A case of subacute thyroiditis after influenza vaccination.

Summary: A 40-year-old Japanese woman presented to the outpatient clinic with fever and palpitations 2 days after receiving the influenza vaccine (Influenza HA Vaccine 'KMB'®) following the second dose of coronavirus disease 2019 (COVID-19) vaccine (COVID-19 vaccine Moderna intramuscular injection®). At the first visit, the patient presented with a swollen thyroid gland with mild tenderness, and she was diagnosed with subacute thyroiditis (SAT) based on the presence of thyrotoxicosis (free T3: 5.42 pg/mL; free T4: 2.34 ng/dL; and thyroid-stimulating hormone (TSH): <0.01 µIU/mL), a high C-reactive protein level (5.77 mg/dL), a negative TSH receptor antibody, and characteristic ultrasound findings. The patient's human leukocyte antigen types were A2, A11, B35, B51, DR4, and DR1403. Prednisolone (15 mg/day) was given as an initial dose, after which the fever subsided, and the dose was tapered and discontinued after 6 weeks. The patient was thought to have developed SAT due to influenza vaccination. SAT after influenza vaccination may be overlooked. For patients with SAT, it is necessary to obtain information regarding their vaccination history. Learning points: After influenza vaccination, subacute thyroiditis (SAT) may develop. If persistent fever, anterior neck pain, swelling, tenderness of the thyroid gland, and symptoms of thyrotoxicosis are observed immediately after vaccination for several viruses, including influenza, an examination to rule out the onset of SAT is recommended. Human leukocyte antigen type A2 (HLA-A2) and HLA-B35 may be linked to the development of SAT following influenza vaccination. The two doses of the coronavirus disease 2019 (COVID-19) vaccine given before the influenza vaccine may affect the onset of SAT.

Prolonged postoperative hypoaldosteronism related to hyperkalemia in patients with aldosterone-producing adenoma.

Hyperkalemia is developed in a part of patients with aldosterone-producing adenoma (APA) after adrenalectomy, suspected to be due to the insufficiency of aldosterone secretion. The purpose of this study is to determine the frequency and characteristics of prolonged postoperative hypoaldosteronism (PPHA) using chemiluminescent enzyme immunoassay (CLEIA). We studied 58 patients with APA with long time after adrenalectomy and whose PAC was measured using a CLEIA kit. The PAC value measured using CLEIA was significantly lower than that of using RIA between two consecutive visits before and after the shift of measuring method of PAC (median [interquantile range], 123.0 [99.8-164.0] vs. 39.5 [15.8-64.2] pg/mL, p < 0.01). PAC was below the minimum limit of quantification (4.0 pg/mL) of the CLEIA kit at least once in nine patients (15.5%) who had PPHA. The PPHA group were older (mean ± standard deviation, 61.3 ± 8.5 vs. 50.5 ± 10.1 years, p < 0.01) and had lower eGFR (60.3 ± 14.0 vs. 82.3 ± 22.8 mL/min/1.73 m2, p < 0.01) than the non-PPHA group. The frequency of postoperative hyperkalemia (maximum serum potassium >5.5 mEq/L) was higher in the PPHA group than in the non-PPHA group (55.6% vs. 8.2%, p < 0.01). In conclusion, a few patients with APA long time after adrenalectomy had unmeasurable PAC using CLEIA. PPHA is likely to develop in patients with APA after adrenalectomy who are older and have impaired renal function. Additionally, PPHA is related to the occurrence of postoperative hyperkalemia.

Subacute thyroiditis associated with thyrotoxic periodic paralysis after COVID-19 vaccination: a case report.

