The Chemical Biology Core Facility consists of a multidisciplinary team of researchers whose aim is to initiate and advance collaborations within NIDDK chemical and biological laboratories. The goal of these partnerships is the discovery of new medicinal agents with therapeutic potential. The Chemical Biology Core Facility will advise, support and often develop each endeavor between laboratories, adding its expertise as needed, either in the synthetic development of novel molecules or in the pharmacological evaluation of existing compounds. Utilizing state-of-the-art methods in organic synthesis and pharmacological analysis the Chemical Biology Core Facility makes available numerous resources to support the laboratories of the NIDDK. ? ? The post-translational modification of protein surface serine and threonine residues is not limited to phosphate addition and removal by kinases and phosphatases. The manipulation of macromolecular substrates by the O-linked adornment of N-acetlyglucosamine by O-GlcNAc transferase and O-GlcNAcase are additional examples of such post-translational alteration that the cell exploits. Control of O-GlcNAc transferase and O-GlcNAcase may result in the management of ailments such as diabetes. With these considerations in mind, we have entered into collaborative efforts with John A. Hanover (NIDDK, NIH) to develop small molecules that display potent and specific control over both O-GlcNAc transferase and O-GlcNAcase. Our most successful efforts have surrounded the small molecule designated PUGNAc (a known inhibitor of the hexosaminadase class of enzymes) (Figure 1). Of particular interest was our extension of the N-acetyl group methyl moiety to larger alkyl chains. The continued development of small molecules that are capable of controlling these cellular functions will continue to aid our ability to dissect the complex phenotype associated with diabetes. ? ? The luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) are heterodimeric glycoprotein hormones that are involved in various roles within reproduction and maturation. Cellular response to all three of the glycoprotein hormones is mediated via separate G-protein coupled receptors (GPCRs); i.e. the LHR, FSHR and TSHR. Further, the seven-transmembrane domain of each receptor is noteworthy due to the high degree of homology between all three receptors. The disruption of the native cellular response of the LHR, FSHR and TSHR has been implicated in numerous disorders. We have entered into collaboration with Marvin C. Gershengorn (NIDDK, NIH) to develop small molecules to control the functions of these receptors. ? Several non-peptide small molecule agonists and antagonists for the LHR have been developed and continue to be of clinical interest including the potent LHR agonist Org 41841. Our interest lay in the intriguing potential to confer the binding affinity of 3 at the LHR to the highly homologus TSHR. It is our hope to re-engineer the small molecule using chemical synthesis and molecular modeling into a potent and specific modulator of TSHR function. To date, we have synthesized numerous analogues of 3, several of which display an altered binding affinity for the TSHR versus LHR. Of particular interest are several analogues that act as potent antagonists at the TSHR. The continued development of this class of small molecules will provide powerful tools for our ability to delineate the roles and deficiencies associated with TSHR function.? ? Cytokines are key regulators of cell development and homeostasis with particular importance concerning an organism?s immune response. Their actions are mediated by a class of nonreceptor tyrosine kinases known as the Janus Kinases (JAK). Over the past several years evidence has mounted that specific mutations on one particular JAK, the JAK 3 variant, are a primary cause of severe combined immune deficiency (SCID). This has lead researchers to consider inhibitors of JAK 3 as potential immunosuppression agents. A 2003 report by scientists at Pfizer, Stanford and the NIH detailed the discovery of a small molecule (CP-690,550) with potent and specific inhibition of JAK 3 and the resulting prevention of allograft rejection in non-human primates. This report detailed the selectivity of this small molecules inhibitory profile at over 30 kinases. The selectivity for JAK 3 was high relative to non-JAK kinases, however its inhibition at JAK 3 was only 20 fold greater than JAK2 and 100 fold greater than JAK 1.
Our aims are to further the inhibitory specificity in collaboration with both John J. O?Shea (NIAMS, NIH) and David M. Harlan (NIDDK, NIH) and we have taken up the synthesis of CP-690,550 and plan to co-crystallize this small molecule within the kinase binding domain of JAK3. Following this, we plan to utilize molecular modeling to detail the environmental differences that CP-690,550 will encounter at JAK3 versus JAK1 and JAK2. This information will allow us to re-engineer the small molecule to more exclusively bind to JAK3 and provide a more specific, less promiscuous kinase inhibitor.
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