2-Aminoethyl Diphenylborinate (2-APB) Analogues: Part 3-Regulators of

Huntington aggregation inhibitory activities and transglutaminase inhibitory activities of 2APB analogues were measured. 2-APB analogues regulated the Huntington aggregation. This fact provided an example that 2-APB analogues can regulate cellular process. Diphenyl (aminoacidonate N,O) boranes, which are effective regulator of Ca2+ release and cellular process, were effective regulators of Huntington aggregation. It was also found that many of 2-APB analogues have moderate transglutaminase inhibition activities 2-Aminoethyl di(4-trifluorophenyl) borinate is a good transglutaminase regurator. 2-Aminoethyl Diphenylborinate (2-APB) Analogues: Part 3 Regulators of Huntington Aggregation and Transglutaminase

This time, we report about 2 tests how these 2APB analogues show the ability to regulator Ca 2+ related enzyme inhibitory activity and regulation of cellular process. One test is a regulation of transglutaminase, Second test is a regulation of Huntington aggregation using 2APB analogs having various Ca 2+ release-related activities: SOCE. We wish to tell the relations of trangltaminase activity (TG), Huntington aggregation inhibition (x-Fold) and SOCE and chemical structures of compounds.
We measured transglutaminase (TG) inhibitory activities .and Huntington aggregation inhibitory activities (x-Fold) of 276 2APB analogues. And we analyzed the result and relation of TG, x-Fold and SOCE IC 50.

Transglutaminase inhibitory activities measurement
TG (TransGlutaminase) inhibiting activity assay: Inhibition of TG enzyme was determined by assaying the enzyme activity in accordance with an optionally modified version of the enzyme activity in accordance with an optionally modified version of the method of Lorand et al. [53]. An enzyme reaction solution (0.1 ml) (100 mM HEPES-NaOH, pH 7.5, 1 mM CaCl 2 , 20 M, monodansyl cadaverine, 0.05 mg/ml N,N-dimethylcasein, 5 μg/ml TGase) was introduced into wells of a 86-well plate (Nunc, 96 well Black Plate with Clear Bottom). A test compound was added in concentration of 100 μM. The plate was set in the fluorescence drug screening system FDSS3000 (hamamatsu Photonics K.K) TGase-inhibiting activity of the compound was calculated by assaying changes in fluorescence wavelength (at 340 nm) per unit time. The assay level at which a fluorescence change was observed with the addition of DMSO (1 μl) used as control instead of the test compound was designated as 100. The assay level at which TGase activity decreased by half in the presence of the test compound was designated as TG 50. Ratio of aggregated cell to total cells (x-Fold) was measured. Without test compounds, ratio of aggregated cell to total cell x-Fold is 1. The smaller the x-Fold, the aggregation inhibition is stronger.

SOCE inhibition activities measurement
Inhibitory activities of the 2APB analogues for SOCE were measured using our improved assays as described previously [50].

Results and Discussion
We measured transglutaminase inhibitory activities (TG) and Huntington aggregation inhibition activity (x-Fold), and SOCE inhibition activities IC 50

Relations of chemical structures and activities
Active compounds of 2APB analogues, nitrogen atom must come in this order B-O-C-C-N as 2-APB C 6 H 5 B(OCH 2 CH 2 NH 2 )C 6 H 5 . The compounds having other order like B-O-C-N, or B-O-C-C-C-N have no activities. When phenyl group is substituted with aliphatic or aryaliphatic group, they lost their activities. When compared mono-boron, bis-boron and poly-boron compounds, mono-boron compounds were best and bis-boron compounds come next.

Relation of SOCE IC 50 value and TG and x-Fold
Relation of SOCE IC 50 and x-Fold: When look at Figure S1, the compounds having strong Ca 2+ release activity, low IC 50 value (IC 50is <1) except aminothiol adduct compounds, showed strong Huntington aggregation inhibiting activity The compounds having weak Ca 2+ releasing activity (IC 50 is >10) showed weak or no transglutaminase inhibiting activity (TG is near 100), and showed weak or no Hunting aggregation inhibiting activity (x-Fold is near 1). These results indicated that 2APB analogues were effective as regulators of cellular process.

Relation of SOCE IC 50 and TG. Relation of TG and x-Fold:
The compounds having strong Ca 2+ release activity, low IC 50 value (IC 50is <1) except aminothiol adduct compounds, showed no transglutaminase inhibition activities, x-Fold is near 1.00 even through IC 50 is 0.2. Because our assay of transglutaminase inhibition is done in vitro and the assay is not cellular process. Calcium release cannot be expected. Therefore we cannot find any relation between TG and SOCE IC 50 value also we cannot find any relation between TG and x-Fold. If we assay transglutaminase inhibition in vivo system, other results would be expected.      [76,77] or thienopyrimidines IC 50 : 0.13 μM reported by Duval [74]. Transglutaminase is necessary enzyme for our lives. Strong inhibitor will give toxicity. Moderate activity is required for clinical use.. We are reporting many TG inhibitors .differing activities. People will be able to choose most suitably active compounds. Some of these compounds were shown to inhibit the calcium dependent enzyme transglutaminase [43]. Transglutaminase inhibitors block the abnormal cross-link of protein [43,[76][77][78] and they may slow down or even stop the progression of disease caused by over cross-linked proteins, such as Huntington`s disease.

Conclusion
2-APB analogues regulated the Huntington aggregation has provided an example that 2-APB analogues can regulate cellular process. Diphenyl (aminoacidonate N,O) boranes are effective regulators of Huntington aggregation. It was also found that many of 2-APB analogues had moderate transglutaminase inhibition activities.