Protein arginine methyltransferases (PRMTs) are (S)-adenosylmethionine (SAM)-dependent methyltransferases that catalyze the post-translational methylation of Arg residues in a variety of different proteins involved in transcriptional regulation and RNA splicing (e.g., histones H2A, H3, and H4). Herein, we describe the use of an N-mustard, 5'-(diaminobutyric acid)-N-iodoethyl-5'-deoxyadenosine ammonium hydrochloride (AAI), to generate a bisubstrate analogue inhibitor of PRMT1. Using the approach outlined in this communication, it should be possible to generate bisubstrate analogue-based inhibitors of PRMT isozymes that are potent and highly selective for a particular isozyme. The fact that PRMT1 catalyzes AAI transfer is also significant because with appropriate modifications (e.g., functionalization with pendant azido or alkyne functionalities) this compound could be used for proteomic applications to identify novel PRMT substrates.

Isothiocyanates (ITCs) are one of the many classes of breakdown products of glucosinolates found in crucifers such as broccoli and are thought to be partially responsible for the reduced risk of degenerative diseases associated with the consumption of vegetables. The production of ITCs such as L-sulforaphane is dependent on the hydrolytic bioactivities of myrosinase, localized both within vegetable tissues and within flora of the human GI tract, and is associated with important cancer chemopreventive activities. We hypothesized that novel isothiocyanates with enhanced chemopreventive properties relative to L-sulforaphane could be identified and that their glucosinolate precursors could be synthesized. From a library of 30 synthetic ITCs, we identified several with bioactivities equal or superior to those of L-sulforaphane. The corresponding non-natural glucosinolate precursors to these novel ITCs were constructed and found to be substrates for myrosinase. By utilizing a novel RP-HPLC assay to monitor myrosinase-dependent hydrolysis reactions, 2,2-diphenylethyl glucosinolate and (biphenyl-2-yl)methyl glucosinolate were shown to exhibit 26.5 and 2.8 %, respectively, of the relative activity of sinigrin and produced their corresponding ITCs in varying yields. These data support the notion that non-natural glucosinolates can act as prodrugs for novel ITCs, with a mechanism of action reliant on their hydrolytic cleavage by myrosinase. Such non-natural glucosinolates may serve as very economical chemopreventive agents for individuals at risk for cancers of and around the GI tract.

This paper describes the construction of a combinatorial artificial receptor array (CARA) and the application of the array to differentiation of proteins based on their binding patterns. Microarrays displaying 5035 unique binding environments were prepared using a library of 19 small molecule building blocks. Each building block was equipped with a carboxylic acid handle, allowing mixtures of the building blocks to be spotted onto the surface of an amine functionalized glass slide for covalent immobilization as subunits of the binding environments. This strategy employs the microarray surface as the receptor synthesis platform, which allows for flexibility in array preparation and agility in application. An advantage of the CARA strategy is the enormous flexibility it enables in the construction of alternate microarray configurations, which facilitates rapid access to the breadth and depth of binding space. Four fluorescently labeled proteins, ubiquitin, myoglobin, alpha-1-acid glycoprotein and lysozyme, were incubated with the arrays to demonstrate the reproducibility of binding and the level of differentiation that can be achieved. The binding environments are stable, scalable, and adaptable to other formats.