Molecular forceps

Building a Bridge Between Chemistry and Biology - Molecular Forceps that Inhibit the Farnesylation of RAS... 

We used this library to find receptors for the CaaX-box of the H-RAS protein. Because selecting molecular forceps from libraries for H-RAS binding does not ensure these forceps will recognize the carboxy terminus of H-RAS, however, we decided to take a two-pronged screening approach - in addition to screening the library with the protein, we sought to screen with the isolated carboxy-terminal peptide from H-RAS to select molecular forceps that bind this epitope specifically. 

The re-synthesized molecular forceps (MF1 through MF8, except MF5 which was not soluble) were tested for inhibition of farnesylation by yeast FTase [25], As expected, MF8, our negative selection, did not inhibit farnesylation of H-RAS protein. MF6 and MF7 from the protein screening also had no effect neither did MF1 that we had obtained from peptide screening. MF4 had a weak impact on the far-... 

To confirm further that inhibition of farnesylation of GFP-CaaX-proteins is because of the binding of the molecular forceps to their CaaX-sequence, and not because of inhibition of FTase, we performed in-vitro binding assays with FTase. Whereas MF3 clearly interacts with H-RAS at a concentration of 250 gM, two and a half times the observed IC50, we did not observe binding to Ftase (Figure 3.1.4b). Because the forceps bind to RAS and fail to bind FTase, we concluded that MF3 could not act as an enzyme inhibitor, but that its activity is caused by its interaction with the substrate [26]. 

By using a simple receptor design we generated a library of molecular forceps and screened them against the farnesylation site of the RAS protein. Our results prove the validity of our assumptions. While the actual numbers of molecules tested... 

Dong DLY, Liu R, Sherlock R, Wigler MH, Nestler HP. Molecular forceps from combinatorial libraries prevent the farnesy-lation of ras by binding to its carboxy-terminus. Chem. Biol. 1999 6 133-141. 

Fukase K, Wakao M, Izumi M, Ueno A, Oikawa M, Suda Y, Kusumoto S, Nestler HP, Sherlock R, Liu R. Identification of molecular forceps that recognize immunostimulating glycoconju-gate lipid A from encoded combinatorial hbraries. In Proc. International Electronic Conferences on Synthetic Organic Chemistry, 5th, 6th, 2001. pp. 374-379. 

Unfortunately, biopsy sampling using AFB-LIFE may remove entire patches of clonal cells [69]. Many lesions were <1.5 mm in diameter and about 50% of these lesions were smaller than the biopsy forceps. Twenty-seven of the 69 paired biopsies obtained at 6-month intervals showed one or more molecular changes in the initial biopsy specimens, 86% had no abnormality after re-biopsy and 24% lacked the initial changes found after repeat biopsy. So, the natural history of minute lesions cannot be studied because of complete mechanical removal during baseline sampling. 

Mix up a small ahquot of fluorescent dextran. Use Molecular Probes product numbers D-3308 and D-3306 for tetramethylrhodamine and fluorescein fluorescence, respectively. The author keeps a small frozen stock of dextran made up at a concentration of lOOmg/mL in distilled water from which he transfer a very small drop onto a Sylgard surface. As the water evaporates off, the dextran becomes sticky and is easily picked up on to the tip of a stainless-steel or tungsten micropin or onto the tips of watchmaker s forceps. Simply add more water to the drop of dextran if it dries out too much. 

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