Microfabricated microfluidic platforms for creating microlens array.

The paper presents a novel and economic manufacturing process for microlens arrays (MLAs). This process uses micromilling machining, PDMS casting, and hybrid bonding between a glass substrate and PDMS membrane to create a microfluidic chip which is used for manufacturing MLAs on a PDMS substrates. MLAs of various diameters were fabricated for experiments, including 1000 µm, 500 µm, and 200 µm. The sag height of the MLAs is easily adjusted by controlling the pressure inside the microchannel to deform the PDMS membrane. Multiple experiments were conducted to characterize the performance of MLAs, the results of which demonstrate: (1) this fabrication process is able to manufacture MLAs with various dimensions and the diameter of an MLAs is determined by the size of micromilling bit and cutting path; (2) the sag height and curvature of MLAs can be controlled by the PDMS membrane thickness and the hydraulic pressure inside the microchannel; (3) an optical system was built to investigate the uniformity of MLAs and the experiment results showed uniform focal length of MLAs; (4) the resulting MLAs magnify tiny objects and significantly enhance the fluorescence signal emitted from the microchannel.

Design and synthesis of α-conotoxin GID analogues as selective α4ß2 nicotinic acetylcholine receptor antagonists.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is an important target for currently approved smoking cessation therapeutics. However, the development of highly selective α4ß2 nAChR antagonists remains a significant challenge. α-Conotoxin GID is an antagonist of α4ß2 nAChRs, though it is significantly more potent toward the α3ß2 and α7 subtypes. With the goal of obtaining further insights into α-conotoxin GID/nAChR interactions that could lead to the design of GID analogues with improved affinity for α4ß2 nAChRs, we built a homology model of the GID/α4ß2 complex using an X-ray co-crystal structure of an α-conotoxin/acetylcholine binding protein (AChBP) complex. Several additional interactions that could potentially enhance the affinity of GID for α4ß2 nAChRs were observed in our model, which led to the design and synthesis of 22 GID analogues. Seven analogues displayed inhibitory activity toward α4ß2 nAChRs that was comparable to GID. Significantly, both GID[A10S] and GID[V13I] demonstrated moderately improved selectivity toward α4ß2 over α3ß2 when compared with GID, while GID[V18N] exhibited no measurable inhibitory activity for the α3ß2 subtype, yet retained inhibitory activity for α4ß2. In this regard, GID[V18N] is the most α4ß2 nAChR selective α-conotoxin analogue identified to date.

Mixture-based combinatorial libraries from small individual peptide libraries: a case study on α1-antitrypsin deficiency.

The design, synthesis and screening of diversity-oriented peptide libraries using a "libraries from libraries" strategy for the development of inhibitors of α1-antitrypsin deficiency are described. The major buttress of the biochemical approach presented here is the use of well-established solid-phase split-and-mix method for the generation of mixture-based libraries. The combinatorial technique iterative deconvolution was employed for library screening. While molecular diversity is the general consideration of combinatorial libraries, exquisite design through systematic screening of small individual libraries is a prerequisite for effective library screening and can avoid potential problems in some cases. This review will also illustrate how large peptide libraries were designed, as well as how a conformation-sensitive assay was developed based on the mechanism of the conformational disease. Finally, the combinatorially selected peptide inhibitor capable of blocking abnormal protein aggregation will be characterized by biophysical, cellular and computational methods.

Oxidative folding and preparation of α-conotoxins for use in high-throughput structure-activity relationship studies.

α-Conotoxins are peptide neurotoxins that selectively inhibit various subtypes of nicotinic acetylcholine receptors. They are important research tools for studying numerous pharmacological disorders, with profound potential for developing drug leads for treating pain, tobacco addiction, and other conditions. They are characterized by the presence of two disulfide bonds connected in a globular arrangement, which stabilizes a bioactive helical conformation. Despite extensive structure-activity relationship studies that have produced α-conotoxin analogs with increased potency and selectivity towards specific nicotinic acetylcholine receptor subtypes, the efficient production of diversity-oriented α-conotoxin combinatorial libraries has been limited by inefficient folding and purification procedures. We have investigated the optimized conditions for the reliable folding of α-conotoxins using simplified oxidation procedures for use in the accelerated production of synthetic combinatorial libraries of α-conotoxins. To this end, the effect of co-solvent, redox reagents, pH, and temperature on the proportion of disulfide bond isomers was determined for α-conotoxins exhibiting commonly known Cys loop spacing frameworks. In addition, we have developed high-throughput 'semi-purification' methods for the quick and efficient parallel preparation of α-conotoxin libraries for use in accelerated structure-activity relationship studies. Our simplified procedures represent an effective strategy for the preparation of large arrays of correctly folded α-conotoxin analogs and permit the rapid identification of active hits directly from high-throughput pharmacological screening assays.

