Locked nucleic acid -anti- let-7a induces apoptosis and necrosis in macrophages infected with Leishmania major.

BACKGROUND: Protozoan parasites of the genus Leishmania are etiologic agents which are intracellular pathogens of vertebrates and replicate inside infected macrophages. Leishmania have developed complex strategies to reverse host immune responses in favor of it. One of the major species causing cutaneous involvements is Leishmania major. MicroRNAs (miRNA) are non-coding small RNAs encoding 22-nucleotide (nt) long RNAs. miRNAs affect diverse biological processes, including cell cycle, proliferation, differentiation, growth and development, metabolism, aging, apoptosis, gene expression and immune regulation. This study aimed at evaluating apoptosis and necrosis after transfection locked nucleic acid (LNA) inhibitor of let-7a in the human macrophages miRNAs upon infectionwith L. major. MATERIALS AND METHODS: Inhibition of let-7a in macrophages was derived originally from the human monocytes (MDM), using locked nucleic acid (LNA) antagomir. The total cellular RNA was extracted 24 and 48 h post transfection. The levels o Let-7a expression was measured by qPCR Real Time using specific primer. Annexin-V/Propidium Iodide staining method was performed to detect apoptosis and necrosis in the MDM cells. Data were analyzed using the Kruskal-Wallis and Mann-Whitney tests. RESULTS: Let-7a inhibition increased the MDM cells apoptosis and necrosis using flow cytometry method. CONCLUSIONS: The results suggested that inhibition of let-7a could be a new approach in treatment of leishmaniasis.

Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry.

Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.

Using distal-site mutations and allosteric inhibition to tune, extend, and narrow the useful dynamic range of aptamer-based sensors.

Here we demonstrate multiple, complementary approaches by which to tune, extend, or narrow the dynamic range of aptamer-based sensors. Specifically, we employ both distal-site mutations and allosteric control to tune the affinity and dynamic range of a fluorescent aptamer beacon. We show that allosteric control, achieved by using a set of easily designed oligonucleotide inhibitors that competes against the folding of the aptamer, allows rational fine-tuning of the affinity of our model aptamer across 3 orders of magnitude of target concentration with greater precision than that achieved using mutational approaches. Using these methods, we generate sets of aptamers varying significantly in target affinity and then combine them to recreate several of the mechanisms employed by nature to narrow or broaden the dynamic range of biological receptors. Such ability to finely control the affinity and dynamic range of aptamers may find many applications in synthetic biology, drug delivery, and targeted therapies, fields in which aptamers are of rapidly growing importance.

Physiologically based pharmacokinetic (PBPK) modeling of RNAi therapeutics: Opportunities and challenges.

Physiologically based pharmacokinetic (PBPK) modeling is a powerful tool with many demonstrated applications in various phases of drug development and regulatory review. RNA interference (RNAi)-based therapeutics are a class of drugs that have unique pharmacokinetic properties and mechanisms of action. With an increasing number of RNAi therapeutics in the pipeline and reaching the market, there is a considerable amount of active research in this area requiring a multidisciplinary approach. The application of PBPK models for RNAi therapeutics is in its infancy and its utility to facilitate the development of this new class of drugs is yet to be fully evaluated. From this perspective, we briefly discuss some of the current computational modeling approaches used in support of efficient development and approval of RNAi therapeutics. Considerations for PBPK model development are highlighted both in a relative context between small molecules and large molecules such as monoclonal antibodies and as it applies to RNAi therapeutics. In addition, the prospects for drawing upon other recognized avenues of PBPK modeling and some of the foreseeable challenges in PBPK model development for these chemical modalities are briefly discussed. Finally, an exploration of the potential application of PBPK model development for RNAi therapeutics is provided. We hope these preliminary thoughts will help initiate a dialogue between scientists in the relevant sectors to examine the value of PBPK modeling for RNAi therapeutics. Such evaluations could help standardize the practice in the future and support appropriate guidance development for strengthening the RNAi therapeutics development program.

In situ entry of oligonucleotides into brain cells can occur through a nucleic acid channel.

