Cell biology,chemogenomics and chemoproteomics – application to drug discovery

Cell biology has added immensely to the understanding of basic biologic concepts. However, scientists need to use cell biology more in the proteomic–genomic revolution. The authors have developed two novel techniques: transitional structural chemogenomics (TSCg) and transitional structural chemoproteomics (TSCp). TSCg is used to regulate gene expression by using ultrasensitive small-molecule drugs that target nucleic acids. By using chemicals to target transitional changes in the helical conformations of single-stranded (ss) and double-stranded (ds) DNA (e.g., B- to Z-DNA) and RNA (e.g., A- to Z-RNA), gene expression can be regulated (i.e., turning genes ‘on/off’ and variably controlling them). Alternative types of ds- and ssDNA and RNA (e.g., cruciform DNA) and other multistranded nucleic acids (e.g., triplex-DNA) are also targeted by this method. The authors’ second technique, TSCp, targets a protein before, during or after post-translational modifications, which alters the protein’s structure and function. These novel methods represent the next step in the evolution of chemical genomics and chemical proteomics. In addition, a novel multi-stranded (alternative) DNA, RNA and plasmid microarray has been developed that allows for the immobilization of intact, non-denatured dsDNA, alternative (i.e., exotic) and other multiple-stranded nucleic acids. This represents the next generation of nucleic acid microarrays, which will aid in the characterization of nucleic acids, studying the ageing process and improving the drug discovery process. The authors discuss how cell biology can be used to enhance genomics and proteomics. Cell biology will play a greater role during the postgenomic age and will help to enhance the omics/omes and drug discovery. It is the authors’ hope that these novel approaches can be used together with cellular biologic techniques to make major contributions towards understanding and manipulating different genomes.     

Patent Evaluation: Biotinylated nucleotide analogues as inhibitors of TNF hyperproduction

Novelty: Nucleotide analogues having a photoreactive group and a biotinyl group which covalently attach to nucleotide dependent protein binding sites are provided. Protein structurefunction studies are significantly facilitated by receptor binding site directed labelling. The invention has particular application to the characterisation of the active sites of nucleotide dependent enzymes. The use of the method for detection and removal of undesired DNA or RNA from cells (e.g. AIDS viral RNA) is briefly described.

Biology: Analogue modified proteins are detected by avidin-linked peroxidase or alkaline phosphatase methods. The modified protein can be purified by strepavidin-linked or avidinlinked affinity chromatography. After elution of unmodified protein, modified proteins are released and collected from the avidin column by raising the pH to hydrolyze the ester linkage by which the biotin is bound to the ribose hydroxyl group.

Chemistry: The biotin radical is attached to the ribose moiety of the nucleotide through an ester linkage. Upon photo irradiation the biotinylated photoaffinity analogues covalently bond to the nucleotide binding site.     

Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics

Antisense technology constitutes development of sequence-specific DNA or RNA analogs that can block the activity of selected single-stranded genetic sequences and offer the potential of high specificity lacking in many current drug treatments. The sequencing of the human genome has greatly increased the potential of this approach. Antisense oligonucleotides, the most commonly used antisense approach, are unmodified or chemically modified single stranded RNA or DNA molecules specifically designed to hybridize to corresponding RNA by Watson-Crick binding. Phosphorodiamidate Morpholino oligomers (PMO) are a novel class of non-ionic antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. PMOs have shown excellent efficiency and safety profile via various routes of administration in multiple animal and human studies. This review will summarize the preclinical studies with PMOs on the road to their development as therapeutic agents with particular emphasis on in vivo biodistribution and pharmacokinetics.     

