagonists of the corticotropin releasing factor (CRF or CRH) receptor have shown promise for the treatment of anxiety, depression, and irritable bowel syndrome. In the present article, medicinal chemistry developments surrounding small molecule CRF receptor antagonists are reviewed, focusing on publications and patents from mid-2004 through the first quarter of 2006. While the CRF type 2 receptor remains an intractable target, incremental progress has been made in the search for drug-like antagonists of the CRF type 1 receptor. Most recent work has not ventured far from previously-established pharmacophoric topologies. A common theme in recent patent disclosures is the addition of novel polar substituents to known heterocyclic core structures to reduce overall lipophilicity. New disclosures of pharmacokinetic (PK) data for several series of antagonists reveal that achieving appropriate PK remains a challenge for the field. The recent publication of selection patents and patents relating to salt and crystal forms of particular compounds suggests that several second generation compounds are nearing or have entered clinical development.     

In vitro and in vivo inhibition of human flavin-containing monooxygenase form 3 (FMO3) in the presence of dietary indoles.

The effect of consumption of glucosinolate-containing Brussels sprouts on flavin-containing monooxygenase functional activity in humans was investigated in 10 healthy, male, non-smoking volunteers. After a 3-week run-in period, 5 volunteers continued on a glucosinolate-free diet for 3 weeks (control group), and 5 others consumed 300 g of cooked Brussels sprouts per day (sprouts group). Human flavin-containing monooxygenase activity was measured by determining the levels of urinary trimethylamine and trimethylamine N-oxide. In the control group similar trimethylamine to trimethylamine N-oxide ratios were observed, while in the sprouts group the trimethylamine to trimethylamine N-oxide ratios were increased 2.6- to 3.2-fold, and thus flavin-containing monooxygenase functional activity was decreased significantly. To investigate the molecular basis for the in vivo inhibition of functional human flavin-containing monooxygenase activity, in vitro studies were carried out examining the effect of acid condensation products of indole-3-carbinol, anticipated to be formed after transit of Brussels sprouts through the gastrointestinal system, on the prominent cDNA-expressed human flavin-containing monooxygenase form 3 enzymes. Two indole-containing materials were observed to be potent inhibitors of human flavin-containing monooxygenases, having Ki values in the low micromolar range. The results suggested that acid condensation products expected to be formed upon transit of Brussels sprouts materials through the gastrointestinal system were potent competitive inhibitors of human flavin-containing monooxygenase form 3 enzymes. The findings indicate that daily intake of Brussels sprouts may lead to a decrease in human flavin-containing monooxygenase activity, and this may have consequences for metabolism of other xenobiotics or dietary constituents.     

Kiiv, an in vivo parameter for predicting the magnitude of a drug interaction arising from competitive enzyme inhibition.

The goal of the study was to test the assumption that a competitive inhibition constant determined in vivo, Kiiv, like its corresponding in vitro counterpart, Ki, is independent of inhibitor concentration. Inhibition of the CYP2C9-dependent formation of (S)-7-hydroxy-warfarin from (S)-warfarin was measured in seven healthy subjects at three different doses of fluconazole. Prothrombin time measurements showed increasing anticoagulant activity with increasing fluconazole dose. The pharmacokinetic parameters calculated from the (S)- and (R)-warfarin plasma levels were consistent with previous studies. Fluconazole reduced the clearance of (S)-warfarin to a greater extent than that of (R)-warfarin. The decrease in clearance of both warfarin enantiomers was fluconazole dose-dependent. The formation of (S)-7-hydroxy-warfarin was inhibited by 31, 55, and 77% at the 100, 200, and 300 mg daily doses of fluconazole, respectively. Kiiv, values calculated from these data based on plasma fluconazole levels at each dose and data from earlier work at 400-mg daily doses of fluconazole were 30.7 +/- 23.7, 19.6 +/- 3.8, 17.9 +/- 7.5, and 19.8 +/- 3.5 microM, respectively. These results confirm the hypothesis that Kiiv is independent of inhibitor concentration.     

