Ehrlich II –2nd World Conference on Magic Bullets

Assessing the Metabolic Liabilities of Aromatic Amines using In Vitro Metabolism and Mass Spectral Techniques WANG J, LU W, MUTILIB A and TALAAT R Drug Safety & Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA Background: The purpose of this study was to establish a combination of in vitro metabolism and mass spectral techniques that could potentially be used to assess metabolic liabilities of aromatic amines in mutagenicity. An attempt was made to correlate the Ames results with formation of the deoxyguanosine adducts. Methods: 2-Aminofluorene (2AF) was used as a model compound that was bioactivated in the presence of rat (aroclor induced) liver S9 fortified with NADPH and N-acetyl CoA. Deoxy-guanosine (dG) was used as a nucleophilic trapping agent. A covalent adduct of 2AF-dG, the formation of which depended on the presence of CYP1A2 and NAT2 in the incubations, was characterized by LC/MS. By employing the technique 11 model compounds, their mutagenicity have been evaluated by Ames assay, were studied. Results: 1) The mass spectral data suggested the covalent binding of aniline nitrogen of 2AF with C8 of deoxyguanosine, consistent with the bioactivation pathways described for this compound in the literature, which suggests that the technique is valid in trapping bioactivated aromatic amine. 2) Quantitative LC/MS analysis of the deoxy-guanosine adducts formed with the 11 target compounds demonstrated that the correlations were 0.85 and 0.54 in ranking the reactivity of the model compounds by the peak area of dG of LC/MS vs by revertrants/nmol of TA100 and TA98 of Ames assay, respectively. 3) The quantitative data also demonstrated that electron withdrawing substituents and stereo hindrance substantially decreased the levels of the trapped adduct. These preliminary results suggest that this technique could potentially be applied to guide synthetic efforts in mitigating bioactivation of such compounds.	Effect of Genetic Polymorphisms in ABCG2 (BCRP) on Inhibition and Potential Drug Resistance WANG M, EMERICK RM, SAWADA GA, PAK YA, RAUB TJ, HILLGREN KM Drug Disposition, Eli Lilly and Company, Indianapolis, IN 46285 (* currently at Bristol-Myers Squibb Company, Pennington, NJ) ABCG2 (Breast cancer resistance protein, BCRP, MXR) is an adenosine triphosphate-binding cassette transporter that effluxes drugs and metabolites out of cells such as anticancer compounds. Single nucleotide polymorphisms (SNPs) for ABCG2, V12M (g34a) and Q141K (c421a), have a high frequency in the population. In addition, Q141K is reported to significantly affect the pharmacokinetics of diflomotecan. Further, an amino acid change at position 482 (R482G, a1444g) that occurs in some drug-resistant cell lines has differing substrate affinity compared to the wild type (WT) protein. However, R482G has not been identified in normal population to date. All of these SNPs are known to lead to changes in protein level or activity of the transporter. Therefore, understanding role of the polymorphisms on changes in substrate and inhibitor affinity is key to unraveling their impact on clinical outcomes To explore the transport kinetics of these SNPs against various ABCG2 substrates and inhibitors, we have created cell lines that functionally express ABCG2 WT, V12M, Q141K or R482G. The expression levels of WT and polymorphic alleles are similar based on both western blot and semi-quantitative RT-PCR. Confocal microscopy demonstrated that all the ABCG2 variants were localized to the cell membrane. A fluorescent inhibition assay was developed and was utilized to measure IC50 against 13 compounds for ABCG2 WT and variants. The results showed that SNPs can have different IC50 against certain inhibitors, indicating that SNPs can play a potential role in drug resistance, and when using a drug that is an ABCG2 substrate/inhibitor, the impact of genetic variation should be considered.
