S-nitrosylation of UCHL1 induces its structural instability and promotes α-synuclein aggregation.

Ubiquitin C-terminal Hydrolase-1 (UCHL1) is a deubiquitinating enzyme, which plays a key role in Parkinson's disease (PD). It is one of the most important proteins, which constitute Lewy body in PD patient. However, how this well folded highly soluble protein presents in this proteinaceous aggregate is still unclear. We report here that UCHL1 undergoes S-nitrosylation in vitro and rotenone induced PD mouse model. The preferential nitrosylation in the Cys 90, Cys 152 and Cys 220 has been observed which alters the catalytic activity and structural stability. We show here that nitrosylation induces structural instability and produces amorphous aggregate, which provides a nucleation to the native α-synuclein for faster aggregation. Our findings provide a new link between UCHL1-nitrosylation and PD pathology.

Thermal stability and hydration behavior of ritonavir sulfate: A vibrational spectroscopic approach.

Ritonavir sulfate is a protease inhibitor widely used in the treatment of acquired immunodeficiency syndrome. In order to elucidate the inherent stability and sensitivity characteristics of ritonavir sulfate, it was investigated under forced thermal and hydration stress conditions as recommended by the International Conference on Harmonization guidelines. In addition, competency of vibrational (infrared and Raman) spectroscopy was assessed to identify structural changes of the drug symbolizing its stress degradation. High performance liquid chromatography was used as a confirmatory technique for both thermal and hydration stress study, while thermogravimetric analysis/differential thermal analysis and atomic force microscopy substantiated the implementation of vibrational spectroscopy in this framework. The results exhibited high thermal stability of the drug as significant variations were observed in the diffuse reflectance infrared Fourier transform spectra only after the drug exposure to thermal radiations at 100 °C. Hydration behavior of ritonavir sulfate was evaluated using Raman spectroscopy and the value of critical relative humidity was found to be >67%. An important aspect of this study was to utilize vibrational spectroscopic technique to address stability issues of pharmacological molecules, not only for their processing in pharmaceutical industry, but also for predicting their shelf lives and suitable storage conditions.

Raman spectroscopic evaluation of DNA adducts of a platinum containing anticancer drug.

Mechanistic understanding of the interaction of drugs with their target molecules is important for better understanding of their mode of action and to improve their efficacy. Carboplatin is a platinum containing anticancer drug, used to treat different type of tumors. In the present work, we applied Raman spectroscopy to study the interaction of carboplatin with DNA at molecular level using different carboplatin-DNA molar ratios. These Raman spectroscopic results provide comprehensive understanding on the carboplatin-DNA interactions and indicate that DNA cross-linked adducts formed by carboplatin are similar to cisplatin adducts. The results indicate that guanine N7 and adenine N7 are the putative sites for carboplatin interaction. It is observed that carboplatin has some affinity toward cytosine in DNA. Phosphate sugar backbone of DNA showed conformation perturbation in DNA which were easily sensible at higher concentrations of carboplatin. Most importantly, carboplatin interaction induces intermediate A- and B-DNA conformations at the cross-linking sites.

Role of minor groove width and hydration pattern on amsacrine interaction with DNA.

Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove) takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.

FTIR and circular dichroism spectroscopic study of interaction of 5-fluorouracil with DNA.

5-Fluorouracil (5FU) is an anticancer chemotherapeutic drug which exerts cytotoxic effect by inhibiting cellular DNA replication. In the present study, we explore the binding of 5FU with DNA and resulting structural and conformational changes on DNA duplex. UV-visible, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic techniques were employed to explore these interactions. A constant concentration of calf thymus DNA was incubated with varying concentrations of 5FU. UV-visible and FTIR spectroscopic results revealed that intercalation is the primary mode of interaction between 5FU and nitrogenous bases of the nucleic acid. The binding constant was found to be 9.7×10(4); which is indicative of moderate type of interaction between 5FU and DNA duplex. It was also observed that 5FU intercalates slightly more between AT base pairs compared to GC pairs. FTIR and circular dichroism spectroscopic results revealed that 5FU disturbs native B-conformation of DNA though, DNA remains in its B conformation even at higher concentrations of 5FU.

Analysis of ovarian tumor pathology by Fourier Transform Infrared Spectroscopy.

BACKGROUND: Ovarian cancer is the second most common cancer among women and the leading cause of death among gynecologic malignancies. In recent years, infrared (IR) spectroscopy has gained attention as a simple and inexpensive method for the biomedical study of several diseases. In the present study infrared spectra of normal and malignant ovarian tissues were recorded in the 650 cm-1 to 4000 cm-1 region. METHODS: Post surgical tissue samples were taken from the normal and tumor sections of the tissue. Fourier Transform Infrared (FTIR) data on twelve cases of ovarian cancer with different grades of malignancy from patients of different age groups were analyzed. RESULTS: Significant spectral differences between the normal and the ovarian cancerous tissues were observed. In particular changes in frequency and intensity in the spectral region of protein, nucleic acid and lipid vibrational modes were observed. It was evident that the sample-to-sample or patient-to-patient variations were small and the spectral differences between normal and diseased tissues were reproducible. CONCLUSION: The measured spectroscopic features, which are the spectroscopic fingerprints of the tissues, provided the important differentiating information about the malignant and normal tissues. The findings of this study demonstrate the possible use of infrared spectroscopy in differentiating normal and malignant ovarian tissues.

DNA interaction studies of an anticancer plant alkaloid, vincristine, using Fourier transform infrared spectroscopy.

The binding of vincristine with DNA has been investigated using Fourier transform infrared spectroscopy. Various changes in the double helical structure of DNA after addition of vincristine have been examined. It is evident from Fourier transform infrared results that vincristine-DNA interaction occurs through guanine and cytosine base pairs. External binding of vincristine with phosphate backbone of the DNA is also observed. Vincristine perturbs guanine band at 1714 cm(-1), cytosine band at 1488 cm(-1), and the phosphate vibrations at 1225 and 1086 cm(-1). The UV-visible spectra of vincristine-DNA complex show hypochromic and bathochromic shifts, indicating the intercalation of vincristine into the double helical structure of DNA. Both intercalative and external binding modes are observed for vincristine binding with DNA, with an estimated binding constant K = 1.0 × 10(3) M(-1).

Development and validation of an infrared spectroscopy-based method for the analysis of moisture content in 5-fluorouracil.

The determination of moisture content in pharmaceuticals is very important as moisture is mainly responsible for the degradation of drugs. Degraded drugs have reduced efficacy and could be hazardous. The objective of the present work is to replace the Karl Fischer (KF) titration method used for moisture analysis with a method that is rapid, involves no toxic materials and is more effective. Diffuse reflectance infrared (IR) spectroscopy, which is explored as a potential alternative to various approaches, is investigated for moisture analysis in 5-fluorouracil, an anticancer drug. A total of 150 samples with varying moisture content were prepared in laboratory by exposing the drug at different relative humidities, for different time intervals. Infrared spectra of these samples were collected with a Fourier transform infrared (FTIR) spectrophotometer using a diffuse reflectance accessory. Reference moisture values were obtained using the Karl Fischer titration method. A number of calibration models were developed using the partial least squares (PLS) regression method. A good correlation was obtained between predicted IR values and reference values in the calibration and validation set. The derived calibration curve was used to predict moisture content in unknown samples. The results show that IR spectroscopy can be used successfully for the determination of moisture content in the pharmaceutical industry.