Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin-K Activity Detection, Revealing Nuclear Activity.

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

Cu(II)-Based Coordination Polymer as a Pristine Form Usable Electrocatalyst for Oxygen Reduction Reaction: Experimental Evaluation and Theoretical Insights into Biomimetic Mechanistic Aspects.

As the postsynthesis-processed metal-organic material-based catalysts for energy applications add additional cost to the whole process, the importance of developing synthesized usable pristine catalysts is quite evident. The present work reports a new Cu-based coordination polymer (Cu-CP) catalyst to be used in its pristine form for oxygen reduction reaction (ORR) application. The catalyst was characterized using single-crystal X-ray diffraction, field emission scanning electron microscopy, and X-ray photoemission spectroscopy. The Cu-CP exhibits admirable electrocatalytic ORR activity with an onset potential of 0.84 V versus RHE and a half wave potential of 0.69 V versus RHE. As revealed by the density functional theory-based computational mechanistic investigation of the electrocatalytic ORR process, the electrochemically reduced Cu(I) center binds to the molecular O2 through an exergonic process (ΔG = -6.8 kcal/mol) and generates the Cu(II)-O2•- superoxo intermediate. Such superoxo intermediates are frequently encountered in the catalytic cycle of the Cu-containing metalloenzymes in their O2 reduction reaction. This intermediate undergoes coupled proton and electron transfer processes to give OH- in an alkaline medium involving H2O2 as the intermediate. The electrocatalytic performance of Cu-CP remained stable even up to 3000 cycles. Overall, the newly developed Cu-CP-based electrocatalyst holds promising potential for efficient biomimetic ORR reactivity, which opens new possibilities toward the development of robust coordination polymer-based electrocatalysts.

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.

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.

Preferential intermolecular interactions lead to chiral recognition: enantioselective gel formation and collapse.

Interesting self-assembly of an amino acid based molecular material into a hydrogel in the presence of selective enantiomeric chiral amines leading to chiral recognition has been demonstrated. Moreover, collapse of a metallogel formed from the same material in the presence of selective enantiomers validated its enantioselective affinity. Importantly, in addition to relevant experimental techniques, DFT studies have been successfully explored to establish chiral recognition through enantioselective gelation.

Renal Clearable New NIR Probe: Precise Quantification of Albumin in Biofluids and Fatty Liver Disease State Identification through Tissue Specific High Contrast Imaging in Vivo.

Development of a highly photostable, renal clearable, and nontoxic new NIR probe (CyG) for precise quantification of albumin in different biofluids and liver targeted in vivo albumin visualization is demonstrated. CyG's inherent property to interact selectively with albumin among different biomolecules in intracellular environment with high degree of sensitivity helps CyG in targeted liver imaging. In addition to its long excitation/emission wavelengths (λex = 740 nm, λem = 804 nm), which are much above the biological tissue opaque window (400-700 nm) ensuring better photon penetration, diminished tissue autofluorescence and high contrasts, its molecular mass and size are far below the renal cutoff and hence, CyG qualifies as imaging material for clinical studies. We anticipate that CyG will provide new strategies to overcome the pitfall of present day albumin detection methods as well as accelerate the detection process at relatively lower costs without compromising the accuracy of detection. Moreover, the renal excretion kinetic and intrahepatic albumin binding affinity of CyG can further be used to differentiate between fatty liver from healthy liver in an experimentally arrived mouse model using noninvasive technique.

Selenium Incorporated Cationic Organochalcogen: Live Cell Compatible and Highly Photostable Molecular Stain for Imaging and Localization of Intracellular DNA.

Successful integration of selenium unit into a newly designed cationic chemical architecture led to the development of a highly photostable molecular maker PA5 to be used in fluorescence microscopy as cellular nucleus staining agent for longer duration imaging under continuous laser illumination. Adaptation of a targeted single-atom modification strategy led to the development of a series of proficient DNA light-up probes (PA1-PA5). Further, their comparative photophysical studies in the presence of DNA revealed the potential of electron rich heteroatoms of chalcogen family in improving binding efficiency and specificity of molecular probes toward DNA. The findings of cell studies confirmed the outstanding cell compatibility of probe PA5 in terms of cell permeability, biostability, and extremely low cytotoxicity. Moreover, the photostability experiment employing continuous laser illumination in solution phase as well as in cell assay (both fixed and live cells) revealed the admirable photobleaching resistance of PA5. Finally, while investigating the phototoxicity of PA5, the probe was found not to exhibit light-induced toxicity even when irradiated for longer duration. All these experimental results demonstrated the promising standing of PA5 as a futuristic cell compatible potential stain for bioimaging and temporal profiling of DNA.

Optical signaling in biofluids: a nondenaturing photostable molecular probe for serum albumins.

The systematic investigation of the interaction of a new class of molecular materials with proteins through structure-optical signaling relationship studies has led to the development of efficient fluorescent probes that can detect and quantify serum albumins in biofluids without causing any denaturation.

Functional molecular lumino-materials to probe serum albumins: solid phase selective staining through noncovalent fluorescent labeling.

Selective staining of human serum albumin protein in gel electrophoresis over wide range of other protein(s) is extremely important because it contains more than 60% volume of serum fluid in human body. Given the nonexistence of suitable dye materials for selective staining of serum albumins in gel electrophoresis, we report a new class of easy synthesizable and low molecular weight staining agents based on 3-amino-N-alkyl-carbazole scaffold for selective staining of serum albumins in solid phase. A detailed structure-efficiency relationship (SER) study enabled us to develop two such potent functional molecular probes which stain both human and bovine serum albumin selectively in gel electrophoresis in the presence of other proteins and enzymes. The present gel staining process was found to be very simple and less time-consuming as compared to the conventional coomassie blue staining which in turn makes these probes a new class of serum albumin-specific staining materials in proteome research. Moreover, these molecular lumino-materials can detect serum albumins at subnanomolar level in the presence of broad spectrum of other proteins/enzymes in aqueous buffer (99.9% water, pH = 7.3) keeping the protein secondary structure intact. Our experimental and the docking simulation results show that these probes bind preferentially at 'binding site I' of both the serum proteins.

Cysteamine-based cell-permeable Zn(2+)-specific molecular bioimaging materials: from animal to plant cells.

Structure-interaction/fluorescence relationship studies led to the development of a small chemical library of Zn(2+)-specific cysteamine-based molecular probes. The probe L5 with higher excitation/emission wavelengths, which absorbs in the visible region and emits in the green, was chosen as a model imaging material for biological studies. After successful imaging of intracellular zinc in four different kinds of cells including living organisms, plant, and animal cells, in vivo imaging potential of L5 was evaluated using plant systems. In vivo imaging of translocation of zinc through the stem of a small herb with a transparent stem, Peperomia pellucida, confirmed the stability of L5 inside biological systems and the suitability of L5 for real-time analysis. Similarly, fluorescence imaging of zinc in gram sprouts revealed the efficacy of the probe in the detection and localization of zinc in cereal crops. This imaging technique will help in knowing the efficiency of various techniques used for zinc enrichment of cereal crops. Computational analyses were carried out to better understand the structure, the formation of probe-Zn(2+) complexes, and the emission properties of these complexes.