Broad-spectrum antiviral activity of adenosine analogues

In recent years certain aliphatic and carbocyclic adenosine analogues have been developed which are of potential clinical importance as antiviral agents. This includes (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA] and carbocyclic 3-deazaadenosine (C-c3Ado). (S)-DHPA and C-c3Ado are remarkably similar in their antiviral spectrum in that they are particularly active against (-) RNA viruses such as measles, para-influenza, respiratory syncytial virus, rabies virus, vesicular stomatitis virus and (+/-)RNA viruses such as reo- and rotavirus, whereas (+)RNA viruses such as polio, coxsackie and (+/-)DNA viruses such as herpes simplex are only minimally affected or not inhibited at all. In contrast with (S)-DHPA and C-c3Ado which are quite selective in their antiviral action, other adenosine analogues, i.e., 3-deazaadenosine and 7-deazaadenosine (tubercidin), exhibit little, if any, selectivity as antiviral agents. Also, tubercidin has a broader activity spectrum, encompassing (+)RNA viruses as well as herpes simplex in addition to the (-)RNA viruses. Considering the high antiviral potency of tubercidin, attempts have been undertaken to increase its selectivity, i.e., by chemical substitutions at C-5 of the pyrrolo(2,3-d)pyrimidine ring. These attempts have been partially successful.     

Drug bioactivation, covalent binding to target proteins and toxicity relevance

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targets of reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.     

Abasic site recognition in DNA as a new strategy to potentiate the action of anticancer alkylating drugs?

Inhibition of abasic site repair in the cell seems an attractive strategy to potentiate the action of antitumor DNA alkylating drugs. Molecules that bind specifically and strongly to the abasic site are possible candidates to achieve such inhibition. We explored this strategy by preparing molecule 4 that incorporates (1) an aminoacridine intercalator for DNA binding, (2) an adenine moiety for abasic site recognition, and (3) a linker containing two guanidinium functions to increase binding to DNA without inducing cleavage at the base-sensitive abasic site. Compound 4 was compared to analogues containing secondary amines, i.e., 1. We report on synthesis of the new heterodimer 4. We show by physicochemical studies-including determination of association constants with calf-thymus DNA, T(m) measurements, and high-field NMR examination of the complexes formed with abasic DNA duplexes-that 4 binds specifically and more strongly to the abasic site than the analogues. Compound 4 does not cleave abasic plasmid DNA. Compound 4 shows apparent synergy with the anticancer bischloroethylnitrosourea (BCNU) in L1210 and A549 cell lines in vitro. It potentiates BCNU in the in vivo tests. The results favor the pertinence of the strategy.     

Assessing activity and toxicity of drugs in silico based on DNA structure

One of the biggest challenges in pharmaceutical development is finding drug candidates with a desired activity or efficacy balanced with low toxicity or side-effects. Despite the enormous effort and cost required to get drugs to market, numerous drugs have been abandoned due to unanticipated, untoward effects. Clearly, technologies are needed that can identify safe and effective pharmaceutical candidates early in the drug pipeline. Our laboratory has developed a computer modeling technology that can be used to screen small-molecule ligands for certain desired activities as well as certain toxicities. The technology utilizes modeling of the intercalation of molecules into DNA to create efficacy and toxicity search engines and is grounded by two fundamental observations. First, intercalation has been shown to be integral in the action of drugs that act in concert with nuclear enzymes, e.g., topoisomerases. Second, evidence is mounting that intercalation facilitated by nuclear receptors bound to natural ligands is a critical part of their genomic mode of action. To date, two classes of search engines have been created, i.e., those that can be used to identify: (1) efficacious molecules, e.g., antibiotics, estrogens, androgens, glucocorticoids, thyroid drugs, antidepressants, antihistamines, and sedatives, and (2) toxic molecules, e.g., certain carcinogens and genotoxins. Here, we describe the creation of two prototype search engines (the estrogen efficacy and arene oxide genotoxicity search engines) and illustrative results of searches of three-dimensional databases. Of particular interest is the specificity of the search engines and their capacity to identify widely different, and in some cases obscure, structures having the same activities. Taken as a whole, future drug discovery research is likely to focus on methods to assess DNA intercalation as a salient feature of selecting safe and effective drug candidates.     

Semiautomated fluorometric analysis of nucleic acids in tissue homogenates

This report describes a technique that was developed to provide an efficient and accurate estimation of RNA:DNA ratios. These ratios have been used as an instantaneous measure of recent growth of individual aquatic organisms where morphometrics are not appropriate (e.g., field-collected species) or insufficiently sensitive (e.g., small life stages or species). In this semiautomated, sensitive method, ethidium bromide fluorescence was used to quantitate total nucleic acids in crude homogenates. Individual concentrations of RNA and DNA were determined by differences in fluorescence before and after elimination of RNA by digestion with RNase. Efficiency of the procedure was enhanced using a computer-driven multiwell plate scanning system (CYTOFLUOR, Millipore Corporation

