A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A.

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC50 values ⩽1µM and 11 effective at ⩽2µM in an ex vivo assay.

In vivo genotoxicity of a novel heterocyclic amine, aminobenzoazepinoquinolinone-derivative (ABAQ), produced by the Maillard reaction between glucose and l-tryptophan.

We recently demonstrated that a novel heterocyclic amine, 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ), is produced from glucose and l-tryptophan by the Maillard reaction at physiological temperature and pH, and that ABAQ was strongly mutagenic for Salmonella strains in the presence of S9 mix. Here, we present the results of three in vivo genotoxicity assays of ABAQ. The comet assay revealed that DNA damage was significantly increased in the livers, kidneys, lungs, and bone marrows of ICR mice, 3h after i.p. injection of ABAQ (50mg/kg body weight (bw)). To evaluate clastogenicity, the peripheral blood micronucleus test was performed, also in ICR mice. ABAQ induced micronucleated reticulocytes (MNRETs) in a dose-dependent manner; the frequency of MNRETs was significantly elevated at all i.p. doses (12.5, 25, and 50mg/kg bw) after 48h. To investigate the mutagenicity of ABAQ in vivo, gpt delta transgenic mice were treated with five consecutive administrations of ABAQ by gavage at doses of 25 or 50mg/kg per week for 3 weeks. The frequencies of gpt mutations (MF) in the liver of mice increased significantly compared with controls, in a dose-dependent manner. No significant increase of gpt MF was detected in the kidneys. Base substitutions predominated; both G:C→A:T and A:T→C:G mutations were significantly increased by ABAQ. The Spi(-) MF was also significantly increased in the liver after ABAQ treatment. If formed in vivo, ABAQ may give rise to adverse genotoxic effects.

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker.

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.

Anti human immunodeficiency virus type 1 (HIV-1) agents 4. Discovery of 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline sulfonates as new HIV-1 inhibitors in vitro.

To search for compounds with superior anti-human immunodeficiency virus type 1 (HIV-1) activity, ten 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline sulfonates (4a-j) were synthesized and preliminarily evaluated as HIV-1 inhibitors in vitro for the first time. Some compounds demonstrated anti-HIV-1 activity, especially 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline p-ethylbenzenesulfonate (4g) and 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline p-chlorobenzenesulfonate (4i) showed the more potent anti-HIV-1 activity with 50% effective concentration (EC(50)) values of 2.59 and 4.01 microg/ml, and therapeutic index (TI) values of 31.77 and 24.51, respectively.

Site-activated multifunctional chelator with acetylcholinesterase and neuroprotective-neurorestorative moieties for Alzheimer's therapy.

A novel strategy to develop site-activated multifunctional chelators for targeting multiple etiologies of Alzheimer's disease is reported. The novel prochelator HLA20A with improved cytotoxicity shows little affinity for metal ions until it is activated by binding and inhibiting acetylcholinesterase (AChE), releasing an active chelator HLA20 that modulates amyloid precursor protein (APP) regulation and beta-amyloid (Abeta) reduction, suppresses oxidative stress, and passivates excess metal ions (Fe, Cu, and Zn) in the brain.

Genotoxicity of plumbagin and its effects on catechol and NQNO-induced DNA damage in mouse lymphoma cells.

Plumbagin, a naphtoquinone present in the roots of Plumbago zeylanica, has been reported to have many beneficial effects such as antibacterial, antifungal, anticancer, antimutagenic and antioxidant effects, but this compound has also been reported to have many side effects. Given the wide use of P. zeylanica in traditional medicine and the various potential therapeutic uses of plumbagin, the present study was carried out to further elucidate the potential genotoxicity and antigenotoxicity of plumbagin in mouse lymphoma L5178Y cells, using the comet assay. Without affecting the cell viability, plumbagin itself was found to induce significant DNA damage at concentrations as low as 0.25 ng/ml. When the cells were exposed to non-DNA damaging concentrations of plumbagin, together with NQNO (known to interact with DNA in many different ways) or catechol (known to induce oxidative DNA damage), plumbagin was found to significantly reduce the catechol-induced DNA damage, but to be without protective effect against the NQNO-induced damage. The fact that non-DNA damaging concentrations of plumbagin diminished the DNA damage induced by catechol, provides further support for the idea that plumbagin may act as an antioxidative agent at low concentrations.

