An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins.

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.

Blended polar/nonpolar peptide conjugate interferes with human insulin amyloid-mediated cytotoxicity.

Insulin, a peptide hormone and a key regulator of blood glucose level, is routinely administered to type-I diabetic patients to achieve the required glycemic control. Insulin aggregation and ensuing amyloidosis has been observed at repeated insulin injection sites and in injectable formulations. The latter occurs due to insulin agglomeration during shipping and storage. Such insulin amyloid leads to enhanced immunogenicity and allow potential attachment to cell membranes leading to cell permeability and apoptosis. Small molecule inhibitors provide useful interruption of this process and inhibit protein misfolding as well as amyloid formation. In this context, we report the propensity of a palmitoylated peptide conjugate to inhibit insulin aggregation and amyloid-mediated cytotoxicity, via designed interference with polypeptide interfacial interactions.

The ABC of Insulin: The Organic Chemistry of a Small Protein.

Insulin is a small protein crucial for regulating the blood glucose level in all animals. Since 1922 it has been used for the treatment of patients with diabetes. Despite consisting of just 51 amino acids, insulin contains 17 of the proteinogenic amino acids, A- and B-chains, three disulfide bridges, and it folds with 3 α-helices and a short ß-sheet segment. Insulin associates into dimers and further into hexamers with stabilization by Zn2+ and phenolic ligands. Selective chemical modification of proteins is at the forefront of developments in chemical biology and biopharmaceuticals. Insulin's structure has made it amenable to organic and inorganic chemical reactions. This Review provides a synthetic organic chemistry perspective on this small protein. It gives an overview of key chemical and physico-chemical aspects of the insulin molecule, with a focus on chemoselective reactions. This includes N-acylations at the N-termini or at LysB29 by pH control, introduction of protecting groups on insulin, binding of metal ions, ligands to control the nano-scale assembly of insulin, and more.

Supramolecular Photoactivatable Anticancer Hydrogels.

A photoactivatable dopamine-conjugated platinum(IV) anticancer complex (Pt-DA) has been incorporated into G-quadruplex G4K+ borate hydrogels by using borate ester linkages (Pt-G4K+B hydrogel). These were characterized by 11B NMR, attenuated total reflection Fourier transform infrared spectroscopy, circular dichroism, scanning electron microscopy and transmission electron microscopy. Microscopy investigations revealed the transformation of an extended fiber assembly into discrete flakes after incorporation of Pt-DA. Pt-DA showed photocytotoxicity against cisplatin-resistant A2780Cis human ovarian cancer cells (IC50 74 µM, blue light) with a photocytotoxic index <2, whereas Pt-G4K+B hydrogels exhibited more potent photocytotoxicity (IC50 3 µM, blue light) with a photocytotoxic index >5. Most notably, Pt-DA and Pt-G4K+B hydrogels show selective phototoxicity for cancer cells versus normal fibroblast cells (MRC5).

Inhibition of human and bovine insulin fibril formation by designed peptide conjugates.

The aggregation of insulin, to afford amyloidogenic fibers, is a well-studied phenomenon, which has interesting biological ramifications and pharmaceutical implications. These fibers have been ascribed an intriguing role in certain disease states and stability of pharmaceutical formulations of this hormone. The present study describes the design and inhibitory effects of novel peptide conjugates toward fibrillation of insulin as investigated by thioflavin T assay, circular dichroism (CD), and atomic force microscopy (AFM). Possible interaction of insulin with peptide-based fibrillation inhibitors is also probed by other solution phase studies, which reveal an important role of aromatic π-π interactions in the inhibition process. CD studies suggest that a freshly prepared solution of insulin, rich in α-helices, transforms into a ß-sheet structure upon aggregation, which gets perturbed in the presence of synthesized inhibitors. Therefore, these newly designed peptides could serve as potential leads as inhibitors of insulin aggregation.

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies.

Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.

COVID-19 image classification using deep learning: Advances, challenges and opportunities.

Corona Virus Disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2), is a highly contagious disease that has affected the lives of millions around the world. Chest X-Ray (CXR) and Computed Tomography (CT) imaging modalities are widely used to obtain a fast and accurate diagnosis of COVID-19. However, manual identification of the infection through radio images is extremely challenging because it is time-consuming and highly prone to human errors. Artificial Intelligence (AI)-techniques have shown potential and are being exploited further in the development of automated and accurate solutions for COVID-19 detection. Among AI methodologies, Deep Learning (DL) algorithms, particularly Convolutional Neural Networks (CNN), have gained significant popularity for the classification of COVID-19. This paper summarizes and reviews a number of significant research publications on the DL-based classification of COVID-19 through CXR and CT images. We also present an outline of the current state-of-the-art advances and a critical discussion of open challenges. We conclude our study by enumerating some future directions of research in COVID-19 imaging classification.

Metal-ion coordinated self-assembly of human insulin directs kinetics of insulin release as determined by preclinical SPECT/CT imaging.