1 Reference to trade names does not imply endorsement.

) to measure fluorescence at timed intervals and perform data manipulations. Routinely, detection limits of 0.1 μg DNA and 0.4 μg RNA were achieved, allowing the analysis of small, individual organisms. Fluorescence results of split samples were comparable with those obtained using a standard spectro-photometric method to quantitate nucleic acids. Coefficients of variation for replicate samples within an assay (1.6%) and for samples within replicate assays (5.6%) indicated good test reproducibility. Quantitative recoveries of nucleic acid standards spiked into tissue homogenates were generally high, averaging 91.0% for DNA and 119.0% for RNA. Factors affecting the fluorescence of ethidium bromide stained nucleic acids—e.g., nucleic acid source, crude homogenate components, and buffer composition—are discussed relative to assay performance. This method provides a rapid and reliable assessment of individual growth, an important sublethal toxicological end point, that is suitable for both laboratory and field studies. © 1994 by John Wiley & Sons, Inc..     

Dual topoisomerase I/II inhibitors in cancer therapy

While the majority of topoisomerase (topo) inhibitors show selectivity against either topo I or topo II, a small class of compounds can act against both enzymes. These can be divided into three classes. The first and largest class comprise drugs that bind to DNA by intercalation and include the clinically-evaluated acridine DACA, the benzopyridoindole intoplicine, the indenoquinolinone TAS-103, the benzophenazine XR11576, and the pyrazoloacridine NSC 366140. The second category comprises hybrid molecules, prepared by physically linking separate inhibitors of topo I and topo II, or by linking pure topo inhibitors to other DNA-interactive carriers. While several derivatives (e.g., camptothecin-epipodophyllotoxin and ellipticine-distamycin hybrids) have been prepared, there have been no detailed studies. The third category are less well defined as a structural class, but apparently recognize structural motifs that are present in both topo I and II enzymes. These include a series of benzoisoquinolinium quaternary salts such as NK 109, and more interestingly modified versions of classical topo I or topo II inhibitors; e.g., the modified camptothecin BN 80927 and the modified epipodophyllotoxin tafluposide (F-11782). There is as yet no detailed understanding of the factors that result in selective or dual inhibition, but structure-activity studies in several classes show that structural changes can influence topo I/II selectivity. DNA intercalation mode also appears to play a part. The basis for the high antitumor activity of some topo inhibitors is not yet understood but may depend on the complex pattern of activities that include both inhibition and poisoning of the two enzymes.     

Synthesis and biological properties of actinomycin D chromophoric analogues substituted at carbon 7 with aziridine and cyclopropyl functions

The growing importance of functionalized tricyclic rings, e.g., cyclopropyl and aziridine, in numerous organic biomolecules led us to develop syntheses of novel actinomycin D (AMD) analogues substituted with aziridine and cyclopropyl functions. Reaction of 7-hydroxyactinomycin D with 1-aziridineethyl iodide and bromomethylcycloporopane afforded the desired 7-[2-(1-aziridinyl)ethoxy] and cyclopropylmethoxy analogues, respectively. Calf thymus DNA binding of these analogues was comparable to that of AMD as examined by UV-vis difference spectral measurements, CD techniques, and relaxation of supercoiled closed circular SV40 DNA, indicating an intercalative mode of binding to the DNA duplex. Thermal denaturation of DNA experiments employing higher temperatures than room temperature exhibit a thermal lability of the DNA analogue complexes, suggestive of a probable covalent bond formation with DNA bases. The analogues were found to be 1/4-1/40 as cytotoxic to human lymphoblastic CCRF-CEM leukemia and B16 melanoma cells in vitro as AMD, with ID50 values in the nanomolar concentration range.     