Brain tissue has become a challenging therapeutic target, in part because of failure of conventional treatments of brain tumors and a gradually increasing number of neurodegenerative diseases. Because antisense oligonucleotides are readily internalized by neuronal cells in culture, these compounds could possibly serve as novel therapeutic agents to meet such a challenge. In previous in vitro work using cell culture systems, we have demonstrated that intracellular delivery requires a vector such as cationic liposomes since free oligonucleotides remain largely trapped in the endocytic pathway following cellular uptake. Here we studied the cellular uptake properties of oligonucleotides by explants of rat brain (brain slices), and by in vivo brain tissue after administration of oligonucleotides by bolus injection. In contrast to in vitro uptake, we show that in brain slices oligonucleotides were taken up by neuronal and nonneuronal cells, irrespective of their assembly with cationic liposomes. In either case, a diffuse distribution of oligonucleotides was seen in the cytosol and/or nucleus. Uptake of oligonucleotides by brain slices as a result of membrane damage, potentially arising from the isolation procedure, could be excluded. Interestingly, internalization was inhibited following treatment of the tissue with antibody GN-2640, directed against a nucleic acid channel, present in rat kidney cells. Our data support the view that an analogous channel is present in brain tissue, allowing entry of free oligonucleotides but not plasmids. Indeed, for delivery of the latter and accomplishment of effective transfection, cationic lipids were needed for gene translocation into both brain slices and brain tissue in vivo. These data imply that for antisense therapy to become effective in brain, cationic lipid-mediated delivery will only be needed for specific cell targeting but not necessarily for delivery per se to accomplish nuclear deposition of oligonucleotides into brain cells and subsequent down-regulation of disease-related targets.

The polyamines spermidine and spermine retard the platination rate of single-stranded DNA oligomers and plasmids.

The presence of polyamines in living cells is crucial for survival. Due to their high net charge at physiological pH, polyamines effectively charge neutralize the phosphodiester backbone of DNA in an interaction that also may protect the DNA from external damage. We here present a study illustrating the influence of spermidine and spermine on the platination reactions of the model oligonucleotides d(T(6)GT(6)), d(T(12)GT(12)), and d(T(24)GT(24)), and the pUC18 DNA plasmid. The aquated forms of the anticancer active compounds cisplatin (cis-[Pt(NH(3))(2)Cl(2)]) and the major Pt(II) metabolite of JM216 (cis-[PtCl(2)(NH(3))(c-C(6)H(11)NH(2))], JM118) were used as platination reagents. The study shows that the kinetics for formation of the coordinative Pt-DNA adduct are strongly influenced by the presence of sub-millimolar polyamine concentrations. At polyamine concentrations in the muM-range, the reactions remain salt-dependent. In contrast, platination of pUC18 is effectively prevented at mM concentrations of both spermidine and spermine with the latter as the more potent inhibitor. The results suggest that variations of intracellular polyamine concentrations may have a profound influence on the efficacy by which cationically charged reagents interfere with DNA function in vivo by modulation of the preassociation conditions.

Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication.

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.

Novel inhibitors of HIV integrase: the discovery of potential anti-HIV therapeutic agents.

The viral enzyme, HIV integrase (MW 32 kDa), is one of the three key enzymes of the pol gene of HIV. HIV integrase is involved in the integration of HIV DNA into host chromosomal DNA. There is apparently no functional equivalent of this enzyme in human cells. Integration of HIV DNA into the host cell genome apparently occurs by a carefully defined sequence of DNA tailoring (3'-processing) and coupling (joining or integration) reactions. In spite of some effort in this area targeted at the discovery of therapeutically useful inhibitors of this viral enzyme, there are no drugs for HIV/AIDS in clinical use where the mechanism of action is inhibition of HIV integrase. It is clear that new knowledge on inhibitors of this enzyme is of critical importance in the anti-HIV drug discovery area. This review focuses on the major classes of compounds that have been discovered as inhibitors of HIV integrase. Some of these compounds are non-specific inhibitors of the enzyme while evidence suggests that others may possess some specificity. The various classes include nucleotides, oligonucleotides, dinucleotides, and miscellaneous small molecules including heterocyclic systems, natural products, diketo acids and sulfones. A major focus of the review is on discoveries from my laboratory in the area of non-natural, nuclease-resistant dinucleotide inhibitors of HIV integrase.