Biomolecular interactions and tools for their recognition: focus on the quartz crystal microbalance and its diverse surface chemistries and applications.

Interactions between molecules are ubiquitous and occur in our bodies, the food we eat, the air we breathe, and myriad additional contexts. Although numerous tools are available for the recognition of biomolecular interactions, such tools are often limited in their sensitivity, expensive, and difficult to modify for various uses. In contrast, the quartz crystal microbalance (QCM) has sub-nanogram detection capabilities, is label-free, is inexpensive to create, and can be readily modified with a number of diverse surface chemistries to detect and characterize diverse interactions. To maximize the versatility of the QCM, scientists need to know available methods by which QCM surfaces can be modified. Therefore, in addition to summarizing the various tools currently used for biomolecular recognition, explicating the fundamental principles of the QCM as a tool for biomolecular recognition, and comparing the QCM with other acoustic sensors, we systematically review the numerous types of surface chemistries-including hydrophobic bonds, ionic bonds, hydrogen bonds, self-assembled monolayers, plasma-polymerized films, photochemistry, and sensing ionic liquids-used to functionalize QCMs for various purposes. We also review the QCM's diverse applications, which include the detection of gaseous species, detection of carbohydrates, detection of nucleic acids, detection of non-enzymatic proteins, characterization of enzymatic activity, detection of antigens and antibodies, detection of cells, and detection of drugs. Finally, we discuss the ultimate goals of and potential barriers to the development of future QCMs.

Small-molecule peptides inhibit Z alpha1-antitrypsin polymerization.

The Z variant of 1-antitrypsin (AT) polymerizes within the liver and gives rise to liver cirrhosis and the associated plasma deficiency leads to emphysema. In this work, a combinatorial approach based on the inhibitory mechanism of (alpha1)-AT was developed to arrest its pathogenic polymerization. One peptide, Ac-TTAI-NH(2), emerged as the most tight-binding ligand for Z (alpha1)-AT. Characterization of this tetrapeptide by gel electrophoresis and biosensor analysis revealed its markedly improved binding specificity and affinity compared with all previously reported peptide inhibitors. In addition, the peptide is not cytotoxic to lung cell lines. A model of the peptide-protein complex suggests that the peptide interacts with nearby residues by hydrogen bonds, hydrophobic interactions, and cavity-filling stabilization. The combinatorially selected peptide not only effectively blocks the polymerization but also promotes dissociation of the oligomerized (alpha1)-AT. These results are a significant step towards the potential treatment of Z (alpha1)-AT related diseases.

Discovery of a potent and selective α3ß4 nicotinic acetylcholine receptor antagonist from an α-conotoxin synthetic combinatorial library.

α-Conotoxins are disulfide-rich peptide neurotoxins that selectively inhibit neuronal nicotinic acetylcholine receptors (nAChRs). The α3ß4 nAChR subtype has been identified as a novel target for managing nicotine addiction. Using a mixture-based positional-scanning synthetic combinatorial library (PS-SCL) with the α4/4-conotoxin BuIA framework, we discovered a highly potent and selective α3ß4 nAChR antagonist. The initial PS-SCL consisted of a total of 113 379 904 sequences that were screened for α3ß4 nAChR inhibition, which facilitated the design and synthesis of a second generation library of 64 individual α-conotoxin derivatives. Eleven analogues were identified as α3ß4 nAChR antagonists, with TP-2212-59 exhibiting the most potent antagonistic activity and selectivity over the α3ß2 and α4ß2 nAChR subtypes. Final electrophysiological characterization demonstrated that TP-2212-59 inhibited acetylcholine evoked currents in α3ß4 nAChRs heterogeneously expressed in Xenopus laevis oocytes with a calculated IC50 of 2.3 nM and exhibited more than 1000-fold selectivity over the α3ß2 and α7 nAChR subtypes. As such, TP-2212-59 is among the most potent α3ß4 nAChRs antagonists identified to date and further demonstrates the utility of mixture-based combinatorial libraries in the discovery of novel α-conotoxin derivatives with refined pharmacological activity.

