NSAID targets SIRT3 to trigger mitochondrial dysfunction and gastric cancer cell death.

Gastric cancer (GC) is a deadly malignancy that demands effective therapeutic intervention capitalizing unique drug target/s. Here, we report that indomethacin, a cyclooxygenase non-selective non-steroidal anti-inflammatory drug, arrests GC cell growth by targeting mitochondrial deacetylase Sirtuin 3 (SIRT3). Interaction study revealed that indomethacin competitively inhibited SIRT3 by binding to nicotinamide adenine dinucleotide (NAD)-binding site. The Cancer Genome Atlas data meta-analysis indicated poor prognosis associated with high SIRT3 expression in GC. Further, transcriptome sequencing data of human gastric adenocarcinoma cells revealed that indomethacin treatment severely downregulated SIRT3. Indomethacin-induced SIRT3 downregulation augmented SOD2 and OGG1 acetylation, leading to mitochondrial redox dyshomeostasis, mtDNA damage, respiratory chain failure, bioenergetic crisis, mitochondrial fragmentation, and apoptosis via blocking the AMPK/PGC1α/SIRT3 axis. Indomethacin also downregulated SIRT3 regulators ERRα and PGC1α. Further, SIRT3 knockdown aggravated indomethacin-induced mitochondrial dysfunction as well as blocked cell-cycle progression to increase cell death. Thus, we reveal how indomethacin induces GC cell death by disrupting SIRT3 signaling.

Optical imaging for early detection of cervical cancer: state of the art and perspectives.

Significance: Cervical cancer is one of the major causes of death in females worldwide. HPV infection is the key cause of uncontrolled cell growth leading to cervical cancer. About 90% of cervical cancer is preventable because of the slow progression of the disease, giving a window of about 10 years for the precancerous lesion to be recognized and treated. Aim: The present challenges for cervical cancer diagnosis are interobserver variation in clinicians' interpretation of visual inspection with acetic acid/visual inspection with Lugol's iodine, cost of cytology-based screening, and lack of skilled clinicians. The optical modalities can assist in qualitatively and quantitatively analyzing the tissue to differentiate between cancerous and surrounding normal tissues. Approach: This work is on the recent advances in optical techniques for cervical cancer diagnosis, which promise to overcome the above-listed challenges faced by present screening techniques. Results: The optical modalities provide substantial measurable information in addition to the conventional colposcopy and Pap smear test to clinically aid the diagnosis. Conclusions: Recent optical modalities on fluorescence, multispectral imaging, polarization-sensitive imaging, microendoscopy, Raman spectroscopy, especially with the portable design and assisted by artificial intelligence, have a significant scope in the diagnosis of premalignant cervical cancer in future.

Naringenin-Functionalized Gold Nanoparticles and Their Role in α-Synuclein Stabilization.

Misfolding and self-assembly of several intrinsically disordered proteins into ordered ß-sheet-rich amyloid aggregates emerged as hallmarks of several neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Here we show how the naringenin-embedded nanostructure effectively retards aggregation and fibril formation of α-synuclein, which is strongly associated with the pathology of Parkinson's-like diseases. Naringenin is a polyphenolic compound from a plant source, and in our current investigation, we reported the one-pot synthesis of naringenin-coated spherical and monophasic gold nanoparticles (NAR-AuNPs) under optimized conditions. The average hydrodynamic diameter of the produced nanoparticle was ∼24 nm and showed a distinct absorption band at 533 nm. The zeta potential of the nanocomposite was ∼-22 mV and indicated the presence of naringenin on the surface of nanoparticles. Core-level XPS spectrum analysis showed prominent peaks at 84.02 and 87.68 eV, suggesting the zero oxidation state of metal in the nanostructure. Additionally, the peaks at 86.14 and 89.76 eV were due to the Au-O bond, induced by the hydroxyl groups of the naringenin molecule. The FT-IR analysis further confirmed strong interactions of the molecule with the gold nanosurface via the phenolic oxygen group. The composite surface was found to interact with monomeric α-synuclein and caused a red shift in the nanoparticle absorption band by ∼5 nm. The binding affinity of the composite nanostructure toward α-synuclein was in the micromolar range (Ka∼ 5.02 × 106 M-1) and may produce a protein corona over the gold nanosurface. A circular dichroism study showed that the nanocomposite can arrest the conformational fluctuation of the protein and hindered its transformation into a compact cross-ß-sheet conformation, a prerequisite for amyloid fibril formation. Furthermore, it was found that naringenin and its nanocomplex did not perturb the viability of neuronal cells. It thus appeared that engineering of the nanosurface with naringenin could be an alternative strategy in developing treatment approaches for Parkinson's and other diseases linked to protein conformation.