Magic bullet and magic shield: a new strategy to target Survivin in human cancers WANG Q, BEREZOV A, FREUDENBERG JA, CAI Z, ZHANG H, MURALI R, GREENE MI Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Background: Survivin belongs to the Inhibitor of Apoptosis Protein (IAP) family and plays a critical role in modulating the spindle checkpoint control during mitosis. verexpression of Survivin is commonly found in various forms of human cancers and is implicated in increasing aneuploidy, as well as resistance to genotoxic agents. Conversely, down-regulation of Survivin may sensitize cancer cells to genotoxic treatment. The goal of this study is to demonstrate that targeting Survivin can be used as a strategy to treat human malignancy. Methods: Small molecule Survivin anatgonists have been developed based on our recently demonstrated Cavity-Induced Allosteric Modification (CIAM) approach, which evaluates surface cavities and clefts as potential binding sites for pseudoallosteric molecules. The binding properties of the Survivin-targeting molecules have been evaluated with isothermal titration calorimetry (ITC). The biological activities of these molecules have been examined with cell-based assays and with in vivo animal models. Results: Our in silico screen identified small molecules that target the Survivin protein at a site important for its function. By site directed mutagenesis and isothermal titration calorimetry analysis, our data indicate the Survivin-targeting molecules bind to intended interface. In addition, the Survivin anatgonists elicit mitotic arrest and causes apoptosis in a cell cycle dependent manner. Finally, we have demonstrated that the Survivin-targeting molecules can inhibit tumor growth in the mouse xenograft model. Conclusions: 1) We have identified small molecules that target the dimerization interface of Survivin. 2) The Survivin-targeting molecules arrest cells in the early stage of mitosis. 3) Disruption of Survivin functions by the small molecules causes apoptosis in a cell cycle dependent manner. 4) The Survivin-targeting molecules inhibit proliferation of cancer cells in vitro and tumor growth in vivo.	Functional characterization of outer membrane proteome in response to antibiotic resistance SAN-YING WANG^2, XIANG-MINLIN^1, CHANG-XIN XU^2,HUI LI^1, XUAN-XIAN PENG^1 ^1Center for Proteomics, State key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, University City, Guangzhou, 510006, China; ^2School of Life Sciences, Xiamen University, Xiamen, 361005, China Background: The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. To understand the mechanisms of the resistance is extremely important to the control of these bacteria. A line of evidence has indicated that four mechanisms are probably involved in the antibiotic resistance. They are modification or hydrolysis of enzymes, modification of targets, activation of efflux pump systems, and reduction of outer membrane (OM) permeability. The permeability and the pump systems are mainly controlled by OM proteins in Gram-negative bacteria. The decrease of OM permeability prevents the influx of antibiotics, and the activation of the efflux pump systems pumps the noxious small molecule substances out of cells. However, little is known about OM proteome and the two-component regulating system of these proteins involved in the NA resistance of E. coli. Recently, we have systematically investigated altered OM proteins of E. coli in response to ampicilin, kanamycin, tetracycline, nalidixic acid, streptomycin resistance. Methods: 1) A differential OM sub-proteome in response to these antibiotics is achieved with the use of 2-DE proteomics and Western blotting methods; 2) Key OM proteins in response to these antibiotics are determined by analysis of functional characterization of the altered OM proteins using their genetic modified strains; 3) Two-component system regulation and a network of the altered OM proteins are investigated using Western blotting and/or bacterial survival capability analyses. Results: Several differential expressed proteins are determined in each of the antibiotics tested: Up-regulated FimD, Tsx, OmpW, OmpC and TolC, and down-regulated NlpB for ampiciline; up-regulated FimD, Tsx, OmpW, OmpC and TolC, and down-regulated NipB and LamB for tetracycline; up-regulated TolC,OmpT and LamB, and down-regulated FadL, OmpW and Dps for streptomycin (SM). up-regulated TolC, OmpT, OmpC and OmpW,and down-regulated FadL for nalidixic acid (NA). Up-regulation and down-regulation of OmpC were observed respectively, in NA-R and ∆ompF, and ∆envZ and ∆ompR cultured with or without NA. Meanwhile, OmpC level in ∆envZ and ∆ompR was significantly lower in cultures with 1/2 MIC NA than the cultures without NA. the interaction network among the altered OM proteins was characterized based on effect in absence of each of the six altered proteins on the other five in SM-resistant genetic modified strains with gene deletion. Of the six altered proteins, TolC mainly regulates other proteins, and OmpT, LamB and Dps may be middle factors at a center of the network. Conclusions: 1) Alteration of OM proteins in resposne to antibiotics is related to chemical characteristics of the antibiotics used, but the OM proteins shared by all antibiotic-resistant strains exist., suggesting universal and specific antibiotic-resistant mechanisms for OM proteins against antibiotics. 2) EnvZ/OmpR two-component system plays an important role of in the regulation of NA resistance. 3) the present study demonstrates that a regulating network exists among the altered OM proteins. In the network, TolC mainly regulates other proteins, and OmpT, LamB and Dps may be middle factors at a center of the network. These results may suggest that a biological feedback network exists in E. coli with SM resistance. This work was sponsored by grants from “973” project (2006AA09Z432), NSFC project (30530610), Guangzhou Key Project (2006Z3-E0251), the Foundation of Guangdong for Natural Sciences (7117645).
