Maxim Berezovski
Project #1
NECEEM-based methods for drug discovery and diagnostic development
Capillary electrophoresis (CE) has proved to be a very efficient analytical tool with high resolution, high sensitivity, high speed, and very low volume of sample required. The research is based on a new method - Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM)-which was invented by Prof. Krylov and me (Berezovski, M.; Krylov, S. N . J. Am. Chem. Soc. 2002, 124, 13764-13765). NECEEM is, in turn, based on our understanding that equilibrium and non-equilibrium conditions can be mixed in a single capillary electrophoresis procedure to provide the foundation of three practical methods. Conceptually, in NECEEM, the equilibrium mixture of molecules T and L is prepared by mixing them and allowing them to reach equilibrium. The concentrations of L, T, and L·T in the equilibrium mixture are defined by the equilibrium biding constant Kd. A short plug of the equilibrium mixture is then injected onto the capillary by pressure and subjected to electrophoresis under non-equilibrium conditions, in the separation buffer that does not contain L or T. The separation conditions are chosen so that at least two of the three components, L, T and L·T, are effectively separated. Under such conditions at least one of L and T is continuously removed from the electrophoretic zone of L·T and such removal causes continuous monomolecular decay of L·T with the rate constant koff. Due to a mixed equilibrium/non-equilibrium nature of NECEEM, electrophoretic data contain accurate information about both Kd and koff. This feature of NECEEM is essential for the three NECEEM-based methods.
Method 1 has been demonstrated in our resent work (Krylov, S. N.; Berezovski, M. Analyst, 2003, 128, 571-575). It allows for the determination of Kd and koff of complex formation from a single NECEEM experiment. Another unique feature of the method is its extremely high sensitivity; if laser-induced fluorescence is used then as few as 10^-18 moles of T is sufficient to determine Kd and koff of its interaction with L. If a series of experiments is performed at different temperatures then a number of thermodynamic parameters (activation energies of forward and reverse reactions, the reaction enthalpy, and the change of entropy) can be determined.
Method 2 has been used to provide a quantitative analysis of proteins using affinity probes whose complexes with proteins decay partially or completely during separation. When the probes are fluorescently labeled then method 2 is characterized by extremely low mass limits of detection (Berezovski, M.; Nutiu, R.; Li, Y.; Krylov, S.N. Analytical Chemistry 2003 , 75, 1382-1386).
We intend to use Method 3 as a new and powerful approach to select target-binding molecules from complex mixtures. Unique capabilities of Method 3 include but not limited by: (a) the selection of ligands with specified ranges of kinetic and thermodynamic parameters of target-ligand interactions, (b) the selection of ligands present in minute amounts in complex mixtures of biological or synthetic compounds, and (c) the selection of ligands for targets available in very low amounts. The method advantageously does not require monitoring the shift of the peak of the target. Furthermore, it does not require the presence of the detectable amounts of the target in the equilibrium mixture in detectable amounts. This allows for selecting very tight ligands with very low values of Kd (e.g. Kd < 10 nM). The method facilitates repetitive refinement procedures that can lead to a series of ligands with very narrow ranges of Kd, kon, and koff or even to a single ligand with desirable Kd, kon, and koff. The three methods can be used for discovery and characterization of drug candidates and the development of new diagnostic methods.
Project#2
Chemical cytometry for studying prenyltransferase activity in single cancer cells.
My first research project is concerned with the study of prenyltransferase activity and activity of farnesyltransferase inhibitors in single cancer cells. In this work I am using a new method, chemical cytometry, and image cytometry to correlate the formation of metabolic products with the cell cycle. The enzymatic activity is monitored in individual cells at different stages of the cell cycle. This assay strategy should be applicable to the analysis of a broad range of intracellular enzymes in single cells.
The timely diagnosis of cancer is very important for the treatment of the disease. It is well known that before morphological changes are detectable, cytochemical changes have long undergone gradual transformation. The concentrations and activities of many molecular markers or biomarkers (e.g. growth factors, proteins, enzymes, DNA adducts, etc.) are constantly changing during carcinogenesis. Much effort therefore has been and will continuously be put into finding and defining potential molecular markers for different kinds of cancers. Among such markers, the enzymes attracted much attention because they control the balance of cytochemicals and actively participate in the cell proliferation process. As investigated previously, enzymes might show different activity in tumour cells compared to that in normal cells, and some isoenzymes even showed different patterns.
Protein prenyltransferases (farnesyltransferase and geranylgeranyltransferases type I and II) are very important for the maturation and cellular function of many proteins. Intense interest in this field was sparked by the observation that mammalian Ras proteins are farnesylated, and that this modification is necessary for both membrane localization and the cell-transforming activity of Ras. Ras mutants have been implicated in approximately 30% of human cancers. Cell culture studies have shown that inhibition of Ras farnesylation results in the reversion of transformed cells to their normal phenotype. Many inhibitors of the protein farnesyltransferase have remarkably low cytotoxicity, making them promising anti-tumour pharmaceutics.
In my work I measure prenyltransferase activity using capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). A fluorescently labeled peptide substrate is synthesized. Classically, this assay would be performed with an unlabeled protein and a radioactive prenyl diphosphate substrate; mobility shifts would be detected by gel electrophoresis and autoradiography. However these techniques are not sensitive enough to be applied to individual cell analysis. Clearly, the CE-LIF is much more faster and accurate. With these characteristics it allows to analyze biochemical reactions at the single-cell level.
References:
Berezovski, M.; Li, W.-P.; Poulter, C.D.; Krylov, S.N. (2002) Measuring the activity of farnesyltransferase by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis. 23: 3398-3403.