Human insulin (HI) has fascinating metal-facilitated self-assembly properties that are essential for its biological function. HI has a natural Zn2+ binding site and we have previously shown that covalently attached abiotic ligands (e.g., bipyridine, terpyridine) can lead to the formation of nanosized oligomeric structures through the coordination of metal ions. Here we studied the hypothesis that metal ions can be used to directly control the pharmacokinetics of insulin after covalent attachment of an abiotic ligand that binds metal ions. We evaluated the pharmacokinetics (PK) and biodistribution of HI self-assemblies directed by metal ion coordination (i.e., Fe2+/Zn2+, Eu3+/Zn2+, Fe2+/Co3+) using preclinical SPECT/CT imaging and ex vivo gamma counting. HI was site-specifically modified with terpyridine (Tpy) at the PheB1 or LysB29 position to create conjugates that bind either Fe2+ or Eu3+, while its natural binding site (HisB10) preferentially coordinates with either Zn2+ or Co3+. HI was also functionalized with trans-cyclooctene (TCO) opposite to Tpy at PheB1 or LysB29, respectively, to allow for tetrazine-TCO coupling via a tetrazine-modified DTPA followed by 111In-radiolabeling for SPECT/CT imaging. When the 111In-B29Tpy-HI conjugate was coordinated with Fe2+/Zn2+, its retention at the injection site 6 h after injection was ~8-fold higher than the control without the metal ions, while its kidney accumulation was lower. 111In-B1Tpy-HI showed comparable retention at the injection site 6 h after injection and slightly increased retention at 24 h. However, higher kidney accumulation and residence time of degraded 111In-B1Tpy-HI was observed compared to that of 111In-B29Tpy-HI. Quantitative PK analysis based on SPECT/CT images confirmed slower distribution from the injection site of the HI-metal ion assemblies compared to control HI conjugates. Our results show that the Tpy-binding site (i.e., PheB1 or LysB29) on HI and its coordination with the added metal ions (i.e., Fe2+/Zn2+ or Fe2+/Co3+) directed the distribution half-life of HI significantly. This clearly indicates that the PK of insulin can be controlled by complexation with different metal ions.

Automated detection of COVID-19 from CT scan using convolutional neural network.

Under the prevailing circumstances of the global pandemic of COVID-19, early diagnosis and accurate detection of COVID-19 through tests/screening and, subsequently, isolation of the infected people would be a proactive measure. Artificial intelligence (AI) based solutions, using Convolutional Neural Network (CNN) and exploiting the Deep Learning model's diagnostic capabilities, have been studied in this paper. Transfer Learning approach, based on VGG16 and ResNet50 architectures, has been used to develop an algorithm to detect COVID-19 from CT scan images consisting of Healthy (Normal), COVID-19, and Pneumonia categories. This paper adopts data augmentation and fine-tuning techniques to improve and optimize the VGG16 and ResNet50 model. Further, stratified 5-fold cross-validation has been conducted to test the robustness and effectiveness of the model. The proposed model performs exceptionally well in case of binary classification (COVID-19 vs. Normal) with an average classification accuracy of more than 99% in both VGG16 and ResNet50 based models. In multiclass classification (COVID-19 vs. Normal vs. Pneumonia), the proposed model achieves an average classification accuracy of 86.74% and 88.52% using VGG16 and ResNet50 architectures as baseline, respectively. Experimental results show that the proposed model achieves superior performance and can be used for automated detection of COVID-19 from CT scans.

A colored hydrophobic peptide film based on self-assembled two-fold topology.

Structural colors are abundant in nature and bear advantages over pigment-based colors, such as higher durability, brilliance and often physical hydrophobicity, thus underlying their vast potential for technological applications. Recently, biomimetics of complex natural topologies resulting in such effects has been extensively studied, requiring advanced processing and fabrication techniques. Yet, artificial topologies combining structural coloration and hydrophobicity have not been reported. Herein, we present the bottom-up fabrication of short self-assembling peptides as surface covering films, resulting in an easily achievable multilevel morphology of primary structures in a foam-like enclosure, producing structural colors and hydrophobicity. We demonstrate simple techniques allowing controlled coloration of different surfaces while maintaining an >100° water contact angle (WCA). The new artificial topology is much simpler than the natural counterparts and is not limited to a specific peptide, thus allowing the design of modular materials with unparalleled multifunctionalities and potential for further tuning and modifications.

Controlling the fractal dimension in self-assembly of terpyridine modified insulin by Fe2+ and Eu3+ to direct in vivo effects.

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at LysB29 with the tridentate chelator 2,2':6',2''-terpyridine (Tpy) is described. SA of this new insulin analog (LysB29Tpy-HI) in the presence of the metal ions Fe2+ and Eu3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe2+ yielded small magenta-colored discrete non-native assemblies, while Eu3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu3+ coordination geometry that included two water molecules. SAXS suggested that LysB29Tpy-HI with Fe2+ oligomerized to a discrete, roughly octameric species, while LysB29Tpy-HI with Eu3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu3+ concentration. We propose that this is a consequence of Eu3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. LysB29Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.