New multifunctional pharmacophores and biocompatible nanocomposites for targeting drug delivery and cancer diagnosis Wang X State Key Lab of Bioelectronics, Southeast University, Nanjing 210096, China It is well known that the failure of chemotherapy to the malignant tumor is usually induced by multidrug resistanc (MDR),and the development of effective anti-MDR agents for efficient drug delivery plays an important role in the tumor therapy. Multidrug resistance (MDR) is the ability of disease-causing organisms to withstand a wide-variety of structurally and functionally distinct drugs or chemicals. MDR involves the expression of membrane proteins which mediate the active extrusion of drugs from the cell and it is a major limiting factor to the chemotherapy of cancer. Because of the importance of MDR in clinical oncology, an extensive search for the new MDR reversal agents is still an unceasing challenge. Many compounds known to have other pharmacological sites of action initially were used to reverse MDR in cancer cells grown in culture and several underwent pilot clinical trials. A large number of small molecules capable of modulating P-gp mediated MDR have been described in the literature. Among the potentially pharmaceutical functional molecules, the remarkable chemical and thermal stability and hydrophobic character allow the carborane to be used as a promising pharmacophore in biologically active moleculesfor anti-drug resistance and anti-cancer application. Our recent studies indicate that when co-administered with a cytotoxic agent, these nontoxic modulators enhance net accumulation of relevant cytotoxic drugs within the tumor cells. In recent years, nanomaterials, which show unique physical and chemical properties, have attracted much attention in various fields and have been widely applied in biological and biomedical engineering. In this report we will pre-sent some of our recent research progress in combining the design of multifunctional biocompatible promising pharmacophore and nanomaterials with targeted and efficient drug delivery system. These studies demonstrate that with the combination of multifunctional pharmacophore or nano-interface, we can realize the synergistic effect on the efficient cytotoxicity suppression in drug sensitive and drug resistance cancer cells and enhance intracellular drug accumulation of anticancer drug into target cancer cells. Meanwhile, our observations also indicate that due to the convenient surface functionalization, the biocompatible nanomaterials can play versatile roles for fabrication of high sensitive probes and biosensors in cancer diagnosis, which show great promise in the design of point-of-care-testing devices in the future. Acknowledgements: This work has been supported by National Natural Science Foundation of China (90713023, 20675014) and Ministry of Science & Technology of China (2007AA022007).	Temozolomide Acid Hexyl Ester (TMZA-HE) as a Topical Bullet for Skin and Cervical Cancers WANG YF^1, LI DX^2, SUPPASANSATORN P^1, CONWAY BR^1, WANG GC^2 ^1Univ. ASTON, BIRMINGHAM, UNITED KINGDOM; ^2TASLY GROUP, TIANJIN, CHINA Background: Temozolomide (TMZ) has been considered as the first choice for treatment of glioma; however, its phase III trials failed to show a significant improvement over dacarbazine in treatment of melanoma. This failure may be due to its virtual insolubility in organic and aqueous media, resulting in inadequate drug distribution into skin. A group of temozolomide acid (TMZA) esters were designed in order to 1) investigate if the esters are prodrugs with the ability to penetrate through skin; 2) verify bioactivities of the ester prodrugs against cancer cell lines and in an animal model; 3) explore topically applicable formulations of the ester prodrug for skin and other cancers. Methods: TMZA methyl to octyl esters were synthesized and fully characterized. NMR was applied in monitoring enzymatic hydrolysis of the ester in vitro. The skin penetration potency of the esters was assessed with silicone membrane, rat skin and human skin. Bioactivities of TMZ, TMZA and TMZA esters were measured against cancer cell lines. Developing a formulation for TMZA-HE is challenging because its sensitivity to a nucleophilic attack restricts the use of many popular excipients. Bioactivities of a TMZA-HE microemulsion were