Current Pharmaceutical Biotechnology - Volume 6, Issue 2, 2005

Recent technological advances in NMR methods and instrumentation are having a significant impact in structural biology. These innovations are also impacting pharmaceutical biotechnology as it is now possible to use NMR spectroscopy to rapidly characterize a growing number of prospective protein drugs and protein drug targets. This review provides a general summary of how solution-state NMR can be used to determine protein structures. It also focuses on exploring how advances in solution state NMR are changing the way in which protein structures can be determined and protein-ligand interactions can be characterized. Recent innovations in protein sample preparation, in instrumentation and data collection, in spectral assignment and in structure generation are highlighted. The impact of solution-state NMR on pharmaceutical biotechnology is also discussed, with a special emphasis on describing how NMR has been used to study a number of pharmaceutically important proteins and how NMR is currently being used to rapidly screen and to map the binding sites of small molecules to a range of protein targets.

Cycloheximide, a protein synthesis inhibitor, was encapsulated in cross-linked gelatin nanoparticles (Type B, Bovine skin, 75 Bloom) of 168nm diameter with 26% entrapment efficiency. In-vitro release kinetics of the drug from the nanoparticles was done in phoshate buffer saline (PBS) at pH 7.4 and pH 5.8. The release kinetics showed a bi-phasic curve. Interestingly, the release of drug is approx 90% in acidic pH as compared to 50% release in neutral pH. The particle size was determined by Dynamic Light Scattering (DLS) technique, and size distribution spectra at different pH were observed to vary inversely with increase in pH. These drug loaded nanoparticles were found to be stable in whole blood showing negligible haemolysis. Cytotoxicity in HBL-100 and MCF-7, breast cancer cell lines was done in a 24-72 hrs assay, showing increased anti-tumour activity over a period of time indicating slow release. Dose dependant cytotoxicity was observed after 24 hours upto 72 hours of incubation of nanoparticles while the drug per se (<4μg) showed 93% toxicity within 24 hours. Phase contrast microscopy of nanoparticle-cell interaction, clearly indicated aggregation along the lipid cell-membrane. Electron Microscopy (TEM, SEM) studies revealed its size and spherical shape. The stability of the particle, the slow and controlled release of drug from the gelatin nanoparticles indicate that it is a good candidate to deliver bio-pharmaceuticals. These behave as “;intelligent” carriers for drug delivery, and can be exploited to empty their drug load in acidic medium. The paper focuses on the release kinetics of the gelatin nanoparticles that can be successfully exploited to treat solid tumors.

The membrane-bound folate receptor (FR) is overexpressed on a wide range of human cancers, such as those originating in ovary, lung, breast, endometrium, kidney and brain. The vitamin folic acid is a high affinity ligand of the FR which retains its receptor binding properties when conjugated to other molecules. Consequently, “folate targeting” technology has successfully been applied for the delivery of protein toxins, chemotherapeutic agents, radio-imaging and therapeutic agents, MRI contrast agents, liposomes, gene transfer vectors, antisense oligonucleotides, ribozymes and immunotherapeutic agents to FR-positive cancers. These folate-bearing delivery systems have produced major enhancements in cancer cell specificity and selectivity over their non-targeted formulation counterparts. Hence, it is hopeful that this targeting strategy will lead to improvements in the safety and efficacy of clinically-relevant anti-cancer agents. Therefore, the focus of this review will be to highlight the current status of folate-targeted technology with particular emphasis on the recent advances in this field as well as possible directions for future development.

Due to its non-invasive character, fluorescence correlation spectroscopy (FCS) is particularly suited for the investigation of diffusion behavior of proteins in living cells. In this study we have investigated the diffusion properties of CFP-labeled gap junction hemichannels in the plasma membrane of living HeLa cells. Gap junction hemichannels or connexons are the precursors for the cell-cell- or gap junction channels that form large plaques at the contact areas between two adjacent cells. It has been proposed that new channels are recruited into a gap junction structure from a pool of hemichannels that can freely diffuse over the entire plasma membrane. The statistical approach shows that the geometry of the membrane within the focus is the most important property for the form of the autocorrelation curve and in turn for the determination of the diffusion coefficient. On the other hand binding-unbinding events which lead to anomalous diffusion have only a minor effect to the position and shape of the correlation curve compared to the geometry of the membrane.

The biophysical investigation of living cells is currently possible by single molecular detection methods such as fluorescence correlation spectroscopy (FCS). FCS is applied for measuring the dynamic mobility of target molecules in living cells; however, the conventional FCS systems still lack quantitative analysis for many regions of interests (ROI) in real time. To improve this situation, we have developed a novel multipoint FCS system (M-FCS) that can measure multipoint correlation functions in the cell simultaneously. To evaluate its performance, we measured correlation functions for rhodamine 6G (Rh6G) in homogeneous conditions and for green fluorescence protein (GFP) in HeLa cells. We conclude that M-FCS possesses reliable performance. As a pharmacological application, glucocorticoid receptor protein fused GFP (GR-GFP) was transfected in HeLa cells and FCS measurements were carried out in the cytoplasm and the nucleus simultaneously. The translocation of GR-GFP from the cytoplasm to the nucleus by ligand stimulation was observed with laser scanning microscopy (LSM) and M-FCS. Particularly in the nucleus, the slower diffusion of GR-GFP suggested molecular interactions after the translocation. These data imply that M-FCS can be applied for quantitative analysis of kinetic processes in living cells.

An infectious particle, termed prion, composed largely and perhaps solely of a single protein, is the likely causative agent of prion disease. It produces lethal decline of cognitive and motor function. The responsible protein arrives at a pathogenic state by misfolding from a normal form that has ubiquitous tissue distribution. Prion diseases are often called spongiform encephalopathies.Probably most mammalian species develop these diseases. Specific examples in various animals are -Scrapie, Transmissible Mink Encephalopathy (TME ), Chronic Wasting Disease(CWD) and bovine spongiform encephalopathy (BSE). Humans are also susceptible to several prion diseases: Creutzfeld-Jacob Disease (CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS), Fatal Familial Insomnia (FFI), Kuru and Alpers Syndrome. This paper reviews transmission of this diseases, protein involvement, nature of protein, the conversion process from PrPc to PrPSc, conversion of prion protein in vitro, the different proposed models for the conversion of PrPc to PrPSc, prion and other amyloid diseases, prion strains, structure of PrPc, the particular process that may induce prion disease, and immunization against these diseases.