Kainic Acid-Based Agonists of Glutamate Receptors: SAR Analysis and Guidelines for Analog Design.

A comprehensive survey of kainic acid analogs that have been tested for their biological activity is presented. Specifically, this review (1) gathers and compares over 100 kainoids according to a relative activity scale, (2) exposes structural features required to optimize affinity for kainate receptors, and (3) suggests design rules to create next-generation KA analogs. Literature SAR data are analyzed systematically and combined with the most recent crystallographic studies. In view of the renewed interest in neuroactive molecules, this review aims to help guide the efforts of organic synthesis laboratories, as well as to inform newcomers to KA/GluK research.

Characterization of novel kainic acid analogs as inhibitors of select microglial functions.

Alzheimer's disease (AD) is characterized by abnormal accumulation of extracellular amyloid beta protein (Aß) plaques and intracellular neurofibrillary tangles, as well as by a state of chronic inflammation in the central nervous system (CNS). Adverse activation of microglia, the brain immune cells, is believed to contribute to AD pathology including excessive neuronal death. Thus, normalizing immune functions of microglia could slow neurodegeneration, and identification of novel compounds capable of modifying microglial functions is an important goal. Since kainic acid (KA) has been shown to modulate microglial morphology and immune functions, we synthesized six new KA analogs (KAAs) and tested their effects on select microglial functions by using three different cell types as microglia models. Four of the KAAs at low micromolar concentrations inhibited secretion of cytotoxins, monocyte chemoattractant protein (MCP)-1, reactive oxygen species and nitric oxide (NO) by immune-stimulated microglia-like cells. We hypothesize that the effects of the novel KAAs on microglia-like cells are not mediated by KA receptors since their biological activity was distinct from that of KA in all assays performed. A structural similarity search identified aldose reductase (AR) as a potential target for the novel KAAs. This hypothesis was supported by use of AR inhibitor zopolrestat, which abolished the inhibitory effects of two KAAs on microglial secretion of NO. Since the newly developed KAAs inhibited pro-inflammatory and cytotoxic functions of microglia, they should be further investigated for their potential beneficial effect on neuroinflammation and neurodegeneration in AD animal models.

Visible Light Photoinitiation of Cell-Adhesive Gelatin Methacryloyl Hydrogels for Stereolithography 3D Bioprinting.

We present the first cell-attachable and visible-light-crosslinkable bioinks based on gelatin methacryloyl (GelMA) with eosin Y (EY) photoinitiation for stereolithography three-dimensional (3D) bioprinting. To develop a visible-light-crosslinkable hydrogel, we systematically studied five combinations of GelMA and EY photoinitiator with various concentrations. Their mechanical properties, microstructures, and cell viability and confluency after encapsulation were investigated rigorously to elucidate the effects of the EY and GelMA macromer concentrations on the characteristics of the hydrogel. Experimental results show that the compressive Young's modulus and pore size are positively affected by the concentration of EY, whereas the mass swelling ratio and cell viability are negatively affected. Increasing the concentration of GelMA helps in improving the compressive Young's modulus and cell attachment. We further employed the developed visible-light-based stereolithography bioprinting system to print the patterned cell-laden hydrogels to demonstrate the bioprinting applications of the developed hydrogel. We observed good cell proliferation and the formation of a 3D cellular network inside the printed pattern at day 5, which proves the great feasibility of using EY-GelMA as the bioinks for biofabrication and tissue engineering.

Synthesis of Kainoids and C4 Derivatives.

A unified stereoselective synthesis of 4-substituted kainoids is reported. Four kainic acid analogues were obtained in 8-11 steps with up to 54% overall yields. Starting from trans-4-hydroxy-l-proline, the sequence enables a late-stage modification of C4 substituents with sp2 nucleophiles. Stereoselective steps include a cerium-promoted nucleophilic addition and a palladium-catalyzed reduction. A 10-step route to acid 21a was also established to enable ready functionalization of the C4 position.

A Novel, Well-Resolved Direct Laser Bioprinting System for Rapid Cell Encapsulation and Microwell Fabrication.

A direct laser bioprinting (DLBP) system is introduced in this work. The DLBP system applies visible-laser-induced photo-crosslinking at a wavelength of 405 nm using the photoinitiator VA-086. It is shown that such a system can fabricate vertical structures with fine features (less than 50 µm) and high cell viability (greater than 95%). Experimental characterizations and theoretical simulations are presented, and good agreement is seen between the experiments and theory. The DLBP system is applied to the fabrication of (1) cell-laden hydrogel microgrids, (2) hydrogel microwells, as well as a test of (3) cell encapsulation, and (4) cell seeding. The DLBP system is found to be a promising tool for bioprinting.

Comparative study of gelatin methacrylate hydrogels from different sources for biofabrication applications.

Gelatin methacrylate (GelMA) hydrogel is a promising bioink for biofabrication applications due to its cost-effectiveness, ease of synthesis and biocompatibility to allow cell adhesion. However, the GelMA synthesized from a widely used porcine skin gelatin has a thermal gelation problem at room temperature. Here, we present thermally stable GelMA hydrogels at room temperature while maintaining the mechanical and biological properties comparable to porcine GelMA. The novel GelMA hydrogels were synthesized from fish skin and cold soluble gelatin. We systematically characterized the properties of the GelMA hydrogels from different sources. The properties include the degree of methacrylation, compressive Young's modulus, mass swelling ratio, viscosity, and cell adhesion and proliferation in 2D and 3D microenvironments. It has been found that the cold soluble GelMA was comparable to the porcine skin GelMA but could offer low viscosity and thermal stability at room temperature. We performed a droplet generation experiment to demonstrate the benefit of using the cold soluble GelMA for biofabrication. The cold soluble GelMA showed a more reliable and stable droplet fabrication process. Taken together, the cold soluble GelMA is a promising bioink solution and may greatly benefit the research in biofabrication.

An unnatural amino acid based fluorescent probe for phenylalanine ammonia lyase.

A fluorescent probe (2a-LP) based on an unnatural amino acid (UAA) is developed for the detection of phenylalanine ammonia lyase (PAL). In the presence of PAL, 2a-LP is catalytically deaminated to ortho-amino-transcinnamic acid (o-a-CA), which shows a remarkable "off­on" fluorescence signal. Thus, the probe 2a-LP enables direct visualization of the PAL activity in tomato under UV illumination and has potential in vitro assays.