assessed in BALB/c nude mice, inoculated with MV3 human melanoma, A431 human skin Basal cell carcinoma, HCT116 human colon carcinoma and HeLa4 human cervical cancer. Results: In vitro the TMZA ester was enzymatically hydrolyzed into TMZA rapidly under incubation condition. The ester prodrugs showed superior ability for skin penetration and readily converted into TMZA within the skin. TMZA-HE demonstrated an adequate balance of skin permeation and retention. IC50 of TMZ, TMZA and TMZA-HE are equal for cancer cell lines. A microemulsion formulation of TMZA-HE with a satisfactory shelf-life was developed using a novel excipient. Growth of MV3 human melanoma and HeLa4 human cervical cancer in BALB/c nude mice was significantly inhibited by the formulated TMZA-HE product. Conclusions: 1) TMZA esters are prodrugs of TMZA (TMZ) with skin penetrating potency. 2) The adequate balance of skin permeation and retention of TMZA-HE warranted promising further developments. 3) The topically applicable TMZA-HE formulation showed promise for treatment of melanoma and cervical cancer.
From Bark to Bullet: A Personal History of the Discovery and Development of Taxol WANI MC Research Triangle Institute, Research Triangle Park, NC 27709, USA Taxol is a secondary metabolite obtained from the wood bark of Taxus brevifolia, found in the Pacific Northwest coastal region of the United States. It was isolated by the bioassay-guided fractionation of the crude plant material. The structure of Taxol was established by single crystal x ray analysis. Taxol has a unique mechanism of antitumor activity. It inhibits cancer cell growth via stabilization of microtubules. Currently, Taxol is approved for clinical use in the USA by the FDA for the treatment of refractory ovarian, breast, and non-small cell lung cancers and Kaposi’s Sarcoma. This presentation will describe the 30-year efforts which transformed this compound from an interesting plant secondary metabolite to a life saving chemotherapeutic agent.	Increased PGC-1 expression exhibits protective effects against age-related neurodegenerative and metabolic disease processes WARD WF^1^,3, LIANG H^2.3, RICHARDSON A^2,3,4, VAN REMMEN H^2,3,4 Departments of Physiology^1 and Cellular & Structural Biology^2. The Barshop Institute for Longevity and Aging Studies^3, The University of Texas Health Science Center at San Antonio and the South Texas Veterans Health Center^4. San Antonio, TX, USA, 78229 Background: It is generally accepted that mitochondrial function declines with age, as evidenced by the increased level of cellular oxidative stress due to increased mitochondrial production of reactive oxygen species (ROS). It is also widely accepted that age-related alterations of mitochondrial function constitute a major component of the aging process. The transcription co-activator peroxisome proliferator-activated receptor  coactivator-1 (PGC-1) is known to be a powerful stimulator of mitochondrial biogenesis. We were therefore interested in determining if stimulation of mitochondrial biogenesis would lead to an improvement in mitochondrial function and thereby modulate the aging process. Methods and Results: To this end a unique transgenic animal model has been developed in which the human PGC-1 gene has been inserted into the C57Bl6/J mouse genome. Ubiquitous 2 to 3 fold increases in the expression of human PGC-1 mRNA and protein was obtained. These changes were accompanied by 2 to 4 fold increases in mRNA and protein of transcription factors and other proteins associated with mitochondrial biogenesis (mTFA, COX II, etc.). Unexpectedly, we found that the transgenic animals exhibited an improvement in the glucose tolerance response. Use of the euglycemic-hyperinsulinemic clamp technique demonstrated that this is due to increased skeletal muscle insulin sensitivity. Another unexpected finding was that cross-breeding the PGC-1 animals with the G93A mouse model of amylotrophic lateral sclerosis (ALS) exhibited a rescuing effect as evidenced by the extension of life span and marked improvement in RotoRod performance. This finding is consistent with the reports that increased expression of PGC-1 exhibits a rescuing effect in a murine model of Huntington’s disease. Conclusions: Alterations of PGC-1expression, or activity, may therefore provide a mechanism for altering the progress of a metabolic disease such as Type II diabetes mellitus as well as in neurodegenerative diseases such as ALS and Huntington’s disease. (Supported by NIH grant AG028294)
