A Novel, Nonpeptidic, Orally Active Bivalent Inhibitor of Human ß-Tryptase.

ß-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

Evaluation of end-capped DNA modules for pRNA capture and functionalization.

The ability of packaging RNA (pRNA) from the phi29 DNA packaging motor to form nanoassemblies and nanostructures has been exploited for the development of the nascent field of RNA nanotechnology and subsequent applications in nanomedicine. For applications in nanomedicine, it is necessary to modify the pRNA structure for the conjugation of active molecules. We have investigated end-capped double-stranded DNA segments as reversible capture reagents for pRNA. These capture agents can be designed to allow the conjugation of any desired molecule for pRNA functionalization. The results of model studies presented in this report show that 5- to 7-nucleotide overhangs on a target RNA can provide efficient handles for the high-affinity association to capped double-stranded DNA.

Optimized method for the synthesis and purification of adenosine--folic acid conjugates for use as transcription initiators in the preparation of modified RNA.

We present an optimized synthetic strategy for the attachment of molecules to 5'-adenosine monophosphate (AMP), which can then be used to label the 5'-end of RNA by T7 RNA polymerase mediated in vitro transcription. Through the use of a boronate affinity gel, we have developed an efficient route to the preparation of folate conjugated AMP with high yields and purity. Affi-Gel boronate is an affinity resin that selectively binds nucleoside and nucleoside derivatives at pH>7.5 and releases them at pH<6.5. This resin is used to efficiently bind and purify ribonucleotides such as AMP. This allows for the addition of a large excess of reactants and reagents in order to drive the reaction to completion and then allow easy purification without HPLC. The synthesis can be successfully scaled up to produce large quantities of AMP conjugates.

1,2,4-trisubstituted cyclopentanes as platforms for diversity.

Despite their simplicity, relatively few examples of 1,2,4 (1,3,4)-amino-, azido-, and hydroxy-substituted cyclopentanes are reported in the literature. We found that cyclopent-3-en-1-ol can be transformed into a significant variety of compounds of this class by relatively common and efficient synthetic procedures. Stereochemical control of epoxidation of the cyclopentene double bond can be achieved by varying the substitutents at C4. The C4 substituent and epoxide functional group can be converted into a variety of intermediates with differential protection designed for use in applications requiring regiospecific control for further elaboration of the cyclopentane scaffold.

Silicon field effect transistors as dual-use sensor-heater hybrids.

We demonstrate the temperature mediated applications of a previously proposed novel localized dielectric heating method on the surface of dual purpose silicon field effect transistor (FET) sensor-heaters and perform modeling and characterization of the underlying mechanisms. The FETs are first shown to operate as electrical sensors via sensitivity to changes in pH in ionic fluids. The same devices are then demonstrated as highly localized heaters via investigation of experimental heating profiles and comparison to simulation results. These results offer further insight into the heating mechanism and help determine the spatial resolution of the technique. Two important biosensor platform applications spanning different temperature ranges are then demonstrated: a localized heat-mediated DNA exchange reaction and a method for dense selective functionalization of probe molecules via the heat catalyzed complete desorption and reattachment of chemical functionalization to the transistor surfaces. Our results show that the use of silicon transistors can be extended beyond electrical switching and field-effect sensing to performing localized temperature controlled chemical reactions on the transistor itself.

Properties of double-stranded oligonucleotides modified with lipophilic substituents.

We have synthesized a series of short, self-complementary oligonucleotide sequences modified at their 5'- and/or 3'- termini with a lipophilic dodecane (C12); these systems serve as models to assess the biophysical properties of double-stranded DNA (dsDNA) equipped with potentially stabilizing lipophilic substituents. Addition of C12 to the 5'-termini of self-complementary 10 nucleotide sequences increased their duplex melting temperatures (T(m)) by approximately 4-8 degrees C over their corresponding unmodified sequences. C12 functionalities added to both the 3'- and 5'-termini increased T(m) values by approximately 10-12 degrees C. The observed increases in T(m) correlated with greater duplex stabilities as determined by the free energy values (DeltaG) derived from T(m) plots. There is a greater degree of stabilization when C12 is positioned with a C.G base pair at the termini, and the stabilizing effect of lipophilic groups far exceeds the effect seen in adding an additional base pair to both ends of DNA. Stable, short dsDNA sequences are of potential interest in the development of transcription factor decoy oligonucleotides as possible therapeutic agents and/or biological tools. These results suggest that the stability of short dsDNA sequences are improved by lipophilic substituents and can be used as the basis for the design of dsDNAs with improved biological stabilities and function under physiological conditions.

