Design, synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor.

An absolute or relative deficiency of pancreatic ß-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect ß-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces ß-cell replication and attenuate ß-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic ß-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional ß-cell mass.

GPER induces mitochondrial fission through p44/42 MAPK - Drp1 pathway in breast cancer cells.

Understanding GPER biology in breast cancer is rather limited in compassion to the classic estrogen receptors. Mitochondrial dynamics play a critical role in determining cell survival and death under various microenvironmental conditions. We present evidence that GPER-induce mitochondrial fission in breast cancer cells. GPER mediated mitochondrial fission through activating Drp1 by phosphorylating S616 residue and down-regulates fusion proteins Mfn1 and Mfn2 levels. GPER-induced Drp1 activation mediated by p44/42 MAPK and inhibition of this signalling axis completely reverse the mitochondrial fission induced by GPER. Further, mitochondrial fission is required for GPER-induced cell death in breast cancer cells. To conclude, GPER induces mitochondrial fission through p44/42 MAPK - Drp1 signalling, and mitochondrial fission is critical for GPER-induced cell death in breast cancer cells. GENERAL SIGNIFICANCE: First time we report GPER's role in mitochondrial dynamics in cancer cells. Mitochondrial dynamics play a critical role in cancer progression including tamoxifen resistance. Exploring a detailed mechanistic understanding of GPER signalling may help to design new therapy for advanced cancers.

The emergence and advancement of Tsuji-Trost reaction triggered carbon monoxide recognition and bioimaging.

Considering that carbon monoxide is both a vital gasotransmitter and an obnoxious gas, tremendous efforts have been dedicated toward its recognition through various methods. However, the fluorescent light-up approach through the exploration of optical markers remains one of the most convenient methods owing to its several advantages. Amongst the different approaches towards the development of CO responsive optically active molecular markers, the Tsuji-Trost reaction-based CO recognition strategy has remained one of the most significant areas of interest across researchers working in this field. However, there have been no attempts to exclusively summarize the commendable work done in this area yet. The current review, therefore, attempts to summarize the developments of various optical probes following this reaction strategy until the year 2022. This review provides detailed mechanistic insights into the Tsuji-Trost mediated CO detection strategy. Besides, discussions on the strategic development and employment of probes based on various allyl derivatives - allyl carbamate/carbonate/ethers - will provide a thorough understanding of the detection method. The significant advancements of the Tsuji-Trost reaction as an interesting strategy that is accepted and extensively explored for monitoring CO in various media including air, aqueous solutions and living systems have been elaborately discussed. Various potential applications and utilization of these developed fluorogenic probes for tracing CO in different living systems have been examined systematically. Moreover, monitoring of exogenous/endogenous CO levels, modulation of intracellular CO concentration under various induced conditions and bioimaging of CO in in vivo models have also been detailed here. Briefly, this review summarizes the current prospects of this detection method and the future directions in related fields.

Sortase A: A chemoenzymatic approach for the labeling of cell surfaces.

Sortase A, a transpeptidase enzyme is present in many Gram-positive bacteria and helps in the recruitment of the cell surface proteins. Over the last two decades, Sortase A has become an attractive tool for performing in vivo and in vitro ligations. Sortase A-mediated ligation has continuously been used for its specificity, robustness, and highly efficient nature. These properties make it a popular choice among protein engineers as well as researchers from different fields. In this review, we give an overview of Sortase A-mediated ligation of various molecules on the cell surfaces, which can have diverse applications in interdisciplinary fields.

Sensing cholesterol-induced rigidity in model membranes with time-resolved fluorescence spectroscopy and microscopy.