N-[3-(1H-imidazol-4-ylmethyl)phenyl]ethanesulfonamide (ABT-866, 1),(1) a novel alpha(1)-adrenoceptor ligand with an enhanced in vitro and in vivo profile relative to phenylpropanolamine and midodrine

N-[3-(1H-Imidazol-4-ylmethyl)phenyl]ethanesulfonamide (ABT-866, 1) is a novel alpha(1) agent having the unique profile of alpha(1A) (rabbit urethra, EC(50) = 0.60 microM) agonism with alpha(1B) (rat spleen, pA(2) = 5.4) and alpha(1D) (rat aorta, pA(2) = 6.2) antagonism. An in vivo dog model showed 1 to be more selective for the urethra over the vasculature than A-61603 (2), ST-1059 (3, the active metabolite of midodrine), and phenylpropanolamine (4).     

Malathion toxicity: In vivo inhibition of acetylcholinesterase in the fish Brachydanio rerio (Cyprinidae)

Acetylcholinesterase (AChE) activity in the nervous tissue (brain) of the fish Brachydanio rerio was significantly inhibited by exposure to the organophosphorus (OP) pesticide, malathion. The inhibition was dose- as well as time-dependent. The fish survived even when the activity of the enzyme was inhibited by 90%. There was a significant recovery in the activity of AChE when malathion stress was lifted.     

Important role of 3-methoxytyramine in the inhibition of cocaine sensitization by 1-methyl-1,2,3,4-tetrahydroisoquinoline: an in vivo microdialysis study

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous compound with neuroprotective and antidopaminergic activities. Our previous research has shown that 1MeTIQ prevents morphine addiction and abates the expression of the reinstatement of cocaine self-administration. The current study investigated the mechanism of action of 1MeTIQ that is responsible for its considerable anticraving potential. Accordingly, we performed behavioral tests that measured the influence of 1MeTIQ on the locomotor activity of rats (Wistar) after a single cocaine (15 mg/kg, i.p.) dose and during cocaine sensitization (15 mg/kg, i.p.). In a neurochemical study, we examined the influence of 1MeTIQ on dopamine release in the rat striatum after a single cocaine administration and during cocaine sensitization using an in vivo microdialysis methodology. The data showed that 1MeTIQ (50 mg/kg, i.p.) only slightly inhibited cocaine-induced hyperactivity but completely antagonized the expression of locomotor cocaine sensitization. The in vivo microdialysis study demonstrated that the administration of 1MeTIQ before the acute cocaine injection intensified the cocaine-induced increase in dopamine release and produced a huge and long-lasting elevation of the extraneuronal concentration of dopamine (by approximately 1400%, p < 0.01) in the rat striatum. A significant increase in 3-methoxytyramine (3-MT) (by approximately 400%, p < 0.01) was also observed. During the expression of cocaine sensitization, the administration of 1MeTIQ before the reminder dose of cocaine produced an additional elevation of dopamine release but considerably more strongly increased the concentration of 3-MT in the synaptic cleft (by about 800%, p < 0.01). In light of these data and of our earlier in vitro and in vivo experiments showing a physiological role of 3-MT in the inhibitory regulation of excessive stimulation, we suggest that locomotor hyperactivity is dependent not only on dopamine concentration in the extracellular space, but also on the ratio of [DA/3-MT]. 1MeTIQ administered before the reminder dose of cocaine to cocaine-experienced rats plainly normalized the [DA/3-MT] ratio, which was increased by cocaine, and this effect may be responsible for its anti-addictive action. The results strongly support the view that 1MeTIQ may have a more general anti-abuse potential, and the extraneuronal metabolite of dopamine, 3-MT, may play a crucial role in its anti-craving effects.     

Small molecule inhibitors of retinoic acid 4-hydroxylase (CYP26): synthesis and biological evaluation of imidazole methyl 3-(4-(aryl-2-ylamino)phenyl)propanoates

The synthesis and potent inhibitory activity of novel imidazole methyl 3-(4-(aryl-2-ylamino)phenyl)propanoates in a MCF-7 CYP26A1 microsomal assay is described. The induction of CYP26A1 mRNA was used to evaluate the ability of the compounds to enhance the biological effects of all-trans retinoic acid (ATRA) in a retinoid-responsive neuroblastoma cell line. The most promising inhibitor, 3-imidazol-1-yl-2-methyl-3-[4-(naphthalen-2-ylamino)-phenyl]-propionic acid methyl ester (20), with an IC(50) of 3 nM (compared with liarozole IC(50) of 540 nM and R116010 IC(50) of 10 nM) was further evaluated for CYP selectivity using a panel of CYP enzymes, mutagenicity (Ames screen), and hepatic stability.     