Mobilization of iron from cells by hydroxyquinoline-based chelators.

With the aim of identifying an iron (Fe) chelator which is effective at mobilizing intracellular Fe, two novel ligands were synthesized and tested. Hydroxyquinoline is known to possess a high affinity for Fe and was thus chosen as the Fe binding motif for the hexadentate chelators, C1 (2,2'-[ethane-1,2-diylbis(iminomethylene)]diquinolin-8-ol) and C2 (2,2'-[cyclohexane-1,2-diylbis(iminomethylene)]diquinolin-8-ol). Both chelators are lipophilic, with Fe3+ complexes slightly more hydrophilic than the free ligands. C1 and C2 were equally toxic to K562 cells, and partial protection was afforded by supplementing the culture medium with human holotransferrin, suggesting that some of the toxicity of the ligands is due to cellular Fe depletion. Micromolar concentrations of both ligands effectively mobilized 59Fe from reticulocytes and K562 cells. In reticulocytes, 50 microM C1 caused the release of 60% of the cells' initial 59Fe uptake after a 4h incubation. Under the same conditions, C2 revealed a release of 50% of the 59Fe. Overall, both ligands merit in vivo study for oral activity. Their effectiveness at low concentrations makes them candidates for therapeutic use.

Effects of a direct injection of liposoluble iron into rat striatum. Importance of the rate of iron delivery to cells.

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical (*OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 microM FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of *OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced *OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with *OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular *OH in damage associated with intracerebral iron injection.

Enhancement of iron toxicity in L929 cells by D-glucose: accelerated(re-)reduction.

It has recently been shown that an increase in the cellular chelatable iron pool is sufficient to cause cell damage. To further characterize this kind of injury, we artificially enhanced the chelatable iron pool in L929 mouse fibroblasts using the highly membrane-permeable complex Fe(III)/8-hydroxyquinoline. This iron complex induced a significant oxygen-dependent loss of viability during an incubation period of 5 h. Surprisingly, the addition of L-glucose strongly enhanced this toxicity whereas no such effect was exerted by L-glucose and 2-deoxyglucose. The assumption that this increase in toxicity might be due to an enhanced availability of reducing equivalents formed during the metabolism of L-glucose was supported by NAD(P)H measurements which showed a 1.5-2-fold increase in the cellular NAD(P)H content upon addition of L-glucose. To assess the influence of this enhanced cellular reducing capacity on iron valence we established a new method to measure the reduction rate of iron based on the fluorescent iron(II) indicator PhenGreen SK. We could show that the rate of intracellular iron reduction was more than doubled in the presence of L-glucose. A similar acceleration was achieved by adding the reducing agents ascorbate and glutathione (the latter as membrane-permeable ethyl ester). Glutathione ethyl ester, as well as the thiol reagent N -acetylcysteine, also caused a toxicity increase comparable with L-glucose. These results suggest an enhancement of iron toxicity by L-glucose via an accelerated (re-)reduction of iron with NAD(P)H serving as central electron provider and ascorbate, glutathione or possibly NAD(P)H itself as final reducing agent.

Differential sensitivity to lung tumorigenesis following transplacental exposure of mice to polycyclic hydrocarbons, heterocyclic amines, and lung tumor promoters.

Research conducted by this laboratory over the past decade has demonstrated the high susceptibility of the fetus to lung tumor formation following in utero exposure of the resistant C57BL/6 and DBA/2N strains of mice to 3-methylcholanthrene (MC). In this review, we describe our more recent studies on the effects of MC and cotreatment with the lung tumor promoter, butylated hydroxytoluene (BHT), on lung tumor formation in the intermediately susceptible BALB/c strain of mice, and the determination of the potential carcinogenicity of the heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in resistant mouse strains. BALB/c mice showed a similar incidence of lung tumors, both in terms of percentage of mice with tumors and number of tumors per mouse, as found in the resistant [D2 x B6D2F1]F2 mice. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung lesions compared with an incidence of 79% in [D2 x B6D2F1]F2 mice. BALB/c lung lesions demonstrated a similar association of Ki-ras mutations with tumor stage. Interestingly, a strain-dependent difference was observed in the mutational spectrum, where 62% and 38% of the lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast with the 16% and 84% incidences observed in [D2 x B6D2F1]F2 mice. BHT had no statistically significant effect on tumor incidence, multiplicity, or Ki-ras mutational spectrum in BALB/c mice treated in utero with MC, although a trend toward increased tumor multiplicity was observed. Finally, experiments initiated to assess the transplacental carcinogenicity of IQ in D2B6F1 mice demonstrated that 1 year after birth, no macroscopically or microscopically visible liver, lung, or colon tumors were found in the transplacentally treated offspring, nor was induction of Cyp1a1, Cyp1b1, or glutathione S-transferases (GSTs) in fetal lung and liver tissues observed. This implies that at least under these experimental conditions, IQ may not be an important transplacental carcinogen. Overall, these data demonstrate that mutagenic damage to Ki-ras is a critical early event mediating murine lung tumorigenesis in both sensitive and resistant strains. Strain-dependent differences in the Ki-ras mutational spectrum may be associated with their differential susceptibility to lung tumor initiation.