Loop Dynamics and Conformational Flexibility in Dengue Serine Protease Activity: Noninvasive Perturbation by Solvent Exchange.

Molecular mechanics play an important role in enzyme action and understanding the dynamics of loop motion is key for designing inhibitors of an enzyme, particularly targeting the allosteric sites. For the successful creation of new protease inhibitors targeting the dengue serine protease, our current investigation detailed the intricate structural dynamics of NS2B/NS3 dengue protease. This enzyme is one of the most essential enzymes in the life cycle of the dengue virus, which is responsible for the activation/processing of viral polyprotein, thus making it a potential target for drug discovery. We showed that the internal dynamics of two regions, fingers 1 and 2 (R24-G39 and L149-A164, respectively) adjacent to the active site triad of this protease, control the enzyme action. Each of these regions is composed of two antiparallel ß-strands connected by ß-turn/hairpin loops. The correlated bending and rocking motions in the two ß-turns on either side of the active site were found to modulate the activity of the enzyme to a large extent. With increasing concentration of cosolvent dimethyl sulfoxide, correlated motions in the finger 2 region get diminished and bending of finger 1 increases, which are also reflected in the loss of enzyme activity. Decreasing temperature and mutations in neighboring nonsubstrate binding residues show similar effects on loop motion and enzyme kinetics. Therefore, in vitro noninvasive perturbation of these motions by the solvent exchange as well as cold stress in combination with in silico molecular dynamics simulations established the importance of the two ß-turns in the functioning of dengue virus serotype 2 NS2B/NS3 serine protease.

Engineering Approaches for Breast Cancer Diagnosis: A Review.

Breast cancer is a leading cause of mortality among women. The patient's survival rate is uncertain due to the limitations in the accuracy of diagnosis and effective monitoring during cancer treatment. The key to efficaciously controlling cancer on a larger scale is effective diagnosis at an early stage of cancer by distinguishing the vital signatures of the diseased from the normal breast tissue. The breast tissue is a heterogeneous turbid media that exhibits multifaceted bulk tissue properties. Various sensing modalities can yield distinct tissue behavior for cancer and adjacent normal tissues, serving as a basis for cancer diagnosis. A novel multimodal diagnostic tool that can concurrently assess the optical, electrical, and mechanical bulk tissue properties can substantially augment the clinical findings such as histopathology, potentially aiding the clinician to establish an accurate and rapid diagnosis of cancer. This review aims to discuss the clinical and engineering aspects along with the unmet challenges of these physical sensing modalities, primarily in the field of optical, electrical, and mechanical. The challenges of combining two or more of these sensing modalities that can significantly enhance the effectiveness of the clinical diagnostic tools are further investigated.

Toward the development of a polarimetric tool to diagnose the fibrotic human ventricular myocardium.

