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The self-assembly system of the rod-shaped tobacco mosaic virus (TMV) has been studied extensively for nanoscale applications. TMV coat protein assembly is modulated by intersubunit carboxylate groups whose electrostatic repulsion limits the assembly of virus rods without incorporating genomic RNA. To engineer assembly control into this system, we reprogrammed intersubunit carboxylate interactions to produce self-assembling coat proteins in the absence of RNA and in response to unique pH and ionic environmental conditions. Specifically, engineering a charge attraction at the intersubunit E50-D77 carboxylate group through a D77K substitution stabilized the coat proteins assembly into virus-like rods. In contrast, the reciprocal E50K modification alone did not confer virus-like rod assembly. However, a combination of R46G/E50K/E97G substitutions enabled virus-like rod assembly. Interestingly, the D77K substitution displays a unique pH-dependent assembly-disassembly profile, while the R46G/E50K/E97G substitutions confer a novel salt concentration dependency for assembly control. In addition, these unique environmentally controlled coat proteins allow for the directed assembly and disassembly of chimeric virus-like rods both in solution and on substrate-attached seed rods. Combined, these findings provide a controllable means to assemble functionally discrete virus-like rods for use in nanotechnology applications.

In this work, we introduce an electrowetting-assisted 3-D biofabrication process allowing both complete and localized functionalization of bionanoreceptors onto densely arranged 3-D microstructures. The integration of biomaterials with 3-D microdevice components offers exciting opportunities for communities developing miniature bioelectronics with enhanced performance and advanced modes of operation. However, most biological materials are stable only in properly conditioned aqueous solutions, thus the water-repellent properties exhibited by densely arranged micro/nanostructures (widely known as the Cassie-Baxter state) represent a significant challenge to biomaterial integration. Here, we first investigate such potential limitations using cysteine-modified tobacco mosaic virus (TMV1cys) as a model bionanoreceptor and a set of Au-coated Si-micropillar arrays (μPAs) of varying densities. Furthermore, we introduce a novel biofabrication system adopting electrowetting principles for the controlled localization of TMV1cys bionanoreptors on densely arranged μPAs. Contact angle analysis and SEM characterizations provide clear evidence to indicate structural hydrophobicity as a key limiting factor for 3-D biofunctionalization and for electrowetting as an effective method to overcome this limitation. The successful 3-D biofabrication is confirmed using SEM and fluorescence microscopy that show spatially controlled and uniform assemblies of TMV1cys on μPAs. The increased density of TMV1cys per device footprint produces a 7-fold increase in fluorescence intensity attributed to the μPAs when compared to similar assemblies on planar substrates. Combined, this work demonstrates the potential of electrowetting as a unique enabling solution for the controlled and efficient biofabrication of 3-D-patterned micro/nanodomains.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective Na V 1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective Na V 1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

A capillary microfluidics-integrated sensor system is developed for rapid assembly of bionanoreceptor interfaces on-chip and label-free biosensing. Genetically engineered Tobacco mosaic virus (TMV) virus-like particles (VLPs), displaying thousands copies of identical receptor peptides FLAG-tags, are utilized as nanoceptors for antibody sensing. Controlled and accelerated assembly of VLP receptor layer on impedance sensor has been achieved using capillary action and surface evaporation from an open-channel capillary microfluidic system. VLPs create a dense and localized receptor monolayer on the impedance sensor using only 5 μL of VLP sample solution (0.2 mg/mL) in only 6 min at room temperature. The VLP-functionalized impedance sensor is capable of label-free detection of target antibodies down to 55 pM concentration within 5 min. These results highlight the significant potentials of an integrated microsystem for rapid and controlled receptor-transducer interface creation and the nanoscale VLP-based sensors for fast, accurate, and decentralized pathogen detection.

Visceral leishmaniasis is a severe parasitic disease that is one of the most neglected tropical diseases. Treatment options are limited, and there is an urgent need for new therapeutic agents. Following an HTS campaign and hit optimization, a novel series of amino-pyrazole ureas has been identified with potent in vitro antileishmanial activity. Furthermore, compound 26 shows high levels of in vivo efficacy (>90%) against Leishmania infantum, thus demonstrating proof of concept for this series.

A series of 4-substituted pyrimido[4,5-d]azepines that are potent, selective 5-HT2C receptor partial agonists is described. A rational medicinal chemistry design strategy to deliver CNS penetration coupled with SAR-based optimization of selectivity and agonist potency provided compounds with the desired balance of preclinical properties. Lead compounds 17 (PF-4479745) and 18 (PF-4522654) displayed robust pharmacology in a preclinical canine model of stress urinary incontinence (SUI) and no measurable functional agonism at the key selectivity targets 5-HT2A and 5-HT2B in relevant tissue-based assay systems. Utilizing recent advances in the structural biology of GPCRs, homology modeling has been carried out to rationalize binding and agonist efficacy of these compounds.