Potent renin inhibitory peptides containing hydrophilic end groups

A previously reported renin inhibitor, Boc-Pro-Phe-N(Me)His-Leu psi [CHOHCH2]Val-Ile-Amp (U-71038), was altered by the incorporation of polar, hydrophilic moieties at either end, e.g., tris(hydroxymethyl)aminomethane (THAM) or glucosamine urea groups at the N-terminus, and the THAM amide or aminomethylpyridine N-oxide at the C-terminus. These modified analogues, with dramatically improved water solubility, all retained the potent renin inhibitory activity of U-71038 in vitro. The fact that good activity was maintained in these new analogues, which possess hydrophilicity and steric bulk considerably different from the parent compound, suggests that neither end of these molecules is critical for recognition and binding of the inhibitors by renin. These modified analogues were evaluated in a rat model, and several exhibited hypotensive activity after both oral and iv administration which was greater in magnitude and longer in duration than that caused by equimolar doses of U-71038. Furthermore, unlike U-71038, the oral activity of these analogues was not dependent upon administration in a citric acid vehicle.     

The impact of nucleic acid secondary structure on PNA hybridization

Hybridization of oligonucleotides and their analogues to complementary DNA or RNA sequences is complicated by the presence of secondary and tertiary structure in the target. In particular, folding of the target nucleic acid imposes substantial thermodynamic penalties to hybridization. Slower kinetics for hybridization can also be observed, relative to an unstructured target. The development of high affinity oligonucleotide analogues such as peptide nucleic acid (PNA) can compensate for the thermodynamic and kinetic barriers to hybridization. Examples of structured targets successfully hybridized by PNA oligomers include DNA duplexes, DNA hairpins, DNA quadruplexes and an RNA hairpin embedded within a mRNA.     

Peptide-nucleic acids (PNAs): a tool for the development of gene expression modifiers

Peptide nucleic acids (PNAs) represent nucleic acid analogues with unique biochemical properties and of great interest for the development of therapeutic agents. The firstly designed and tested PNAs are molecules in which the sugar-phosphate backbone of DNA was replaced with a pseudopeptide chain constituted by N-(2-aminoethyl) glycine monomers. Nucleobases can be linked to this backbone through a carboxymethyl moiety, which allows to maintain a two atom spacer between the backbone and the bases. Since the first reports on PNAs based on N-(2-aminoethyl) glycine backbone, other PNA analogues have been synthesized, with the main purpose of improve biological activities as well as stability and efficient delivery to target cells. Of great interest are chiral PNAs, PNA analogues bearing phosphate groups (PHONA), PNA-DNA and PNA-peptide chimeras, PNA linked to non-peptide vectors. PNAs hybridize to DNA and RNA with high efficiency following the Watson-Crick hybridization rules, forming highly stable PNA/DNA and PNA/RNA duplexes. In addition, homopyrimidine PNAs, as well as PNAs containing a high pyrimidine:purine ratio, are able to bind to DNA or RNA forming highly stable (PNA)(2)-DNA triple helices. Accordingly, therapeutic PNA and PNA analogues could act as antigéne as well as antisense molecules. In addition, recent studies provide evidences for the possible use of PNA-based therapeutic molecules as artificial promoters, as decoy or ribozyme facilitator. Among the therapeutic applications of PNA-based molecules, the most pomising include anti-cancer and anti-viral experimental strategies, but activity of PNAs against bacteria and medically important parasitic organisms have been also reported.     

Strand-specific silencing of a picornavirus by RNA interference: evidence for the superiority of plus-strand specific siRNAs

RNA interference triggered by small interfering RNAs (siRNAs) can be used to effectively contain viral spread. Here, we report on the mechanism of action of siRNAs targeting the medically important coxsackievirus B3 (CVB-3) as a typical representative of viruses with a non-segmented RNA genome in positive-strand orientation. Antiviral siRNAs can be designed to target the genomic (+)-strand, the (-)-strand that occurs as a replication intermediate, or both. In the present study, two complementary and systematic approaches are presented providing direct evidence that silencing of the viral (+)-strand is the key to inhibit CVB-3: first, we used rational siRNA design to direct silencing activity specifically against either of the two viral strands. As a second approach, we employed siRNA containing modified nucleotides to render them specific for one of the virus RNAs. Experiments with infectious coxsackievirus revealed that the inhibitory efficiency correlates exclusively with the activity of the siRNAs directed against the viral (+)-strand. Our finding that only (+)-strand specific siRNAs exert significant antiviral potency may hold true for other RNA viruses with (+)-stranded genomes as well and may therefore be helpful in the development of efficient strategies to inhibit virus propagation.     