SIGNIFICANCE: Optical polarimetry is an emerging modality that effectively quantifies the bulk optical properties that correlate with the anisotropic structural properties of cardiac tissues. We demonstrate the application of a polarimetric tool for characterizing healthy and fibrotic human myocardial tissues efficiently with a high degree of accuracy. AIM: The study was aimed to characterize the myocardial tissues from the left ventricle and right ventricle of N = 7 control and N = 10 diseased subjects. The diseased subjects were composed of two groups: N = 7 with rheumatic heart disease (RHD) and N = 3 with myxomatous valve (MV) disease. APPROACH: A portable, affordable, and accurate linear polarization-based diagnostic tool is developed to measure the degree of linear polarization (DOLP) of the myocardial tissues while working at a wavelength of 850 nm. RESULTS: The sensitivity, specificity, and accuracy of the polarimetric tool in distinguishing the control group from the RHD group were found to be 73.33%, 76.92%, and 75%, respectively, and from the MV group were 91.6%, 62.5%, and 80%, respectively, which demonstrates the efficacy of the polarimetric tool to distinguish the healthy myocardial tissues from diseased tissues. CONCLUSIONS: We have successfully developed a polarimetric tool that can aid cardiologists in characterizing the myocardial tissues in conjunction with endomyocardial biopsy. This work should be followed up with experiments on a large cohort of control and diseased subjects. We intend to create and develop a probe to quantify the DOLP of in vivo heart tissue during surgery.

Toward the development of portable light emitting diode-based polarization spectroscopy tools for breast cancer diagnosis.

A robust, affordable and portable light emitting diode-based diagnostic tools (POLS-NIRDx) using a polarization-sensitive (linear as well as circular polarization) technique were designed and developed to quantify the degree of linear polarization (DOLP), degree of circular polarization (DOCP). The study was performed on malignant (invasive ductal carcinoma) and adjacent normal ex-vivo biopsy tissues excised from N = 10 patients at the operating wavelengths of 850 and 940 nm. The average DOLP and DOCP values were lower for malignant than adjacent normal while operating at 850 and 940 nm. The highest accuracy was observed for DOLP (100%) and DOCP (80%) while operating at 850 nm, which reduced (80% for DOLP and 65% for DOCP) at 940 nm. This pilot study can be utilized as a differentiating factor to delineate malignant tissues from adjacent normal tissues.

Evaluation of therapeutic effect of Premna herbacea in diabetic rat and isoverbascoside against insulin resistance in L6 muscle cells through bioenergetics and stimulation of JNK and AKT/mTOR signaling cascade.

BACKGROUND: Premna herbacea Roxb., a perennial herb is well documented for its therapeutic uses among the traditional health care-givers of Assam, India. Scientific validation on the traditional use of the medicinal plant using modern technology may promote further research in health care. PURPOSE: This study evaluates the therapeutic potential of methanolic extract of P. herbacea (MEPH) against type 2 diabetes mellitus (T2DM) and its phytochemical(s) in ameliorating insulin resistance (IR), thereby endorsing the plant bioactives as effective anti-hyperglycemic agents. METHODS: The anti-diabetic potential of the plant extract was explored both in L6 muscle cells and high fructose high fat diet (HF-HFD) fed male Sprague Dawley (SD) rats. Bioactivity guided fractionation and isolation procedure yielded Verbascoside and Isoverbascoside (ISOVER) as bioactive and major phytochemicals in P. herbacea. The bioenergetics profile of bioactive ISOVER and its anti-hyperglycemic potential was validated in vitro by XFe24 analyzer, glucose uptake assay and intracellular ROS generation by flourometer, FACS and confocal microscopy. The potential of ISOVER was also checked by screening various protein markers via immunoblotting. RESULTS: MEPH enhanced glucose uptake in FFA-induced insulin resistant (IR) L6 muscle cells and decreased elevated blood glucose levels in HF-HFD fed rats. Isoverbascoside (ISOVER) was identified as most bioactive phytochemical for the first time from the plant in the Premna genus. ISOVER activated the protein kinase B/AMP-activated protein kinase signaling cascades and enhanced glucose uptake in IR-L6 muscle cells. ISOVER decreased the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) and increased that of mammalian target of rapamycin (mTOR), thereby attenuating IR. However, molecular docking revealed that ISOVER increases insulin sensitivity by targeting the JNK1 kinase as a competitive inhibitor rather than mTOR. These findings were further supported by the bioenergetics profile of ISOVER. CONCLUSION: This study for the first time depicts the functional properties of ISOVER, derived from Premna herbacea, in ameliorating IR. The phytochemical significantly altered IR with enhanced glucose uptake and inhibition of ROS through JNK-AKT/mTOR signaling which may pave the way for further research in T2DM therapeutics.