“The legacy of Paul Ehrlich To antimalarial chemotherapy” WARHURST D London School of Hygiene and Tropical Medicine Review: Ehrlich's work forms an essential mechanistic background for current views of antimalarial chemotherapy. This is exemplified here by the 4-aminoquinoline chloroquine [CQ], and the arylaminoalcohol quinine [(-)QN] and related compounds. These are hydropathic weak bases which become concentrated in the acid content of the lysosome (digestive vacuole:DV) of blood stage malaria parasites by protonation to CQ2H+ or QNH+ (haptophore effect). These magic bullets bind a haematin target, released during digestion of host haemoglobin, and they prevent detoxication to malaria pigment. (toxophile effect). In CQ-resistant (CQ-R) Plasmodium falciparum, since target haematin cannot change, access of drug is diminished. In CQ-R, PfCRT protein in the DV membrane differs from CQ-S by 2 residue changes, K76T in transmembrane helix 1 (TMH-1), which removes a positive charge in a putative CQ2H+ efflux channel, and A220S in TMH-6. The effect of K76T in TMH-1 is increased in SE Asian and related African isolates by introduction of a negative charge (N to E) on 75, which with 72 and 76 lines the channel, and M74I, hydropathy increase on the membrane side. TMH-1 in many Asian, Oceanian and S. American CQ-R isolates shows only C72S in addition to K76T, giving increased polarity, reducing the resistance-reversing effect of verapamil and enhancing resistance to desethylamodiaquine (D-AQ). CQ-S P. falciparum is more sensitive to (-)QN diastereomer (+) quinidine than to (-)QN. In parasites with experimental replacements of PfCRT K76, this differential effect is maintained: (T), (N), or reversed: (I ), and this also applies to in vitro QN-verapamil potentiation seen in CQ-R, indicating that the QN magic bullet has a stereochemical interaction with a second target, the PfCRT channel. The pH-modulated lipid distribution coefficient LogD (from pKa values and LogP) has been important in understanding how more hydropathic 4-aminoquinolines such as AQ and D-AQ are able to retain activity in CQ-R. They show an increase in LogD giving higher concentrations in lipid and allowing binding to the more hydropathic lining of the CQ-R efflux channel. The bis 4-aminoquinoline piperaquine, effective in CQ-R, achieves a LogD (pH4.8) of 0.97 ([medium]: [vacuolar lipid] = 1:973492) while retaining a high concentration in lysosomal water ([medium]: [vacuolar water] = 1:104378) because the number of haptophore-like protonatable amino groups on the molecule is increased from 2 to 4.	Vascular Directed Tumour Therapy by a Novel Pyrazole that Inhibits the Ras-Net (Elk-3) Pathway and Affects Microtubules Wasylyk B^1^,, Wasylyk C^1, Zheng H^1, Castell C^2, Debussche L^2, Multon M-C^2 ^1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch, France; ^2Sanofi-Aventis, Vitry-sur-seine, France Background: The Growth Factor-Ras-Erk signalling pathway is frequently perturbed in human cancers, and is a target for the development of tumour therapies. The Ras-ERK signalling pathway targets various cellular effectors, including the Net (Elk-3/SAP-2/Erp) transcription factor, which is phosphorylated and activated by ERK, and is involved in wound healing, angiogenesis and tumour growth. Methods: A cell based screen for small molecule inhibitors of Ras activation of Net transcriptional activity was used for a high throughput screen for small molecule inhibitors of the pathways. Selected molecules were tested for their activity, specificity and mechanisms of action using in-vitro and in-vivo molecular and cellular based approaches. Results: We identified a novel pyrazole XRP44X. XRP44X inhibits FGF-2 induced Net phosphorylation by the Ras-ERK signalling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerises microtubules, stimulates cell membrane blebbing and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, that produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signalling. This differs from other classes of agents that target microtubules which have either little effect (Vincristine), or no effect (Docetaxel and Nocodazole), on the Ras-Net pathway.