Targeting the lateral interactions of transmembrane domain 5 of Epstein-Barr virus latent membrane protein 1.

The lateral transmembrane protein-protein interaction has been regarded as "undruggable" despite its importance in many biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein, we report a small molecule agent, NSC 259242 (compound 1), to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of compound 1 are essential for its inhibitory activity. Furthermore, cell-based assays revealed that compound 1 inhibits full-length LMP-1 signaling in EBV infected B cells. These studies demonstrated a new strategy for identifying small molecule disruptors for investigating transmembrane protein-protein interactions.

Evidence that opioids may have toll-like receptor 4 and MD-2 effects.

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

Development of agents that modulate protein-protein interactions in membranes.

Membrane proteins account for approximately one third of all proteins in eukaryotic and prokaryotic cells. These proteins are critical in a diverse array of cellular functions. Despite their obvious importance, the effectiveness of research tools to study the structure and function of integral membrane proteins lags behind that of water-soluble proteins. This is due in part to the lack of probing agents that can specifically and selectively recognize these targets. This review focuses on methods developed to overcome the obstacles of studying membrane proteins. We describe TM protein properties as well as biophysical properties of amino acids within the membrane bilayer. We also summarize the known characteristics of membrane regions in their distinctive environments and generate a summary of current research approaches that succeed in probing interactions of TM proteins within their native setting. This allows further insight into protein-protein interactions in a hydrophobic environment as it pertains to drug development.

Design, synthesis, and evaluation of biotinylated opioid derivatives as novel probes to study opioid pharmacology.

A generally applicable strategy of chemically labeling (-)-morphine (1) is described. The synthesis starts from commercially available starting materials and can be completed in two steps with an overall yield of 23%. In silico simulation and NMR results show that the binding of (-)-morphine to one of its molecular targets, toll-like receptor 4 (TLR4), was not affected by the modification. Secreted embryonic alkaline phosphatase (SEAP) reporter assay results demonstrate that C(3) biotinylated and unmodified (-)-morphine show similar biological activities in live cells. To our knowledge, these studies provide the first practical and concise method to label various opioid derivatives, a group of important therapeutics in pain management, for biochemical/pharmacological studies.