Dabrafenib, idelalisib and nintedanib act as significant allosteric modulator for dengue NS3 protease.

Dengue virus (DENV) encodes a unique protease (NS3/NS2B) essential for its maturation and infectivity and, it has become a key target for anti-viral drug design to treat dengue and other flavivirus related infections. Present investigation established that some of the drug molecules currently used mainly in cancer treatment are susceptible to bind non-active site (allosteric site/ cavity) of the NS3 protease enzyme of dengue virus. Computational screening and molecular docking analysis found that dabrafenib, idelalisib and nintedanib can bind at the allosteric site of the enzyme. The binding of the molecules to the allosteric site found to be stabilized via pi-cation and hydrophobic interactions, hydrogen-bond formation and π-stacking interaction with the molecules. Several interacting residues of the enzyme were common in all the five serotypes. However, the interaction/stabilizing forces were not uniformly distributed; the π-stacking was dominated with DENV3 proteases, whereas, a charged/ionic interaction was the major force behind interaction with DENV2 type proteases. In the allosteric cavity of protease from DENV1, the residues Lys73, Lys74, Thr118, Glu120, Val123, Asn152 and Ala164 were involved in active interaction with the three molecules (dabrafenib, idelalisib and nintedanib). Molecular dynamics (MD) analysis further revealed that the molecules on binding to NS3 protease caused significant changes in structural fluctuation and gained enhanced stability. Most importantly, the binding of the molecules effectively perturbed the protein conformation. These changes in the protein conformation and dynamics could generate allosteric modulation and thus may attenuate/alter the NS3 protease functionality and mobility at the active site. Experimental studies may strengthen the notion whether the binding reduce/enhance the catalytic activity of the enzyme, however, it is beyond the scope of this study.

Hybrid Spectral-IRDx: Near-IR and Ultrasound Attenuation System for Differentiating Breast Cancer From Adjacent Normal Tissue.

OBJECTIVE: While performing surgical excision for breast cancer (lumpectomy), it is important to ensure a clear margin of normal tissue around the cancer to achieve complete resection. The current standard is histopathology; however, it is time-consuming and labour-intensive requiring skilled personnel. METHOD: We describe a Hybrid Spectral-IRDx - a combination of the previously reported Spectral-IRDx tool with multimodal ultrasound and NIR spectroscopy techniques. We show how this portable, cost-effective, minimal-contact tool could provide rapid diagnosis of cancer using formalin-fixed (FF) and deparaffinized (DP) breast biopsy tissues. RESULTS: Using this new tool, measurements were performed on cancerous/fibroadenoma and its adjacent normal tissues from the same patients (N = 14). The acoustic attenuation coefficient (α) and reduced scattering coefficient (µ's) (at 850, 940, and 1060 nm) for the cancerous/fibroadenoma tissues were reported to be higher compared to adjacent normal tissues, a basis of delineation. Comparing FF cancerous and adjacent normal tissue, the difference in µ's at 850 nm and 940 nm were statistically significant (p = 3.17e-2 and 7.94e-3 respectively). The difference in α between the cancerous and adjacent normal tissues for DP and FF tissues were also statistically significant (p = 2.85e-2 and 7.94e-3 respectively). Combining multimodal parameters α and µ's (at 940 nm) show highest statistical significance (p = 6.72e-4) between FF cancerous/fibroadenoma and adjacent normal tissues. CONCLUSION: We show that Hybrid Spectral-IRDx can accurately delineate between cancerous and adjacent normal breast biopsy tissue. SIGNIFICANCE: The results obtained establish the proof-of-principle and large-scale testing of this multimodal breast cancer diagnostic platform for core biopsy diagnosis.

Thermo-optic measurements and their inter-dependencies for delineating cancerous breast biopsy tissue from adjacent normal.