Summary: We report a 26-year-old Japanese man who visited our outpatient clinic presenting fever immediately after i.m. injection of the second dose of a coronavirus disease 2019 (COVID-19) vaccine (Moderna®). At the first visit, the patient had a fever of 37.7°C and a swollen thyroid gland with mild tenderness. He was diagnosed with subacute thyroiditis (SAT) based on the presence of thyrotoxicosis (free tri-iodothyronine, 32.3 pg/mL; free thyroxine, >7.77 ng/dL; and thyroid-stimulating hormone (TSH) < 0.01 µIU/mL), high C-reactive protein level (7.40 mg/dL), negative TSH receptor antibody, and characteristic ultrasound findings. His HLA types were A*02:01/24:02, B*15:11/35:01, Cw*03:03, DRB1*09:01/12:01, DQB1*03:03, and DPB1*05: 01/41:01. He was initially administered prednisolone 15 mg/day, following which the fever subsided. After 10 days, he developed limb weakness and could not walk. The serum potassium level decreased to 1.8 mEq/L, which confirmed the diagnosis of thyrotoxic periodic paralysis (TPP). Potassium supplementation was initiated. The muscle weakness gradually decreased. Prednisolone therapy was terminated 6 weeks after the first visit. His thyroid function returned to normal 5 months after the first visit, through a hypothyroid state. To our knowledge, this is the first reported case of TPP-associated SAT following COVID-19 vaccination. Persistent fever following vaccination should be suspected of SAT. Additionally, TPP may be associated with SAT in Asian male patients. Learning points: Following coronavirus disease 2019 (COVID-19) vaccination, subacute thyroiditis may develop regardless of the vaccine type. If persistent fever, anterior neck pain, swelling and tenderness of thyroid gland, and symptoms of thyrotoxicosis are observed immediately after the COVID-19 vaccination, examination in consideration of the onset of subacute thyroiditis is recommended. HLA-B35 may be associated with the onset of subacute thyroiditis after the COVID-19 vaccination. Although rare, subacute thyroiditis can be associated with thyrotoxic periodic paralysis, especially in Asian men. Glucocorticoid therapy for subacute thyroiditis may induce thyrotoxic periodic paralysis through hypokalemia.

Improved time in range and postprandial hyperglycemia with canagliflozin in combination with teneligliptin: Secondary analyses of the CALMER study.

AIMS/INTRODUCTION: We recently reported the beneficial effect of the combination of sodium-glucose cotransporter 2 inhibitor and dipeptidyl peptidase-4 inhibitor on daily glycemic variability in patients with type 2 diabetes mellitus. Additional favorable effects of combination therapy were explored in this secondary analysis. MATERIALS AND METHODS: The CALMER study was a multicenter, open-label, prospective, randomized, parallel-group comparison trial for type 2 diabetes mellitus involving continuous glucose monitoring under meal tolerance tests. Patients were randomly assigned to switch from teneligliptin to canagliflozin (SWITCH group) or to add canagliflozin to teneligliptin (COMB group). The continuous glucose monitoring metrics, including time in target range, were investigated. RESULTS: All 99 participants (mean age 62.3 years; mean glycated hemoglobin 7.4%) completed the trial. The time in target range was increased in the COMB group (71.2-82.7%, P < 0.001). The extent of the reduction in time above target range was significantly larger in the COMB group compared with the SWITCH group (-14.8% vs -7.5%, P < 0.01). Area under the curve values for glucose at 120 min after all meal tolerance tests were significantly decreased in the COMB group compared with the SWITCH group (P < 0.05). CONCLUSIONS: Sodium-glucose cotransporter 2 inhibitor combined with dipeptidyl peptidase-4 inhibitor improved the quality of glycemic variability and reduced postprandial hyperglycemia compared with each monotherapy.

Impact of endogenous insulin secretion on the improvement of glucose variability in Japanese patients with type 2 diabetes treated with canagliflozin plus teneligliptin.

AIMS/INTRODUCTION: To identify the effect of combination therapy with a dipeptidyl peptidase-4 inhibitor and a sodium-glucose cotransporter 2 inhibitor compared with switching from a dipeptidyl peptidase-4 inhibitor to a sodium-glucose cotransporter 2 inhibitor on improving the glucose variability in patients with or without impaired endogenous insulin secretion. MATERIALS AND METHODS: A secondary analysis regarding the relationship between endogenous insulin secretion and the change in mean amplitude of glycemic excursions (ΔMAGE) was carried out in a multicenter, prospective, randomized, parallel-group comparison trial that enrolled patients with type 2 diabetes who had been taking teneligliptin and were treated by switching to canagliflozin (SWITCH) or adding canagliflozin (COMB). Participants were categorized into the following four subgroups: SWITCH or COMB and high or low fasting C-peptide (CPR) divided at baseline by the median. RESULTS: ΔMAGE in the COMB group was greatly improved independent of a high or low CPR (-29.2 ± 28.3 vs -20.0 ± 24.6, respectively; P = 0.60). However, ΔMAGE was not ameliorated in the low CPR SWITCH group, and the ΔMAGE was significantly smaller than that in the high CPR COMB group (P < 0.01). CONCLUSIONS: COMB would be a better protocol rather than switching teneligliptin to canagliflozin to improve daily glucose variability in patients with impaired endogenous insulin secretion.