Synthesis and activity of substituted 2-phenylquinolin-4-amines, antagonists of immunostimulatory CpG-oligodeoxynucleotides.

Fifty-seven 2-phenylquinolines substituted at the phenyl group and C4 of the quinoline were synthesized and analyzed for inhibition of the immunostimulatory effect of oligodeoxynucleotides with a CpG-motif. The Fujita-Ban variant of the classical Free-Wilson analysis gave a highly significant correlation for a series of 48 relatively small molecules demonstrating that (i) the partial contributions of substituents to biological activity (EC(50)) are additive and (ii) assuming similar bioavailability for all quinolines studied, the larger molecules cannot be accommodated within a still unknown biological receptor. The results suggest interaction of a basic antagonist molecule with weakly acidic groups in the antagonist-receptor complex. N-[2-(Dimethylamino)ethyl]-2-[4-(4-methylpiperazino)phenyl]quinolin-4-amine (50) is the most effective antagonist found in this study (EC(50) = 0.76 nM).

Immunospecific reduction of antioligonucleotide antibody-forming cells with a tetrakis-oligonucleotide conjugate (LJP 394), a therapeutic candidate for the treatment of lupus nephritis.

A discrete tetravalent conjugate, 7a (LJP 394), consisting of four oligonucleotides attached to a common carrier or platform was prepared. Single-stranded oligonucleotide 20-mers consisting of alternating deoxycytidine-deoxyadenosine nucleotides, (CA)10, were attached to a tetrabromoacetylated platform by displacement with sulfhydryl-terminated linkers. The tetrabromoacetylated platform 3a was synthesized in three steps using triethylene glycol bis-(chloroformate). The single-stranded conjugate was characterized by polyacrylamide gel electrophoresis, DNA sequencing, phosphate analysis, carbon and nitrogen combustion analysis, and correlation of stoichiometry to conversion in the conjugation process. HPLC and capillary electrophoretic methods were developed to evaluate purity. The tetrakis, single-stranded conjugate was annealed with a stoichiometric amount of a complementary single-stranded oligonucleotide 20-mer consisting of alternating thymidine-deoxyguanosine nucleotides, (TG)10. The double-stranded conjugate LJP 394 was characterized by melt temperature and hyperchromicity, phosphate analysis, and carbon and nitrogen combustion analysis. LJP 394 inhibits binding of DNA to anti-double-stranded oligonucleotide antibodies and reduces anti-oligonucleotide-specific plaque (antibody)-forming cells in an immunized mouse model by a proposed mechanism involving cross-linking B cell surface immunoglobins.

Programmable oligomers targeting 5'-GGGG-3' in the minor groove of DNA and NF-kappaB binding inhibition.

A series of hairpin oligomers containing benzimidazole (Bi) and imidazopyridine (Ip) rings were synthesized and screened to target 5'-WGGGGW-3', a core sequence in the DNA-binding site of NF-kappaB, a prolific transcription factor important in biology and disease. Five Bi and Ip containing oligomers bound to the 5'-WGGGGW-3' site with high affinity. One of the oligomers (Im-Im-Im-Im-gamma-Py-Bi-Py-Bi-beta-Dp) was able to inhibit DNA binding by the transcription factor NF-kappaB.

CpG oligodeoxynucleotides induce cyclooxygenase-2 in human B lymphocytes: implications for adjuvant activity and antibody production.

Synthetic CpG oligodeoxynucleotides (ODN), similar to DNA sequences found in certain microorganisms, have shown promise as adjuvants for humans by enhancing immune responses. Since antibodies are often indicators of successful vaccination, it is important to understand how CpG ODNs affect human B cells and influence antibody production. Treatment of human B cells with synthetic CpG ODN sequences increased both steady-state Cox-2 mRNA levels and protein expression. B cell receptor stimulation in concert with CpG ODN treatment induced Cox-2 expression and production of prostaglandin E(2), well above that seen with CpG ODN alone. Importantly, CpG-induced human B cell IgM and IgG production was attenuated by dual Cox-1/Cox-2 inhibitors and Cox-2-selective inhibitors. Our findings support a key role for CpG ODN-induced human B cell Cox-2 in the production of IgM and IgG antibodies, revealing that drugs that attenuate Cox-2 activity have the potential to reduce optimal antibody response to adjuvants/vaccination.