The histopathological diagnosis of cancer is the current gold standard to differentiate normal from cancerous tissues. We propose a portable platform prototype to characterize the tissue's thermal and optical properties, and their inter-dependencies to potentially aid the pathologist in making an informed decision. The measurements were performed on 10 samples from five subjects, where the cancerous and adjacent normal were extracted from the same patient. It was observed that thermal conductivity (k) and reduced-scattering-coefficient (µ's ) for both the cancerous and normal tissues reduced with the rise in tissue temperature. Comparing cancerous and adjacent normal tissue, the difference in k and µ's (at 940 nm) were statistically significant (p = 7.94e-3), while combining k and µ's achieved the highest statistical significance (6.74e-4). These preliminary results promise and support testing on a large number of samples for rapidly differentiating cancerous from adjacent normal tissues.

Intriguing Biomedical Applications of Synthetic and Natural Cell-Derived Vesicles: A Comparative Overview.

The significant role of a vesicle is well recognized; however, only lately has the advancement in biomedical applications started to uncover their usefulness. Although the concept of vesicles originates from cell biology, it later transferred to chemistry and material science to develop nanoscale artificial vesicles for biomedical applications. Herein, we examine different synthetic and biological vesicles and their applications in the biomedical field in general. As our understanding of biological vesicles increases, more suitable biomimicking synthetic vesicles will be developed. The comparative discussion between synthetic and natural vesicles for biomedical applications is a relevant topic, and we envision this could enable the development of a proper approach to realize the next-generation treatment goals.

Biophysical and theoretical studies of the interaction between a bioactive compound 3,5-dimethoxy-4-hydroxycinnamic acid with calf thymus DNA.

3,5-Dimethoxy-4-hydroxycinnamic acid commonly known as Sinapic acid is a well-known derivative of hydroxycinnamic acids, is commonly present in human diet. Due to its wide variety of pharmacological activities like antioxidant, antimicrobial, anti-inflammatory, anticancer, and anti-anxiety, it has attracted much attention for the researchers. In our previous published work we have already analyzed the interaction between sinapic acid (SA) with a model transport protein. In this work our aim is to demonstrate a detailed investigation of the binding interaction between sinapic acid with another carrier of genetic information in a living cell, the DNA. Here we have used calf thymus DNA (ct-DNA) as a model. The binding characteristic of SA with ct-DNA was investigated by different spectroscopic and theoretical tools. The spectroscopic investigation revealed that quenching of intrinsic fluorescence of SA by ct-DNA occurs through dynamic quenching mechanism. The thermodynamic parameters established the involvement of hydrogen bonding and weak van der Waals forces in the interaction. Further, the circular dichroism, competitive binding experiment with ethidium bromide and potassium iodide quenching experiment suggested that SA possibly binds to the groove position of the ct-DNA. Finally, molecular docking analysis established the SA binds to minor groove position of ct-DNA in G-C rich region through hydrogen bonding interaction. Additionally, gel electrophoresis analysis has been performed to determine the protective efficacy of SA against UVB induced DNA damage and 50 µM of SA was found to protect the DNA from UVB induced damage. We hope that our study could provide the validation of SA on behalf of therapeutics and development of next generation therapeutic drug as well as designing new efficient drug molecule and methodology for the interaction study of the drug with DNA.

The Role of Imidazolium-Based Surface-Active Ionic Liquid to Restrain the Excited-State Intramolecular H-Atom Transfer Dynamics of Medicinal Pigment Curcumin: A Theoretical and Experimental Approach.