Studies on the cyanamide-ethanol interaction: Dimethylcyanamide as an inhibitor of aldehyde dehydrogenase in vivo

Administration of dimethylcyanamide (DMC) to rats caused a marked elevation in ethanol-derived blood acetaldehyde (AcH) and depressed the specific activity of the low K_m mitochondrial aldehyde dehydrogenase (AlDH) by 90% at 12–24 hr, coincident with depletion of hepatic glutathione levels. Comparison of the relative efficacy of DMC and cyanamide in elevating blood AcH measured at 2hr and 1 hr post-drug treatment, respectively, indicated that DMC was at least one-fifth as active as cyanamide. However, since the comparison was not made at optimal times for DMC (12–24 hr), it is likely that its activity in vivo approaches that of cyanamide itself. DMC was essentially inactive in vitro as an inhibitor of the low K_m AlDH isozyme in intact rat liver mitochondria. Although methylcyanamide, the product of N-demethylation of DMC, was too unstable to be prepared for this evaluation, the higher monoalkyl cyanamide, n-propylcyanamide, was synthesized chemically and was shown to be a good inhibitor of the mitochondrial enzyme in vitro. These results suggest that DMC must be N-demethylated before being converted to a reactive species that inhibits AlDH activity.     

In vivo acute toxicological studies of an antioxidant extract from Mangifera indica L. (Vimang)

Mango (Mangifera indica L.) stem bark aqueous extract (MSBE) is a natural product with antioxidant, anti-inflammatory, analgesic, and immunomodulatory effects. Its formulations (e.g., tablets, capsules, syrup, vaginal oval, and suppositories) are known by the brand name of Vimang®. In view of the ethnomedical, preclinical, and clinical uses of this extract and the necessity to assess its possible toxicological effect on man, a toxicological analysis of a standard extract is reported in this paper. Acute toxicity was evaluated in mice and rats by oral, dermal, and intraperitoneal (i.p.) administration. The extract, by oral or dermal administration, showed no lethality at the limit doses of 2,000 mg/kg body weight and no adverse effects were found. Deaths occurred with the i.p. administration at 200, but not 20 mg/kg in mice. MSBE was also studied on irritant tests in rabbits, and the results showed that it was nonirritating on skin, ocular, or rectal mucosa. The extract had minimal irritancy following vaginal application.     

Protriptyline induced inhibition of the in vivo 3H-noradrenaline synthesis from 3H-l-Dopa in the rat brain

Summary ³H-l-Dopa was given intraperitoneally, after a peripheral decarboxylase inhibitor Ro 4-4602 (50 mg/kg), to male Wistar rats and the effect of protriptyline pretreatment (10 mg/kg, 30 min) on the formation and metabolism of the brain ³H-catecholamines, dopamine and noradrenaline and all their metabolites was investigated.Protriptyline produced a strong decrease of labelled noradrenaline and its metabolites normetanephrine, free and conjugated 3-methoxy-4-hydroxyphenyl-eneglycol and 3,4-dihydroxyphenyleneglycol 60 and 120 min after ³H-l-Dopa. ³H-noradrenaline was also decreased 30 and 45 min after ³H-l-Dopa. In rats and mice the pretreatment with protriptyline (10 mg/kg, 30 min) induced also a significant decrease in brain ³H-noradrenaline but not ³H-dopamine synthesized from ³H-l-tyrosine. Protriptyline (10 mg/kg) produced no effect on endogenous dopamine and noradrenaline in the rat or mouse brain.The present findings strongly indicate that the acute treatment with protriptyline inhibits the ³H-noradrenaline formation from ³H-l-Dopa. This effect seems most likely to be related to an interaction of protriptyline with the precursor(s) ³H-l-Dopa or ³H-dopamine at sites of ³H-noradrenaline biosynthesis.A preliminary report of the present study was presented at the Scandinavian Pharmacological Society, June 1972, Uppsala, Sweden and the VIII C.I.N.P. Congress, August 1972, Copenhagen, Denmark.