Dissecting Tn5 transposition using HIV-1 integrase diketoacid inhibitors.

Diketoacid (DKA) compounds have been shown to inhibit HIV-1 integrase by a mechanism that involves sequestration of the active site metals. Because HIV-1 integrase and Tn5 transposase have similar active site architectures and catalytic mechanisms, we investigated whether DKA analogues would inhibit Tn5 transposase activity and provide a model system to explore the mechanisms of action of these inhibitors. A screen of several hundred DKA analogues identified several with activity against Tn5 Tnp. Six DKA inhibitors used in this study manifested a variety of effects on different transposition steps suggesting that different analogues may have different binding contacts with transposase. All DKA compounds inhibited paired end complex (PEC) formation in which the nucleoprotein complex required for catalysis is assembled. Dissociation of PECs by some DKA compounds indicates that these inhibitors can decrease PEC stability. Four DKA compounds inhibited the two cleavage steps releasing transposon DNA from flanking DNA, and one of these four compounds preferentially inhibited the second cleavage step. The differential effect of this inhibitor on the second cleavage event indicates that cleavage of the two transposon-donor DNA boundaries is a sequential process requiring a conformational change. The requirement for a conformational change between cleavage events was also demonstrated by the inability of transposase to perform second cleavage at 25 degrees C. Finally, all six compounds inhibit strand transfer, the final step of Tn5 transposition. Two of the compounds that inhibited strand transfer have no effect on DNA cleavage. The strand transfer inhibition properties of various DKA compounds was sensitive to the structure of the 5'-non-transferred strand, suggesting that these compounds bind in or near the transposase active site. Other results that probe compound binding sites include the effects of active site mutations and donor DNA on DKA compound inhibition activities. Thus, DKA inhibitors will provide an important set of tools to investigate the mechanism of action of transposases and integrases.

Structure-activity relationship analysis of substituted 4-quinolinamines, antagonists of immunostimulatory CpG-oligodeoxynucleotides.

On the basis of a systematic SAR analysis of substituted quinolines, a derivative 32 was synthesized that shows half-maximal inhibition of the immunostimulatory effect of CpG-oligodeoxynucleotides in vitro at the concentration of 0.24 nM.

Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity of priming in reverse transcription. In the conventional method, two-base anchored oligo(dT) primers (5'dT16VN3', where N is any base and V is G, A or C) are used to make windows for the display of transcripts. However, reverse transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT16VN primers, we have developed two new technologies. One is higher temperature priming with reverse transcriptase thermoactivated by the disaccharide trehalose. The other is the use of competitive oligonucleotide blockers that hybridize to the non-selectively primed mRNAs, preventing the mispriming from the VN site. These methods were combined to improve restriction landmark cDNA scanning (RLCS), resulting in the elimination of the redundant signals that appear in different windows. This was achieved by the increased specificity of initiation of reverse trans-cription from the beginning of poly(A) sites. This method paves the way for the precise visualization of transcripts to allow expression profiles in individual tissues and at each developmental stage to be understood.

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. II. 3',5'-blocked oligonucleotides.

The pharmacokinetics and metabolism of four radiolabeled phosphodiester oligonucleotides with 3'- and 5'-blocked ends were studied in mice and compared with previously studied, unblocked, all-phosphodiester and all-phosphorothioate oligonucleotides. The radiolabel was a tritiated methyl group enzymatically attached at an internal cytidine. The ends of the blocked phosphodiester oligonucleotides were protected by cyclization or by incorporation of either phosphorothioate or methylphosphonate linkages. Although these modifications protected the blocked oligonucleotides from degradation by exonucleases present in mouse serum, degradation initiated by endonucleases was 50% complete in 0.5-5 hr. After intravenous injection, the blocked oligonucleotides were much less stable than the all-phosphorothioate oligonucleotide and only marginally more stable than the previously studied, unblocked phosphodiester oligonucleotide. Even a "chimeric" blocked oligonucleotide with 16 phosphorothioate linkages and eight contiguous phosphodiester linkages was rapidly degraded. Despite the favorable serum binding, tissue accumulation, and stability observed with phosphorothioate oligonucleotides, these properties did not provide the chimeric oligonucleotide access to a compartment where its phosphodiester linkages were stable. In other respects, the blocked and chimeric phosphodiester oligonucleotides also resembled the unblocked phosphodiester oligonucleotide; radiolabel was cleared rapidly from the blood, there was little evidence of tissue accumulation, high performance liquid chromatographic analysis of tissue extracts showed extremely rapid degradation to mononucleotides, and only mononucleotide metabolites were present in urine. In summary, blocked phosphodiester oligonucleotides are rapidly attacked by endonucleases present in mice. Unless this problem is less serious in primates, such blocked oligonucleotides will be relatively unattractive candidates for drug development.