Prediction of unilateral hyperaldosteronism on adrenal vein sampling using captopril challenge test in patients with primary aldosteronism.

Captopril challenge test (CCT) is a simple and safe confirmatory test for primary aldosteronism (PA). We investigated the effectiveness of the indices after captopril administration for prediction of unilateral hyperaldosteronism (UHA) on adrenal vein sampling (AVS). We studied 238 patients with PA who had CCT and successful AVS between July 2007 and December 2019 in Sapporo City General Hospital. Receiver operating characteristic (ROC) curve analysis showed that the diagnostic performance for prediction of UHA on AVS in regard to the reduction rate of plasma aldosterone concentration (PAC) after captopril administration was inferior to aldosterone to renin ratio (ARR) and PAC (area under the ROC curve 0.72 vs. 0.84, 0.72 vs. 0.89, respectively, both p < 0.01). Based on the optimal cut-off values in ARR (897 pg/mL/ng/mL/h, sensitivity 64.6%, specificity 93.0%) and PAC (203 pg/mL, sensitivity 73.9%, specificity 93.0%) after captopril administration, the patients were divided into three groups: (1) both positive, (2) one positive, and (3) both negative. The prevalence of UHA on AVS in the three groups were 90.0%, 52.9%, and 7.3%, respectively. In the first group, 31 of 32 patients with unilateral nodular lesion on CT had an ipsilateral unilateral AVS. In conclusion, the combination of post-captopril ARR and PAC is useful for prediction of laterality diagnosis on AVS. AVS is strongly recommended in patients with both positive or one positive results for the optimal cut-off values of post-captopril ARR and PAC and is weakly recommended in patients with both negative results.

Initial dip predicts renal protective effects after the administration of sodium-glucose cotransporter 2 inhibitors in patients with type 2 diabetes and chronic kidney disease with normoalbuminuria.

INTRODUCTION: We investigated the renoprotective effects of sodium-glucose cotransporter 2 inhibitors (SGLT2is) on renal function in patients with type 2 diabetes and chronic kidney disease (CKD) with normoalbuminuria. METHODS: A retrospective review of clinical records of Japanese participants with type 2 diabetes and CKD (estimated glomerular filtration rate [eGFR] < 60 mL/min/1.73 m2) with normoalbuminuria (urine albumin to creatinine ratio < 30 mg/g Cr and/or urinary protein to creatinine ratio < 150 mg/g Cr) was conducted. Participants were categorized into two groups depending on whether they had started using SGLT2is. The main study outcome was a comparison of the change in renal function evaluated by eGFR after 1 year between the two groups. Then, we identified predictors that were associated with the outcome. RESULTS: Among the 46 participants, 21 were treated with SGLT2is (SGLT2 group) and 25 were treated with other antidiabetic medications (control group). Although eGFR was significantly decreased at 1 year in the control group, the decline in eGFR was not observed in the SGLT2 group. The decrease in eGFR was significantly smaller in the SGLT2 group than in the control group. Additionally, multiple linear regression analysis showed that an initial dip was an independent factor associated with the worsening of renal function in the SGLT2 group. CONCLUSIONS: Although more favorable effects of SGLT2is on renal function were observed in patients with type 2 diabetes and CKD with normoalbuminuria, the higher initial dip was a possible marker of worsening renal function after the initiation of SGLT2is.

Favourable effect of the sodium-glucose co-transporter-2 inhibitor canagliflozin plus the dipeptidyl peptidase-4 inhibitor teneligliptin in combination on glycaemic fluctuation: An open-label, prospective, randomized, parallel-group comparison trial (the CALMER study).

