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.

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.

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.

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.

4-Aminocyclopentane-1,3-diols as platforms for diversity: synthesis of anandamide analogs.

Starting from cyclopentadiene, two racemic mixtures of 4-aminocyclopentane-1,3-diols were prepared in 8 steps and characterized. Structure determination proved the anticipated trans-orientation of the two oxygen atoms with respect to the plane of the ring. The fragment-like new compounds are small and hydrophilic, devoid of rotatable bonds, and offer stereochemically defined attachment points for substituents. Thus, these platforms for diversity are suitable starting points for the construction of combinatorial libraries of lead-like 4-amidocyclopentane-1,3-diols or natural product analogs. As a proof of concept, cyclopentanoid anandamide analogs were prepared using these molecular platforms and evaluated as tools for the investigation of unresolved issues in the molecular biology of anandamide.

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.

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.

Label-free imaging of semiconducting and metallic carbon nanotubes in cells and mice using transient absorption microscopy.

As interest in the potential biomedical applications of carbon nanotubes increases, there is a need for methods that can image nanotubes in live cells, tissues and animals. Although techniques such as Raman, photoacoustic and near-infrared photoluminescence imaging have been used to visualize nanotubes in biological environments, these techniques are limited because nanotubes provide only weak photoluminescence and low Raman scattering and it remains difficult to image both semiconducting and metallic nanotubes at the same time. Here, we show that transient absorption microscopy offers a label-free method to image both semiconducting and metallic single-walled carbon nanotubes in vitro and in vivo, in real time, with submicrometre resolution. By using appropriate near-infrared excitation wavelengths, we detect strong transient absorption signals with opposite phases from semiconducting and metallic nanotubes. Our method separates background signals generated by red blood cells and this allows us to follow the movement of both types of nanotubes inside cells and in the blood circulation and organs of mice without any significant damaging effects.

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.

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.

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.

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.

Unnatural nucleosides with unusual base pairing properties.

Synthetic modified nucleosides designed to pair in unusual ways with natural nucleobases have many potential applications in biology and biotechnology. This overview lays the foundation for future protocol units on synthesis and application of unnatural bases, with particular emphasis on unnatural base analogs that mimic natural bases in size, shape, and biochemical processing. Topics covered include base pairs with alternative H-bonding schemes, dimensionally expanded base pairs, hydrophobic base pairs, metal-ligated bases, degenerate bases, universal nucleosides, and triplex constituents.

Conjugation of (E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine to hydrophilic microspheres: development of a mobile microscale UV light actinometer.

( E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine was biotinylated through a diisopropylsilylacetal linkage and attached to the surface of hydrophilic streptavidin-coated microspheres through the high-affinity noncovalent interaction between biotin and streptavidin. The functionalized microspheres form a stable suspension in water. Upon UV irradiation, the nonfluorescent ( E)-5-[2-(methoxycarbonyl)ethenyl]cytidine on the microspheres undergoes photocyclization to produce highly fluorescent 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine. The fluorescence intensity of the microspheres can be correlated to the particle-specific UV doses applied at different suspension concentrations. The microspheres allow one to measure the UV dose (fluence) distribution in high-throughput water disinfection systems.

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.

Efficient primer strand extension beyond oxadiazole carboxamide nucleobases.

Two oxadiazole carboxamide deoxyribonucleoside analogues are described that can be incorporated and efficiently extended by Taq DNA polymerase. The primer strand extension beyond oxadiazole nucleoside analogues occurs at rates similar to the values observed for the canonical Watson-Crick base pairs irrespective of the template nucleobase. These distinctive chemical effects in DNA polymerase extensions are attributed to the smaller size and unique electronic properties of the oxadiazole nucleobase.

(E)-5-[2-(methoxycarbonyl)ethenyl]cytidine as a chemical actinometer for germicidal UV radiation.

(E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine (S) was examined for use as a chemical actinometer for germicidal UV radiation. Its photoproduct, 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine (P), is strongly fluorescent with excitation and emission maxima at 330 and 385 nm, respectively. Experiments were conducted to characterize the dynamic behavior of aqueous solutions of S and P when subjected to UV radiation. UV sources used for these experiments included a low-pressure mercury lamp, a XeBr excimer lamp, and a KrCI excimer lamp; all three sources were mounted in collimating devices to provide incident beams that could be easily and accurately characterized by radiometry. These three sources each yielded essentially monochromatic outputwith characteristic wavelengths of 254, 282, and 222 nm, respectively. At practical concentrations, it was found that the absorbance of the actinometer solution was neither high enough to make the actinometer solutions optically opaque nor low enough to be optically transparent to UV. In addition, the photoproduct displayed a molar absorption coefficient that was similar in magnitude to that of the parent compound, thereby resulting in competitive absorption of UV energy between Sand Pduring irradiation. For purposes of evaluation of the results of irradiation, a mathematical model was developed to accountforthe nonideal optical characteristics of the system. The model is based on a description of local photochemical kinetics; predictions of overall reactor performance were developed by spatial and temporal integration of model results. The model was used to analyze the dynamic behavior of actinometer solutions during UV irradiation and to estimate the quantum yield for photoproduction of Pfrom S. This modeling approach is potentially applicable to other photochemical processes in which multiple compounds are present that absorb photoactive radiation; however, general application of this modeling approach to photochemical reactor systems will require inclusion of othertermsto describe relevanttransport behavior within the system.

Alternative nucleic acid analogues for programmable assembly: hybridization of LNA to PNA.

Complementary locked nucleic acid (LNA) and peptide nucleic acid (PNA) hexamers bind to each other with significantly higher affinity than each binds to DNA, and with far greater affinity than DNA binds to complementary DNA. The hybridization is highly specific with a single mismatch causing decreases in T(m) values ranging from 12 (G/T) to 30 degrees C (A/A). Importantly, the hybridization of an LNA oligomer to a PNA oligomer is unaffected by the ionic strength of the buffer. These properties make the LNA/PNA pair an attractive candidate as a replacement for DNA in programmable assembly.