Targeting SxIP-EB1 interaction: An integrated approach to the discovery of small molecule modulators of dynamic binding sites.

End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule (MT) growing ends, which has a fundamental role in MT polymerisation. EB1 is an important protein target as it is involved in regulating MT dynamic behaviour, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved four amino acid motif, (serine-X-isoleucine-proline) which exists within an intrinsically disordered region. Here we report the use of a multidisciplinary computational and experimental approach for the discovery of the first small molecule scaffold which targets the EB1 recruiting domain. This approach includes virtual screening (structure- and ligand-based design) and multiparameter compound selection. Subsequent studies on the selected compounds enabled the elucidation of the NMR structures of the C-terminal domain of EB1 in the free form and complexed with a small molecule. These structures show that the binding site is not preformed in solution, and ligand binding is fundamental for the binding site formation. This work is a successful demonstration of the combination of modelling and experimental methods to enable the discovery of compounds which bind to these challenging systems.

Microbial transformation of hydroxy metabolites of 1-oxohexyl derivatives of theobromine by Cunninghamella echinulata NRRL 1384.

AIM: The biotransformation of pentoxifylline (PTX), propentofylline (PPT) and their racemic hydroxy metabolites ((+/-)-OHPTX and (+/-)-OHPPT) by using the fungus Cunninghamella echinulata NRRL 1384. METHODS AND RESULTS: A fungus Cunninghamella echinulata NRRL 1384 was used to catalyse the (S)-selective oxidation of the racemic hydroxy metabolites: (+/-)-OHPTX and (+/-)-OHPPT and for reduction of PTX and PPT. The first oxidation step appears to be selective and relatively fast while the second reduction step is slower and more selective with PTX. Modifications involving supplementing the bioconversion with glucose give yields and enantiomeric excess (ee) values similar to those obtained without glucose. CONCLUSIONS: The bioconversion of (+/-)-OHPTX gave an (R)-enantiomer (LSF-lisofylline) with a higher enantiopurity (maximum approximately 93% ee) compared to the bioconversion of (+/-)-OHPPT, when the maximum ee value for (R)-OHPPT was recorded at 83%. SIGNIFICANCE AND IMPACT OF THE STUDY: The conversion of (+/-)-OHPTX and (+/-)-OHPPT using Cunninghamella echinulata can be recognized as a process, which may be recommended as an alternative to the methods used to obtain (R)-OHPTX and (R)-OHPPT.

Enadoline, a selective kappa-opioid receptor agonist shows potent antihyperalgesic and antiallodynic actions in a rat model of surgical pain.

Enadoline is a highly selective and potent kappa-opioid receptor agonist. This report describes and compares the activities of enadoline and morphine in a rat model of postoperative pain. A 1 cm incision through the muscle and skin of the plantar surface of the right hind paw induced thermal hyperalgesia as well as static and dynamic allodynia lasting at least 2 days. Postoperative testing was carried out using the plantar test for thermal hyperalgesia, von Frey hairs for static allodynia and light stroking with a cotton bud for dynamic allodynia. A single i.v. dose of enadoline 15 min before surgery dose-dependently (1-100 microg/kg) blocked the development of thermal hyperalgesia as well as static and dynamic allodynia for over 24 h with respective MEDs of < or = 1, 10 and 10 microg/kg. The administration of enadoline (100 microg/kg, i.v.), 1 h after surgery, completely blocked the maintenance of the hyperalgesic and allodynic responses, but its duration of action was much shorter (2 h) than when administered before surgery. Previous studies have shown that administration of morphine (1-6 mg/kg, s.c.) 0.5 h before surgery can prevent the development of thermal hyperalgesia with a MED of < or =1 mg/kg, but it has little effect on static allodynia. In the present study similar administration of morphine (1-3 mg/kg), unlike enadoline, had no effect on the development of dynamic allodynia. Morphine dose-dependently (1-6 mg/kg, s.c.) potentiated isoflurane-induced sleeping time and respiratory depression in the rat. However, whilst enadoline also (1-1000 microg/kg, i.v.) potentiated isoflurane-induced sleeping time, it did not cause respiratory depression. It is suggested that enadoline may possess therapeutic potential as a pre-emptive antihyperalgesic and antiallodynic agent.

Stereoinversions using microbial redox-reactions.

This paper aims to provide a summary of the recent literature on the use of redox enzymes to carry out stereoinversion reactions on chiral secondary alcohols. Emphasis has been placed on biotransformations which result in the deracemization of a racemic substrate to give high value synthetic intermediates in a theoretical 100% yield. Most of the biocatalysts which are competent to carry out such transformations are whole cell systems, which contain the necessary cofactor recycling machinery to facilitate this otherwise entropically disfavoured process. The first section deals with deracemization of compounds such as mandelic acid and pantoyl lactone using two microorganisms which display enantiocomplementary stereospecificity. The deracemization of chiral alcohols such as beta-hydroxyesters, aryl ethanols and terminal 1,2-diols with single microorganisms will then be discussed and the influence of growth and reaction conditions on the selectivity observed will be emphasised. Then the ability of several microorganisms to deracemize by double stereoinversion substrates with two stereocentres such as cyclohexan-1,2-diol, cis and trans indan-1,2-diol and pentan-2,4-diol will be presented and some mechanistic rationale proposed. Lastly enzymes known as epimerases which are important in sugar and deoxysugar biosynthesis will be discussed with reference to some recent work on the mechanism of UDP-glucose epimerase.