Blocking formation of large protein aggregates by small peptides.

Abnormal protein aggregation is responsible for a variety of human disorders, including Alzheimer's disease, Creutzfeldt-Jakob disease, systemic amyloidosis, and α1-antitrypsin deficiency (AATD). These diseases are collectively termed conformational diseases and many of them are lethal and have no cure to date. The pathogenesis of these clinical conditions shares a common mechanism of disease, i.e., formation of large protein tangles through intermolecular linkages, cross-ß-sheets in particular. These tenacious aggregates are difficult to eradicate and accumulate in cells or tissues over affected individual's lifetime, which eventually leads to devastating consequences. The chronic process is commonly underdiagnosed at an early stage and the prevalence of conformational disease is higher than generally realized. AATD is a typical conformational disease causing both lung and liver disorders, and the World Health Organization (WHO) has highlighted the healthcare problem in 1996. The mechanism of AATD has been unraveled and it serves as an excellent model for the study of conformational disease. Point mutations render α1-antitrypsin susceptible to self-aggregation and form stacked ß-sheets, which is the hallmark of AATD and other conformational diseases. The key to attenuating these diseases is searching for small molecules capable of preventing the formation of large aggregates and dissociating the pre-existing oligomers. To this end, a chemical approach has been developed to tackle the biological problem.

The chemical synthesis of α-conotoxins and structurally modified analogs with enhanced biological stability.

α-Conotoxins are peptide neurotoxins isolated from the venom ducts of carnivorous marine cone snails that exhibit exquisite pharmacological potency and selectivity for various nicotinic acetylcholine receptor subtypes. As such, they are important research tools and drug leads for treating various diseases of the central nervous system, including pain and tobacco addiction. Despite their therapeutic potential, the chemical synthesis of α-conotoxins for use in structure-activity relationship studies is complicated by the possibility of three disulfide bond isomers, where inefficient folding methods can lead to a poor recovery of the pharmacologically active isomer. In order to achieve higher yields of the native isomer, especially in high-throughput syntheses it is necessary to select appropriate oxidative folding conditions. Moreover, the poor biochemical stability exhibited by α-conotoxins limits their general therapeutic applicability in vivo. Numerous strategies to enhance their stability including the substitution of disulfide bond with diselenide bond and N-to-C cyclization via an oligopeptide spacer have successfully overcome these limitations. This chapter describes methods for performing both selective and nonselective disulfide bond oxidation strategies for controlling the yields and formation of α-conotoxin disulfide bond isomers, as well as methods for the production of highly stable diselenide-containing and N-to-C cyclized conotoxin analogs.

Targeting serpins in high-throughput and structure-based drug design.

Native, metastable serpins inherently tend to undergo stabilizing conformational transitions in mechanisms of health (e.g., enzyme inhibition) and disease (serpinopathies). This intrinsic tendency is modifiable by ligand binding, thus structure-based drug design is an attractive strategy in the serpinopathies. This can be viewed as a labor-intensive approach, and historically, its intellectual attractiveness has been tempered by relatively limited success in development of drugs reaching clinical practice. However, the increasing availability of a range of powerful experimental systems and higher-throughput techniques is causing academic and early-stage industrial pharmaceutical approaches to converge. In this review, we outline the different systems and techniques that are bridging the gap between what have traditionally been considered distinct disciplines. The individual methods are not serpin-specific. Indeed, many have only recently been applied to serpins, and thus investigators in other fields may have greater experience of their use to date. However, by presenting examples from our work and that of other investigators in the serpin field, we highlight how techniques with potential for automation and scaling can be combined to address a range of context-specific challenges in targeting the serpinopathies.

Development of two alginate-based wound dressings.