抗菌肽buforin Ⅱ衍生物抑制细菌核酸合成的机制研究

目的探究抗菌肽buforin Ⅱ的衍生物buforin Ⅱ-A(BF2-A)和buforin Ⅱ-B(BF2-B)对细菌的胞内抑菌作用机制。方法体外用原子力显微镜观察抗菌肽与基因组DNA的结合情况,荧光光谱分析肽与基因组DNA的结合方式。体内用透射电镜观察抗菌肽作用后金黄色葡萄球菌细胞膜超微结构的变化,流式细胞仪分析肽对金黄色葡萄球菌细胞周期的影响。最后通过凝胶阻滞实验推测肽与金黄色葡萄球菌DNA合成相关基因的结合引起了细胞周期的改变。结果肽与基因组DNA发生了结合,与溴化乙锭(EB)竞争性嵌入基因组DNA;BF2-A/BF2-B与金黄色葡萄球菌作用后几乎在不破坏细胞膜的情况下渗透进入胞内,与DNA合成相关基因发生了结合,特异性阻滞细胞周期中的DNA合成阶段;肽与DNA合成相关基因发生了结合。另外,所有的实验结果显示了BF2-B穿透细胞、与DNA结合的能力及对细胞周期的影响强于BF2-A。结论 BF2-A/BF2-B与DNA穿过金黄色葡萄球菌细胞膜后在细胞内通过与DNA结合,特异性的将细胞阻滞在了细胞周期的DNA合成期而发挥了抑菌作用,而且BF2-B的上述作用强于BF2-A。     

The optimal conditions for the estimation of DNA methylation levels using high throughput microarray derived DNA immunoprecipitation (MeDIP)-enrichment in human bloods

Genomic-wide mapping of epigenetic changes is essential for understanding the mechanisms involved in chemical-induced gene regulation. The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is a recently devised method for the extraction of methylated DNA from a sample of interest and used to determine the distribution of DNA-methylation within functional regions (e.g., promoters) or in the entire genome robustly and cost-efficiently. This approach is based on the enrichment of methylated DNA with an antibody that specifically binds to 5-methyl-cytosine (5mC) and can be combined with PCR and microarray. Here we summarize the experimental procedure of MeDIP followed by high throughput microarray and provides a comprehensive summary of quality control strategies.     

Recent developments in the use of vitamin D analogues

The non-classical effects of 1alpha,25-dihydroxyvitamin D(3) (1alpha, 25(OH)(2)D(3)) create possible therapeutic applications for immune modulation (e.g., autoimmune diseases and graft rejection), inhibition of cell proliferation (e.g., psoriasis, cancer) and induction of cell differentiation (e.g., cancer). The major drawback related to the use of 1alpha,25(OH)(2)D(3) is its calcaemic effect, which prevents the application of pharmacological concentrations. Intensive research has led to the development of analogues of 1(2)D(3) characterised by a clear dissociation of the antiproliferative and prodifferentiating capacity from the calcaemic effects. Due to this dissociation, these analogues can be used not only for the treatment of bone disorders but also for non-classical applications. In the present review, a summary is given on the use of the 1alpha,25(OH)(2)D(3) analogues for the treatment of cancer, skin and immune disorders and for the prevention of graft rejection. Moreover a brief overview is given on the use of analogues for secondary hyperparathyroidism.     

Identification of HIV-1 inhibitors targeting the nucleocapsid protein

The HIV-1 nucleocapsid (NC) is a RNA/DNA binding protein encoded within the Gag polyprotein, which is critical for the selection and chaperoning of viral genomic RNA during virion assembly. RNA/DNA binding occurs through a highly conserved zinc-knuckle motif present in NC. Given the necessity of NC-viral RNA/DNA interaction for viral replication, identification of compounds that disrupt the NC-RNA/DNA interaction may have value as an antiviral strategy. To identify small molecules that disrupt NC-viral RNA/DNA binding, a high-throughput fluorescence polarization assay was developed and a library of 14,400 diverse, druglike compounds was screened. Compounds that disrupted NC binding to a fluorescence-labeled DNA tracer were next evaluated by differential scanning fluorimetry to identify compounds that must bind to NC or Gag to impart their effects. Two compounds were identified that inhibited NC-DNA interaction, specifically bound NC with nanomolar affinity, and showed modest anti-HIV-1 activity in ex vivo cell assays.