This multicentre, prospective, randomized, open-label, blinded-endpoint, parallel-group, short-term (4-5 weeks) controlled trial was conducted to investigate the superiority of the effect of reducing mean amplitude of glycaemic excursions (MAGE) during meal tolerance tests (MTTs) for the combination of dipeptidyl peptidase-4 (DPP-4) inhibitor and sodium-glucose co-transporter-2 (SGLT2) inhibitor compared with SGLT2 inhibitor monotherapy. Ninety-nine patients with type 2 diabetes who were taking teneligliptin (20 mg/d) were randomized to one of the following two groups: those who switched to 100 mg/d of canagliflozin (SWITCH group) or those who added 100 mg/d of canagliflozin (COMB group). MAGE in the COMB group was significantly decreased compared with that in the SWITCH group (COMB 117.5 ± 39.8 to 92.2 ± 28.0 mg/dL vs SWITCH 110.7 ± 29.8 to 104.2 ± 27.6 mg/dL; P<0.01). Mean blood glucose decreased significantly during MTTs in both groups, although the extent of the reduction was significantly greater in the COMB group (COMB 142.3 ± 28.7 to 119.5 ± 25.1 mg/dL vs SWITCH 146.4 ± 25.5 to 135.5 ± 22.4 mg/dL; P < 0.01). SGLT2 inhibitor combined with DPP-4 inhibitor therapy strongly reduced glycaemic fluctuation compared with SGLT2 inhibitor monotherapy.

A head-to-toe dimerization has physiological relevance for ligand-induced inactivation of protein tyrosine receptor type Z.

Protein-tyrosine phosphatase (PTPase) receptor type Z (PTPRZ) has two receptor isoforms, PTPRZ-A and -B, containing tandem intracellular PTP-D1 and -D2 domains, with only D1 being active. Pleiotrophin (PTN) binding to the extracellular PTPRZ region leads to inactivation of its PTPase activity, thereby facilitating oligodendrocyte precursor cell (OPC) differentiation and myelination in the central nervous system. However, the mechanisms responsible for PTN-induced PTPRZ inactivation remain unclear. We herein report that the crystal structure of the intracellular region of PTPRZ (PTPRZ-ICR) shows a "head-to-toe"-type dimer conformation, with D2 masking the catalytic site of D1. MS analyses revealed that PTPRZ-ICR proteins remain in monomer-dimer equilibrium in aqueous solution and that a substrate-derived inhibitory peptide or competitive inhibitor (SCB4380) specifically bind to the monomer form in a 1:1 ratio. A D2 deletion (ΔD2) or dimer interface mutation (DDKK) disrupted dimer formation, but SCB4380 binding was maintained. Similar to WT PTPRZ-B, monomer-biased PTPRZ-B-ΔD2 and PTPRZ-B-DDKK variants efficiently dephosphorylated p190RhoGAP at Tyr-1105 when co-expressed in BHK-21 cells. The catalytic activities of these variants were not suppressed by PTN treatment, but were inhibited by the cell-permeable PTPase inhibitor NAZ2329. Of note, the PTN treatment did not enhance OPC differentiation in primary cultured glial cells from ΔD2 or PTPase-inactive PTPRZ-B (CS) mutant knock-in mice. Our results thus indicate that PTN-induced PTPRZ inactivation results from dimer formation of the intracellular tandem PTP domains in a head-to-toe configuration, which is physiologically relevant to the control of OPC differentiation in vivo.

Discovery and structure-activity relationship of imidazolinylindole derivatives as kallikrein 7 inhibitors.

A series of imidazolinylindole derivatives were discovered as novel kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Structure-activity relationship (SAR) studies led to the identification of potent human KLK7 inhibitors. By further modification of the benzenesulfonyl moiety to overcome species differences in inhibitory activity, potent inhibitors against both human and mouse KLK7 were identified. Furthermore, the complex structure of 25 with mouse KLK7 could explain the SAR and the cause of the species differences in inhibitory activity.

Structure-based drug design to overcome species differences in kallikrein 7 inhibition of 1,3,6-trisubstituted 1,4-diazepan-7-ones.

A series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were prepared as kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Previously reported compounds 1-3 were potent human KLK7 inhibitors; however, they did not exhibit inhibitory activity against mouse KLK7. Comparison of the human and mouse KLK7 structures reveals the cause of this species differences; therefore, compounds that could inhibit both KLK7s were designed, synthesized, and evaluated. Through this structure-based drug design, compound 22g was identified as an inhibitor against human and mouse KLK7, and only one of the enantiomers, (-)-22g, exhibited potent inhibitory activity. Furthermore, the crystal structure of mouse KLK7 complexed with 22g enabled the elucidation of structure-activity relationships and justified 22g as a valuable compound to overcome the species differences.

Structure-based drug design of 1,3,6-trisubstituted 1,4-diazepan-7-ones as selective human kallikrein 7 inhibitors.

A novel series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were investigated as human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Based on the X-ray co-crystal structure of compound 1 bound to human KLK7, the derivatives of this scaffold were designed, synthesized, and evaluated. Through structure-activity relationship studies focused on the side chain located in the prime site region of the enzyme, representative compounds 15, 33a, and 35a were identified as highly potent and selective inhibitors of human KLK7.