Synthesis and evaluation of new spacers for use as dsDNA end-caps.

A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl-protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. End-caps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. End-caps containing either a terthiophene or a naphthalene tetracarboxylic acid diimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A*T base pair. Spacers containing only methylene (-CH(2)-) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together.

Novel, Self-Assembling Dimeric Inhibitors of Human ß Tryptase.

ß-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.

Localized heating on silicon field effect transistors: device fabrication and temperature measurements in fluid.

We demonstrate electrically addressable localized heating in fluid at the dielectric surface of silicon-on-insulator field-effect transistors via radio-frequency Joule heating of mobile ions in the Debye layer. Measurement of fluid temperatures in close vicinity to surfaces poses a challenge due to the localized nature of the temperature profile. To address this, we developed a localized thermometry technique based on the fluorescence decay rate of covalently attached fluorophores to extract the temperature within 2 nm of any oxide surface. We demonstrate precise spatial control of voltage dependent temperature profiles on the transistor surfaces. Our results introduce a new dimension to present sensing systems by enabling dual purpose silicon transistor-heaters that serve both as field effect sensors as well as temperature controllers that could perform localized bio-chemical reactions in Lab on Chip applications.

Target-Directed Self-Assembly of Homodimeric Drugs Against ß-Tryptase.

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC50 (0.19 ± 0.08 µM) over controls (5.50 ± 0.09 µM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

Oligodeoxyribonucleotide association with single-walled carbon nanotubes studied by SPM.

Studies have been performed on both as-received and chemically oxidized single-walled carbon nanotubes (SWCNTs) grown by two different growth methods to better understand the preferential association of the oligodeoxyribonucleotide T30 (ODN) with SWCNTs. Samples of T30 ODN:SWCNT were examined under ambient conditions using non-contact scanning probe microscope (SPM) techniques. The resulting images show different morphologies ranging from tangled networks of SWCNTs to individual, well-dispersed isolated SWCNTs as the sonication time is increased. SPM images of well-dispersed, as-received SWCNTs reveal isolated features that are 1.4 to 2.8 nm higher than the bare SWCNT itself. X-ray photoemission spectroscopy (XPS) confirmed these features to be T30 ODN in nature. Chemically oxidizing the SWCNTs before sonication is found to be an effective way to increase the number of T30 ODN features.

Protein-DNA footprinting by endcapped duplex oligodeoxyribonucleotides.

Oligodeoxyribonucleotides (5'-phosphorylated) of varying lengths were capped using a polyamide linker to form thermodynamically stable, endcapped DNA duplexes containing 8-14 bp. We have employed these endcapped DNA duplexes as tools to determine the DNA footprint of T4 DNA ligase. By high-performance liquid chromatography and PAGE analysis of the ligation mixtures of the endcapped DNA duplexes, we have found that by varying the lengths and the position of the nick, we can determine the minimal DNA-binding site as well as the mode of binding (symmetrical or asymmetrical binding) by the enzyme. The results of the study revealed that a 11 bp endcapped duplex was the shortest duplex effectively ligated. Dependence of ligation efficiency on nick position demonstrates that T4 DNA ligase bound asymmetrically to its DNA substrate. The use of a set of thermodynamically stable endcapped deoxyribonucleoside duplexes as a tool to elucidate the DNA footprint provides an efficient strategy for footprinting, which avoids ambiguities associated with chemical and biochemical footprinting methods.

Fluoride-cleavable biotinylation phosphoramidite for 5'-end-labeling and affinity purification of synthetic oligonucleotides.