Linomide-mediated protection of oligodendrocytes is associated with inhibition of nitric oxide production and IL-1beta expression in Lewis rat glial cells.

Linomide is a synthetic immunomodulator that down-regulates autoimmune response without inducing systemic immunosuppression. Linomide effectively inhibits severe experimental autoimmune diseases, like experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis (MS). Here we report that Linomide suppresses nitric oxide (NO) production by microglia and astrocytes derived from newborn rats and prevented oligodendrocyte damage. Linomide strongly inhibited interleukin (IL) 1 betamRNA expression on glial cells, suggesting a potential mechanism for inhibition of NO production by Linomide. These results demonstrate that Linomide-mediated inhibition of NO production by glial cells could explain the preventive and therapeutic effects of Linomide in EAE and perhaps also MS. However, Linomide at higher dose [correction of doss] (10(-5) M) resulted in direct oligodendrocyte damage.

Elevated ferritin production, iron containment, and oxidant resistance in hemin-treated leukemia cells.

Hemin (ferriprotoporphyrin IX), the oxidized prosthetic group of hemoglobin, is a source of potentially cytotoxic iron, but in chronic low doses can induce cytoprotection against iron-stimulated oxidative stress. The latter property of hemin has been examined, using murine L1210 cells and three different oxidant generating systems: (i) glucose/glucose oxidase, (ii) near-ultraviolet irradiation, and (iii) dye-mediated photodynamic action. Cells treated with the lipophilic iron donor ferric-8-hydroxyquinoline, Fe(HQ)2 (1 microM, 30 min) were found to be more sensitive to oxidative killing than nontreated controls. However, cells challenged after long-term (20-24 h) exposure to hemin (10 microM) were substantially more resistant than controls and were sensitized far less by Fe(HQ)2. Immunoblot analyses of 24-h hemin-treated cells indicated that the ferritin heavy (H) subunit was elevated 12- to 15-fold, whereas the light (L) subunit was essentially unchanged. Experiments carried out with 55Fe(HQ)2 showed that iron uptake capacity of cells was greatly enhanced after hemin treatment. More specifically, hemin-stimulated cells were found to contain approximately 9 times more immunoprecipitable ferritin iron after incubation with saturating levels (4-5 microM) of 55Fe(HQ)2 and approximately 3 times more iron per ferritin molecule compared with nonstimulated controls. The nonferritin iron content of the latter was estimated to be approximately 40 times greater than that of the former following low-level (0.5 microM) 55Fe(HQ)2 treatment. These results are consistent with the idea that induced ferritin, enriched in H-chain, sequesters redox active iron rapidly and copiously, thereby enhancing cellular resistance to oxidants.

Phase II study of roquinimex in myelodysplastic syndrome.

A Phase II clinical trial was undertaken using roquinimex (Linomide) in patients with myelodysplastic syndromes (MDS). Roquinimex is an orally active drug with immunostimulating activities demonstrated in vitro and clinically. Seventeen patients with MDS were enrolled in the study. Eligibility was limited to cytopenic patients with <20% marrow blasts. The drug was given orally twice weekly for 12 weeks with frequent monitoring of clinical, hematologic, and immunologic parameters. An increase in CD8+ and CD56+/CD3- cells was detected by 3 weeks. There was, however, no augmentation of natural killer or lymphokine-activated killer cell activity; progenitor cells were unchanged. Four patients had improvement in neutrophil counts, and two patients had improvement in platelet counts. Despite this improvement, the responses were transient or not maintained after discontinuation of therapy. One patient with RAEB, who was red cell transfusion dependent, experienced a complete remission that has persisted 14 months after completion of therapy. Adverse events developed in >25% of patients and included arthralgia, fever, headache, and myalgia. These side effects led to early withdrawal of therapy in five patients. These findings suggest that roquinimex may be of occasional benefit to patients with myelodysplastic syndromes.