Structure-based design, synthesis, and binding mode analysis of novel and potent chymase inhibitors.

Based on insight from the X-ray crystal structure of human chymase in complex with compound 1, a lactam carbonyl of the diazepane core was exchanged with O-substituted oxyimino group, leading to amidoxime derivatives. This modification resulted in highly potent chymase inhibitors, such as O-phenylamidoxime 5f. X-ray crystal structure analysis indicated that compound 5f induced movement of the Leu99 and Tyr94 side chains at the S2 site, and the increase in inhibitory activity of O-phenyl amidoxime derivatives suggested that the O-phenyl moiety interacted with the Tyr94 residue. Surface plasmon resonance experiments showed that compound 5f had slower association and dissociation kinetics and the calculated residence time of compound 5f to human chymase was extended compared to that of amide compound 1.

Discovery and structure-activity relationship study of 1,3,6-trisubstituted 1,4-diazepane-7-ones as novel human kallikrein 7 inhibitors.

Compound 1, composed of a 1,3,6-trisubstituted 1,4-diazepane-7-one, was discovered as a novel human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme, SCCE) inhibitor, and its derivatives were synthesized and evaluated. Structure-activity relationship studies of the amidoxime unit and benzoic acid part of this new scaffold led to the identification of 25 and 34, which were more potent than the hit compound, 1. The X-ray co-crystal structure of compound 25 and human KLK7 revealed the characteristic interactions and enabled explanations of the structure-activity relationship.

A randomized controlled trial comparing the effects of dapagliflozin and DPP-4 inhibitors on glucose variability and metabolic parameters in patients with type 2 diabetes mellitus on insulin.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) inhibitors and sodium-glucose co-transporter 2 (SGLT2) inhibitors improve hyperglycemia, and the usefulness of co-administration of DPP-4 inhibitors and insulin therapy has been well established. However, it has been still uncertain whether combination therapy of SGLT2 inhibitors and insulin is superior to that of DPP-4 inhibitors and the latter. Therefore, we investigated the superiority of dapagliflozin on glucose fluctuation compared with DPP-4 inhibitors in patients with type 2 diabetes mellitus (T2DM) on insulin using a continuous glucose monitoring (CGM) system. METHODS: In this prospective, randomized, open-label controlled trial, 36 patients with T2DM and treated with DPP-4 inhibitors and insulin therapy, were enrolled and allocated into two groups. The patients either switched their DPP-4 inhibitors to dapagliflozin 5 mg for 12 weeks, or continued their DPP-4 inhibitors for the same period. CGM analyses and metabolic markers were assessed before and after treatment periods. RESULTS: In total, data from 29 patients were analyzed. There were no significant differences in the mean amplitude of glycemic excursions and other CGM profiles in either group after treatment. Within the dapagliflozin treatment group, significant reductions of body mass index and albuminuria, and increases of HbA1c, hemoglobin and hematocrit were observed, but improvement of albuminuria was not significant if compared with the DPP-4 continuation group. CONCLUSIONS: Combination therapy of dapagliflozin and insulin was not superior in glucose fluctuation to DPP-4 inhibitors on insulin. However, dapagliflozin may in part provide favorable effects on metabolism in patients with T2DM treated with insulin therapy. Trial registration UMIN-CTR: UMIN000015033. Registered 2 September 2014.

Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells.

The R5 subfamily of receptor-type protein tyrosine phosphatases (RPTPs) comprises PTPRZ and PTPRG. A recent study on primary human glioblastomas suggested a close association between PTPRZ1 (human PTPRZ) expression and cancer stemness. However, the functional roles of PTPRZ activity in glioma stem cells have remained unclear. In the present study, we found that sphere-forming cells from the rat C6 and human U251 glioblastoma cell lines showed high expression levels of PTPRZ-B, the short receptor isoform of PTPRZ. Stable PTPRZ knockdown altered the expression levels of stem cell transcription factors such as SOX2, OLIG2, and POU3F2 and decreased the sphere-forming abilities of these cells. Suppressive effects on the cancer stem-like properties of the cells were also observed following the knockdown of PTPRG. Here, we identified NAZ2329, a cell-permeable small molecule that allosterically inhibits both PTPRZ and PTPRG. NAZ2329 reduced the expression of SOX2 in C6 and U251 cells and abrogated the sphere-forming abilities of these cells. Tumor growth in the C6 xenograft mouse model was significantly slower with the co-treatment of NAZ2329 with temozolomide, an alkylating agent, than with the individual treatments. These results indicate that pharmacological inhibition of R5 RPTPs is a promising strategy for the treatment of malignant gliomas.