Oligomerization of uridine phosphorimidazolides on montmorillonite: a model for the prebiotic synthesis of RNA on minerals.

The 5'-phosphorimidazolide of uridine reacts on Na(+)-montmorillonite 22A in aqueous solution to give oligomers as long as 7 mers. The maximum chain length increases to 9 mers and the overall oligomer yield increases when 9:1 ImpU, A5' ppA mixtures react under the same conditions. The oligomer yield and maximum chain length decreases with the structure of the added pyrophosphate in the order A5' ppA > A5' ppU > U5' ppU. Structure analysis of individual oligomer fractions was performed by selective enzymatic hydrolyses followed by HPLC analysis of the products. The regioselectivity for 3',5'-bond formation is 80-90% in the 9:1 ImpU, A5' ppA reaction, a percentage comparable to that observed in the 9:1 ImpA, A5' ppA reaction. Oligomerization of ImpU is inhibited by addition of dA5' ppdA, and MeppA. No oligomers containing A5' ppU were products of the 9:1 ImpU,A5' ppA reaction, a finding consistent with the simple addition of the ImpU to the A5' ppA and not the rearrangement of an ImpU-A5' ppA adduct. Concentrations of lysine or arginine which were close to that of the ImpU did not inhibit oligomer formation. Treatment of Na(+)-montmorillonite with 1 M arginine yielded arginine-montmorillonite, an amino acid-mineral adduct which did not catalyze ImpU oligomerization. Neither the 4-9 mers formed in the 9:1 ImpU, A5' ppA reaction nor the 4-9 mers formed by the base hydrolysis of poly(U) served as templates for the formation of oligo(A)s.

Quantitating tertiary binding energies of 2' OH groups on the P1 duplex of the Tetrahymena ribozyme: intrinsic binding energy in an RNA enzyme.

Binding of the Tetrahymena ribozyme's oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme's internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2' OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2' OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using "substrate inhibition". This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2' OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to -2.9 kcal/mol. The energetic contributions of specific 2' OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A "threshold" model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G x U wobble at the cleavage site and the individual 2' OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energetic cost for docking the P1 duplex into the active site that is paid for by the "intrinsic binding energy" of groups on the P1 duplex. Substrates that lack sufficient tertiary binding energy to overcome this energetic barrier exhibit reduced reactivities. Thus, the ribozyme appears to use the intrinsic binding energy of groups on the P1 duplex for catalysis. This intrinsic binding energy may be used to position reactants within the active site and to induce electrostatic destabilization of the substrate, relative to its interactions in solution.

Bis-4-aminoquinolines: novel triple-helix DNA intercalators and antagonists of immunostimulatory CpG-oligodeoxynucleotides.

Six dimeric 2-(2-naphthyl)quinolin-4-amines with a linker between the amino groups and eight dimeric 2-(4-anilino)quinolin-4-amines linked between the anilino groups were synthesized and evaluated for their interaction with duplex/triplex DNA's and as antagonists of immunostimulatory oligodeoxynucleotides with a CpG-motif (CpG-ODN). The most powerful triple-helix DNA intercalator known to date, with high affinity toward T.A.T triplets and triplex/duplex selectivity, was found. The potent antagonism of immunostimulatory CpG-ODN by several bis-4-aminoquinolines is not related to their DNA interactions.