A fluoride-cleavable phosphoramidite for biotinylation was designed, synthesized and coupled efficiently to the 5'-end of DNA on an automatic synthesizer. The diisopropylsilyl acetal functionality was used to link the biotin moiety through a tertiary hydroxide group to the 5'-end of DNA. This linkage proved to be completely stable under certain post-synthetic DNA cleavage/deprotection conditions [0.05 M K(2)CO(3) in MeOH, room temperature, 24 h and MeNH(2) (approximately 40%)/NH(4)OH (approximately 29%), 1:1 v/v, 65 degrees C, 30 min] while it can be readily broken by fluoride ion, releasing unmodified DNA. To demonstrate the use of this DNA biotinylation method, we applied this method in affinity purification of synthetic DNA. As revealed by HPLC analysis, biotinylated full-length DNA can be efficiently attached to NeutrAvidin coated microspheres, and failure sequences can be readily removed. Subsequent treatment of the microspheres with pyridine/HF released high quality full-length unmodified DNA in good yield.

Orthogonal base pairs continue to evolve.

Recent developments in the design and construction of unusual analogs of the natural nucleic acid bases have reached a milestone with the report (in this issue of Chemistry & Biology) of a new orthogonal base pair that allows site-specific introduction of a photo-crosslinkable modified base into an RNA molecule by T7 RNA polymerase-mediated transcription of DNA containing the base-pairing partner.

DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs.

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1-5; ) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases. The results demonstrate how nucleoside triphosphate substrate selection by DNA polymerase is a complex phenomenon involving electrostatic interactions in addition to hydrogen bonding and shape recognition. These azole nucleobase analogs offer unique molecular tools for probing nonbonded interactions dictating substrate selection and fidelity of DNA polymerases.

Synthesis and structural analysis of oxadiazole carboxamide deoxyribonucleoside analogs.

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2'-endo ("south") conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

Polysulfonates derived from metal thiolate complexes as inhibitors of HIV-1 and various other enveloped viruses in vitro.

Sodium 2-mercaptoethanesulfonate reacts with the metal ions Pd(II), Pt(II), Ag(I), Cd(II) and Zn(II) to yield complexes containing multiple anionic sulfonate sites. On the basis of spectroscopic and other analytical data the complexes were assigned the tentative molecular formulas: Pd6(SCH2CH2SO3Na)12, Ptn(SCH2CH2SO3Na)2n+2, Agn(SCH2CH2SO3Na)n, Na2Zn4(SCH2CH2SO3Na)10, and Na2Cd4(SCH2CH2SO3Na)10. The complexes displayed a variety of differences in activity towards DNA and RNA viruses. The platinum complex showed no measurable cytotoxicity and exhibited a spectrum of antiviral activity resembling that of dextran sulfate. It was active against HIV-1 and HIV-2, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), thymidine kinase-deficient HSV-1, human cytomegalovirus, vesicular stomatitis virus (VSV), influenza A virus, respiratory syncytial virus (RSV), Sindbis virus, Junin virus and Tacaribe virus. The palladium complex also showed no measurable cytotoxicity, but was completely inactive against most viruses, with one notable exception: both HIV-1 and HIV-2 were substantially inhibited by the palladium complex. The silver complex showed significantly less antiviral activity and greater cytotoxicity than the platinum complex but did show some selectivity against RSV. The zinc complex showed only modest activity against VSV, RSV, Junin virus, and Tacaribe virus, and like the silver compound was more cytotoxic than either the platinum or palladium complex. The cadmium complex was toxic to all of the cell lines used for in vitro evaluation of antiviral activity. Based on these results, the platinum and palladium compounds appear to be promising candidates for further studies, that is, as vaginal microbicides in the prevention of genital HIV and/or HSV transmission.

Development of a nucleoside analog UV light sensor.

Conjugation of the photosensitive nucleoside (E)-5-(2-methoxycarbonylethenyl)cytidine to biotin provided a means to attach this analogue to microparticles for dosimetry applications that require UV sensor mobility.

Endcaps for stabilizing short DNA duplexes.

The syntheses of endcaps for covalently linking the 3' and 5' hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.