Self-assembly of a Sequence-shuffled Short Peptide Amphiphile Triggered by Metal Ions into Terraced Nanodome-like Structures.

We highlight the structural diversity of strategically designed two short peptide amphiphiles (sPAs) and describe their structure-function relationship studies. The shuffling of two key amino acids, that is, tyrosine and phenylalanine, in a designed sPA lead to a pair of constitutional isomers. Such small and strategic alteration can bring a substantial change in the self-assembling pattern. Inspired from the naturally occurring metallopeptides, bioactive transition-metal ions were used for constructing the unusual nanostructures. Use of appropriate metal ions created bigger differences between the properties of these isomers and hence the self-assembly. Coordination of appropriate transition metal ions modifies the internal nanoscale structures of sPA, thus leading to the formation of vertically stacked terraced layers with decreasing size, which possess a high degree of dimensional regularity. We propose that such metal-induced terraced nanodome-like hierarchical self-assembly may have relevance for specific biotechnology applications.

Synthesis, characterization and in vitro antitrypanosomal activities of new carboxamides bearing quinoline moiety.

The reported toxicities of current antitrypanosomal drugs and the emergence of drug resistant trypanosomes underscore the need for the development of new antitrypanosomal agents. We report herein the synthesis and antitrypanosomal activity of 24 new amide derivatives of 3-aminoquinoline, bearing substituted benzenesulphonamide. Nine of the new derivatives showed comparable antitrypanosomal activities at IC50 range of 1-6 nM (melarsoprol 5 nM). Compound 11n and 11v are more promising antitrypanosomal agents with IC50 1.0 nM than the rest of the reported derivatives. The novel compounds showed satisfactory predicted physico-chemical properties including oral bioavailability, permeability and transport properties.

Combining Experimental and Simulation Techniques to Understand Morphology Control in Pentapeptide Nanostructures.

A controlled route toward biocompatible nanostructures has immense relevance for drug delivery and tissue engineering. We present an experimental-computational study identifying factors that govern the formation of well-defined aggregates by self-assembled pentapeptides, using single amino acid substitution. A subtle interplay between peptide rigidity/flexibility, hydrogen-bonding capacity, partitioning of aromatic side chains, and influence of dimerization determines the formation of ordered and disordered aggregate structures, and shifts the balance between the emergence of spherical or ill-defined morphologies.

Modulating peptide amphiphile morphology by gold nanocolloids.

We report facile synthesis and structural study of ditryptophan-based short peptide amphiphilic (sPA) constructs, which were used to synthesize gold nanostructures, in the presence of sunlight. This process occurs concurrently with morphological transformation, followed by encapsulation of gold nanostructures to afford hybrid scaffolds of interest.

Thermoplasmonic effect of silver nanoparticles modulates peptide amphiphile fiber into nanowreath-like assembly.

This study demonstrates the beneficial role of di-tryptophan containing short peptide amphiphiles (sPA), for the synthesis and stabilization of AgNPs in the presence of sunlight followed by garlanding of AgNPs along the fibrous network of sPA. Such hybrid structures were precisely and selectively moulded into a nanowreath-type morphology due to the thermoplasmonic effect of AgNPs, and can be used for several bio-nanotechnological applications.

Controlling in Vitro Insulin Amyloidosis with Stable Peptide Conjugates: A Combined Experimental and Computational Study.

Insulin aggregation, to afford amyloidogenic polypeptide fibrils, is an energetically driven, well-studied phenomenon, which presents interesting biological ramifications. These aggregates are also known to form around insulin injection sites and in diabetic patients suffering from Parkinson's disease. Such occurrences force considerable reduction in hormone activity and are often responsible for necrotic deposits in diabetic patients. Changes in physicochemical environment, such as pH, temperature, ionic strength, and mechanical agitation, affect insulin fibrillation, which also presents intrigue from the structural viewpoint. Several reports have tried to unravel underlying mechanisms concerning the aggregation process taking into account a three aromatic amino acid patch Phe(B24)-Phe(B25)-Tyr(B26) located in the C-terminal part of the B chain, identified as a key site for human insulin-receptor interaction. The present study describes design and inhibitory effects of novel peptide conjugates toward fibrillation of insulin as investigated by thioflavin T assay, circular dichroism, and AFM. Possible interaction of insulin with peptide-based fibrillation inhibitors reveals an important role of hydrophobic interactions in the inhibition process. Molecular dynamics simulation studies demonstrate that inhibitor D4 interacts with insulin residues from the helix and the C-terminal extended segment of chain B. These studies present a novel approach for the discovery of stable, peptide-based ligands as novel antiamyloidogenic agents for insulin aggregation.