Here, we report the characterization of cholesterol levels on membrane fluidity with a twisted intramolecular charge transfer (TICT) membrane dye, namely DI-8-ANEPPS, using fluorescence lifetime techniques such as time-correlated single photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM). The characterized liposomes comprised a 3 : 1 ratio of POPC and POPG, respectively, 1% DI-8-ANEPPS, and increasing cholesterol levels from 0% to 50%. Fluorescence lifetime characterization revealed that increasing the cholesterol levels from 0% to 50% increases the fluorescence lifetime of DI-8-ANEPPS from 2.36 ns to 3.65 ns, a 55% increment. Such lengthening in the fluorescence lifetime is concomitant with reduced Stokes shifts and higher quantum yield, revealing that localized excitation (LE) dominates over TICT states with increased cholesterol levels. Fluorescence anisotropy measurements revealed a less isotropic environment in the membrane upon increasing cholesterol levels, suggesting a shift from liquid-disorder (Lα) to liquid-order (LO) upon adding cholesterol. Local electrostatic and dipole characterization experiments revealed that changes in the zeta-potential (ζ-potential) and transmembrane dipole potential (Ψd) induced by changes in cholesterol levels or the POPC : POPG ratio play a minimal role in the fluorescence lifetime outcome of DI-8-ANEPPS. Instead, these results indicate that the cholesterol's effect in restricting the degree of movement of DI-8-ANEPPS dominates its photophysics over the cholesterol effect on the local dipole strength. We envision that time-resolved spectroscopy and microscopy, coupled with TICT dyes, could be a convenient tool in exploring the complex interplay between membrane lipids, sterols, and proteins and provide novel insights into membrane fluidity, organization, and function.

NIR-I emissive cyanine derived molecular probe for selective monitoring of hepatic albumin levels during hyperglycemia.

In this study, we report a small molecule optical marker BI-CyG derived from the structural engineering of a cyanine scaffold. The developed probe offers suitable advantages over existing cyanine-based albumin specific probes in terms of its excitation and emission wavelengths, which are 760 and 830-832 nm, respectively. Structural tuning of the cyanine architecture leading to extended π-conjugation and resulting in a suitable bathochromic shift in the emission wavelength of the probe is represented in this study. The probe besides emitting in the NIR region, also possesses the desirable characteristics of being a potential target selective optical marker, as established from various biophysical studies. Molecular modelling and simulation studies provided critical insights into the binding of the probe in the protein microenvironment, which was further supported by experimental studies. The probe displayed intracellular albumin selectivity and was utilized for demonstrating alteration in albumin levels in pathological states such as hyperglycemia in hepatic cells. The present study also sheds some light on using BI-CyG as an imaging probe and on the role of metformin as a suitable drug for balancing hyperglycemia-induced reduced intra-hepatic albumin levels. The study, thus, attempts to highlight the structural derivatization of cyanine to afford a potential probe for serum albumin and its deployment to image altering albumin levels in an induced pathological condition, hyperglycemia.

Near-infrared emissive cyanine probes for selective visualization of the physiological and pathophysiological modulation of albumin levels.

With the promising advantages of the near-infrared region (NIR) emissive markers for serum albumin becoming very prominent recently, we devised CyG-NHS as the cyanine derived longest NIR-I emissive optical marker possessing albumin selective recognition ability in diverse biological milieu. Multiscale modeling involving molecular docking, molecular dynamics, and implicit solvent binding free energy calculations have been employed to gain insights into the unique binding ability of the developed probe at domain-I of albumin, in contrast to the good number of domain IIA or IIIA binding probes available in the literature reports. The binding free energy was found to be -31.8 kcal mol-1 majorly predominated by hydrophobic interactions. Besides, the conformational dynamics of CyG-NHS in an aqueous medium and the albumin microenvironment have been comprehensively studied and discussed. The potentiality of this optical platform to monitor the intracellular albumin levels in human hepatoma (HepG2) cells in different pathophysiological states has been demonstrated here. Also, the competency of the phenformin drug in restoring the albumin levels in chronic hyperinsulinemic and hypercholesterolemic in vitro models has been established through the visualization approach. Altogether, the findings of this study throw light on the significance of the development of a suitable optical marker for the visualization of critical bioevents related to albumin.