The coupling of aromatic moieties with saturated heterocyclic partners is currently an area of significant interest for the pharmaceutical industry. Herein, we present a procedure for the metal-free coupling of 4-, 5-, and 6-membered saturated heterocyclic p-methoxyphenyl (PMP) sulfonylhydrazones with aryl and heteroaromatic boronic acids. This procedure enables a simple, two-step synthesis of a range of functionalized sp(2)-sp(3) linked bicyclic building blocks, including oxetanes, piperidines, and azetidines, from their parent ketones.

A robust convergent synthesis of the prodrugs of HCV replicase inhibitors 1-5 is described. The central 5H-imidazo[4,5-d]pyridazine core was formed from acid-catalyzed cyclocondensation of an imidazole-4,5-dicarbaldehyde (20) and a α-hydrazino ester, generated in situ from the bis-BOC-protected precursors 25 and 33. The acidic conditions not only released the otherwise unstable α-hydrazino esters but also were the key to avoid facile decarboxylation to the parent drugs from the carboxylic ester prodrugs 1-5. The bis-BOC α-hydrazino esters 25 and 33 were prepared by addition of ester enolates (from 23 and 32) to di-tert-butyl azodicarboxylate via catalysis with mild inorganic bases, such as Li2CO3. A selective aerobic oxidation with catalytic 5% Pt-Bi/C in aqueous KOH was developed to provide the dicarbaldehyde 20 from the diol 27.

Uniform dimensions and genetic tractability make filamentous viruses attractive templates for the display of functional groups used in materials science, sensor applications, and vaccine development. However, active virus replication and recombination often limit the usefulness of these viruses for such applications. To circumvent these limitations, genetic modifications of selected negatively charged intersubunit carboxylate residues within the coat protein of tobacco mosaic virus (TMV) were neutralized so as to stabilize the assembly of rod-shaped virus-like particles (VLPs) within bacterial expression systems. Here we show that TMV-VLP nanorods are easily purified, stable, and can be programmed in a variety of configurations to display functional peptides for antibody and small molecule binding.

Ion channels are membrane proteins expressed in almost all living cells. The sequencing of the human genome has identified more than 400 putative ion channels, but only a fraction of these have been cloned and functionally tested. The widespread tissue distribution of ion channels, coupled with the plethora of physiological consequences of their opening and closing, makes ion-channel-targeted drug discovery highly compelling. However, despite some important drugs in clinical use today, as a class, ion channels remain underexploited in drug discovery and many existing drugs are poorly selective with significant toxicities or suboptimal efficacy. This Perspective seeks to review the ion channel family, its structural and functional features, and the diseases that are known to be modulated by members of the family. In particular, we will explore the structure and properties of known ligands and consider the future prospects for drug discovery in this challenging but high potential area.

A novel series of potent and selective sulfonamide derived β(2)-adrenoreceptor agonists are described that exhibit potential as inhaled ultra-long-acting bronchodilators for the treatment of asthma and chronic obstructive pulmonary disease. Analogues from this series mediate very long-lasting smooth muscle relaxation in guinea pig tracheal strips. The sulfonamide agonist headgroup confers high levels of intrinsic crystallinity that could relate to the acidic sulfonamide motif supporting a zwitterionic form in the solid state. Optimization of pharmacokinetic properties was achieved through targeted introduction of a phenolic moiety to support rapid phase II clearance, thereby minimizing systemic exposure following inhalation and reducing systemically mediated adverse events. Compound 38 (PF-610355) is identified as a clinical candidate from this series, with in vivo duration of action studies confirming its potential for once-daily use in humans. Compound 38 is currently in advanced phase II clinical studies.

The relevance of the melanocortin system to sexual activity is well established, and nonselective peptide agonists of the melanocortin receptors have shown evidence of efficacy in human sexual dysfunction. The role of the MC4 receptor subtype has received particular scrutiny, but the sufficiency of its selective activation in potentiating sexual response has remained uncertain owing to conflicting data from studies in preclinical species. We describe here the discovery of a novel series of small-molecule MC4 receptor agonists derived from library hit 2. The addition of methyl substituents at C3 and C5 of the 4-phenylpiperidin-4-ol ring was found to be markedly potency-enhancing, enabling the combination of low nanomolar potencies with full rule-of-five compliance. In general, the series shows only micromolar activity at other melanocortin receptors. Our preferred compound 40a provided significant systemic exposure in humans on both sublingual and oral administration and was safe and well tolerated up to the maximum tested dose. In a pilot clinical study of male erectile dysfunction, the highest dose of 40a tested (200 mg) provided a similar level of efficacy to sildenafil.

The influence of nanoscale out-of-plane roughness on the ordering of submicron spheres during evaporative deposition from colloidal suspension was examined using shallow corrugated substrates possessing optical wavelengths and nanoscale amplitude. Under conditions in which spheres were embedded in a liquid layer with thickness on the order of the sphere diameter, it was observed that the spheres overwhelmingly deposited in the valleys of the surface corrugations rather than on their peaks. This behavior persisted to surprisingly shallow corrugation amplitudes, sometimes 100 times smaller than the sphere diameter. An analysis of the capillary forces on the spheres explains this behavior and also yields a critical corrugation amplitude below which a substrate will appear "flat" to depositing spheres. The observation that substrate features significantly smaller than the sphere diameter can influence deposition morphology may lead to simple methods to create large domains of order in colloidal crystals.