Ipragliflozin effectively reduced visceral fat in Japanese patients with type 2 diabetes under adequate diet therapy.

To investigate if ipragliflozin, a novel sodium-glucose co-transporter 2 inhibitor, alters body composition and to identify variables associated with reductions in visceral adipose tissue in Japanese patients with type 2 diabetes mellitus. This prospective observational study enrolled Japanese participants with type 2 diabetes mellitus. Subjects were administered ipragliflozin (50 mg/day) once daily for 16 weeks. Body composition, visceral adipose tissue volume and plasma variables were measured at 0, 8, and 16-weeks. The subjects' lifestyle habits including diet and exercise were evaluated at baseline and 16 weeks. The primary endpoint was defined as the decrease of visceral adipose tissue mass. Twenty-four of 26 enrolled participants completed the study. The visceral adipose tissue decreased significantly (110 ± 33 to 101 ± 36 cm(2), p = 0.005) as well as other parameters for metabolic insufficiency including hemoglobin A1c. Seventy-one % of the total body weight reduction (-2.49 kg) was estimated by a decrease in fat mass (-1.77 kg), and the remaining reduction (22%) by water volume (-0.55 kg). A minor but significant reduction in the skeletal muscle index was also observed. Correlation analyses were performed to identify variables associated with changes in visceral adipose tissue and the only significant variable identified was diet therapy (Spearman's r = -0.416, p = 0.043). Ipragliflozin significantly decreased visceral adipose tissue, and improved parametres for metabolic dysfunction. Adequate diet therapy would be necessary to induce and enhance the therapeutic merit.

Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.

Protein tyrosine phosphatase receptor-type Z (PTPRZ) is aberrantly over-expressed in glioblastoma and a causative factor for its malignancy. However, small molecules that selectively inhibit the catalytic activity of PTPRZ have not been discovered. We herein performed an in vitro screening of a chemical library, and identified SCB4380 as the first potent inhibitor for PTPRZ. The stoichiometric binding of SCB4380 to the catalytic pocket was demonstrated by biochemical and mass spectrometric analyses. We determined the crystal structure of the catalytic domain of PTPRZ, and the structural basis of the binding of SCB4380 elucidated by a molecular docking method was validated by site-directed mutagenesis studies. The intracellular delivery of SCB4380 by liposome carriers inhibited PTPRZ activity in C6 glioblastoma cells, and thereby suppressed their migration and proliferation in vitro and tumor growth in a rat allograft model. Therefore, selective inhibition of PTPRZ represents a promising approach for glioma therapy.

Discovery of novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as human chymase inhibitors using structure-based drug design.

A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones were explored as human chymase inhibitors using structure-based drug design according to the X-ray cocrystal structure of chymase and compound 1. The optimization focused on the prime site led to the attainment of compounds that showed potent inhibitory activity, and among them, 18R shows a novel interaction mode.

Structural insight into the reaction mechanism and evolution of cytokinin biosynthesis.

The phytohormone cytokinin regulates plant growth and development. This hormone is also synthesized by some phytopathogenic bacteria, such as Agrobacterium tumefaciens, and is as a key factor in the formation of plant tumors. The rate-limiting step of cytokinin biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferase (IPT). Agrobacterium IPT has a unique substrate specificity that enables it to increase trans-zeatin production by recruiting a metabolic intermediate of the host plant's biosynthetic pathway. Here, we show the crystal structures of Tzs, an IPT from A. tumefaciens, complexed with AMP and a prenyl-donor analogue, dimethylallyl S-thiodiphosphate. The structures reveal that the carbon-nitrogen-based prenylation proceeds by the SN2-reaction mechanism. Site-directed mutagenesis was used to determine the amino acid residues, Asp-173 and His-214, which are responsible for differences in prenyl-donor substrate specificity between plant and bacterial IPTs. IPT and the p loop-containing nucleoside triphosphate hydrolases likely evolved from a common ancestral protein. Despite structural similarities, IPT has evolved a distinct role in which the p loop transfers a prenyl moiety in cytokinin biosynthesis.