Two types of new alginate-based wound dressings, Type-AP and Type-AE, were fabricated by the EDC-activated crosslinking of alginate with Polyethyleneimine and Ethylenediamine, respectively. As compared with the commercial non-woven wound dressing, Kaltostat, both Type-AP and Type-AE dressings had higher degradation temperature, lower calcium content, and a sponge-like macroporous structure. In addition, these two alginate-based dressings had higher mechanical stress (12.37 +/- 1.72 and 6.87 +/- 0.5 MPa for Type-AP and -AE, respectively) and higher water vapor transmission rates (both about 3,500 g/m2/day) than Kaltostat (0.87 +/- 0.12 MPa and 2,538 g/m2/day). Fibroblasts proliferated faster on these two newly developed wound dressings at a higher rate as compared with that on Kalostat dressing. The results of animal study showed that the wounds treated with either Type-AP or Type-AE dressings healed faster than Kaltostat with less encapsulation of residuals by fibrous tissue and more neo-capillary formation. These two newly developed Type-AP and Type-AE porous wound dressings thus have great potential for clinical applications.

Quantitative measurements of vancomycin binding to self-assembled peptide monolayers on chips by quartz crystal microbalance.

This paper describes direct binding of a small vancomycin to peptide ligands immobilized on a sensor chip using quartz crystal microbalance. In this study, the binding ligands were composed of three components: a molecular recognition element (peptide), a conformationally flexible and hydrophilic linker, and a long-chain alkanethiol. These peptide ligands were used to establish the well-packed, self-assembled monolayers on quartz chips and could be readily synthesized using conventional organic chemistry protocols. Results of quartz crystal microbalance measurements showed that vancomycin specifically associated with the d-Ala-d-Ala-containing peptide with an affinity of 3.2+/-0.3 microM and was, as expected, completely inactive to the self-assembled monolayer presenting l-Ala-l-Ala peptide. The dissociation constant obtained correlated well with values reported in literature and was further confirmed by surface plasmon resonance measurement (2.7+/-0.7 microM). The technique used in this study should be applicable to both peptidyl and nonpeptidyl ligands of greater complexity than that used here. This method is practical, it provides quantitative binding information, and complicated analysis is avoided.

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip.

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.

Identification of a 4-mer peptide inhibitor that effectively blocks the polymerization of pathogenic Z alpha1-antitrypsin.

alpha(1)-Antitrypsin (AT) is a major proteinase inhibitor within the lung. The Z variant of AT (E342K) polymerizes within the liver and lung, resulting in hepatic aggregation of AT and tissue deficiency, predisposing to early onset of cirrhosis and emphysema, respectively. Polymerization of the aberrant protein can be prevented in vitro by specific peptides such as FLEAIG. This peptide serves as a lead molecule to design a shorter peptide that may be effective as a therapeutic agent. In this study we employed a systematic chemical approach using alanine scanning of Ac-FLEAIG-OH and subsequent peptide shortening to study the binding of shorter peptides to Z-AT. While two additional 6-mer peptides Ac-FLAAIG-OH and Ac-FLEAAG-OH were found to bind to Z-AT, their daughter peptides Ac-FLEAA-NH(2) and Ac-FLAA-NH(2) also bound avidly to Z-AT and prevented polymerization of the protein. Further comparative studies revealed that the binding of Ac-FLAA-NH(2) was more specific for Z-AT. The peptide-AT complex formation was enhanced by the presence of C-terminal amide group on the peptide, and circular dichroism analysis demonstrated that a random coil rather than a beta-helical conformation favored binding of the peptide to AT. In summary, this study has identified novel small peptides that inhibit Z-AT polymerization, and are a significant advance towards the treatment of Z-AT-related cirrhosis and emphysema.

Using surface plasmon resonance to directly determine binding affinities of combinatorially selected cyclopeptides and their linear analogs to a streptavidin chip.

In our recent report, several HPQ-containing streptavidin ligands were identified from a structurally constrained combinatorial library, and the relative affinities in IC(50) of these tight-binding ligands were revealed by a captured enzyme-linked immunosorbent assay. In the present work, surface plasmon resonance was employed to directly evaluate the binding affinities between immobilized streptavidin and combinatorially selected ligands. The equilibrium dissociation constants and kinetic on/off rates of a previously identified N-to-side chain and newly synthesized N-to-C cyclopeptides were readily deduced using Scatchard analysis and computational simulation. It was found that both cyclopeptides bound streptavidin far more tightly than its linear counterpart ( approximately 1000-fold), while the reversed (QPH) linear and cyclic peptidyl ligands were hardly recognized by streptavidin. Consequently, not only was the binding specificity of synthetic ligands distinguished qualitatively but also the entropic advantage of conformationally constrained cyclopeptides over their linear forms was demonstrated quantitatively by surface plasmon resonance.