The naturally occurring polyphenolic compound curcumin has shown various medicinal and therapeutic effects. However, there are various challenges associated with curcumin, which limits its biomedical applications, such as its high degradation rate and low aqueous solubility at neutral and alkaline pH. In the present study, efforts have been directed towards trying to resolve such issues by encapsulating curcumin inside the micelles formed by imidazolium-based surface-active ionic liquid (SAIL). The shape and size of the micelles formed by the SAIL have been characterized by using DLS analysis as well as TEM measurements. The photo-physics of curcumin in the presence of ionic liquid (IL) and also with the addition of salt (NaCl) has been explored by using different optical spectroscopic tools. The time-dependent absorption studies have shown that there is relatively higher suppression in the degradation rate of curcumin after encapsulation by the imidazolium-based SAIL in an aqueous medium. The TCSPC studies have revealed that there is deactivation in the nonradiative intramolecular hydrogen transfer process of curcumin in the presence of IL micelles as well as with the addition of salt. Furthermore, the time-dependent fluorescence anisotropy measurement has been carried out to figure out the location of curcumin inside the micellar system. In order to correlate all experimental findings, density functional theory (DFT) and classical molecular dynamics (MD) simulations at neutral pH media have been performed. It has been found that the van der Waals force of interactions plays a major role in the stabilization of curcumin in the micelles rather than the coulombic forces. It also has been observed that the van der Waals interactions remain unaffected in the presence of salt. However, as revealed by the MD simulation results, the micelles are found to be more compact in size after the addition of salt. The RMSD results show that the micelles formed by the SAIL achieve greater stability after a particular time constraint. Our results have divulged that the SAIL could act as a promising drug delivery system.

Towards a Portable Platform Integrated With Multispectral Noncontact Probes for Delineating Normal and Breast Cancer Tissue Based on Near-Infrared Spectroscopy.

Currently, the confirmation of diagnosis of breast cancer is made by microscopic examination of an ultra-thin slice of a needle biopsy specimen. This slice is conventionally formalin-fixed and stained with hematoxylin-eosin and visually examined under a light microscope. This process is labor-intensive and requires highly skilled doctors (pathologists). In this paper, we report a novel tool based on near-infrared spectroscopy (Spectral-IRDx) which is a portable, non-contact, and cost-effective system and could provide a rapid and accurate diagnosis of cancer. The Spectral-IRDx tool performs absorption spectroscopy at near-infrared (NIR) wavelengths of 850, 935, and 1060 nm. We measure normalized detected voltage (Vdn) with the tool in 10 deparaffinized breast biopsy tissue samples, 5 of which were cancer (C) and 5 were normal (N) tissues. The difference in Vdn at 935 nm and 1060 nm between cancer and normal tissues is statistically significant with p-values of 0.0038 and 0.0022 respectively. Absorption contrast factor (N/C) of 1.303, 1.551, and 1.45 are observed for 850, 935, and 1060 nm respectively. The volume fraction contrast (N/C) of lipids and collagens are reported as 1.28 and 1.10 respectively. Higher absorption contrast factor (N/C) and volume fraction contrast (N/C) signifies higher concentration of lipids in normal tissues as compared to cancerous tissues, a basis for delineation. These preliminary results support the envisioned concept for noninvasive and noncarcinogenic NIR-based breast cancer diagnostic platform, which will be tested using a larger number of samples.

DNA Damage and Apoptosis Induction in Cancer Cells by Chemically Engineered Thiolated Riboflavin Gold Nanoassembly.

Herein we have engineered a smart nuclear targeting thiol-modified riboflavin-gold nano assembly, RfS@AuNPs, which accumulates selectively in the nucleus without any nuclear-targeting peptides (NLS/RGD) and shows photophysically in vitro DNA intercalation. A theoretical model using Molecular Dynamics has been developed to probe the mechanism of formation and stability as well as dynamics of the RfS@AuNPs in aqueous solution and within the DNA microenvironment. The RfS@AuNPs facilitate the binucleated cell formation that is reflected in the significant increase of DNA damage marker, γ-H2AX as well as the arrest of most of the HeLa cells at the pre-G1 phase indicating cell death. Moreover, a significant upregulation of apoptotic markers confirms that the cell death occurs through the apoptotic pathway. Analyses of the microarray gene expression of RfS@AuNPs treated HeLa cells show significant alterations in vital biological processes necessary for cell survival. Taken together, our study reports a unique nuclear targeting mechanism through targeting the riboflavin receptors, which are upregulated in cancer cells and induce apoptosis in the targeted cells.

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides.

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two É£ H of curcumin heptadiene chain are closely positioned to the A16-H8 and A17-H8, while G12-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.