Differential cytoprotection by glycine against oxidant damage to proximal tubule cells.

Tert-butyl hydroperoxide (tBHP) injured freshly isolated proximal tubules in an Fe-dependent fashion that was ameliorated by a lipophilic antioxidant, diphenyl-p-phenylenediamine (DPPD), but was only minimally affected by glycine. Menadione-induced injury was Fe-independent and was unaffected by DPPD, but was strongly blocked by glycine. Fe was highly toxic when intracellular loading was facilitated by concomitant treatment with hydroxyquinoline (HQ). This toxicity was blocked by DPPD or chelating the Fe, but not by glycine. All of the lesions were characterized by severe depletion of glutathione and other soluble thiols. Menadione induced large increases in protein associated with the Triton-insoluble cytoskeleton and decreases in protein thiol content, consistent with extensive cross linking, but did not increase thiobarbituric acid reactive substances (TBARS). tBHP and HQ + Fe had either no effect or only moderate, delayed effects on cytoskeletal proteins, but induced substantial increases of TBARS. Glycine did not the alter changes in cytoskeletal proteins, thiols, or TBARS produced by any of the agents. Protection against tBHP toxicity by deferoxamine and DPPD was accompanied by substantial suppression of TBARS accumulation. Superimposition of hypoxia during tBHP exposure reduced TBARS accumulation and restored cytoprotective activity to glycine. Thus, in contrast to its consistently strong cytoprotection against a number of other insults, glycine is only variably cytoprotective against oxidant lesions in freshly isolated proximal tubules. Extensive oxidative crosslinking of proteins is compatible with maintenance of glycine cytoprotection against lethal membrane damage. Fe-induced injury to proximal tubules associated with lipid peroxidation as manifested by TBARS formation is a relatively glycine-insensitive insult.

The immunomodulator LS-2616 (linomide) more effectively than sensitization of the donor enhances graft-versus-host reaction after small bowel transplantation.

The effects of the immunomodulating substance LS-2616 (linomide) on graft-versus-host reaction (GVHR) were investigated in a semi-syngeneic small bowel transplantation model. The entire bowel of Lewis donors were transplanted heterotopically into (Lewis x BN) F1 hybrids. Both untreated animals and animals treated with LS-2616, in a daily dose of 160 mg/kg, developed a lethal GVHR. The median survival time in untreated animals was 14.5 days while in LS-2616 treated animals it was just eight days (p < 0.01). LS-2616 in combination with cyclosporin A (CyA), 15 mg/kg given orally on days 0-20, did not seem to alter the survival times compared with CyA treatment alone; 56% of the animals treated with CyA survived for more than 100 days and after combined treatment with CyA/LS-2616 there were 50% permanent survivors. Also the effect of earlier sensitization of the donor on the course of GVHR was investigated. Hearts from BN rats were transplanted heterotopically to the neck vessels of Lewis rats. The hearts were rejected on about day six; five days later the bowels were harvested and transplanted into (Lewis x BN) F1 hybrids. The median survival time in this group was 12.5 days. Taken together our results, in combination with earlier findings, suggest that, at the level of effector mechanisms, GVHR is not an exact mirror image of rejection. Also, LS-2616 appears to be a useful tool for further studies of the mechanisms of action of GVHR.

Comparison of the mutagenicity of quinoline and all monohydroxyquinolines with a series of arene oxide, trans-dihydrodiol, diol epoxide, N-oxide and arene hydrate derivatives of quinoline in the Ames/Salmonella microsome test.