A Novel Near Infrared Spectroscopy Based Device for Albumin Estimation.

In this paper we have proposed a fluorescence based spectroscopy device which can be used to quantitatively estimate the amount of albumin that gets excreted out of our body. Albumin is a significant protein in bio-fluids and performs a wide range of metabolic functions. The dye that has been used as a fluorescent indicator for the presence of albumin in this study has been earlier tested with bovine serum albumin (BSA) and human serum albumin (HSA) with satisfactory results. The method is based on principle of fluorescence in near infrared range (NIR) of 700 to 850 nm by using a novel dye with the test mixture. The chosen near infrared range has a benefit of absence of the auto fluorescence of the bio-molecules present in urine other than the albumin molecules. The system consists of: light source, spectroscopic chamber, sensing and computational unit. The study shows the stability and reproducibility of device so as to avoid fluctuations of voltage and other undesirables. The optimization with bovine serum albumin and human serum albumin has been done and the device can sense as low as 100 nM concentration precisely and accurately.Clinical Relevance-The system being presented is intended for developing a low cost point of care testing device for determining albumin concentration in urine.

Long Range Emissive Water-Soluble Fluorogenic Molecular Platform for Imaging Carbon Monoxide in Live Cells.

Despite its murderous act, carbon monoxide (CO) is found to be a very crucial small gaseous messenger molecule in dictating prime biological and physiological processes. Determination of endogenous or exhaled CO levels can throw significant light on smoking status and can be used as a breath biomarker of inflammatory diseases. Therefore, fluorescence imaging of CO in biofluids will empower one with the minute details of various disease states that involve CO. Unfortunately, such efficient fluorescent probes are less in number and also associated with tedious protocols. This enticed our attention and inspired us to look upon developing perceptive imaging agents for CO in a living system. In this report, a resorufin-based "turn-on" orange emissive molecular probe has been successfully utilized to detect CO in an aqueous system. The mono protection of a resorufin unit with an allyl chloroformate furnished a weakly fluorescent small molecular probe P1. Further, the P1+Pd2+ ensemble has been successfully developed in situ using PdCl2 (as Pd2+) and utilized as a light-up signaling mechanism tool for the sensing of CO at the nanomolar level (62 nM) through deprotection mechanism. The probe selectively detects CO without any interference from other anions, gasotransmitters and fatty acids. The present integrated probe P1+Pd2+ system has been found to be highly sensitive to detect CO in cellular systems as well.

Molecular Scale Optimum Hydrophobicity To Establish an Enhanced Probe-Protein Interaction: Near-Infrared Imaging of Albumin Biosynthesis Modulation.

Albumin is the most abundant serum protein and shows variation in its synthesis rate in different physiological and pathophysiological conditions. Thus, there might be an association expected between serum albumin concentration and body health. A library of NIR probes engineered with the optimum hydrophobicity has been developed and characterized using spectroscopy techniques and was employed to understand the variation of hepatic albumin synthesis rates on physiological and pathophysiological states. Given the importance of hydrophobicity in rendering an effective interaction of small molecules with biomolecules, strategic structure interaction relationship studies led us toward the development of a potent emissive molecular probe through chemical library development. By exploration of these newly developed molecular probes, our study elegantly showed how a pathophysiological condition like the hyperinsulinemic state significantly downregulates albumin biosynthesis in HepG2 cells using fluorescence microscopy as a tool. An excellent correlation between the albumin transcript level and fluorescence intensity inside the cells has been observed. The key role of hydrophobicity resulting in an effective interaction of the probes with albumin, thus leading to strong optical signals, has been experimentally demonstrated in this report. Also, a siRNA interference technique has been utilized to establish the excellent selectivity of the developed probes with excitation as well as emission in the NIR region. We therefore have established through our experimental findings that suitable cell permeable emissive molecular markers with a high degree of albumin specificity can be used as a good optical tool for studying the effect of hyperinsulinemia on albumin biosynthesis modulation.