Formation of Annular Protofibrillar Assembly by Cysteine Tripeptide: Unraveling the Interactions with NMR, FTIR, and Molecular Dynamics.

Both hydrogen-bonding and hydrophobic interactions play a significant role in molecular assembly, including self-assembly of proteins and peptides. In this study, we report the formation of annular protofibrillar structure (diameter ∼500 nm) made of a newly synthesized s-benzyl-protected cysteine tripeptide, which was primarily stabilized by hydrogen-bonding and hydrophobic interactions. Atomic force microscopy and field emission scanning electron microscopy analyses found small oligomers (diameter ∼60 nm) to bigger annular (outer diameter ∼300 nm; inner diameter, 100 nm) and protofibrillar structures after 1-2 days of incubation. Rotating-frame Overhauser spectroscopic (ROESY) analysis revealed the presence of several nonbonded proton-proton interactions among the residues, such as amide protons with methylene group, aromatic protons with tertiary butyl group, and methylene protons with tertiary butyl group. These added significant stability to bring the peptides closer to form a well-ordered assembled structure. Hydrogen-deuterium exchange NMR measurement further suggested that two individual amide protons among the three amide groups were strongly engaged with the adjacent tripeptide via H-bond interaction. However, the remaining amide proton was found to be exposed to solvent and remained noninteracting with other tripeptide molecules. In addition to chemical shift values, a significant change in amide bond vibrations of the tripeptide was found due to the formation of the self-assembled structure. The amide I mode of vibrations involving two amide linkages appeared at 1641 and 1695 cm-1 in the solid state. However, in the assembled state, the stretching band at 1695 cm-1 became broad and slightly shifted to ∼1689 cm-1. On the contrary, the band at 1641 cm-1 shifted to 1659 cm-1 and indicated that the -C═O bond associated with this vibration became stronger in the assembled state. These changes in Fourier transform infrared spectroscopy frequency clearly indicated changes in the amide backbone conformation and the associated hydrogen-bonding pattern due to the formation of the assembled structure. In addition to hydrogen bonding, molecular dynamics simulation indicated that the number of π-π interactions also increased with increasing number of tripeptides participated in the self-assembly process. Combined results envisaged a cross ß-sheet assembly unit consisting of four intermolecular hydrogen bonds. Such noncovalent peptide assemblies glued by hydrogen-bonding and other weak forces may be useful in developing nanocapsule and related materials.

Envisaging Structural Insight of a Terminally Protected Proline Dipeptide by Raman Spectroscopy and Density Functional Theory Analyses.

