Anti-inflammatory activity of non-nucleoside adenosine deaminase inhibitor FR234938.

Adenosine has anti-inflammatory activity. Adenosine deaminase (EC 3.5.4.4) metabolizes extracellular adenosine, resulting in an exacerbation of inflammation. Consequently, it was hypothesized that adenosine deaminase inhibitors produce anti-inflammatory activity by increasing extracellular adenosine concentration. This group recently developed a non-nucleoside adenosine deaminase inhibitor, FR234938, by using rational structure-based drug design. FR234938 inhibits recombinant human adenosine deaminase enzyme competitively. FR234938 inhibits interleukin (IL)-6-dependent immunoglobulin (Ig) M production by SKW6.4 cells, in the presence of adenosine. Inhibitory effect of FR234938/adenosine combination is blocked by an A2a adenosine receptor antagonist. FR234938 also inhibits anti-type II collagen delayed type hypersensitivity (DTH) in a dose-dependent manner, both in the presence and absence of recombinant human adenosine deaminase. Moreover, FR234938 inhibits tumor necrosis factor (TNF)-alpha and IL-10 production in a lipopolysaccharide (LPS)-induced cytokine production model in mice. These results indicate that FR234938 has potential anti-inflammatory activity. Non-nucleoside adenosine deaminase inhibitor FR234938 has good potential as a new type of anti-rheumatic and anti-inflammatory drug, by modulating host-defense concentrations of adenosine.

FR177391, a new anti-hyperlipidemic agent from Serratia. IV. Target identification and validation by chemical genetic approaches.

Natural products with distinct biological activities are very promising molecular probes to dissect the novel pathways of biology. FR177391, a product of bacteria, was obtained as a natural compound possessing anti-hyperlipidemic effects. FR177391 enhances differentiation of mouse 3T3-L1 fibroblasts to adipocytes and reduces the circulating levels of triglyceride in C57BL/KsJ-db/db mice, a obese non-insulin-dependent diabetes mellitus animal model, although its mechanism of actions remained to be unknown. We report here that the target protein for FR177391 was identified to be protein phosphatase 2A (PP2A) by employing the method of affinity chromatography. FR177391 potently inhibited PP2A activity at nano molar concentration, and shared its binding pocket with a phosphatase inhibitor, okadaic acid. In addition to the phenotypic alterations, the enhancement for phosphorylation of extracellular signal-regulated kinase (ERK) protein was observed in the FR177391-treated 3T3-L1 cells. These results suggest that prolonged activation of ERK protein due to inhibition of its dephosphorylation by PP2A plays an important role in adipocyte maturation and regulation of the blood revels of lipids.

The significance of the flexible loop in the azurin (Az-iso2) from the obligate methylotroph Methylomonas sp. strain J.

The obligate methylotroph Methylomonas sp. strain J produces two azurins (Az-iso1 and Az-iso2) as candidates for electron acceptor from methylamine dehydrogenase (MADH) in the electron-transfer process involving the oxidation of methylamine to formaldehyde and ammonia. The X-ray crystallographic study indicated that Az-iso2 gives two types of crystals (form I and form II) with polyethylene glycol (PEG4000) and ammonium sulfate as the precipitants, respectively. Comparison between the two Az-iso2 structures in forms I and II reveals the remarkable structural changes at the top surface of the molecule around the copper atom. Az-iso2 possesses Gly43 instead of Val43 or Ala43, which is unique among all other azurins around the copper ligand His46, inducing the remarkable structural change in the loop region from Gly37 to Gly43. When the structure of Az-iso2 is superimposed on that of amicyanin in the ternary complex composed of MADH, amicyanin, and cytochrome c(551), the loop of Az-iso2 deeply overlaps with the light subunit of MADH. However, the Az-iso2 molecule is probably able to avoid any steric hindrance with the cognate MADH to form the complex for intermolecular electron-transfer reaction, since the loop containing Gly43 is flexible. We discuss why the electron-transfer activity of Az-iso2 is fivefold higher than that of Az-iso1.

Structure-based de novo design of non-nucleoside adenosine deaminase inhibitors.

We searched for non-nucleoside inhibitors of adenosine deaminase by rational structure-based de novo design and succeeded in the discovery of 1-(1-hydroxy-4-phenyl-2-butyl)imidazole-4-carboxamide (FR221647: K(i)=5.9 microM to human ADA) as a novel inhibitor with moderate activity and good pharmacokinetics compared with the known inhibitors pentostatin and EHNA.

Structure of bovine adenosine deaminase complexed with 6-hydroxy-1,6-dihydropurine riboside.

The crystal structure of adenosine deaminase (ADA) from bovine intestine complexed with a transition-state analogue, 6-hydroxy-1,6-dihydropurine riboside (HDPR), was solved at 2.5 A resolution by the molecular-replacement method using a homology model based on the crystal structure of mouse ADA. The final refinement converged to a crystallographic R factor of 20.7%. The C(alpha) backbone of bovine ADA is mostly superimposable on that of mouse ADA, although mouse ADA itself did not lead to a solution by molecular replacement. HDPR tightly interacts with ADA by means of six hydrogen bonds and is entirely enclosed within the active site. The lid of the envelope consists of two components: one contains two leucine residues, Leu55 and Leu59, and the other contains the backbone atoms Asp182 and Glu183. The C(delta) atoms of the two leucine residues are 3.5 A from the respective N atoms of the backbone. A weak interaction, similar to CH-pi binding, might make it possible to open the lid. Taking account of the movement and observation of this structural feature, the aim is to design novel ADA inhibitors.