Design of Nucleotidomimetic Chemical Probes

Design of Nucleotidomimetic

Translational Control

Translation initiation can occur by internal ribosomal entry or by cap-dependent means. The first steps in this process depends on the assembly of a trimolecular cap-binding complex designated eIF4F, which consists of eIF4E, eIF4G, and eIF4A. Translational control is usually exerted by regulation of the abundance and activity of eIF4E, which binds the 5' methylguanosine cap of mRNA (7-methyl-G(5')ppp(5')N where N is any nucleotide). Since the abnormal over-expression of eIF4E has been associated with tumor tissues, the down regulation of cap-dependent translation should revert the transformed phenotype.

Our laboratory had designed the only cell permeable inhibitor, 4eI1, of eIF4e. With this inhibitor we have been able to demonstrate that inhibtion of eIF4e chemosensitizes cancer cells to non-toxic doses of gemcitibine. In addition, 4eI1 has become a useful probe for determining the importance of weak verses strong mRNAs on tumor cell transformation and T-cell activation. Currently, we are designing newer more potent analogs of 4eI1 that can be used as potential IV and orally dosed chemosensitization agents for a variety of cancers.

Role of HINT1 in Pain

Pain is highly dependent on the glutamatergic N-Methyl-D-Aspartate Receptor (NMDAR) to control neurotransmission and synaptic plasticity. NMDAR is a calcium channel that can be either positively or negatively regulated by a wide range of G protein-coupled receptors (GPCRs) such as the Mu-opioid receptor (MOR).

Recently, our team has demonstrated that HINT1 is a key mediator of MOR-NMDAR cross-regulation by means of a series of molecular and mouse knock-out studies. In particular, HINT1 directs the association of MOR with NMDAR by co-associating with the C-terminus of both receptors. Upon binding with morphine and activation of analgesia, Zn²⁺ is mobilized, resulting in Protein Kinase C g (PKCg) recruitment. NMDAR phosphorylation by PKCg results in activation of the channel and Calmodulin-Dependent Kinase II (CaMKII) suppression of MOR. Thus, preventing the recruitment of PKCg by HINT1 to the MOR-NMDAR complex could be a promising strategy for overcoming opioid tolerance and developing non-opioid drugs for pain

Over the past decade, our laboratory has carried out extensive biochemical, structural and pharmacological studies of bacterial and human HINT1 enzymes. Substrate specificity analyses have revealed that HINT1s are nucleoside phosphoramidate monoester and acyl-adenylate hydrolases with a moderate preference for substrates containing ribose rings and purines. We have also elucidated the essential active site requirements for binding and catalysis. Using knowledge gained from this enzymology, we prepared a highly stable 5’-nucleoside carbamate-based inhibitor, 5’-tryptamine guanosine carbamate (TpGc) of hHINT1. We have demonstrated via x-ray crystallography that TpGc binds directly to the hHINT1 active site by interacting with amino acid residues governing catalysis. Using mouse models, we have demonstrated that TpGc can both prevent and rescue morphine tolerance and block neuropathic pain. Taken together, our results demonstrate that the HINT1 active site is intimately involved in the regulation of the observed behavior and thus a potential novel pain target. Currently, we are designing newer more potent and selective inhibitors of HINT1 as chemical probes of its role in the regulation of pain.