Fourteen new quinoline derivatives were synthesised and their mutagenicity compared in the Ames test using Salmonella typhimurium TA100 as indicator strain with and without (Aroclor-induced) S9 mix. None of the synthesised quinoline derivatives had to our knowledge been examined before in the Ames test. Quinoline and the monohydroxyquinolines were included as reference compounds. Three of the new derivatives, i.e., quinoline 7,8-oxide, N-methyl-quinoline 5,6-oxide and trans-quinoline-5,6,7,8-dioxide appeared to be mutagenic. Quinoline 7,8-oxide was positive only in the presence of S9 mix, the specific mutagenicity amounting to 2498 +/- 96 and 1289 +/- 120 revertants per mumole with 20 and 10% S9 in the mix, respectively. Both N-methyl-quinoline 5,6-oxide and trans-quinoline-5,6,7,8-dioxide were weakly positive, the former only in the presence of the S9 mix, and the latter irrespective of the presence of S9 mix, the specific mutagenicity amounting to 134 +/- 6 and 123 +/- 10 revertants per mumole, respectively. The mutagenic potency of quinoline 7,8-oxide was of the same order as that of quinoline itself and was distinctly lower than that of 8-hydroxyquinoline. Inconclusive results were obtained with trans-7,8-dihydroxy-7,8-dihydroquinoline, 5,6-dihydroxy-7,8-epoxy-5,6,7,8-tetrahydroquinoline and 8-hydroxyquinoline-N-oxide; if these compounds are mutagenic their mutagenic potency would be at least 20-30 times lower than that of the parent compounds. None of the other chemically synthesised quinoline derivatives showed mutagenic activity with TA100 either in the presence or in the absence of S9 mix. The results obtained with the reference compounds were in accordance with literature data.

Genotoxicity of two metabolites of benzene: phenol and hydroquinone show strong synergistic effects in vivo.

Possible interactions between hydroquinone (HQ) and phenol (PHE), 2 known benzene metabolites, in inducing micronuclei in mouse bone marrow cells were investigated. HQ and PHE administered alone gave weak and negative results, respectively, at the doses tested. However, simultaneous administration of both compounds caused a considerable increase in the induction of micronuclei as well as an increase in bone marrow toxicity. Using 3 different statistical methods, it was shown that the observed joint effect was significantly higher than additive interaction, and was close to multiplicative interaction. These findings bring further support to the hypothesis that the toxic and genotoxic effects of benzene are produced by several metabolites acting synergistically.

Selective neurotoxicity of clioquinol on the function of the posterior column nuclei.

In the gracile nucleus of clioquinol-treated rats, the presynaptic inhibition was remarkably diminished, and the excitatory synaptic transmission was less intensely inhibited by a conditioning sural nerve volley. These changes may be the pathophysiology responsible for paresthesia and/or dysesthesia in patients with subacute optico-myelo-neuropathy (SMON).

In vivo antimutagenic effect of ascorbic acid against mutagenicity of the common antiamebic drug diiodohydroxyquinoline.

We have previously shown that the common antiamebic drug diiodohydroxyquinoline (DIHQ) exhibits mutagenic activity in the in vivo micronucleus test in Swiss albino mice. Results of experiments undertaken to study the influence of ascorbic acid (vitamin C) on the mutagenicity of DIHQ in this model system showed that ascorbic acid acts as an antimutagen against DIHQ. The effective antimutagenic doses of ascorbic acid themselves do not show any genotoxic effects in this in vivo system. It will be necessary, however, to elucidate the mechanism of action of ascorbic acid as well as its effects on the therapeutic properties of DIHQ before a practical use of ascorbic acid is contemplated for this purpose.

Genotoxic potency of three quinoline compounds evaluated in vivo in mouse marrow cells.

In vitro assays of the genotoxicity of quinoline compounds have yielded varying indications of their potency, and only limited determinations have been reported following in vivo administrations. We have quantified chromosome aberrations (CA) and sister chromatid exchanges (SCE) in the marrow cells of mice that had been injected with quinoline, 8-hydroxyquinoline, or 4-nitroquinoline-1-oxide up to levels approaching lethal. Treatments with quinoline produced no consistent increase in the number of aberrations at either 17 or 36 hr after treatment and no significant increase in SCE numbers at either 23 or 42 hr. Similarly, 8-hydroxyquinoline had no measurable effect on either CA or SCE but did tend to prolong the cell cycle. 4-Nitroquinoline-1-oxide was a very potent inducer of both CA and SCE as well as an inhibitor of cell division.