Discovery of a pyrazolo[1,5-a]pyrimidine derivative (MT-3014) as a highly selective PDE10A inhibitor via core structure transformation from the stilbene moiety.

We have developed a new class of PDE10A inhibitor, a pyrazolo[1,5-a]pyrimidine derivative MT-3014 (1). A previous compound introduced was deprioritized due to concerns for E/Z-isomerization and glutathione-adduct formation at the core stilbene structure. We discovered pyrazolo [1,5-a] pyrimidine as a new lead scaffold by structure-based drug design utilizing a co-crystal structure with PDE10A. The lead compound was optimized for in vitro activity, solubility, and selectivity against human ether-á-go-go related gene cardiac channel binding. We observed that MT-3014 shows excellent efficacy in rat conditioned avoidance response test and suitable pharmacokinetic properties in rats, especially high brain penetration.

Comparison of changes in urinary and blood levels of biomarkers associated with proximal tubular injury in rat models.

To investigate useful biomarkers associated with proximal tubular injury, we assessed changes in levels of a focused set of biomarkers in urine and blood. Male rats administered a single dose or four doses of gentamicin (GM, 240 mg/kg/day) or a single dose of cisplatin (CDDP, 5 mg/kg) were euthanized on days 2 (the day after initial dosing) 5, or 12. At each time point, histopathological examination of the kidney and immunohistochemistry for biomarkers, kidney injury molecule-1 (Kim-1), lipocalin (NGAL), clusterin (CLU), cystatin C (CysC) and ß2-microglobulin (ß2M) were performed. Biomarker levels were measured in urine and blood. In both treatment groups, degenerated/necrotic proximal tubules and regenerated tubules were mainly observed on days 5 and 12, respectively. At the same time as these tubular injuries, urinary Kim-1, CysC and ß2M levels were increased. Moreover, urinary levels of CysC and ß2M in GM-treated animals and Kim-1 in CDDP-treated animals increased (on day 2) prior to tubular injury on day 5. This was considered to reflect the characteristics of drug toxicity. Although almost all of the biomarkers in blood were not sufficiently sensitive to detect proximal tubular injury, urinary and plasma ß2M levels simultaneously increased. Therefore, in addition to urinary Kim-1, CysC and ß2M levels, plasma ß2M levels were also considered useful for detecting proximal tubular injury.

Enzymatic polymerization catalyzed by immobilized endoglucanase on gold.

Enzymatic polymerization was carried out on gold by immobilized genetically engineered endoglucanase II (EGII) from Trichoderma viride , and the polymerization behavior and the produced cellulose were analyzed in comparison with those by free enzymes. Mutant EGIIs were EGII(core2) and EGII(core2H), which consist of two sequential catalytic core domains with one His-tag (His6) on N-terminal and with totally two His-tags on both terminals, respectively. His-tagged EGIIs were immobilized via Ni chelators of nitrilotriacetic acid (NTA) introduced on gold surface. From SPR measurements, the affinity of EGII(core2H) to the surface was higher than that of EGII(core2), and the molecular occupation area of EGII(core2H) was larger than that of EGII(core2), indicating that EGII(core2H) was immobilized with utilizing two His-tags introduced on both terminals. The hydrolytic activity of the immobilized EGII(core2H) using cellohexaose as substrate was slightly lower than that of free EGII(core2H) when they were compared at the same amount in the hydrolytic system. Enzymatic polymerization catalyzed by both immobilized EGII(core2) and EGII(core2H) proceeded with producing highly crystalline cellulose in comparison with free enzyme. Immobilization of the endoglucanase is thus effective to obtain crystalline cellulose due to the high density of the catalytic domain on gold.

Enzymatic activities of novel mutant endoglucanases carrying sequential active sites.

Novel mutant enzymes of endoglucanase II (EGII) from fungus Trichoderma viride were prepared and their hydrolysis and enzymatic polymerization activities were studied. EGII(core)2 and EGII(core)2-His, which possess sequential two active sites of EGII with a His-tag probe at the N-terminal and with His-tag probes at the N and C terminals, respectively, showed higher hydrolysis activities than EGIIcore with a single active site even in comparison on the active-site concentration basis. These mutant enzymes were applied to the enzymatic polymerization to afford artificial cellulose. The polymerization rates with using EGII(core)2 and EGII(core)2-His were also higher than that with using EGIIcore. The polymerization products were identified as highly crystalline cellulose of type II. The mutant enzymes were also effective to prepare spherulites. EGII(core)2 and EGII(core)2-His are considered to possess higher hydrolysis and polymerization activities than EGIIcore mainly due to the suitably stabilized conformation with the sequential arrangement.

Enzymatic polymerization behavior using cellulose-binding domain deficient endoglucanase II.

A mutant enzyme, EGII(core), in which the cellulose-binding domain was deleted from endoglucanase II from Trichoderma viride, was expressed in yeast, and the secreted enzyme was examined for the enzymatic polymerization to obtain artificial cellulose. EGII(core) polymerized beta-cellobiosyl fluoride to afford crystalline cellulose of type II. Comparison of the polymerization behavior of EGII(core) with that of EGII revealed the following: i) the crystalline product obtained with EGII(core) was stable in the polymerization solution, although the product was readily hydrolyzed in the presence of EGII; ii) the turnover number of EGII(core) was as high as that of EGII; iii) EGII(core) produced highly crystalline cellulose. EGII(core) is therefore advantageous for enzymatic polymerization.

Preparation of fibrous cellulose by enzymatic polymerization using cross-linked mutant endoglucanase II.

A cross-linked mutant endoglucanase II was prepared for enzymatic polymerization to cellulose. The cross-linked enzyme is composed of three mutant enzymes showing polymerization activity. A characteristic feature of the polymerization with this cross-linked enzyme is formation of cellulose fibrils in contrast to plate-like crystals obtained by using a free enzyme.

Immobilization of His-tagged endoglucanase on gold via various Ni-NTA self-assembled monolayers and its hydrolytic activity.

A genetically modified His-tagged endoglucanase, EGII(core2), with two active sites in series was immobilized on gold via three different kinds of anchor molecules, and its hydrolytic activity was studied. Immobilization of EGII(core2) was influenced by the chain length and hydrophilicity of anchor molecules. The hydrolytic activity of the immobilized EGII(core2) was nearly the same on either anchor molecule. Interestingly, the immobilized EGII(core2) apparently retained the inherent hydrolytic activity similarly to free EGII(core2). It is therefore considered that the local high concentration of EGII(core2) on the surface should promote the successive hydrolysis of the transient products to show the high hydrolytic activity despite of immobilization.