Since its discovery and isolation, exogenous insulin has dramatically changed the outlook for patients with diabetes. However, even when patients strictly follow an insulin regimen, serious complications can result as patients experience both hyperglycemic and hypoglycemic states. Several chemically or genetically modified insulins have been developed that tune the pharmacokinetics of insulin activity for personalized therapy. Here, we demonstrate a strategy for the chemical modification of insulin intended to promote both long-lasting and glucose-responsive activity through the incorporation of an aliphatic domain to facilitate hydrophobic interactions, as well as a phenylboronic acid for glucose sensing. These synthetic insulin derivatives enable rapid reversal of blood glucose in a diabetic mouse model following glucose challenge, with some derivatives responding to repeated glucose challenges over a 13-h period. The best-performing insulin derivative provides glucose control that is superior to native insulin, with responsiveness to glucose challenge improved over a clinically used long-acting insulin derivative. Moreover, continuous glucose monitoring reveals responsiveness matching that of a healthy pancreas. This synthetic approach to insulin modification could afford both long-term and glucose-mediated insulin activity, thereby reducing the number of administrations and improving the fidelity of glycemic control for insulin therapy. The described work is to our knowledge the first demonstration of a glucosebinding modified insulin molecule with glucose-responsive activity verified in vivo.diabetes | protein modification | molecular engineering | glucose sensing | smart therapy

A highly site-selective modification of peptides/proteins with aldehydes or carbohydrates under mild conditions was achieved.

The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.

Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormone's hexameric storage form. Removal of this segment (residues B23-B30) in human insulin results in substantial loss of receptor affinity. Here, we found that Con-Ins G1 is monomeric, strongly binds the human insulin receptor and activates receptor signaling. Con-Ins G1 thus is a naturally occurring B-chain-minimized mimetic of human insulin. Our crystal structure of Con-Ins G1 reveals a tertiary structure highly similar to that of human insulin and indicates how Con-Ins G1's lack of an equivalent to the key receptor-engaging residue Phe is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins.

Summary Cytokine-induced beta-cell apoptosis is important to the etiology of type-1 diabetes. Although previous reports have shown that general inhibitors of histone deacetylase (HDAC) activity, such as suberoylanilide hydroxamic acid and trichostatin A, can partially prevent beta-cell death, they do not fully restore beta-cell function. To understand HDAC isoform selectivity in beta cells, we measured the cellular effects of eleven structurally diverse HDAC inhibitors on cytokine-induced apoptosis in the rat INS-1E cell line. All eleven compounds restored ATP levels and reduced nitrite secretion. However, caspase-3 activity was reduced only by MS-275 and CI-994, both of which target HDAC1, 2, and 3. Importantly, both MS-275 and genetic knock-down of Hdac3 alone were sufficient to restore glucose-stimulated insulin secretion in the presence of cytokines. These results suggest that HDAC3-selective inhibitors may be effective in preventing cytokine-induced beta-cell apoptosis.

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone snail venom with moderate human-insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins—a human des-octapeptide insulin analog—as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a novel direction for therapeutic insulin development.

The lasers used were a XeCl laser operating at 308 nm, s pulse » 30 ns (Lambda Physik, EMG 201 MSC), and an Nd:yttrium aluminum garnet (YAG) laser, operating at the second harmonic, k = 532 nm, s pulse » 5 ns (B. M. Industries, Serie 5000).Experimental Methods: For the macroscopic mechanical actuation of the films, the laser beams were weakly focused onto the front surface of the film so that the entire surface area of the film was homogeneously irradiated (spot area: 1.5 mm 2.5 mm). A digital camera, placed at an angle of~85 to the normal to the surface, was used for monitoring the bending of the films.In the fluorescence experiments, the UV laser beam was weakly focused almost perpendicularly onto the sample in a 1.2 mm diameter spot using a quartz spherical lens (f = +500 mm). The green laser beam was also weakly focused onto exactly the same area where the UV beam is focused, almost perpendicularly to the sample. Irradiation of the samples was performed in ambient atmosphere. The fluorescence of the stable trans MC photoproducts was probed by excitation with laser pulses of 532 nm of very low fluence (F laser £ 4 mJ cm ±2 ). Photoproduct formation by the probe beam was negligible, and all recorded fluorescence can be assumed to derive exclusively from photoproducts formed by the preceding pump laser pulses. A relatively long delay (of the order of several seconds) between the pump and probe pulses was employed for ensuring that the fast-formed, metastable isomers had turned into the final/stable isomers. On the other hand, the monitoring of the metastable photoproducts was achieved by collecting the emission signals from the samples with zero delay with respect to the pump laser pulses.The induced emission was collected by an optical fiber placed nearly perpendicular to the substrate and~1±2 cm from its surface. An x±y micrometer ensured accurate positioning of the optical fiber relative to the irradiated spot. The light from the fiber was spectrally analyzed in a 20 cm grating spectrograph and recorded by a gated optical multichannel analyzer (OMA III system, EG&G PARC Model 1406). A gate time value of 1 ls was applied. Highly Efficient UV Organic Light-Emitting Devices Based on Bi(9,9-diarylfluorene)s** By Teng-Chih Chao, Yu-Ting Lin, Chen-Yu Yang, Tsung Shi Hung, Hung-Chieh Chou, Chung-Chih Wu,* and Ken-Tsung Wong*The rapid advancement of organic light-emitting devices (OLEDs) in recent years has extended the emission wavelengths over the whole visible range and has enabled realization of full-color OLED displays. Extending the emission of OLEDs into even shorter ultraviolet wavelengths, however, has not progressed as well, although compact and efficient UV emitters would find many uses in biological/fluorescent sensors, [1±2] in full-color displays by generating blue-to-red emission through pumping luminescent materials, [3] or in high-density information storage devices. [4±6] In addition, wide-gap materials are also of increasing importance in triplet emitters for electrophosphorescence. [7±9] In th...

The application of thiol-yne/thiol-ene reactions to synthesize mono- and bicyclic-stapled peptides and proteins is reported. First, a thiol-ene-based peptide-stapling method in aqueous conditions was developed. This method enabled the efficient stapling of recombinantly expressed coil-coiled proteins. The resulting stapled protein demonstrated higher stability in its secondary structure than the unstapled version. Furthermore, a thiol-yne coupling was performed by using an α,ω-diyne to react with two cysteine residues to synthesize a stapled peptide with two vinyl sulfide groups. The stapled peptide could further react with another biscysteine peptide to yield a bicyclic stapled peptide with enhanced properties. For example, the cell permeability of a stapled peptide was further increased by appending an oligoarginine cell-penetrating peptide. The robustness and versatility of thiol-yne/thiol-ene reactions that can be applied to both synthetic and expressed peptides and proteins were demonstrated.