The proline residue in a protein sequence generates constraints to its secondary structure as the associated torsion angles become a part of the heterocyclic ring. It becomes more significant when two consecutive proline residues link via amide linkage and produce additional configurational constraint to a protein's folding and stability. In the current manuscript we have illustrated conformation preference of a novel dipeptide, (R)-tert-butyl 2-((S)-2-(methoxycarbonyl)pyrrolidine-1-carbonyl)pyrrolidine-1-carboxylate. The dipeptide crystallized in the orthorhombic crystalline state and produced rod-shaped macroscopic material. The analysis of the crystal coordinates showed dihedral angles (φ, ψ) of the interlinked amide groups as (+72°, -147°) and the dihedral angles (φ, ψ) produced with the next carbonyl were (-68°, +151°), indicating polyglycine II (PGII) and polyproline II (PPII)-like helix states at the N- and C-terminals, respectively. These two states, PGII and PPII, are mirror image configurations and are expected to produce similar vibration bands from the associated carbonyl groups. However, the unique atomic arrangement in the molecule produces three carbonyl groups and one of them was very specific, being part of the main peptide linkage that connects both the pyrrolidine rings. The carbonyl group in the peptide bond exhibited a Raman vibration frequency at ∼1642 cm-1 and is considered a signatory Raman marker band for the peptide bond linking two heterochiral proline residues. The carbonyl group (t-Boc) at the N-terminal of the peptide showed a characteristic vibration at ∼1685 cm-1 and the C-terminal carbonyl group as a part of the ester showed a vibration signature at a significantly high frequency (1746 cm-1). Conformation analyses performed with density functional theory (DFT) calculations depicted that the dipeptide was stabilized in vacuum with dihedral angles (+72°, -154°) and (-72°, +151°) at the N- and C-terminals, respectively. Molecular dynamics (MD) simulation also showed that the peptide conformation having dihedral angles around (+75°, -150°) and (-75°, +150°) at the N- and C-terminals, respectively, was reasonably stable in water. Due to unique absence of the amide N-H, the peptide was ineffective in forming any intramolecular hydrogen bonding. MD investigation, however, revealed an intermolecular hydrogen bonding interaction with the water molecules, leading to its stability in aqueous solution. Metadynamics simulation analysis of the dipeptide in water also supported the PGII-PPII-like conformation at the N- and C-terminals, respectively, as the energetically stable conformation among the other possible combinations of conformations. The possible electronic transitions along with the HOMO-LUMO analysis further depicted the stability of the dipeptide in water and their possible absorption pattern. Time-dependent density functional theory (TDDFT) analysis showed strong negative rotatory strength of the dipeptide around 210 nm in water and acetonitrile, and it could be the source of experimentally observed high-amplitude negative absorption in the circular dichroism (CD) spectra around 200-203 nm. The very weak positive band (signature) in the region at ∼228 nm in CD spectra could also be correlated to the positive rotatory strength at 228 nm observed in ECD. To test the effect of such a dipeptide on a living cell, an MTT assay was performed and the result indicated no cytotoxic effect toward human hepatocellular carcinoma Hep G2 cancer cell lines.

Cα-H carries information of a hydrogen bond involving the geminal hydroxyl group: a case study with a hydrogen-bonded complex of 1,1,1,3,3,3-hexafluoro-2-propanol and tertiary amines.

Experimental measurement of the contribution of H-bonding to intermolecular and intramolecular interactions that provide specificity to biological complex formation is an important aspect of macromolecular chemistry and structural biology. However, there are very few viable methods available to determine the energetic contribution of an individual hydrogen bond to binding and catalysis in biological systems. Therefore, the methods that use secondary deuterium isotope effects analyzed by NMR or equilibrium or kinetic isotope effect measurements are attractive ways to gain information on the H-bonding properties of an alcohol system, particularly in a biological environment. Here, we explore the anharmonic contribution to the C-H group when the O-H group of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) forms an intermolecular H-bond with the amines by quantum mechanical calculations and by experimentally measuring the H/D effect by NMR. Within the framework of density functional theory, ab initio calculations were carried out for HFP in its two different conformational states and their H-bonded complexes with tertiary amines to determine the (13)C chemical shielding, change in their vibrational equilibrium distances, and the deuterium isotope effect on (13)C2 (secondary carbon) of HFP upon formation of complexes with tertiary amines. When C2-OH was involved in hydrogen bond formation (O-H as hydrogen donor), it weakened the geminal C2-H bond; it was reflected in the NMR chemical shift, coupling constant, and the equilibrium distances of the C-H bond. The first derivative of nuclear shielding at C2 in HFP was -48.94 and -50.73 ppm Å(-1) for anti and gauche conformations, respectively. In the complex, the values were -50.28 and -50.76 ppm Å(-1), respectively. The C-H stretching frequency was lower than the free monomer, indicating enhanced anharmonicity in the C-H bond in the complex form. In chloroform, HFP formed a complex with the amine; δC2 was 69.107 ppm for HFP-triethylamine and 68.766 ppm for HFP-d2-triethylamine and the difference in chemical shift, the ΔδC2 was 341 ppb. The enhanced anharmonicity in the hydrogen-bonded complex resulted in a larger vibrational equilibrium distance in C-H/D bonds. An analysis with the Morse potential function indicated that the enhanced anharmonicity encountered in the bond was the origin of a larger isotope effect and the equilibrium distances. Change in vibrational equilibrium distance and the deuterium isotope effect, as observed in the complex, could be used as parameters in monitoring the strength of the H-bond in small model systems with promising application in biomacromolecules.