Potent and selective inhibitors of thrombin were sought based on the (R)-Phe-Pro-Arg sequence. The objective was to generate similar binding interactions to those achieved by potent competitive inhibitors of the argatroban type, so eliminating the need for covalent interaction with the catalytic serine function, as utilized by aldehyde and boronic acid type inhibitors. Improving the S(1) subsite interaction by substitution of arginine with a 4-alkoxybenzamidine residue provided potent lead 2 (K(i) = 0.37 nM). Though an amide bond, which H-bonds to the active site, is lost, modeling indicated that a new H-bond is generated between the alkoxy oxygen atom and the catalytic Ser-195 hydroxyl group. Substitution of the benzamidine system by 1-amidinopiperidine then gave compound 4, which provided a further gain in selectivity over trypsin. However, previous work had shown that these compounds were likely to be too lipophilic (Log D +0.4 and +0.2, respectively) and to suffer rapid hepatic extraction, presumably via biliary elimination. Accordingly, both proved short-acting when administered intravenously to rats and showed poor activity when given intraduodenally. The aim was then to reduce lipophilicity below a log D of -1.2, which in a previously reported series had been effective in preventing rapid clearance. It was anticipated that compounds of this type would rely on the cation selective paracellular route of absorption from the gastrointestinal tract. Potent polar analogues with selectivity >1000 over trypsin were obtained. The best in vivo activity was shown by compound 12. However, in the final analysis, its oral bioavilability proved poor, relative to analogues with similar physicochemical properties derived from argatroban, consistent with the hypothesis that molecular shape is an additional important determinant of paracellular absorption.

The preparation and properties are described of complexes resulting from the mono- and diprotonation of the [Co(4)O(4)](4+) unit to give products containing [Co(4)O(3)(OH)](5+) and [Co(4)O(2)(OH)(2)](6+), respectively. Treatment of [Co(4)O(4)(O(2)CC(6)H(4)-p-OMe)(2)(bpy)(4)](2+) (bpy = 2,2'-bipyridine) in MeCN with 70% perchloric acid leads to isolation of [Co(4)O(2)(OH)(2)(O(2)CC(6)H(4)-p-OMe)(2)(bpy)(4)](ClO(4))(4) (5) in 61% yield. Treatment of [Co(4)O(4)(O(2)CC(6)H(4)-p-Me)(2)(bpy)(4)](2+) in MeCN with an acidic solution of (NH(4))(3)[Ce(NO(3))(6)] leads to isolation of [Co(4)O(3)(OH)(O(2)CC(6)H(4)-p-Me)(2)(bpy)(4)][Ce(NO(3))(6)] (6) in 96% yield. Complex 5.3MeCN.H(2)O crystallizes in monoclinic space group C2/c with (at -155 degrees C) a = 33.838(8) Å, b = 13.826(3) Å, c = 29.944(7) Å, beta = 98.84(1) degrees, and Z = 8. Complex 6.PhCN.H(2)O crystallizes in orthorhombic space group Pbca with (at -154 degrees C) a = 22.603(4) Å, b = 34.759(6) Å, c = 18.167(3) Å, and Z = 8. The complexes contain [Co(4)O(2)(OH)(2)](6+) (5) and [Co(4)O(3)(OH)](5+) (6) distorted-cubane cores, the sites of protonation being apparent by (i) the approach of ClO(4)(-) or [Ce(NO(3))(6)](3)(-) anions to within hydrogen-bonding distances (O.O approximately 2.7 Å) and (ii) the longer Co-OH(-) bond lengths compared with Co-O(2)(-) bond lengths. Peripheral ligation is provided by chelating bpy and syn,syn-bridging RCO(2)(-) groups. The Co(III) atoms are six-coordinate and approximately octahedral. Solution studies show that the diprotonated species is a strong diprotic acid analogous to H(2)SO(4); the first deprotonation goes to completion, but the monoprotonated species is a weak acid and is in equilibirium with the nonprotonated species. pH studies in aqueous solution yield a pK(a2) value of 3.15 for the second deprotonation. Electronic and (1)H NMR spectra of the diprotonated complexes in various solvents are consistent with the monoprotonated complex being the major species in solution. The combined results demonstrate that the [Co(4)O(4)](4+) core is capable of acting as a Brønsted base, undergoing either one or two protonation reactions.

The synthesis of a series of N-(mercaptoacyl)-4-substituted-(S)-prolines (2 and 3) is described. These compounds were evaluated in vitro for inhibition of angiotensin-converting enzyme (ACE), and selected compounds were evaluated in vivo for ACE inhibition. The most potent compounds in vitro are 108, 109, 111, 114, and 116, having relative potencies of 1.0, 1.0, 1.3, 1.1, and 2.6 as compared to the potency of captopril. The most potent compounds in vivo intravenously are 108, 111, 114, 116, 117, and 97.